Cellular heterogeneity in cultured human chondrocytes identified by antibodies specific for α2(XI) collagen chains

Abstract

Collagen type XI is a component of hyaline cartilage consisting of α1(XI), α2(XI), and α3(XI) chains; with 5-10% of the total collagen content, it is a minor but significant component next to type II collagen, but its function and precise localization in cartilaginous tissues is still unclear. Owing to the homology of the α3(XI) and α1(II) collagen chains, attempts to prepare specific antibodies to native type XI collagen have been unsuccessful in the past. In this study, we report on the preparation and use for immunohistochemistry of a polyclonal antibody specific for α2(XI denatured collagen chains. The antibody was prepared by immunization with the isolated α2(XI) chain and reacts neither with native type XI collagen nor type I, II, V, or IX by ELISA or immunoblotting, nor with α1(XI) or α3(XI), but with α2(XI) chains. Using this antibody, it was possible to specifically localize α2(XI) in cartilage by pretreating tissue sections with 6 M urea. In double immunofluorescence staining experiments, the distribution of α2(XI) as indicative for type XI collagen in fetal bovine and human cartilage was compared with that of type II collagen, using a monoclonal antibody to α1(II). Type XI collagen was found throughout the matrix of hyaline cartilage. However, owing to cross-reactivity of the monoclonal anti-α1(II) with α3(XI), both antibodies produced the same staining pattern. Cellular heterogeneity was, however, detected in monolayer cultures of human chondrocytes. Numerous cells stained intracellularly for α2(XI) but not for α1(II)/α3(XI) and vice versa, besides the majority of the cells that stained with both antibodies. These findings suggest that (a) chondrocytes may not always simultaneously synthesize α1(II) and α2(XI) collagen chains in cell culture; and (b) that a subpopulation of chondrocytes may exist in vitro that synthesize type XI collagen molecules containing α2(XI) but not α3(XI) subunits.