Binding modes of IgG from pemphigus autoimmune sera onto guinea pig keratinocytes and the fate of bound IgGs.
Pemphigus is an intraepidermal autoimmune blistering disease of humans caused by circulating IgGs. We have investigated the binding mode and the fate of bound antibodies from Pemphigus sera (P-IgG) on guinea pig keratinocytes in suspension in order to find clues to the loss of cell adhesion in vivo (acantholysis). Flow cytometry, following indirect immunofluorescent labeling of the keratinocytes, and dead cells' staining with ethidium bromide, demonstrated the specific surface binding of P-IgG onto living keratinocytes only. This was shown with several Pemphigus sera or purified P-IgG. This technique, used with various Pemphigus sera, showed that the specific binding is increased when the serum titer is higher, and "Km" values for P-IgG were roughly and inversely correlated to the titers. Upon saturation the same average number of Pemphigus IgG sites per cell were found for the sera of different patients. Analysis of the specific binding of [125I]-P-IgG onto Percoll-separated (living) keratinocytes showed the existence of two classes of sites: 2 x 10(6) sites/cell high-affinity sites (Kd = 1.5 x 10(-6) M total IgG) and 25 x 10(6) sites/cell low-affinity sites (Kd = 6 x 10(-5) M total IgG). Cell sorting and flow cytometry of individual cells allowed us to correlate the light-scattering signal, the RNA content, the size and morphology, and the P-IgG binding to the cells. The results indicated that P-IgG binding is homogeneous within the living keratinocytes and increases with cell size (cell maturity). Cell-sorter analysis of cells with membrane-bound P-IgG, coupled to direct determination of P-IgG released in the medium, revealed the fate of bound P-IgG: 40-60% of the P-IgGs were released in the medium within 30 minutes at 37 degrees C. This was accompanied and followed by a much slower, metabolic energy-dependent, internalization process of the membrane-bound P-IgG. The internalization has been confirmed by electron microscopy of bound P-IgG labeled with protein A-gold. Internalized IgGs were seen in the cells in coated membranous vesicles and other endocytic compartments. Similar behavior was also observed with two other membrane ligands: i.e., concanavalin A and multispecific rabbit "antisurface" antibodies.
Milner
Y
Y
Department of Biological Chemistry, Hebrew-University of Jerusalem, Israel.
Sagi
E
E
Timberg
R
R
Michel
B
B
Me'te'zeau
P
P
Goldberg
M
M
D000818:Animals; D000943:Antigens, Differentiation / Q000378:metabolism*; D002448:Cell Adhesion; D000078404:Epidermal Cells; D004817:Epidermis / Q000276:immunology*; D005434:Flow Cytometry; D006168:Guinea Pigs; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism*; D066298:In Vitro Techniques; D007633:Keratins; D008853:Microscopy / Q000379:methods; D010392:Pemphigus / Q000276:immunology* / Q000473:pathology; D011961:Receptors, Fc / Q000378:metabolism*; D017452:Receptors, IgG
D000943:Antigens, Differentiation; D007074:Immunoglobulin G; D011961:Receptors, Fc; D017452:Receptors, IgG; D007633:Keratins