C-Reactive Protein Impairs Dendritic Cell Development, Maturation, and Function: Implications for Peripheral Tolerance.

C-reactive protein (CRP) is the prototypical acute phase reactant, increasing in blood concentration rapidly and several-fold in response to inflammation. Recent evidence indicates that CRP has an important physiological role even at low, baseline levels, or in the absence of overt inflammation. For example, we have shown that human CRP inhibits the progression of experimental autoimmune encephalomyelitis (EAE) in CRP transgenic mice by shifting CD4+ T cells away from the TH1 and toward the TH2 subset. Notably, this action required the inhibitory Fcγ receptor IIB (FcγRIIB), but did not require high levels of human CRP. Herein, we sought to determine if CRP's influence in EAE might be explained by CRP acting on dendritic cells (DC; antigen presenting cells known to express FcγRIIB). We found that CRP (50 µg/ml) reduced the yield of CD11c+ bone marrow-derived DCs (BMDCs) and CRP (≥5 µg/ml) prevented their full expression of major histocompatibility complex class II and the co-stimulatory molecules CD86 and CD40. CRP also decreased the ability of BMDCs to stimulate antigen-driven proliferation of T cells in vitro. Importantly, if the BMDCs were genetically deficient in mouse FcγRIIB then (i) the ability of CRP to alter BMDC surface phenotype and impair T cell proliferation was ablated and (ii) CD11c-driven expression of a human FCGR2B transgene rescued the CRP effect. Lastly, the protective influence of CRP in EAE was fully restored in mice with CD11c-driven human FcγRIIB expression. These findings add to the growing evidence that CRP has important biological effects even in the absence of an acute phase response, i.e., CRP acts as a tonic suppressor of the adaptive immune system. The ability of CRP to suppress development, maturation, and function of DCs implicates CRP in the maintenance of peripheral T cell tolerance.

Enantioselective Synthesis of 4'-Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) via Enzymatic Desymmetrization.

An enantioselective synthesis of the potent anti-HIV nucleoside EFdA is presented. Key features of stereocontrol include construction of the fully substituted 4'-carbon via a biocatalytic desymmetrization of 2-hydroxy-2-((triisopropylsilyl)ethynyl)propane-1,3-diyl diacetate and a Noyori-type asymmetric transfer hydrogenation to control the stereochemistry of the 3'-hydroxyl bearing carbon. The discovery of a selective crystallization of an N-silyl nucleoside intermediate enabled isolation of the desired ß-anomer from the glycosylation step.

Regulation of FcRγ function by site-specific serine phosphorylation.

The common FcRγ, an immunoreceptor tyrosine-based activation motif (ITAM)- containing adaptor protein, associates with multiple leukocyte receptor complexes and mediates signal transduction through the ITAM in the cytoplasmic domain. The presence of multiple serine and threonine residues within this motif suggests the potential for serine/threonine phosphorylation in modulating signaling events. Single-site mutational analysis of these residues in RBL-2H3 cells indicates that each may contribute to net FcRγ-mediated signaling, and mass spectrometry of WT human FcRγ from receptor-stimulated cells shows consistent preferential phosphorylation of the serine residue at position 51. Immunoblot analysis, mass spectrometry, and mutational analyses showed that phosphorylation of serine 51 in the 7-residue spacer between the 2 YxxL sequences regulates FcRγ signaling by inhibiting tyrosine phosphorylation at the membrane proximal Y47 position of the ITAM, but not phosphorylation at position Y58. This inhibition results in reduced Syk recruitment and activation. With in vitro kinase assays, PKC-δ and PKA show preferential phosphorylation of S51. Serine/threonine phosphorylation of the FcRγ ITAM, which functions as an integrator of multiple signaling elements, may explain in part the contribution of variants in PKC-δ and other PKC isoforms to some autoimmune phenotypes.

Estimation of Cell-Type Composition Including T and B Cell Subtypes for Whole Blood Methylation Microarray Data.

