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Background: Our index case was a patient with common variable immunodeficiency (CVID). She had anaphylactoid reactions on administration of intravenous immunoglobulin (IVIg) associated with the presence of IgG antibodies against IgA., Objective: We sought to determine the role of Fcγ receptor (FcγR) IIa in IVIg-induced anaphylactoid reactions., Methods: Neutrophils and PBMCs were isolated from healthy subjects and IVIg-treated patients. FcγRIIa mRNA and DNA were analyzed by using real-time PCR and sequencing. IgG-mediated elastase release and intracellular Ca(2+) mobilization were determined in neutrophils and transfected cell lines, respectively., Results: A novel splice variant of FcγRIIa containing an expressed cryptic exon 6* (FcγRIIa(exon6∗)) was identified in our index patient. This exon is normally spliced out of all FcγRII isoforms, except the inhibitory FcγRIIb1. Compared with healthy control subjects, the heterozygous FCGR2A(c.742+871A>G) mutation was more frequent in patients with CVID (n = 53, P < .013). Expression in patients with CVID was associated with anaphylaxis on IVIg infusion (P = .002). On screening of additional IVIg-treated patient cohorts, we identified 6 FCGR2A(c.742+871A>G) allele-positive patients with Kawasaki disease (n = 208) and 1 patient with idiopathic thrombocytopenia (n = 93). None had adverse reactions to IVIg. Moreover, FcγRIIa(exon6∗) was also demonstrated in asymptomatic family members. Functional studies in primary cells and transfected murine cells demonstrated enhanced cellular activation by FcγRIIa(exon6∗) compared with its native form, as shown by increased elastase release and intracellular calcium mobilization., Conclusion: A novel splice variant, FcγRIIa(exon6∗), was characterized as a low-frequency allele, coding for a gain-of-function receptor for IgG. In the presence of immune complexes, FcγRIIa(exon6∗) can contribute to anaphylaxis in patients with CVID., (Copyright © 2013 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Background: Plasma cells are one of the end products of the B-lymphocyte mediated immune response. These cells normally reside in the bone marrow or some peripheral lymphoid tissues. Increased numbers of plasma cells in the blood, usually indicates pathology such as infection, auto-immunity or haematological malignancy. Therefore, the ability to measure plasma cells (PCs) on an automated cell analyzer might be advantageous., Methods: The performance of the Sysmex XE-5000 leukocyte differential channel [high fluorescent lymphocyte count (HFLC) area] was evaluated for the ability to detect plasma cells in peripheral blood and compared to the detection of plasma cells by flow cytometric analyses., Results: Our results show that the HFL count from the XE-5000 correlates (R(2)=0.8) with the number of PCs in peripheral blood, but detects PCs with moderate to good sensitivity (88.9%) and specificity (87.8%)., Conclusions: The Sysmex XE-5000 is suitable for screening blood samples for the presence of elevated number of plasma cells in peripheral blood, but the actual quantification needs to be confirmed by flow cytometry.

Human Fcgamma receptors (FcgammaRs) are glycoproteins that bind the Fc region of IgG. The genes encoding the low-affinity FcgammaRs are located on chromosome 1q23-24. Beside single nucleotide polymorphisms (SNPs), gene copy number variation (CNV) is now being recognized as an important indicator for inter-individual differences. Recent studies on identifying CNV in the human genome suggest large areas at chromosome 1q23-24 to be involved, and CNV in this region has been associated with manifestations of systemic autoimmune disease. To study both SNPs and CNV of the low-affinity FcgammaRs in one assay, we have developed a Multiplex Ligation-dependent Probe Amplification (MLPA) assay. A novel CNV for FCGR3A was observed. Similar to FCGR3B and FCGR2C, a gene-dosage effect of FCGR3A was found, that seemed to correlate nicely with the FcgammaRIIIa expression on NK cells. Next, we delineated the approximate boundaries of CNV at the FCGR locus. Variation in co-segregation of neighboring FCGR genes was limited to four variants, with patterns of Mendelian inheritance. No CNV of the FCGR2A and FCGR2B genes was observed in over 600 individuals. In conclusion, we report a novel CNV of the FCGR3A gene that correlates with FcgammaRIIIa expression and function on NK cells. Only FCGR3A, FCGR2C and FCGR3B show CNV, in contrast to FCGR2A and FCGR2B., (Copyright 2009 Wiley-Liss, Inc.)

