Antibody to intermediate filaments of the cytoskeleton.

IgM antibodies against cultures of intermediate filaments (IMF) of the cytoskeleton were demonstrated by immunofluorescence in the sera of 94 (80%) of 118 patients with seropositive rheumatoid arthritis. These antibodies reacted with IMF in cultures of both human fetal fibroblasts and laryngeal carcinoma (HEp2) cells. Of 10 patients from whom paired synovial fluids were also available 8 had anti-IMF antibodies in both serum and fluid. In seronegative RA the incidence of anti-IMF was 40%, in ankylosing spondylitis 25%, in osteoarthrosis 16%, and in normal subjects 14%. Only a minority of RA sera positive for anti-IMF antibodies were also positive for smooth muscle antibody. Absorption experiments suggest that in RA anti-IMF is directed at the intermediate filament protein, vimentin.

Intermediate filaments in synovial lining cells in rheumatoid arthritis and other arthritides are of vimentin type.

Cryostat section of synovial tissue from patients with rheumatoid arthritis, ankylosing spondylitis, osteoarthrosis, pigmented villonodular synovitis, and from a normal knee were studied by indirect immunofluorescence with guinea-pig antibodies to the intermediate filament proteins prekeratin, vimentin, and desmin. Staining for vimentin, but absence of prekeratin and desmin, was demonstrated in synovial lining cells. Antivimentin antibody also stained synovial tissue fibroblasts and vascular endothelial lining cells. The intensity of fluorescent staining for vimentin broadly correlated with cellular proliferative activity at these 3 sites.

Cytoplasmic intermediate filaments in cultured glial cells.

Monolayer cultures of rat glial cells derived from 12 to 14 day fetal spinal cords and rat C6 glioma cells were examined for immunofluorescent reactivity with human or rabbit autoantibodies to intermediate filaments and with rabbit antibody to GFAP. The embryonic glial cells and C6 glima cells reacted identically with both. Protoplasmic astrocytes gave immunofluorescent staining of an intricate network of cytoplasmic filaments while fibrous astrocytes and C6 glioma cells gave diffuse cytoplasmic staining of the cell body and cell processes. Cells, pretreated with colchicine or vinblastine, showed staining of thick coils of peri-nuclear, circum-nuclear, or juxtanuclear filaments. The observations suggest that intermediate filaments in glial cells contain unique antigenic determinants as well as antigenic determinants shared with intermediate filaments in other cell types,

Immunoperoxidase EM localisation of cytoplasmic actin in cultured fibroblasts.

Human smooth muscle autoantibody (SMA) of defined anti-actin specificity was tested by the indirect immunoperoxidase staining method on frozen sections of tissues and on cultured rat lung fibroblasts. The serum stained tissue sections and cultured fibroblasts in a pattern identical with that obtained by indirect immunofluorescence. Ultrastructural studies carried out on the immunoperoxidase stained cells showed that the long parallel filaments spanning the long axis of cultured fibroblasts seen by light microscopy correspond with the thick bundles of microfilaments.

Mechanisms of smooth muscle antibody production: a clinical study in children with infections, haemolytic syndromes, and idiopathic thrombocytopenic purpura.

Sera from 530 children suffering from various diseases and from 64 controls were tested for smooth muscle autoantibodies (SMA) by indirect immunofluorescence. A high incidence of SMA (51-86%) was found in patients with viral and bacterial infections (viral hepatitis, infectious mononucleosis, measles, mumps, chickenpox, typhoid fever, and brucellosis), independently of liver invovlvement, and in patients with acute haemolytic anaemia due to G-6-PD deficiency (48%). By contrast, the incidence of SMA from patients with beta-thalassaemia major and idiopathic thrombocytopenic purpura was no higher than in the controls. The discrepancy in incidence in haemolytic anaemias due to different causes may reflect the effect of endogenous and extrinsic agents. In the viral infections, SMA were mainly of the IgM class and gave an 'SMA-V' staining pattern. In bacterial infections (typhoid fever and brucellosis), SMA were either IgG only or IgM and IgG, and the staining pattern was also mainly 'SMA-V'. In infections which affect or may affect the liver (viral hepatitis, infectious mononucleosis, typhoid fever, and brucellosis), SMA was present at high titres (1:80-1:320), whereas in infections not affecting the liver (measles, mumps, and chickenpox) the titres were lower (less than or equal to 1:80). In most patients SMA occurred transiently and without apparent pathogenetic significance. The antigen against which infection-induced SMA is directed is not actin; its nature has yet to be identified.

Immunofluorescence demonstrates the distribution of actin, myosin and intermediate filaments in cultured neuroblastoma cells.

Monolayers of cultured neuroblastoma cells were examined for immunofluorescent reactivity with antibodies directed against actin, myosin or intermediate filaments. In well spread cells, antibody to intermediate filaments stained an intricate cytoplasmic network which extended as filament bundles into cell processes; in poorly spread or rounded cells, the antibody stained thick juxtanuclear filament bundles. By contrast, antibodies to actin or myosin reacted with microspikes and with axonal growth cones. The different topographical distribution of actin, myosin and intermediate filaments suggests that while actin and myosin may have roles in axon elongation, intermediate filaments may function as an internal cytoskeleton as well as in axoplasmic transport. The different distribution of intermediate filaments in well spread compared with rounded cells suggests that the cell makes its filaments prior to axon development and that the filaments subsequently unwind and migrate into the cell processes to form the axon skeleton.

Viral infections and IgM autoantibodies to cytoplasmic intermediate filaments.

Seventy-four out of 113 sera from patients with infectious hepatitis, chickenpox, measles and mumps reacted with both smooth muscle and cytoplasmic filaments in cultured fibroblasts and neuroblastoma. Five out of eighty-five control sera also reacted in this way. That the cytoplasmic structures are intermediate filaments was suggested by their rearrangement into coils of perinuclear filaments in colchicine- or vinblastine-treated fibroblasts, but not in cytochalasin B-treated cells. The idenity of these structures was confirmed by the demonstration that the same structures reacted with the post-viral sera and a rabbit and human anti-intermediate filament antibody. Immunoabsorption studies showed that twenty-seven out of thirty-two positive sera were neutralised by skeletin, the intermediate filament protein from smooth muscle. In all but one of the sera, the antibody was IgM. Antibody titres fell in the second specimen in eleven out of fourteen pairs of acute and convalescent sera. The association between viral infections and autoantibodies suggest that production of antibodies suggests that production of antibody to intermediate filaments may be initiated by viruses.

Distribution of actin and myosin in muscle and non-muscle cells.

Specific anti-actin and anti-myosin antibodies were shown to react in single and double immunofluorescence sandwich tests with identical sites in non-muscle cells in frozen sections of tissues and in cultured cells. In tissues, both antibodies reacted with liver cell membranes, parts of renal glomeruli, brush borders and peritubular fibrils of renal tubules, brain synaptic junctions, and membranes of lymphoid cells in thymic medulla, lymph nodes and spleen. Both antibodies reacted strongly with long parallel cytoplasmic fibrils in cultured fibroblasts, and with disrupted fibrils in cytochalasin-B treated cells. In neuroblastoma cells both antibodies gave prominent staining of growth cones and microspikes. The observation that the distribution of myosin parallels that of actin in non-muscle cells argues strongly in favour of a functional interaction between the two molecules in the generation of contractile activity in non-muscle cells.