HC-HA/PTX3 Complex in AM
In 2006, we first reported that AM stromal matrix exerts an anti-inflammatory action by inducing apoptosis of IFN-γ–activated monocyte/macrophage RAW264.7 cells (See Figure 1) and that such an action is not caused by nitric oxide but instead by the downregulation of anti-apoptotic NF-κB and Akt-FKHR signaling pathways.1 Subsequently in 2008, we demonstrated that such an anti-inflammatory action is retained in soluble AM extract (AME). AME upregulates IL-10, downregulates TNF-α and IL-6, and suppresses the activation of RAW264.7 cells by IFN-γ, LPS and IFN-γ/LPS.2 These findings suggest the key molecule(s) in AM responsible for its anti-inflammatory and other biological actions can be extracted and possibly be identified and isolated.
Indeed, in 2009, we successfully purified a complex designated as “HC-HA/PTX3” from AME.3 HC-HA/PTX3, first found in cumulus-oocyte complex (COC) surrounding the ovulated oocyte, is vital for subsequent fertilization.4,5 It is formed by tight association between pentraxin 3 (PTX3) and HC-HA, which consists of high molecular weight hyaluronic acid covalently linked to heavy chain 1 (HC1) of inter-α-trypsin inhibitor. IαI is mainly secreted by the liver and present in the blood at considerably high concentrations (0.15 to 0.5 mg/ml).6 It is composed of a common light chain and two heavy chains (HC1 and HC2). The light chain is a typical proteoglycan molecule having a single chondroitin four-sulfate chain.
Retains Anti-inflammatory Action
Our studies have also shown that HC-HA/PTX3 purified from AM retains AM’s anti-inflammatory action by exerting the following effects:
1) HC-HA/PTX3 promotes apoptosis of pro-inflammatory but not resting neutrophils and macrophages. During inflammation, neutrophils are among the first recruited to engulf pathogens and damaged tissues before their eventual apoptosis.18-20 Pathologically, delayed neutrophil apoptosis leads to a prolonged inflammation, which is a hallmark of many inflammatory diseases.21,22 We have reported that apoptosis of freshly-isolated neutrophils activated by fMLP or LPS is promoted only when they are treated by soluble HC-HA/PTX3, but not HA or PBS control.23 We also report that soluble HC-HA/PTX3, the same as AM and AME, dose-dependently promotes apoptosis of RAW264.7 cells activated by IFN-γ, LPS, or IFN-γ/LPS.2,3,23
2) HC-HA/PTX3 enhances phagocytosis of apoptotic neutrophils by macrophages. Clearance of apoptotic neutrophils by macrophages is essential for inflammation resolution.24-26 We have reported that soluble HC-HA/PTX3, but not HA, is effective in enhancing phagocytosis of apoptotic neutrophils by resting macrophages (about sevenfold vs. PBS control). Meanwhile, immobilized HC-HA/PTX3 is more potent in promoting phagocytosis of apoptotic neutrophils by LPS-activated macrophages (about 2.5-fold).23
4) HC-HA/PTX3 suppresses activation of CD4+ T cells. Naïve CD4+ T cells can be activated to proliferate and differentiate into Th1, Th2, Th17 or Tregs.34-36 Th1 cells secrete IFN-γ and IL-2 to enhance pro-inflammatory responses.37,38 This action can be downregulated by Tregs that is activated by M2 macrophages.39 Our study shows the soluble HC-HA/PTX3 suppresses the proliferation and production of IFN-γ and IL-2 and expression of T cell activation markers (CD25 and CD69) while significantly promoting the expansion of CD25+/FOXP3+ T cells.31 These data strongly suggest that HC-HA/PTX3 suppresses activation of CD4+ T cells into Th1 cells.
5) HC-HA/PTX3 suppresses the macrophage influx to LPS-elicited corneas and prolongs survival of corneal allografts. While intrastromal injection of LPS elicited notable influx of EGFP+ macrophages to the corneal periphery in Mafia mice from day one to day five,40,41 subconjunctival injection of HC-HA/PTX3 significantly suppresses the infiltrated macrophages. Further, the infiltrated macrophages are polarized towards an M2 phenotype by expressing higher M2 markers (Arg-1 and IL-10) but lower M1 markers (IL-12p35 and IL-12p40).31 Using a murine orthotropic corneal transplantation model,42-45 we have found that subconjunctival injection of HC-HA/PTX3 prolongs the significant survival of allografts compared to PBS injection.31 These data suggest that HC-HA/PTX3 may be used to exert a potent anti-inflammatory action leading to suppression of (alloreactive) immune activation in vivo.
HC-HA/PTX3, a unique matrix component purified from human AM, retains AM’s multifactorial anti-inflammatory actions. It is foreseeable that HC-HA/PTX3 can be used as a new class of biologics to treat ocular inflammatory diseases (e.g., proliferative diabetic retinopathy, trauma and subretinal fibrosis secondary to age-related macular degeneration and other choroidal neovascular processes) as well as similar pathological processes in other parts of the body. REVIEW
Dr. Tseng is a physician scientist and the chief scientific officer of TissueTech Inc, where both Dr. He and Dr. Zhang serve as research scientists. Contact Dr. Tseng at Ocular Surface Center, 7000 SW 97 Ave., Ste. 213, Miami, Fla. 33173. Phone: (305) 274-1299; fax: (305) 274-1297; e-mail: email@example.com.
Supported in part by grants from National Institute of Health, National Eye Institute. Additional support is from a research grant from TissueTech Inc. and an unrestricted grant from Ocular Surface Research & Education Foundation, Miami.
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