Role of MYH9 and APOL1 in African and non-African populations with lupus nephritis.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease characterized by autoantibody production and organ damage. Lupus nephritis (LN) is one of the most severe manifestations of SLE. Multiple studies reported associations between renal diseases and variants in the non-muscle myosin heavy chain 9 (MYH9) and the neighboring apolipoprotein L 1 (APOL1) genes. We evaluated 167 variants spanning MYH9 for association with LN in a multiethnic sample. The two previously identified risk variants in APOL1 were also tested for association with LN in European-Americans (EAs) (N = 579) and African-Americans (AAs) (N = 407). Multiple peaks of association exceeding a Bonferroni corrected P-value of P < 2.03 × 10(-3) were observed between LN and MYH9 in EAs (N = 4620), with the most pronounced association at rs2157257 (P = 4.7 × 10(-4), odds ratio (OR) = 1.205). A modest effect with MYH9 was also detected in Gullah (rs8136069, P = 0.0019, OR = 2.304). No association between LN and MYH9 was found in AAs, Asians, Amerindians or Hispanics. This study provides the first investigation of MYH9 in LN in non-Africans and of APOL1 in LN in any population, and presents novel insight into the potential role of MYH9 in LN in EAs.

A functional haplotype of UBE2L3 confers risk for systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease with diverse clinical manifestations characterized by the development of pathogenic autoantibodies manifesting in inflammation of target organs such as the kidneys, skin and joints. Genome-wide association studies have identified genetic variants in the UBE2L3 region that are associated with SLE in subjects of European and Asian ancestry. UBE2L3 encodes an ubiquitin-conjugating enzyme, UBCH7, involved in cell proliferation and immune function. In this study, we sought to further characterize the genetic association in the region of UBE2L3 and use molecular methods to determine the functional effect of the risk haplotype. We identified significant associations between variants in the region of UBE2L3 and SLE in individuals of European and Asian ancestry that exceeded a Bonferroni-corrected threshold (P<1 × 10(-4)). A single risk haplotype was observed in all associated populations. Individuals harboring the risk haplotype display a significant increase in both UBE2L3 mRNA expression (P=0.0004) and UBCH7 protein expression (P=0.0068). The results suggest that variants carried on the SLE-associated UBE2L3 risk haplotype influence autoimmunity by modulating UBCH7 expression.

The role of HLA-DR-DQ haplotypes in variable antibody responses to anthrax vaccine adsorbed.

Host genetic variation, particularly within the human leukocyte antigen (HLA) loci, reportedly mediates heterogeneity in immune response to certain vaccines; however, no large study of genetic determinants of anthrax vaccine response has been described. We searched for associations between the immunoglobulin G antibody to protective antigen (AbPA) response to Anthrax Vaccine Adsorbed (AVA) in humans, and polymorphisms at HLA class I (HLA-A, -B, and -C) and class II (HLA-DRB1, -DQA1, -DQB1, -DPB1) loci. The study included 794 European-Americans and 200 African-Americans participating in a 43-month, double-blind and placebo-controlled clinical trial of AVA (clinicaltrials.gov identifier NCT00119067). Among European-Americans, genes from tightly linked HLA-DRB1, -DQA1, -DQB1 haplotypes displayed significant overall associations with longitudinal variation in AbPA levels at 4, 8, 26 and 30 weeks from baseline in response to vaccination with three or four doses of AVA (global P=6.53 × 10(-4)). In particular, carriage of the DRB1-DQA1-DQB1 haplotypes (*)1501-(*)0102-(*)0602 (P=1.17 × 10(-5)), (*)0101-(*)0101-(*)0501 (P=0.009) and (*)0102-(*)0101-(*)0501 (P=0.006) was associated with significantly lower AbPA levels. In carriers of two copies of these haplotypes, lower AbPA levels persisted following subsequent vaccinations. No significant associations were observed amongst African-Americans or for any HLA class I allele/haplotype. Further studies will be required to replicate these findings and to explore the role of host genetic variation outside of the HLA region.

Evaluation of the TREX1 gene in a large multi-ancestral lupus cohort.

