Neutrophil chemotaxis and superoxide production are induced by cross-linking FcgammaRII receptors.

Neutrophils express two types of receptor for the Fc region of IgG, FcgammaRII and FcgammaRIIIB. Via these receptors, neutrophils bind IgG complexes that contain more than one IgG molecule. This binding activates functional processes, such as the respiratory burst and chemotaxis. Neutrophils were treated with biotinylated anti-Fc receptor monoclonal antibodies and chemotaxis toward streptavidin, a cross-linking agent, was determined. Cross-linking FcgammaRII and not FcgammaRIIIB induced neutrophil chemotaxis. Superoxide production in response to immobilized anti-Fc receptor antibodies was also examined. Anti-FcgammaRII Fab bound to ELISA plates induced superoxide production, while anti-FcgammaRIIIB Fab did not. Pretreatment of neutrophils with anti-FcgammaRII Fab reduced superoxide generated by immobilized anti-FcgammaRII antibody. The data demonstrate that FcgammaRII and not FcgammaRIIIB are responsible for neutrophil chemotaxis and superoxide production upon Fc receptor activation.

Effect of allotype on activation of neutrophils by FcgammaRIIIB cross-linking.

Human neutrophils constitutively synthesize two receptors for the constant region of IgG, FcgammaRII, and FcgammaRIIIB. Fluo-3-loaded neutrophils were treated with biotinylated Fab fragments of anti-FcgammaR antibodies and cross-linked with streptavidin, and intracellular calcium ([Ca2+](i)) was monitored by flow cytometry. Polymerization of filamentous actin was quantitated by NBD-phallacidin using flow cytometry. Cross-linking of FcgammaRII by monoclonal antibody (mAb) IV.3 induces an increase in [Ca2+](i), superoxide generation, and the polymerization of actin. [Ca2+](i) responses from cross-linking of FcgammaRIIIB by mAb 3G8 varied from minimal to no release. To determine whether discrepancies in 3G8-induced [Ca2+](i) release were due to allotype variation, we selected five donors who were homozygous for the NA1 allotype of FcgammaRIIIB and five who were either heterozygous or homozygous for the NA2 allotype and compared their [Ca2+](i) response and actin polymerization induced by FcgammaRIIIB cross-linking. Cross-linking of FcgammaRIIIB by 3G8 produced minimal [Ca2+](i) release and polymerization of actin irrespective of donor allotype.

Myelinated dorsal root ganglion cultures activate both the alternative and classical pathways of complement.

We used rat myelinated dorsal root ganglion (MDRG) cultures to study antibody and complement-mediated mechanisms of peripheral demyelinating diseases. Heat inactivated serum from a patient (LT) with peripheral neuropathy and a monoclonal IgM reactive with myelin-associated glycoprotein (anti-MAG) and sulfated glucuronosyl glycolipids (anti-SGGL) was used as an antibody source. Incubation of whole human serum (WHS) or WHS and anti-SGGL with MDRGs resulted in reduction of classical and alternative pathway hemolytic activities and the development of abnormal myelin sheaths. Incubation of MDRG cultures with C2-deficient serum showed activation of the alternative complement pathway. Classical pathway hemolytic activity was reduced when Factor B-depleted serum was incubated with MDRG cultures. The rat MDRG culture system provides a good model system of a peripheral nerve and has therefore been used by several investigators to study antibody and complement-mediated demyelination associated with peripheral neuropathies. However, our studies indicate a high degree of complement activation and membrane disruption of cultures incubated with WHS.