Characterization of sequential immune complexes in infective endocarditis by Western blot analysis.

Abstract A patient with cutaneous vasculitis during infective endocarditis due to Lactobacillus casei was studied. Immune complexes (IC) were isolated from serum at the time of diagnosis and after 4 wk of therapy. Purification of IC used differential polyethylene glycol precipitation and competitive binding to staphylococcal protein A. In situ radioiodination of IC was performed, followed by SDS-polyacrylamide gel electrophoresis (PAGE). Anti-IC antisera were raised in rabbits by immunization with purified IC. IC were characterized by SDS-PAGE followed by electrophoretic transfer to nitrocellulose, incubation with antiserum and then with 125I protein A, and autoradiography. Although early and late IC differed quantitatively, there were no differentiating immunochemical features. Both IC contained a 60,000 dalton component that did not react with preimmune serum nor with anti-normal human serum. This component reacted with antiserum rendered specific for L. casei by affinity chromatography. The restricted antigen-antibody representation in IC contrasted with a wider panel of antibody activity in patient serum. The Western blot analysis proves to be an ideal method for the characterization of IC because of its sensitivity, dissociative capability, and preservation of immunoreactivity. IC isolated at a time removed from the original antigenic challenge may provide insight into the nature of the inciting antigen.</jats:p

Binding of immunoglobulin G aggregates and immune complexes in human sera to Staphylococci containing protein A.

Using the Cowan I strain of Staphylococcus aureus, we compared the binding properties of human monomeric immunoglobulin (Ig)G and oligomeric or complexed IgG. Heat-aggregated IgG served as a model for complexed IgG and heat-killed, formalin-fixed S. aureus (StaphA) as a cellular receptor for IgG, in determining the parameters for oligomeric and monomeric binding. Because of its capacity for multipoint attachment, complexed IgG binding was favored over monomeric IgG binding, and this preferential binding was demonstrated kinetically in equivalent forward rates of binding but in a much slower rate of release from StaphA receptors. From binding studies, we determined which conditions maximize complexes IgG binding and minimized monomeric IgG binding and applied them to the development of an assay for aggregated IgG and immune complexes in human sera. The StaphA binding assay that was devised is quantitative, sensitive, and not complement dependent. It is relatively unaffected by factors such as heparin, complement fixation, native antibodies, and immunoglobulin concentrations, but is affected by the presence of rheumatoid factors. It compares favorably with two other complement-dependent assays of immune complexes, the 125I-Clq binding assay and the Raji cell assay, in terms of sensitivity and the size of immune complexes detected. Studies on the potential of the assay for detecting, isolating, and characterizing immune complexes in biological fluids are presented

Differences among immune complexes: association of C1q in SLE immune complexes with renal disease.

Abstract Studies that made use of multiple assay systems demonstrated increased levels of immune complexes (IC) in patients with systemic lupus erythematosus (SLE), but no consistent correlations of IC concentration to patterns or activity of disease have been observed. Furthermore, consistent associations between qualitative differences in IC and disease manifestations have been elusive. IC interaction with erythrocytes and mononuclear phagocytic cells is another variable in SLE that may also mediate some of the biological effects of IC. The present report concerns studies of the composition of purified IC obtained from individuals with SLE and other rheumatic diseases; a 64,000 dalton component identified as the A-B subunit of C1q was detected in purified IC from 27 of 51 SLE patients (53%). The presence of this 64,000 dalton component was not related to either IC concentration or to the serum C1q level. However, the presence of the C1q component in isolated SLE IC did correlate with the presence of renal disease (p less than 0.02). These observations are interpreted relative to a recently described kinetic model of IC clearance.</jats:p

Human Fc gamma RIII (CD16). Isoforms with distinct allelic expression, extracellular domains, and membrane linkages on polymorphonuclear and natural killer cells.

Abstract Human Fc gamma RIII (CD16), a low-affinity receptor expressed on several different cell types, has a polymorphism on polymorphonuclear cells (Fc gamma RIIIPMN) identified by the NA1 and NA2 markers. Inasmuch as this polymorphism has functional consequences, an understanding of the structural biology of Fc gamma RIII may provide important insight into receptor function. We analyzed Fc gamma RIIIPMN by SDS-PAGE and found that receptor from individuals allotyped for either NA1 or NA2 contained only one protein after removal of N-linked glycosylations (19 and 21 kDa respectively) whereas receptor from NA1/2 individuals contained both bands. Because some reports indicate that digestion of Fc gamma RIII on NK cells (Fc gamma RIIINK) with N-glycanase also results in two bands on SDS-PAGE, we investigated Fc gamma RIIINK to explore the possibility of a corresponding allelic polymorphism in this receptor. Contrary to expectation, Fc gamma RIIINK from all donors irrespective of their NA allotype contained two bands (20 and 24 kDa) on SDS-PAGE after deglycosylation. In addition, those distinct epitopes on the extracellular domain of Fc gamma RIIIPMN found with mAb B73.1 and CLB gran 11 in association with the NA allotypic differences are expressed (or not expressed) on Fc gamma RIIINK independent of donor NA allotype. Fc gamma RIIIPMN and Fc gamma RIIINK differ at the protein level as they have different m.w. (glycosylated and deglycosylated), different epitopes in the extracellular domain (not attributable to tissue-specific glycosylation), and differential expression of the NA allelic protein polymorphism. Although the membrane anchor of Fc gamma RIIIPMN is a phosphatidylinositol-specific phospholipase C sensitive glycosyl-phosphatidylinositol linkage, Fc gamma RIIINK is insensitive to phosphatidylinositol-specific phospholipase C. However, a form of Fc gamma RIIINK is released from NK cells upon incubation at 37 degrees C. Thus, the basis for the two bands in Fc gamma RIIINK after N-linked deglycosylation is neither coexpression of two molecular isoforms with different membrane anchors nor an identifiable allelic polymorphism in m.w. restricted to Fc gamma RIIINK (p less than 10(-6)). The differences between the two receptors indicate that, independent of cell anchor type, PMN and mononuclear cells must have different molecular isoforms. The allelic variants, different isoforms, alternative anchor mechanisms and release processes provide for an extensive genetic and regulatory diversity in Fc gamma RIII function.</jats:p