Systemic lupus erythematosus (SLE) is a prototypic autoimmune disorder with a complex pathogenesis in which genetic, hormonal and environmental factors have a role. Rare mutations in the TREX1 gene, the major mammalian 3'-5' exonuclease, have been reported in sporadic SLE cases. Some of these mutations have also been identified in a rare pediatric neurological condition featuring an inflammatory encephalopathy known as Aicardi-Goutières syndrome (AGS). We sought to investigate the frequency of these mutations in a large multi-ancestral cohort of SLE cases and controls. A total of 40 single-nucleotide polymorphisms (SNPs), including both common and rare variants, across the TREX1 gene, were evaluated in ∼8370 patients with SLE and ∼7490 control subjects. Stringent quality control procedures were applied, and principal components and admixture proportions were calculated to identify outliers for removal from analysis. Population-based case-control association analyses were performed. P-values, false-discovery rate q values, and odds ratios (OR) with 95% confidence intervals (CI) were calculated. The estimated frequency of TREX1 mutations in our lupus cohort was 0.5%. Five heterozygous mutations were detected at the Y305C polymorphism in European lupus cases but none were observed in European controls. Five African cases incurred heterozygous mutations at the E266G polymorphism and, again, none were observed in the African controls. A rare homozygous R114H mutation was identified in one Asian SLE patient, whereas all genotypes at this mutation in previous reports for SLE were heterozygous. Analysis of common TREX1 SNPs (minor allele frequency (MAF)>10%) revealed a relatively common risk haplotype in European SLE patients with neurological manifestations, especially seizures, with a frequency of 58% in lupus cases compared with 45% in normal controls (P=0.0008, OR=1.73, 95% CI=1.25-2.39). Finally, the presence or absence of specific autoantibodies in certain populations produced significant genetic associations. For example, a strong association with anti-nRNP was observed in the European cohort at a coding synonymous variant rs56203834 (P=2.99E-13, OR=5.2, 95% CI=3.18-8.56). Our data confirm and expand previous reports and provide additional support for the involvement of TREX1 in lupus pathogenesis.

The glycosyl phosphatidylinositol-linked Fc gamma RIIIPMN mediates transmembrane signaling events distinct from Fc gamma RII.

To investigate the ability of FcgammaRIII(PMN), the GPI-anchored isoform of FcgammaRIII (CD16) in polymorphonuclear leukocytes (PMN), to mediate transmembrane signaling events, we measured changes in membrane potential with DiOC(5) and in intracellular calcium with indo-1. FcgammaR were ligated by anti-FcgammaRIII mAb 3G8 (IgG and Fab), anti-FcgammaRII mAb IV.3 (IgG and Fab), and human IgG aggregates. Cell bound mAbs were also crosslinked by goat F(ab')(2) anti-mouse IgG. 3G8 IgG elicited a rapid change in [Ca(2+)](i), which was unaffected by EGTA, Vibrio cholerae toxin (CT), or Bordetella pertussis toxin (PT), and was abolished by BAPTA . Univalent receptor binding with 3G8 Fab gave no response but crosslinking with F(aV)2 GAM gave a rapid [Ca2,](i) response. Neither IV.3 Fab, IV.3 IgG, nor crosslinking of IV.3 Fab elicited a calcium signal. PI-PLC-treated PMN with the density of FcgammaRIII(PMN) reduced to that of FcgammaRII showed an unattenuated change in [Ca(2+)](i), with a 3G8 stimulus. The effects of IgG aggregates paralleled those of 3G8 mAb. These data indicate that multivalent ligation of FcgammaRIII(PMN) initiates an increase in [Ca(2+)];, derived from intracellular stores, that is distinct from both the FMLP- and FcgammaRII-induced responses. Ligand-dependent interaction with FcgammaRII is not required. Since FcgammaRIII(PMN) can internalize the FcgammaRIII-specific probe Con A-opsonized E and lyse anti-FcgammaRIII heteroantibody-opsonized chick E, this GPI-anchored molecule mediates both signal transduction and integrated cell responses.

Fcgamma receptors: structure, function and role as genetic risk factors in SLE.

Over 30 years ago, receptors for the Fc region of IgG (FcgammaR) were implicated in the pathogenesis of systemic lupus erythematosus (SLE). Since those pioneering studies, our knowledge of the structure and function of these FcgammaRs has increased dramatically. We now know that FcgammaR contributes to the regulation of acquired immunity and to the regulation of innate immune responses where FcgammaRs act as specific receptors for innate opsonins (CRP and SAP). Our understanding of the genomic architecture of the genes encoding the FcgammaR has also witnessed remarkable advances. Numerous functionally relevant single-nucleotide polymorphism (SNP) variants and copy number (CN) variants have been characterized in the FcgammaR genes. Many of these variants have also been shown to associate with risk to development of SLE and some have been associated with disease progression. This review will provide an overview of the FcgammaR in relation to SLE, including consideration of the role of genetic variants in FcgammaR in SLE pathogenesis. The difficulties in assessing genetic variation in these genes will be discussed. To enhance our understanding of the functional roles of these receptors in SLE, future research will need to integrate our knowledge of SNP variants, CN variants and the functional diversity of these receptors.