DNA methylation levels vary markedly by cell-type makeup of a sample. Understanding these differences and estimating the cell-type makeup of a sample is an important aspect of studying DNA methylation. DNA from leukocytes in whole blood is simple to obtain and pervasive in research. However, leukocytes contain many distinct cell types and subtypes. We propose a two-stage model that estimates the proportions of six main cell types in whole blood (CD4+ T cells, CD8+ T cells, monocytes, B cells, granulocytes, and natural killer cells) as well as subtypes of T and B cells. Unlike previous methods that only estimate overall proportions of CD4+ T cell, CD8+ T cells, and B cells, our model is able to estimate proportions of naïve, memory, and regulatory CD4+ T cells as well as naïve and memory CD8+ T cells and naïve and memory B cells. Using real and simulated data, we are able to demonstrate that our model is able to reliably estimate proportions of these cell types and subtypes. In studies with DNA methylation data from Illumina's HumanMethylation450k arrays, our estimates will be useful both for testing for associations of cell type and subtype composition with phenotypes of interest as well as for adjustment purposes to prevent confounding in epigenetic association studies. Additionally, our method can be easily adapted for use with whole genome bisulfite sequencing (WGBS) data or any other genome-wide methylation data platform.

Human FcR polymorphism and disease.

Fc receptors play a central role in maintaining the homeostatic balance in the immune system. Our knowledge of the structure and function of these receptors and their naturally occurring polymorphisms, including single nucleotide polymorphisms and/or copy number variations, continues to expand. Through studies of their impact on human biology and clinical phenotype, the contributions of these variants to the pathogenesis, progression, and/or treatment outcome of many diseases that involve immunoglobulin have become evident. They affect susceptibility to bacterial and viral pathogens, constitute as risk factors for IgG or IgE mediated inflammatory diseases, and impact the development of many autoimmune conditions. In this chapter, we will provide an overview of these genetic variations in classical FcγRs, FcRLs, and other Fc receptors, as well as challenges in achieving an accurate and comprehensive understanding of the FcR polymorphisms and genomic architecture.

The unique cytoplasmic domain of human FcγRIIIA regulates receptor-mediated function.

Ligand specificity characterizes receptors for Abs and many other immune receptors, but the common use of the FcR γ-chain as their signaling subunit challenges the concept that these receptors are functionally distinct. We hypothesized that elements for specificity might be determined by the unique cytoplasmic domain (CY) sequences of the ligand-binding α-chains of γ-chain-associated receptors. Among Fcγ receptors, a protein kinase C (PKC) phosphorylation consensus motif [RSSTR], identified within the FcγRIIIa (CD16A) CY by in silico analysis, is specifically phosphorylated by PKCs, unlike other FcRs. Phosphorylated CD16A mediates a more robust calcium flux, tyrosine phosphorylation of Syk, and proinflammatory cytokine production, whereas nonphosphorylatable CD16A is more effective at activation of the Gab2/PI3K pathway, leading to enhanced degranulation. S100A4, a specific protein-binding partner for CD16A-CY newly identified by yeast two-hybrid analysis, inhibits phosphorylation of CD16A-CY by PKC in vitro, and reduction of S100A4 levels in vivo enhances receptor phosphorylation upon cross-linking. Taken together, PKC-mediated phosphorylation of CD16A modulates distinct signaling pathways engaged by the receptor. Calcium-activated binding of S100A4 to CD16A, promoted by the initial calcium flux, attenuates the phosphorylation of CY, and, acting as a molecular switch, may both serve as a negative feedback on cytokine production pathways during sustained receptor engagement and favor a shift to degranulation, consistent with the importance of granule release following conjugate formation between CD16A(+) effector cells and target cells. This switch mechanism points to new therapeutic targets and provides a framework for understanding novel receptor polymorphisms.

Enantioselective synthesis of ß-aryloxycarboxylic esters via asymmetric hydrogenation of ß-aryloxy-α,ß-unsaturated esters.

