In situ localization by double-labeling immunoelectron microscopy of anti-neutrophil cytoplasmic autoantibodies in neutrophils and monocytes.
Anti-neutrophil cytoplasmic autoantibodies (ANCA) associated with active Wegener's granulomatosis are directed against a soluble 29-Kd protein present in human neutrophils and monocytes. Affinity labeling with tritiated diisopropylfluorophosphate (3H-DFP) suggested that ANCA-antigen is a serine protease. We used immunoelectron microscopy to study the in situ localization of the ANCA-antigen in normal human neutrophils and monocytes using immunoglobulin G (IgG) from ANCA-positive patients and a mouse monoclonal antibody against the ANCA-antigen. Label was observed on the large granules of the neutrophils and in granules of monocytes. Double-labeling, using anti-myeloperoxidase or the peroxidase reaction as markers for azurophil granules and anti-lactoferrin as marker for specific granules, showed that ANCA is colocalized with markers of azurophil granules but not with lactoferrin. Furthermore, elastase and cathepsin G were found in the azurophil granules of neutrophils and in the peroxidase-positive granules of monocytes, colocalized with ANCA-antigen. Cytochalasin-B-treated neutrophils stimulated with N-formyl-methionyl-leucyl-phenylalanine (fMLP) formed large intracellular vacuoles and were partially degranulated. Some vacuoles contained ANCA-antigen, as well as myeloperoxidase, elastase, and cathepsin G, demonstrating release of these enzymes from the azurophil granules into vacuoles. Our results demonstrate that ANCA-antigen is located in myeloperoxidase-containing granules of neutrophils and monocytes, and is packaged in the same granules as elastase and cathepsin G, the two previously identified serine proteases of myeloid leukocytes.
Calafat
J
J
Laboratory of Electron Microscopy, The Netherlands Cancer Institute, Amsterdam.
Goldschmeding
R
R
Ringeling
P L
PL
Janssen
H
H
van der Schoot
C E
CE
D001323:Autoantibodies / Q000276:immunology*; D001324:Autoantigens / Q000276:immunology*; D056649:Cathepsin G; D002403:Cathepsins / Q000378:metabolism; D002451:Cell Compartmentation; D003571:Cytochalasin B / Q000494:pharmacology; D014890:Granulomatosis with Polyangiitis / Q000276:immunology; D006801:Humans; D007124:Immunoenzyme Techniques; D007150:Immunohistochemistry; D008247:Lysosomes / Q000276:immunology; D008854:Microscopy, Electron; D009000:Monocytes / Q000201:enzymology / Q000276:immunology* / Q000648:ultrastructure; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000201:enzymology / Q000276:immunology* / Q000648:ultrastructure; D010196:Pancreatic Elastase / Q000378:metabolism; D009195:Peroxidase / Q000378:metabolism; D012697:Serine Endopeptidases / Q000276:immunology* / Q000378:metabolism; D013347:Subcellular Fractions / Q000276:immunology
D001323:Autoantibodies; D001324:Autoantigens; D003571:Cytochalasin B; D009240:N-Formylmethionine Leucyl-Phenylalanine; D009195:Peroxidase; D002403:Cathepsins; D012697:Serine Endopeptidases; C534094:CTSG protein, human; D056649:Cathepsin G; C534096:Ctsg protein, mouse; D010196:Pancreatic Elastase