Type I collagen or gelatin stimulates mouse peritoneal macrophages to aggregate and produce pro-inflammatory molecules through upregulated ROS levels

This study aims to investigate the effect of col. I, the main component of collagenous protein and its denatured product, gelatin, on mouse peritoneal macrophages (MPMs).

Extracellular matrix (ECM) comprising the environments of multicellular society has a dynamic network structure. Collagen is one of the ubiquitous components of ECM. Collagen affects the inflammatory response by regulating the release of pro-inflammatory cytokines from cells. Gelatin, denatured collagen found temporally in tissues, is supposed to be pathophysiologically involved in tissue remodeling, inflammation caused by tissue damage. Previous reports indicate that, phorbol myristate (PMA)-stimulated human U937 (lymphoma cell line) cells that are often used as macrophage-like cells, show cell aggregations when cultured on type I collagen (col I) or gelatin-coated dishes, accompanying the changes of production and release of proinflammatory factors. However, it still remains to be examined whether collagen and gelatin affects normal macrophages as well.

Col I - or gelatin-coated culture induced the formation of multicellular aggregates and increased production of NF-κB-associated pro-inflammatory molecules in MPMs through up-regulation of ROS levels.

MTT assay, flow cytometric analysis of ROS, biochemical detection of antioxidant levels, ELISA assay, and western blot were used.

MPMs formed multicellular aggregates on col. I - and gelatin-coated dishes with a concentration- and time-dependent manner. Further studies showed that the culture on col. I and gelatin up-regulated the protein expression and secretion of pro-inflammatory molecules such as IL-1β, TNFα and prostaglandin E2 (PGE2) in MPMs. The levels were higher in the cells on gelatin than those on col. I. ROS levels are significantly increased in the cells cultured on both col. I- and gelatin-coated dishes, accompanying decreased levels of antioxidant enzyme catalase (CAT) and anti-oxidant glutathione (GSH), and enhanced nuclear translocation of NF-κB.