Identification of new SLE-associated genes with a two-step Bayesian study design.

In our earlier study, we utilized a Bayesian design to probe the association of approximately 1000 genes (approximately 10,000 single-nucleotide polymorphisms (SNPs)) with systemic lupus erythematosus (SLE) on a moderate number of trios of parents and children with SLE. Two genes associated with SLE, with a multitest-corrected false discovery rate (FDR) of <0.05, were identified, and a number of noteworthy genes with FDR of <0.8 were also found, pointing out a future direction for the study. In this report, using a large population of controls and adult- or childhood-onset SLE cases, we have extended the earlier investigation to explore the SLE association of 10 of these noteworthy genes (109 SNPs). We have found that seven of these genes exhibit a significant (FDR<0.05) association with SLE, both confirming some genes that have earlier been found to be associated with SLE (PTPN22 and IRF5) and presenting novel findings of genes (KLRG1, interleukin-16, protein tyrosine phosphatase receptor type T, toll-like receptor (TLR)8 and CASP10), which have not been reported earlier. The results signify that the two-step candidate pathway design is an efficient way to study the genetic foundations of complex diseases. Furthermore, the novel genes identified in this study point to new directions in both the diagnosis and the eventual treatment of this debilitating disease.

Complement receptor 2 polymorphisms associated with systemic lupus erythematosus modulate alternative splicing.

Genetic factors influence susceptibility to systemic lupus erythematosus (SLE). A recent family-based analysis in Caucasian and Chinese populations provided evidence for association of single-nucleotide polymorphisms (SNPs) in the complement receptor 2 (CR2/CD21) gene with SLE. Here we confirmed this result in a case-control analysis of an independent European-derived population including 2084 patients with SLE and 2853 healthy controls. A haplotype formed by the minor alleles of three CR2 SNPs (rs1048971, rs17615, rs4308977) showed significant association with decreased risk of SLE (30.4% in cases vs 32.6% in controls, P=0.016, OR=0.90 (0.82-0.98)). Two of these SNPs are in exon 10, directly 5' of an alternatively spliced exon preferentially expressed in follicular dendritic cells (FDC), and the third is in the alternatively spliced exon. Effects of these SNPs and a fourth SNP in exon 11 (rs17616) on alternative splicing were evaluated. We found that the minor alleles of these SNPs decreased splicing efficiency of exon 11 both in vitro and ex vivo. These findings further implicate CR2 in the pathogenesis of SLE and suggest that CR2 variants alter the maintenance of tolerance and autoantibody production in the secondary lymphoid tissues where B cells and FDCs interact.

Replication of the BANK1 genetic association with systemic lupus erythematosus in a European-derived population.

Systemic lupus erythematosus (SLE) is an autoimmune disease with highly variable clinical presentation. Patients suffer from immunological abnormalities that target T-cell, B-cell and accessory cell functions. B cells are hyperactive in SLE patients. An adapter protein expressed in B cells called BANK1 (B-cell scaffold protein with ankyrin repeats) was reported in a previous study to be associated with SLE in a European population. The objective of this study was to assess the BANK1 genotype-phenotype association in an independent replication sample. We genotyped 38 single nucleotide polymorphisms (SNPs) in BANK1 on 1892 European-derived SLE patients and 2652 European-derived controls. The strongest associations with SLE and BANK1 were at rs17266594 (corrected P-value=1.97 x 10(-5), odds ratio (OR)=1.22, 95% CI 1.12-1.34) and rs10516487 (corrected P-value=2.59 x 10(-5), OR=1.22, 95% CI 1.11-1.34). Our findings suggest that the association is explained by these two SNPs, confirming previous reports that these polymorphisms contribute to the risk of developing lupus. Analysis of patient subsets enriched for hematological, immunological and renal ACR criteria or the levels of autoantibodies, such as anti-RNP A and anti-SmRNP, uncovers additional BANK1 associations. Our results suggest that BANK1 polymorphisms alter immune system development and function to increase the risk for developing lupus.

