ASSEMBLY OF BASEMENT-MEMBRANE IN-VITRO BY COOPERATION BETWEEN ALVEOLAR EPITHELIAL-CELLS AND PULMONARY FIBROBLASTS

To investigate basement membrane formation by cooperation between pneu mocytes and pulmonary fibroblasts, we cultured type II alveolar epithe lial cells obtained from rats transfected with SV40-large T antigen ge ne (SV40-T2 cells) on type I collagen matrices. On fibroblasts-embedde d gel (T2-Fgel), SV40-T2 cells ultrastructurally formed a continuous a nd thin layer of lamina densa, while on collagen gel without fibroblas ts (T2-gel) SV40-T2 cells produced only discontinuous and diffuse depo sits. Stripping SV40-T2 cells off the tissues by H2O2 treatment reveal ed a continuous and plane surface of lamina densa assembled on the T2- Fgel tissue, whereas only amorphous deposits appeared on the T2-gel ti ssue. Immunolocalization of major basement membrane components showed that type IV collagen, laminin, perlecan and entactin (nidogen) were c ontinuously integrated on the lamina densa in T2-Fgel. In T2-gel, all these components were discontinuously distributed beneath SV40-T2 cell s. The contribution of pulmonary fibroblasts to the assembly of baseme nt membrane through reorganization of collagen matrix and/or soluble f actors was examined by the cultured of SV40-T2 cells on the freeze-tha wed fibroblast-tissue and/or with the fibroblast-conditioned medium. B oth SV40-T2 cells on the freeze-thawed fibroblast-tissue and SV40-T2 c ells in T2-gel in the fibroblast-conditioned medium failed to produce a lamina densa. SV40-T2 cells could assemble a lamina densa only on th e freeze-thawed fibroblast-tissue in the fibroblast-conditioned medium . These results show that the basement membrane components are assembl ed to a lamina densa by combination of the reorganization of collagen matrix and the supply of soluble Factors by pulmonary fibroblasts.