Jerome S. Perlish

The purpose of this study was to determine the frequency, distribution, and nature of cellular infiltrates in 108 skin biopsies from patients with systemic scleroderma (SS) and localized scleroderma (LS). Cellular infiltrates, perivascular or diffuse, were noted in 49% of SS and 84% of LS patients and consisted of lymphocytes, plasma cells, and macrophages. No correlation was noted between the presence or severity of skin cellular infiltrates and serum serologic abnormalities.

Human skin was labeled with purified antibodies against type II and III collagens and against their extension aminopropeptides by using the ferritin technique. Both aminopropeptides were visualized mainly along thin collagenous fibrils (diameter, 20-40 nm) and rarely in nonfibrillar regions of the skin. The labeling showed a periodicity of 60-65 nm resembling the D (67 nm) stagger of collagen molecules. Blocking of antibodies with aminopropeptides and treatment of tissues with procollagen NH2-terminal protease abolished labeling. Antibodies against type I collagen uniformly labeled approximately equal to 80% of the fibrils (diameter, 20-80 nm), while reaction with antibodies against type III collagen was restricted to thin fibrils. It is currently thought that the aminopropeptides of procollagen molecules are cleaved after they are released from the cell and before fibril formation. Our data indicate that aminopropeptides are removed at the fibrillar level and that fibril growth can be regulated by extracellular procollagen processing.

There is some evidence that type I and type III collagens may be present in the same fibril. In order to demonstrate this, double labeling immunofluorescence microscopy and immunoelectron microscopy were performed with antibodies directed against the collagen molecule and the aminopropeptide domains of type I and type III procollagens using embryonic (postabortion) and adult human skin. Double indirect and protein A immunoelectron microscopy were carried out with 5- and 15-nm gold particles. Skin extracts were also studied by immunoblotting. Double immunofluorescence microscopy with antibodies against type I and type III collagen molecules revealed patterns of fluorescence that were identical in both fetal and adult skins. Immunofluorescence microscopy using an antibody directed against the aminopropeptide of type III procollagen labeled the entire dermis in both embryonic and adult skins. In contrast, although the aminopropeptide of type I procollagen was present throughout embryonic dermis, it was markedly reduced in adult dermis, except for the epidermo-dermal junction. Double immunoelectron microscopy of fetal skin revealed labeling of the aminopropeptide of type I and type III procollagens on the same thin (20-30 nm) fibrils. Large type I fibrils (90-100 nm) were coated with type III collagen molecules and their corresponding aminopropeptide but not with the aminopropeptide of type I procollagen. The aminopropeptide of type I procollagen was present on thin fibrils only at the epidermo-dermal junction in adult skin. Immunoblotting of skin extracts revealed the presence of both pN-type III procollagen (collagen plus the aminopropeptide) and pN-type I procollagen in fetal skin, but only pN-type III in adult skin. This study demonstrates that type I and type III collagens coexist within the same fibril and that the aminopropeptide of type III procollagen is present at the surface of type I collagen fibrils that apparently have reached full growth.

Chicken embryo skin of different ages and adult skin were labeled with antibodies against the amino propeptide and carboxyl propeptide of type I collagen and processed for indirect immunoelectron microscopy by the ferritin technique. The results indicate that the formation of thin collagen fibrils involves polymerization of pN-collagen. Fibrils that are thicker than 35-40 nm do not appear to contain the amino propeptide. How fibrils increase in size is not clear, but growth may involve mechanisms such as lateral aggregation of subfibril structures or fusion of thin fibrils. Carboxyl propeptides were localized near or in contact with thin collagen fibrils, but they did not appear to be arranged in a periodic manner along the fibrils. In experiments using antibodies against the amino propeptides of type III collagen, fibrils 20-40 nm in diameter were also labeled in a periodic fashion. pN-Collagen chains were extracted from embryonic skin and identified by NaDodSO4/polyacrylamide gel electrophoresis and by immunoblotting. The presence of significant amounts of pN-collagen in skin from 10- and 12-day chicken embryos agreed well with the labeling of amino propeptides by immunoelectron microscopy. This study provides evidence for the role of the amino propeptide in collagen fibrillogenesis in embryonic skin.