A novel synthesis of ß-aryloxycarboxylic esters via asymmetric hydrogenation of the corresponding ß-aryloxy-α,ß-unsaturated esters has been demonstrated. Bis(norbornadiene)rhodium(I) tetrafluoroborate (1 mol %) and Walphos W008-1 were used to generate the saturated products with high enantioselectivity and in high yield. The tolerability of the reaction to a diverse range of substituents on the aromatic ring was also explored.

Serine phosphorylation of FcγRI cytoplasmic domain directs lipid raft localization and interaction with protein 4.1G.

The high-affinity IgG receptor (CD64, FcγRI) has several special capacities, including the receptor-stimulated cleavage of the cell surface B cell-activating factor of the TNF superfamily (TNFSF13B). With the use of the yeast two-hybrid system, we and others have shown that FcγRI interacts with protein 4.1G (EPB41L2). Our mutational analyses identified two required 4.1G-interacting regions in the FcγRI CY and one FcγRI-interacting site in the C-terminus of protein 4.1G. Herein, we explore mechanism(s) that may regulate the interaction between protein 4.1G and FcγRI CY and influence FcγRI membrane mobility and function. We show that FcγRI CY interacts with protein 4.1G in vitro and that FcγRI coimmunoprecipitates protein 4.1G in freshly isolated human PBMC. With the use of immunostaining, we show that FcγRI colocalizes with protein 4.1G in unstimulated U937 cells, in which the FcγRI CY is constitutively serine-phosphorylated, but significant uncoupling occurs following FcγRI cross-linking, suggesting phosphoserine-regulated interaction. In vitro, protein 4.1G interacted preferentially with CK2-phosphorylated FcγRI CY, and compared with WT FcγRI, a nonphosphorylatable FcγRI mutant receptor was excluded from lipid rafts, suggesting a key role for protein 4.1G in targeting phosphorylated FcγRI to rafts. These data are consistent with a phosphoserine-dependent tethering role for protein 4.1G in maintaining FcγRI in lipid rafts and provide insight into the unique phosphoserine-based regulation of receptor signaling by FcγRI CY.

Human FasL gene is a target of ß-catenin/T-cell factor pathway and complex FasL haplotypes alter promoter functions.

FasL expression on human immune cells and cancer cells plays important roles in immune homeostasis and in cancer development. Our previous study suggests that polymorphisms in the FasL promoter can significantly affect the gene expression in human cells. In addition to the functional FasL SNP -844C>T (rs763110), three other SNPs (SNP -756A>G or rs2021837, SNP -478A>T or rs41309790, and SNP -205 C>G or rs74124371) exist in the proximal FasL promoter. In the current study, we established three major FasL hyplotypes in humans. Interestingly, a transcription motif search revealed that the FasL promoter possessed two consensus T-cell factor (TCF/LEF1) binding elements (TBEs), which is either polymorphic (SNP -205C>G) or close to the functional SNP -844C>T. Subsequently, we demonstrate that both FasL TBEs formed complexes with the TCF-4 and ß-catenin transcription factors in vitro and in vivo. Co-transfection of LEF-1 and ß-catenin transcription factors significantly increased FasL promoter activities, suggesting that FasL is a target gene of the ß-catenin/T-cell factor pathway. More importantly, we found that the rare allele (-205G) of the polymorphic FasL TBE (SNP -205C>G) failed to bind the TCF-4 transcription factor and that SNP -205 C>G significantly affected the promoter activity. Furthermore, promoter reporter assays revealed that FasL SNP haplotypes influenced promoter activities in human colon cancer cells and in human T cells. Finally, ß-catenin knockdown significantly decreased the FasL expression in human SW480 colon cancer cells. Collectively, our data suggest that ß-catenin may be involved in FasL gene regulation and that FasL expression is influenced by FasL SNP haplotypes, which may have significant implications in immune response and tumorigenesis.