Genetic associations of LYN with systemic lupus erythematosus.

We targeted LYN, a src-tyosine kinase involved in B-cell activation, in case-control association studies using populations of European-American, African-American and Korean subjects. Our combined European-derived population, consisting of 2463 independent cases and 3131 unrelated controls, shows significant association with rs6983130 in a female-only analysis with 2254 cases and 2228 controls (P=1.1 x 10(-4), odds ratio (OR)=0.81 (95% confidence interval: 0.73-0.90)). This single nucleotide polymorphism (SNP) is located in the 5' untranslated region within the first intron near the transcription initiation site of LYN. In addition, SNPs upstream of the first exon also show weak and sporadic association in subsets of the total European-American population. Multivariate logistic regression analysis implicates rs6983130 as a protective factor for systemic lupus erythematosus (SLE) susceptibility when anti-dsDNA, anti-chromatin, anti-52 kDa Ro or anti-Sm autoantibody status were used as covariates. Subset analysis of the European-American female cases by American College of Rheumatology classification criteria shows a reduction in the risk of hematological disorder with rs6983130 compared with cases without hematological disorders (P=1.5 x 10(-3), OR=0.75 (95% CI: 0.62-0.89)). None of the 90 SNPs tested show significant association with SLE in the African American or Korean populations. These results support an association of LYN with European-derived individuals with SLE, especially within autoantibody or clinical subsets.

Regulation of phagocytosis and [Ca2+]i flux by distinct regions of an Fc receptor.

The binding of multivalent immunoglobulin G complexes to Fc receptors (Fc gamma Rs) on macrophages activates multiple immune functions. A murine macrophage cell line, but not a fibroblast cell line, that was transfected with human Fc gamma RIIA mediated phagocytosis and an intracellular Ca2+ concentration ([Ca2+]i) flux upon cross-linking of human Fc gamma RIIA. Transfected macrophages that expressed a truncated receptor lacking 17 carboxy-terminal amino acids phagocytosed small antibody complexes. However, only wild-type transfectants phagocytosed labeled erythrocytes and fluxed [Ca2+]i. Thus, the cytoplasmic domain of human Fc gamma RIIA contains distinct functional regions.

Factors associated with arterial vascular events in PROFILE: a Multiethnic Lupus Cohort.

The objective of this study was to determine the factors associated with the occurrence of arterial vascular events in a multiethnic systemic lupus erythematosus (SLE) cohort. The PROFILE cohort, comprised SLE patients (n = 1333) of defined ethnicity from five different US institutions, was studied to determine demographic, clinical and biological variables associated with vascular events. An arterial vascular event (first episode) was either a myocardial infarction, angina pectoris and/or a vascular procedure for myocardial infarction, stroke, claudication and/or evidence of gangrene. Patient characteristics were analyzed by univariable and multivariable Cox proportional hazards regression analyses. One-hundred twenty-three (9.8%) patients had at least one incident arterial event. Age at cohort enrollment (HR = 1.04, 95% CI 1.03-1.06), smoking (HR = 2.20, 95% CI 1.40-3.46) and the CRP2* C alleles (HR = 1.91, 95% CI 1.04-3.49) were associated with a shorter time-to-the occurrence of arterial vascular events. Some clinical manifestations of disease activity were associated with a shorter time-to-occurrence [psychosis (HR = 2.21, 95% CI 1.10-4.44), seizures (HR = 1.85, 95% CI 1.00-3.24) and anaemia (HR = 1.83, 95% CI 1.02-3.31)], but others were not [arthritis (HR = 0.32, 95% CI 0.18-0.58)]. In conclusion, older patients, especially in the context of a predisposing environmental factor (smoking) and severe clinical manifestations, are at higher risk of having arterial vascular events. The genetic contribution of the variation at the CRP locus was not obscured by demographic or clinical variables. Awareness of these factors should lead to more effective management strategies of patients at risk for arterial vascular events.

Interferon regulatory factor-5 is genetically associated with systemic lupus erythematosus in African Americans.