Gene copy number variation (CNV) and single nucleotide polymorphisms (SNPs) count as important sources for interindividual differences, including differential responsiveness to infection or predisposition to autoimmune disease as a result of unbalanced immunity. By developing an FCGR-specific multiplex ligation-dependent probe amplification assay, we were able to study a notoriously complex and highly homologous region in the human genome and demonstrate extensive variation in the FCGR2 and FCGR3 gene clusters, including previously unrecognized CNV. As indicated by the prevalence of an open reading frame of FCGR2C, Fcgamma receptor (FcgammaR) type IIc is expressed in 18% of healthy individuals and is strongly associated with the hematological autoimmune disease idiopathic thrombocytopenic purpura (ITP) (present in 34.4% of ITP patients; OR 2.4 (1.3-4.5), P < .009). FcgammaRIIc acts as an activating IgG receptor that exerts antibody-mediated cellular cytotoxicity by immune cells. Therefore, we propose that the activating FCGR2C-ORF genotype predisposes to ITP by altering the balance of activating and inhibitory FcgammaR on immune cells.

We tested the hypothesis that the ratio between the activating and inhibitory Fcgamma receptor type II (FcgammaRII) in neutrophils determines their responsiveness to immune complexes. We measured mRNA levels of FcgammaRII isoforms and observed differences in the ratio of FcgammaRIIa to FcgammaRIIb2 mRNA in granulocytes of 50 white and 10 black healthy volunteers, and found 4 discrete groups of ratios (ie, 4:1; 3:1, 2:1, or 1:1). The response to either dimeric IgG or aggregated IgG (aIgG) was assessed. Up-regulation of CD11b on the surface as well as the elastase release was significantly more pronounced in neutrophils with a high FcgammaRIIa/FcgammaRIIb2 mRNA ratio of 4:1 compared with a 2:1 or 1:1 ratio. Individual ratios as well as the functional responsiveness of neutrophils were constant over time, as was tested over 12 months. Neutrophil stimulation with various agents in vitro did not alter the FcgammaRIIa/FcgammaRIIb2 mRNA ratio in the neutrophils of these donors, in clear contrast to the findings in their mononuclear cells. We found a strong association between the 2B.4 haplotype of the FCGR2B promoter with increased transcriptional activity in individuals with 1:1 ratios and the more common low-expression 2B.1 haplotype in individuals with FcgammaRIIa/FcgammaRIIb2 mRNA ratios of 2:1, 3:1, or 4:1.

Intravenous Ig preparations (IVIg), originally developed as a substitution therapy for patients with low plasma IgG, are nowadays frequently used in the treatment of various immune diseases. However, the mechanism of action of IVIg in these diseases remains elusive and is often referred to as "immunomodulatory." We hypothesized that monomeric IgG may act as a low-affinity FcgammaR antagonist and sought experimental evidence for this hypothesis. Human neutrophils as well FcgammaRIIa-transfected IIA1.6 cells were used as FcgammaR-positive cells and aggregated IgG (aIgG) or stable dimeric IgG as FcgammaR-specific agonists for these cells. We found that monomeric IgG purified from IVIg at concentrations similar to that of IgG in plasma, diminished the binding of stable dimeric IgG to FcgammaRIIa transfectants, reduced aIgG-induced influx of Ca(2+) ions into the cytosol of neutrophils, and attenuated the aIgG-induced release of elastase. Notably, monomeric IgG by itself did not elicit these responses, nor did it affect these processes in response to fMLP. Absorption of IgG from normal plasma revealed that plasma IgG exerted similar effects as monomeric IgG in IVIg. In addition, adding monomeric IgG to blood of healthy volunteers showed a dose-dependent decrease of aIgG-induced elastase release. Finally, we observed decreased aIgG-induced polymorphonuclear neutrophil responses in two hypogammaglobulinemic patients upon treatment with IVIg. We conclude that monomeric IgG at physiological levels acts as a low-affinity FcgammaR antagonist. Moreover, FcgammaR antagonism constitutes an immunomodulatory effect of IVIg.