FAS mRNA editing in Human Systemic Lupus Erythematosus.

FAS/FASL system plays a central role in maintaining peripheral immune tolerance. Human Systematic Lupus Erythematosus (SLE) is a prototypic systemic autoimmune disease characterized by expansion of autoreactive lymphocytes. It remains unclear whether a defective FAS/FASL system is involved in the pathogenesis of SLE. In this study, we have discovered a novel nucleotide insertion in FAS mRNA. We demonstrate that this novel FAS mutation occurs at mRNA levels, likely through a site-specific mRNA editing process. The mRNA editing mutation is unique for human FAS because the similar mRNA editing event is absent in other human TNF receptor (TNFR) family genes with death domains (DR5, DR6, and TNFR1) and in murine FAS. The adenine insertion mutation in the coding region message causes the alteration of human FAS mRNA reading frame. Functionally, cells expressing the edited FAS (edFAS) were refractory to FAS-mediated apoptosis. Surprisingly, cells from SLE patients produced significantly more edFAS products compared to cells from normal healthy controls. Additionally, we demonstrated that persistent engagement of T-cell receptor increases human FAS mRNA editing in human T cells. Our data suggest that the site-specific FAS mRNA editing mutation may play a critical role in human immune responses and in the pathogenesis of human chronic inflammatory diseases.

Functional expression of IgA receptor FcalphaRI on human platelets.

FcalphaRI (CD89) is a human IgA FcR expressed on cells of myeloid lineage such as neutrophils, monocytes, tissue macrophages, eosinophils, and subpopulations of dendritic cells. FcalphaRI mediates cell activation through Src family kinases and downstream tyrosine-based phosphorylation pathways. However, the role of IgA and the expression and role of its cognate receptor FcalphaRI (CD89) in platelet activation are undefined. In the current study, we demonstrate that human platelets express FcalphaRI mRNAs and proteins. Furthermore, we show that the platelet FcalphaRI is associated with the FcR gamma-chain, and cross-linking of FcalphaRI leads to Syk phosphorylation. Clustering of FcalphaRI induces pre-mRNA splicing and protein production of tissue factor and IL-1beta, suggesting novel roles for human platelet FcalphaRI and serum IgA in thrombosis and inflammation.

A practical synthesis of a gamma-secretase inhibitor.

A practical and scaleable synthesis of the gamma-secretase inhibitor 1 is reported. The inhibitor consists of a central trisubstituted cyclohexane core with appended propionic acid, 2,5-difluorophenyl, and 4-chlorophenylsulfonyl moieties. Two alternative synthetic strategies, proceeding by way of a common disubstituted cyclohexanone derivative 5, were studied. In the preferred route, conjugate reduction of acrylonitrile derivative 4 with L-Selectride configures the desired relative stereochemistry of the cyclohexane core with >99.9:0.1 dr. A second strategy, based on catalyst-controlled hydrogenation of racemic cyclohexene derivative 2, is more convergent but less diastereoselective (up to 75:25 dr). The common cyclohexanone intermediate 5 was constructed by a regioselective Diels-Alder condensation of a 1,1-disubstituted vinyl sulfone 6 with 2-trimethylsiloxybutadiene.

FcalphaRI (CD89) alleles determine the proinflammatory potential of serum IgA.

The human IgA FcR (FcalphaRI; CD89) mediates a variety of immune system functions including degranulation, endocytosis, phagocytosis, cytokine synthesis, and cytokine release. We have identified a common, nonsynonymous, single nucleotide polymorphism (SNP) in the coding region of CD89 (844A-->G) (rs16986050), which changes codon 248 from AGC (Ser(248)) to GGC (Gly(248)) in the cytoplasmic domain of the receptor. The two different alleles demonstrate significantly different FcalphaRI-mediated intracellular calcium mobilization and degranulation in rat basophilic leukemia cells and cytokine production (IL-6 and TNF-alpha) in murine macrophage P388D1 cells. In the absence of FcR gamma-chain association in P388D1 cells, the Ser(248)-FcalphaRI allele does not mediate cytokine production, but the Gly(248)-FcalphaRI allele retains the capacity to mediate a robust production of proinflammatory cytokine. This allele-dependent difference is also seen with FcalphaRI-mediated IL-6 cytokine release by human neutrophils ex vivo. These findings and the enrichment of the proinflammatory Gly(248)-FcalphaRI allele in systemic lupus erythematosus populations in two ethnic groups compared with their respective non-systemic lupus erythematosus controls suggest that FcalphaRI (CD89) alpha-chain alleles may affect receptor-mediated signaling and play an important role in the modulation of immune responses in inflammatory diseases.