Increased expression of interferon (IFN)-inducible genes is implicated in the pathogenesis of systemic lupus erythematosus (SLE). One transcription factor responsible for regulating IFN, interferon regulatory factor-5 (IRF5), has been associated with SLE in genetic studies of Asian, Caucasian and Hispanic populations. We genotyped up to seven polymorphic loci in or near IRF5 in a total of 4870 African-American and Caucasian subjects (1829 SLE sporadic cases and 3041 controls) from two independent studies. Population-based case-control comparisons were performed using the Pearson's chi(2)-test statistics and haplotypes were inferred using HaploView. We observed significant novel associations with the IRF5 variants rs2004640 and rs3807306 in African Americans and replicated previously reported associations in Caucasians. While we identified risk haplotypes, the majority of haplotypic effects were accounted for by one SNP (rs3807306) in conditional analyses. We conclude that genetic variants of IRF5 associate with SLE in multiple populations, providing evidence that IRF5 is likely to be a crucial component in SLE pathogenesis among multiple ethnic groups.

Copy number variants in genetic susceptibility and severity of systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a systemic autoimmune disorder characterized by the presence of auto-antibodies to nuclear antigens, immune complex deposition, and subsequent tissue destruction. Early studies in twins suggested that SLE has, at least in part, a genetic basis, and a role for class II alleles in the major histocompatibility complex has been known for over 30 years. Through both linkage studies and candidate gene studies, numerous additional genetic risk factors have been identified. The recent publication of two SNP-based genome-wide association studies (GWAS) has resulted in the confirmation of a number of previously identified genetic risk loci and has identified new previously unappreciated loci conferring risk for development of SLE. A role for gene copy number variation (CNV) in SLE has also been appreciated through studies of the complement component 4 (C4) loci and more recent work in the IgG Fc receptor loci. The availability of large SNP-based GWAS datasets will undoubtedly lead to the genome-wide analysis and identification of copy number variants related to genetic susceptibility for development of SLE. We review current studies of CNV in SLE susceptibility that include reports of association between SLE and CNV in C4, IgG Fc receptors, TLR7, and CCL3L1.

Fc gamma receptor III on human neutrophils. Allelic variants have functionally distinct capacities.

As a model system to explore the functional consequences of structural variants of human Fc gamma receptors (Fc gamma R), we have investigated Fc gamma R-mediated phagocytosis in relation to the NA1-NA2 polymorphism of Fc gamma RIII (CD16) on neutrophils (Fc gamma RIIIPMN). The neutrophil-specific NA antigen system is a biallelic polymorphism with codominant expression demonstrating a gene dose effect with the anti-NA1 MAb CLB-gran 11 in a large donor population. To explore the impact of this allelic variation of Fc gamma RIIIPMN on phagocytosis, we used two Fc gamma RIII-dependent probes, IgG-sensitized erythrocytes (EA) and concanavalin. A-treated erythrocytes (E-ConA). Comparison of Fc gamma R-mediated phagocytosis by PMN from NA1 subjects and from NA2 subjects showed lower levels of phagocytosis of both probes by the NA2 individuals. The difference was most pronounced with lightly opsonized EA: at the lowest level of sensitization the phagocytic index was 72% lower for NA2 donors, whereas at the highest level of sensitization it was 21% lower (P less than 0.003). Blockade of Fc gamma RII with MAb IV.3 Fab amplified by threefold the difference between NA1 and NA2 donors. NA1 and NA2 individuals had identical phagocytic capacities for the non-Fc gamma RIII probes, serum-treated and heat-treated zymosan. These individuals did not show differential quantitative cell surface expression of Fc gamma RIIIPMN measured by a panel of anti-CD16 MAb (3G8, CLB FcR-gran 1, VEP13, BW209/2) and by Scatchard analysis of 125I-IgG dimer binding. The difference in Fc gamma R-mediated phagocytosis was not explicable on the basis of differential collaboration of Fc gamma RIIIPMN alleles with Fc gamma RII, since (a) the difference in phagocytic capacity between NA1 and NA2 individuals was readily apparent with the E-ConA probe (which is independent of Fc gamma RII) and (b) the difference in phagocytosis of EA was magnified by Fc gamma RII blockade. The demonstration that allelic polymorphisms in Fc gamma R can have significant consequences for physiological functions implies that within the structural complexity of human Fc gamma Rs, including both allelic forms and cell type-specific isoforms, there will be differences in quantitative, and perhaps qualitative, function with potential importance for disease processes.

Preferential expression of human Fc gamma RIIIPMN (CD16) in paroxysmal nocturnal hemoglobinuria. Discordant expression of glycosyl phosphatidylinositol-linked proteins.

