In Vitro Complement-Binding on Cytoplasmic Structures in Normal Human Skin: I. Immunofluorescence Studies

Incubation of cryostat sections of normal human skin with normal human serum (NHS) at 37°C followed by fluorescein isothiocyanate labeled rabbit antihuman C3 (FITC-R/Hu-C3) yields cytoplasmic staining of various cell types including keratinocytes of the upper epidermal layers, melanocytes, fibroblasts, smooth muscle cells, and cells lining vascular structures.Deposition of C3 on the respective cytoplasmic structures is most likely due to activation of the classical complement (C) cascade on these structures since no fluorescent staining is observed when serum of patients with hereditary C4-deficiency is used instead of NHS or when incubation with NHS is performed in the presence of EDTA or EGTA in concentrations known to inhibit classical C pathway activation. Further evidence suggesting the involvement of the classical C pathway comes from the finding that incubation of cryostat skin sections with NHS followed by FITC labeled rabbit antihuman Clq (FITC-R/Hu-Clq) results in a fluorescent staining pattern remarkably similar to that seen after exposure of cryostat skin sections to NHS and FITC-R/ Hu-C3.Although formal proof is lacking, our investigations strongly indicate that binding to and activation of C components on cytoplasmic structures occur independently of the presence of circulating antibodies. This assumption is based on the finding that in 17 out of 20 NHS we were not able to detect any skin reactive antibodies by indirect immunofluorescence (IF) techniques. More conclusive evidence for a direct, antibody-independent interaction between C components and cytoplasmic structures is provided by the observation that incubation of the substrate with purified Clq followed by FITC-R/ Hu-Clq results in cytoplasmic staining of some of the skin cell populations described above.The phenomenon of C-binding adn activation on cytoplasmic structures of normal human skin cells may be a critical event in the initiation of complement mediated pathopysiological reactions of the skin