Expression profile of FcgammaRIIb on leukocytes and its dysregulation in systemic lupus erythematosus.

FcgammaRIIb (CD32B, Online Mendelian Inheritance in Man 604590), an IgG FcR with a tyrosine-based inhibitory motif, plays a critical role in the balance of tolerance and autoimmunity in murine models. However, the high degree of homology between FcgammaRIIb and FcgammaRIIa in humans and the lack of specific Abs to differentiate them have hampered study of the normal expression profile of FcgammaRIIb and its potential dysregulation in autoimmune diseases such as systemic lupus erythematosus (SLE). Using our newly developed anti-FcgammaRIIb mAb 4F5 which does not react with FcgammaRIIa, we found that FcgammaRIIb is expressed on the cell surface of circulating B lymphocytes, monocytes, neutrophils, myeloid dendritic cells (DCs), and at very low levels on plasmacytoid DCs from some donors. Normal donors with the less frequent 2B.4 promoter haplotype have higher FcgammaRIIb expression on monocytes, neutrophils, and myeloid DCs similar to that reported for B lymphocytes, indicating that FcgammaRIIb expression on both myeloid and lymphoid cells is regulated by the naturally occurring regulatory single nucleotide polymorphisms in the FCGR2B promoter. FcgammaRIIb expression in normal controls is up-regulated on memory B lymphocytes compared with naive B lymphocytes. In contrast, in active SLE, FcgammaRIIb is significantly down-regulated on both memory and plasma B lymphocytes compared with naive and memory/plasma B lymphocytes from normals. Similar down-regulation of FcgammaRIIb on myeloid-lineage cells in SLE was not seen. Our studies demonstrate the constitutive regulation of FcgammaRIIb by natural gene polymorphisms and the acquired dysregulation in SLE autoimmunity, which may identify opportunities for using this receptor as a therapeutic target.

Differential gene expression modulated by the cytoplasmic domain of Fc gamma RIa (CD64) alpha-chain.

The cytoplasmic domain (CY) of the ligand-binding alpha-chain of the gamma-chain-associated FcRs can modulate receptor function such as phagocytosis, endocytosis, and intracellular trafficking of receptor-Ag complexes. To assess the potential role of the CY domain of human FcgammaRIa (CD64) alpha-chain in the transcriptional regulation of receptor-induced gene expression, we developed stably transfected murine macrophage cell lines expressing a full-length or a CY deletion mutant (tail-less) of human FcgammaRIa to analyze gene expression in response to receptor-specific cross-linking. Using the Affymetrix murine genome U74Av2 GeneChip array, we observed >100 candidate genes having > or =2-fold difference expression at 1.5 and 3 h after stimulation. Focusing on several immunologically related genes, we confirmed differential expression of M-CSF, macrophage inhibitory cytokine-1, leukocyte-specific protein 1, MIP-2, and IL-1R antagonist by RT-PCR and RNase protection assays. Analysis of mRNA stability indicated that the differential regulation of gene expression by the CY of the CD64 alpha-chain is at the level of gene transcription. Our results indicate that the CY of the CD64 alpha-chain modulates transcriptional activity induced by receptor-specific engagement in macrophages and provides a framework for understanding distinct expression profiles elicited by different Fc gamma-chain-associated receptors.