The isoform of Fc gamma RIII (CD16) expressed on PMN has a GPI membrane anchor, and in paroxysmal nocturnal hemoglobinuria (PNH) there is a deficiency in Fc gamma RIII expression on PMN. Contrary to expectation, however, CD16 expression is preserved (albeit at reduced levels) in all affected PNH PMN that completely lack the GPI-anchored proteins DAF (CD55) and CD59. Fc gamma RIII negative PMN are not observed in any of the six PNH patients examined in this study. Analysis of the molecular weight of both glycosylated and deglycosylated Fc gamma RIII from PMN with reduced Fc gamma RIII expression indicates no variations in size relative to normal donor Fc gamma RIIIPMN. Indeed, the Fc gamma RIII expressed at intermediate levels is phosphatidylinositol-specific phospholipase C (PI-PLC)-sensitive. Thus, there is no evidence suggestive of expression of a transmembrane isoform and all data indicate that Fc gamma RIIIPMN on affected cells in PNH is a GPI-linked isoform. With Fc gamma RIIIPMN expression preserved at reduced levels on affected cells in PNH, PMN from PNH patients retain the capacity to internalize the Fc gamma RIIIPMN-specific probe E-ConA (at reduced levels) as well as IgG-opsonized erythrocytes. Reduced expression of GPI-anchored molecules on PNH PMN is not restricted to Fc gamma RIIIPMN since intermediate levels of CD59 were observed in the PNH PMN that were decay-accelerating factor (DAF)-negative and Fc gamma RIIIPMN intermediate. In addition, discordant expression of GPI-linked molecules in individual cells is not restricted to PMN since DAF+/CD14- monocytes were observed in one PNH patient. These data suggest that, when analyzed on an individual cell level, the GPI anchor defect in PNH is not absolute and must involve either a hierarchy of access of different protein molecules to available GPI anchors, distinct anchor biochemistries for the different proteins, or differential regulation of protein-anchor assembly.

Allelic polymorphisms of human Fc gamma receptor IIA and Fc gamma receptor IIIB. Independent mechanisms for differences in human phagocyte function.

Two different allelic polymorphisms among the isoforms of human Fc gamma receptors have been defined: the low-responder (LR)-high-responder (HR) polymorphism of huFc gamma RIIA expressed on both PMN and monocytes and the NA1-NA2 polymorphism of the neutrophil Fc gamma RIII (huFc gamma RIIIB). To address the issues of whether the LR-HR polymorphism has a significant impact on Fc gamma R-mediated functions in human blood cells and whether any differences in LR-HR might be related to higher Fc gamma R-mediated phagocytosis in NA1 donors, we examined Fc gamma R-specific binding and internalization by donors homozygous for the two huFc gamma RIIA alleles. PMN from LR homozygotes showed consistently higher levels of internalization of erythrocytes opsonized with pooled human IgG (E-hIgG). The absence of an LR-HR phagocytic difference with erythrocytes opsonized with either anti-Fc gamma RIIA MAb IV.3 or rabbit IgG, as opposed to E-hIgG, suggested that the Fc piece of the opsonin might be important for this LR-HR difference. Accordingly, we studied HR and LR homozygotes with human IgG subclass-specific probes. Both PMN (independent of huFc gamma RIIIB phenotype) and monocytes from LR donors bound and internalized erythrocytes coated with human IgG2 (E-hIgG2) efficiently, whereas phagocytes from HR donors did so poorly. E-hIgG2 internalization was completely abrogated by blockade of the ligand binding site of huFc gamma RIIA with IV.3 Fab, indicating that huFc gamma RIIA is essential for the handling of hIgG2 and that the mechanism of the LR-HR phagocytic difference is at the level of ligand binding to huFc gamma RIIA. In contrast, the difference in internalization of E-hIgG between NA1 and NA2 homozygous donors was independent of the huFc gamma RIIA phenotype and did not manifest differences in ligand binding. Thus, the two known allelic polymorphisms of human Fc gamma R have distinct and independent mechanisms for altering receptor function, which may influence host defense and immune complex handling.

In vivo handling of soluble complement fixing Ab/dsDNA immune complexes in chimpanzees.