Fc gamma RIII expressed on cultured monocytes is a N-glycosylated transmembrane protein distinct from Fc gamma RIII expressed on natural killer cells.

Abstract Fc gamma RIII is a family of protein isoforms encoded by at least two distinct, yet highly homologous, genes. Fc gamma RIII on neutrophils is a glycosylphosphatidylinositol-linked protein with an allelic polymorphism (NA1/NA2) while Fc gamma RIII on NK cells (Fc gamma RIIINK) is an exclusively transmembrane protein without the NA polymorphism. The relationship of the isoform of Fc gamma RIII expressed on cultured monocytes (Fc gamma RIIIM phi) to these two forms, however, is unclear because some evidence suggests lowered expression of Fc gamma RIIIM phi in paroxysmal nocturnal hemoglobinuria (unlike Fc gamma RIIINK) and a unique deglycosylated m.w. for Fc gamma RIIIM phi. In this study we demonstrate that, as with Fc gamma RIIINK, Fc gamma RIIIM phi is resistant to the action of phosphatidylinositol-specific phospholipase C and is expressed at normal levels on affected (glycosylphosphatidylinositol-anchor negative) cultured monocytes from patients with paroxysmal nocturnal hemoglobinuria. Fc gamma RIIIM phi is also shed from the cell surface upon incubation at 37 degrees C. However, Fc gamma RIIIM phi and Fc gamma RIIINK have different m.w. as glycosylated proteins despite the same deglycosylated m.w. Thus, each cell type appears to express distinct glycoforms. These differences in glycosylation may influence the functional properties of the receptor.</jats:p

Anti-neutrophil cytoplasmic antibodies engage and activate human neutrophils via Fc gamma RIIa.

Abstract The presence of anti-neutrophil cytoplasmic Abs (ANCA) in many patients with systemic vasculitis suggests that ANCA may play a role in disease pathogenesis. Neutrophils from patients with Wegener's granulomatosis often express ANCA target Ags (myeloperoxidase (MPO) and proteinase 3 (PR3)) on their surface, making these intracellular primary granule enzymes accessible to these autoantibodies. Similarly, normal neutrophils can be induced to translocate MPO and PR3 to the cell surface in vitro, and we demonstrate that murine mAb ANCA IgG, but not IgM, binds to the ANCA target and engages the Fc gamma RIIa ligand-binding site on the surface of human neutrophils. In contrast to ANCA IgM, ANCA IgG also induces an oxidative burst in neutrophils (oxidation of dihydrorhodamine = 91 +/- 15 fluorescence units with anti-PR3 IgG vs 17 +/- 2 with anti-PR3 IgM, p &amp;lt; 0.001). Blockade of the ligand-binding site of Fc gamma RIIa with an antibinding site mAb Fab significantly reduces this ANCA IgG-triggered production of reactive oxygen species (p &amp;lt; 0.01). Similarly, human ANCA bind the ANCA target, engage Fc gamma RIIa, and induce an oxidative burst in neutrophils. The allelic phenotype of Fc gamma RIIa strongly influences the Fc gamma receptor engagement by ligand, and Fc gamma RIIa homozygous donors differ by more than threefold in the quantitative production of reactive oxygen intermediates (ROI) (p &amp;lt; 0.01). Thus, engagement of Fc gamma RIIa by the Fc region of ANCA is one mechanism by which these autoantibodies activate receptor-mediated signal transduction systems in human neutrophils to initiate programs of inflammation and tissue injury. Fc gamma receptor alleles may represent heritable disease risk factors influencing the magnitude of such a process.</jats:p

Identification of bacterial antigens in circulating immune complexes of infective endocarditis.

The presence of circulating immune complexes (IC) in patients with infective endocarditis has been well documented but the contributions of host and bacterial components to these IC have not been defined. To study this question, IC were isolated from serum of a patient with Streptococcus faecalis endocarditis by differential polyethylene glycol precipitation and competitive binding to staphylococcal protein A. A rabbit antiserum raised against the purified IC had reactivity by crossed immunoelectrophoresis primarily with an antigen derived from the cytoplasm of the infective organism. The antigen was a protein with a 12,000-dalton molecular mass. In situ radiolabeling of the IC bound to the protein A demonstrated a component of the same molecular mass as the bacterial antigen recognized by the antiserum. The patient serum had multiple antibody specificities reactive with bacterial antigens, including the antigen recognized by the rabbit anti-IC antiserum. These techniques for isolation and characterization of circulating IC may have value in the study of IC diseases in which the inciting antigens are not known