A novel polymorphic CAAT/enhancer-binding protein beta element in the FasL gene promoter alters Fas ligand expression: a candidate background gene in African American systemic lupus erythematosus patients.

A single-nucleotide polymorphism (SNP), identified at nucleotide position -844 in the 5' promoter of the FasL gene, lies within a putative binding motif for CAAT/enhancer-binding protein beta (C/EBPbeta). Electrophoretic mobility shift and supershift assays confirmed that this element binds specifically to C/EBPbeta and demonstrated that the two alleles of this element have different affinities for C/EBPbeta. In luciferase reporter assays, the -844C genotype had twice the basal activity of the -844T construct, and basal expression of Fas ligand (FasL) on peripheral blood fibrocytes was also significantly higher in -844C than in -844T homozygous donors. FasL is located on human chromosome 1q23, a region that shows linkage to the systemic lupus autoimmune phenotype. Analysis of 211 African American systemic lupus erythematosus patients revealed enrichment of the -844C homozygous genotype in these systemic lupus erythematosus patients compared with 150 ethnically matched normal controls (p = 0.024). The -844C homozygous genotype may lead to the increased expression of FasL, to altered FasL-mediated signaling in lymphocytes, and to enhanced risk for autoimmunity. This functionally significant SNP demonstrates the potential importance of SNPs in regulatory regions and suggests that differences in the regulation of FasL expression may contribute to the development of the autoimmune phenotype.

Fc receptor homologs: newest members of a remarkably diverse Fc receptor gene family.

Newfound relatives of the classical Fc receptors (FcR) have been provisionally named the Fc receptor homologs (FcRH). The recent identification of eight human and six mouse FcRH genes substantially increases the size and functional potential of the FcR family. The extended family of FcR and FcRH genes spans approximately 15 Mb of the human chromosome 1q21-23 region, whereas in mice this family is split between chromosomes 1 and 3. The FcRH genes encode molecules with variable combinations of five subtypes of immunoglobulin (Ig) domains. The presence of a conserved sequence motif in one Ig domain subtype implies Ig Fc binding capability for many FcRH family members that are preferentially expressed by B lineage cells. In addition, most FcRH family members have consensus tyrosine-based activating and inhibitory motifs in their cytoplasmic domains, while the others lack features typical of transmembrane receptors. The FcRH family members, like the classical FcRs, come in multiple isoforms and allelic variations. The unique individual and polymorphic properties of the FcR/FcRH members indicate a remarkably diverse Fc receptor gene family with immunoregulatory function.

The CY domain of the Fcgamma RIa alpha-chain (CD64) alters gamma-chain tyrosine-based signaling and phagocytosis.

Although the cytoplasmic domain of the human FcgammaRIa alpha-chain lacks tyrosine-based phosphorylation motifs, it modulates receptor cycling and receptor-specific cytokine production. The cytoplasmic domain of FcgammaRIa is constitutively phosphorylated, and the inhibition of dephosphorylation with okadaic acid, an inhibitor of type 1 and type 2A protein serine/threonine phosphatase, inhibits both receptor-induced activation of the early tyrosine phosphorylation cascade and receptor-specific phagocytosis. To explore the basis for these effects of the cytoplasmic domain of FcgammaRIa, we developed a series of human FcgammaRIa molecular variants, expressed in the murine macrophage cell line P388D1, and demonstrate that serine phosphorylation of the cytoplasmic domain is an important regulatory mechanism. Truncation of the cytoplasmic domain and mutation of the cytoplasmic domain serine residues to alanine abolish the okadaic acid inhibition of phagocytic function. In contrast, the serine mutants did not recapitulate the selective effects of cytoplasmic domain truncation on cytokine production. These results demonstrate for the first time a direct functional role for serine phosphorylation in the alpha-chain of FcgammaRIa and suggest that the cytoplasmic domain of FcgammaRI regulates the different functional capacities of the FcgammaRIa-receptor complex.