We used soluble, C-fixing antibody/dsDNA IC to investigate immune complex (IC) handling and erythrocyte (E)-to-phagocyte transfer in chimpanzees. IC bound efficiently to chimpanzee E in vitro and showed minimal release with further in vitro incubation in the presence of serum in EDTA (less than or equal to 15% within 1 h). These IC also bound rapidly to E in vivo (70-80% binding within 1 min) and did not show detectable release from E in the peripheral circulation after infusion in vivo (less than or equal to 2% within 1 h). Despite such slow C-mediated release of IC from E, IC were rapidly stripped from E by the mononuclear phagocyte system (T50 for E-IC1500 = 5 min) without sequestration of E. Treatment of the chimpanzees with the anti-Fc gamma RIII MAb 3G8 impaired the clearance of infused IC. This effect was most evident on the fraction of IC500 which did not bind to E and which presumably had captured less C3b (pre-MAb 3G8 T50: 45 min vs. post-MAb 3G8 T50: 180 min). With IC bound in vitro to E before infusion, anti-Fc gamma RIII MAb treatment led to significant amounts of non-E bound IC detectable in the circulation. Thus, the anti-Fc gamma RIII MAb appeared to interfere with the ability of fixed tissue mononuclear phagocytes to take up/or retain IC after their release from E. Both rapid stripping of IC from E, despite slow complement-mediated release of IC from E in the peripheral circulation, and blockade of IC clearance with anti-Fc gamma RIII MAb indicate that the interaction of IC with the fixed tissue phagocyte involves qualitatively different mechanisms than the interaction of IC with E. Fc gamma receptors appear to play an important role in the transfer and retention of IC by the phagocyte.

A novel polymorphism of FcgammaRIIIa (CD16) alters receptor function and predisposes to autoimmune disease.

A novel polymorphism in the extracellular domain 2 (EC2) of FcgammaRIIIA affects ligand binding by natural killer (NK) cells and monocytes from genotyped homozygous normal donors independently of receptor expression. The nonconservative T to G substitution at nucleotide 559 predicts a change of phenylalanine (F) to valine (V) at amino acid position 176. Compared with F/F homozygotes, FcgammaRIIIa expressed on NK cells and monocytes in V/V homozygotes bound more IgG1 and IgG3 despite identical levels of receptor expression. In response to a standard aggregated human IgG stimulus, FcgammaRIIIa engagement on NK cells from V/V (high-binding) homozygotes led to a larger rise in [Ca2+]i, a greater level of NK cell activation, and a more rapid induction of activation-induced cell death (by apoptosis). Investigation of an independently phenotyped normal cohort revealed that all donors with a low binding phenotype are F/F homozygotes, while all phenotypic high binding donors have at least one V allele. Initial analysis of 200 patients with SLE indicates a strong association of the low binding phenotype with disease, especially in patients with nephritis who have an underrepresentation of the homozygous high binding phenotype. Thus, the FcgammaRIIIa polymorphism at residue 176 appears to impact directly on human biology, an effect which may extend beyond autoimmune disease characterized by immune complexes to host defense mechanisms.

C-reactive protein binding to FcgammaRIIa on human monocytes and neutrophils is allele-specific.

C-reactive protein (CRP) is involved in host defense, regulation of inflammation, and modulation of autoimmune disease. Although the presence of receptors for CRP on phagocytes has been inferred for years, their identity was determined only recently. FcgammaRIa, the high-affinity IgG receptor, binds CRP with low affinity, whereas FcgammaRIIa, the low-affinity IgG receptor, binds CRP with high affinity. Because the single nucleotide polymorphism in FcgammaRIIA - which encodes histidine or arginine at position 131 - strongly influences IgG2 binding, we determined this polymorphism's effect on CRP binding. CRP bound with high avidity to monocytes and neutrophils from FcgammaRIIA R-131 homozygotes, and binding was inhibited by the R-specific mAb 41H16. CRP showed decreased binding to cells from FcgammaRIIA H-131 homozygotes (which bind IgG2 with high affinity). However, IFN-gamma enhanced FcgammaRI expression by H-131 monocytes and increased CRP binding. FcgammaRIIa heterozygotes showed intermediate binding. CRP initiated increases in [Ca(2+)](i) in PMN from R-131, but not from H-131 homozygotes. These data provide direct genetic evidence for FcgammaRIIa as the functional, high-affinity CRP receptor on leukocytes while emphasizing the reciprocal relationship between IgG and CRP binding avidities. This counterbalance may affect the contribution of FcgammaRIIA alleles to host defense and autoimmunity.