Constitutive Expression of PTX3 by Human Amniotic Membrane Cells Leads to Formation of the HC-HA/PTX3 Complex [Cell Biology]

March 20th, 2014 by Zhang, S., Zhu, Y.-T., Chen, S.-Y., He, H., Tseng, S. C. G.

HC-HA, a complex formed by the covalent linkage between HC1 from inter-α-trypsin inhibitor (IαI) and hyaluronic acid (HA), purified from human amniotic membrane (AM) is responsible for AM anti-inflammatory, anti-scarring, and anti-angiogenic actions. This HC-HA complex is produced by constitutive expression of TNF-stimulated gene-6 and endogenous production of IαI by AM cells. Pentraxin 3 (PTX3), a prototypic long pentraxin that plays a non-redundant role in innate immunity against selected pathogens also helps stabilize HC-HA to ensure female fertility. Herein, we noted strong positive PTX3 staining in the AM epithelium and compact stroma. PTX3 was constitutively expressed and secreted by cultured AM epithelial and stromal cells and further greatly upregulated by TNF and IL-1β. Using an agarose overlay to trap the HA-containing matrix, HC-HA/PTX3 complex was formed analyzed by Westernblot by AM cells but not human skin fibroblasts, despite being cultured in the presence of serum and TNF. However, exogenous PTX3 helps human skin fibroblasts form HC-HA/PTX3 complex with an agarose overlay. Furthermore, PTX3 can be co-immuneprecipitated with HC-HA complex from agarose-overlaid AM cell extracts by anti-human IαI antibody. Such HC-HA/PTX3 complex can be reconstituted in vitro and exhibit similar effects reported for AM HC-HA/PTX3 on polarization of M2 macrophages. The tight binding between PTX3 and AM HC-HA withstands four runs of CsCl ultracentrifugation in the presence of 4 M GnHCl. These results indicate that PTX3 is constitutively expressed and secreted by AM cells as an integral component of AM HC-HA/PTX3 complex, and contributes to the biological function of AM HC-HA/PTX3.