ABSTRACT
A mouse monoclonal IgG, G82, directed against guinea pig liver transglutaminase recognizes a transglutaminase-related antigen that is associated with the keratin intermediate filament network in some primary mouse keratinocytes. The association can be seen at the resolution of individual keratin tonofibrils following fixation and staining for double-label indirect immunofluorescence. Western blots indicate that G82 reacts with two proteins of 95 kDa and 280 kDa, respectively, in extracts of these cells. The 95-kDa band is also recognized by a polyclonal antibody against purified guinea pig liver transglutaminase, and the 280-kDa protein seems to correspond to a similar protein that was shown to be recognized by G92.1.2 in the intermediate filament fraction of primary mouse fibroblasts. The transglutaminase-related antigen was shown by confocal microscopy to co-localize only with nonbasal cell specific keratin intermediate filaments.
Subject(s)
Epidermis/chemistry , Keratins/analysis , Transglutaminases/analysis , Animals , Antibodies, Monoclonal/immunology , Cell Differentiation , Guinea Pigs , Mice , Mice, Inbred BALB C , Molecular Weight , RabbitsABSTRACT
Smyth line (SL) chickens develop a spontaneous, autoimmune loss of melanocytes (vitiligo) from the feather. To determine whether lymphocyte infiltration into two-week-old regenerating feathers of vitiliginous chickens was accompanied by lymphocyte infiltration into the skin, skin biopsies were conducted in SL chickens at two-week intervals throughout the development of vitiligo (SLV) when the chicks were 6 to 14 weeks of age. Control skin samples were obtained from age-matched, normally-pigmented Light Brown Leghorn (LBL) chickens. Frozen skin sections were labelled with a panel of mouse mAb to identify various lymphocyte populations. There was no lymphocyte infiltration into the skin of SL or control chickens. However, in both lines of chickens, dermal lymphoid aggregates (DLA), consisting primarily of T cells, were observed. In the DLA of SL chickens, the ratio between CD4+ and CD8+ T cells was lower (P = 0.005) and the percentage of gamma delta T cells higher (P = 0.027) than in the controls. Additionally, in SL chickens the lymphocyte populations in the DLA varied depending on the developmental stage of vitiligo. These data suggest a relationship between the DLA and autoimmune vitiligo in the SL model.
Subject(s)
Chickens/immunology , Lymphocytes/immunology , Poultry Diseases/immunology , Skin/immunology , Vitiligo/veterinary , Animals , CD4-CD8 Ratio , Cell Aggregation , Mice , Vitiligo/immunologyABSTRACT
A mouse monoclonal antibody, G92.1.2, raised against guinea pig liver transglutaminase (TGase) recognizes an antigen present in primary mouse dermal fibroblasts. A filamentous pattern, bearing remarkable similarity to the vimentin intermediate filament (IF) network, is seen when these cells are fixed and processed for indirect immunofluorescence with the antibody. Double-label immunofluorescence reveals that the antigen reacting with the antibody colocalizes precisely with vimentin IF and that this colocalization is retained after the treatment of fibroblasts with colchicine, which induces a redistribution of the majority of IFs into perinuclear aggregates. These morphological observations are further supported by the finding that the protein reacting with G92.1.2 is retained in IF-enriched cytoskeletal preparations made by using nonionic detergent-containing high ionic strength solutions. Western blots of the IF fraction show that G92.1.2 recognizes a major band of approximately 280 kDa and does not cross react with vimentin. Furthermore, when the antibody is microinjected into live dermal fibroblasts, it causes a collapse of the vimentin IF network in the majority of injected cells. The results suggest that a form of TGase, or a TGase-related antigen, is closely associated with the vimentin IF network of primary cultures of mouse dermal fibroblasts.
Subject(s)
Intermediate Filaments/enzymology , Transglutaminases/isolation & purification , Animals , Antibodies, Monoclonal/immunology , Antibody Specificity , Antigens/immunology , Apoptosis/physiology , Cells, Cultured , Cross Reactions , Enzyme-Linked Immunosorbent Assay , Fibroblasts/immunology , Fibroblasts/ultrastructure , Fluorescent Antibody Technique , Guinea Pigs , Immunoblotting , Intermediate Filaments/immunology , Liver/enzymology , Mice , Protein Conformation , Skin/cytology , Skin/immunology , Skin/ultrastructure , Subcellular Fractions/enzymology , Transglutaminases/immunology , Vimentin/isolation & purificationABSTRACT
Smyth line (SL) chickens exhibit a spontaneous, autoimmune, depigmentation (SL vitiligo, SLV) in regenerating feather tissue due to the death of pigment cells (melanocytes). It was the purpose of this study to characterize mononuclear leukocytes which infiltrate the feather as SLV develops. Frozen sections of regenerating features from SL and control chickens were labeled with a panel of mAbs specific for chicken mononuclear leukocytes. SL chickens were found to have significantly greater numbers of T cells in the feather pulp prior to, and throughout, the development of SLV. Initially, both SL and control chickens had a CD4/CD8 ratio near 1.0 in their feather pulp. Following the onset of SLV, the CD4/CD8 ratio in SL chickens dropped below 0.4. Throughout the development of SLV, SL chickens had greater proportions of T cells with alpha/beta T cell receptors than controls. Histological evidence strongly supports an important role for T cells in the pathology of SLV.
