Biosynthesis of complement factor P (properdin) by the human pre-monocyte cell line (U-937)

Area of Science:

• Immunology
• Complement System Biology

Background:

• The U-937 cell line, a human monocyte model, differentiates into macrophage-like cells upon stimulation.
• Activated macrophages exhibit increased synthesis of complement components like C2, C3, Factor B, and Factor H.
• Factor P, a crucial component of the alternative complement pathway, plays a role in immune responses.

Purpose of the Study:

• To investigate the biosynthesis of Factor P by monocytoid cells.
• To examine the effects of immunomodulatory agents on Factor P production in U-937 cells.
• To characterize the molecular properties and synthesis pathway of Factor P in this cell model.

Main Methods:

• Culturing U-937 cells and measuring Factor P secretion over 72 hours.
• Assessing Factor P hemolytic activity.
• Utilizing cycloheximide to evaluate de novo synthesis.
• Employing Sephacryl S-300 gel filtration to determine molecular weight.
• Stimulating cells with phorbol myristate acetate (PMA), interferon-gamma (IFN-gamma), and lipopolysaccharide (LPS).
• Performing metabolic labeling and SDS-PAGE with immunoprecipitation to analyze Factor P structure.

Main Results:

• Non-stimulated U-937 cells secreted Factor P in a nearly linear fashion.
• Secreted Factor P exhibited hemolytic activity.
• Factor P synthesis was reversible inhibited by cycloheximide, confirming de novo synthesis.
• Factor P displayed heterogeneous molecular sizes (250-800 kDa) in both cell culture supernatant and plasma.
• PMA, IFN-gamma, and LPS treatment augmented Factor P synthesis by 1.8-, 2.1-, and 2.5-fold, respectively.
• SDS-PAGE revealed a 54 kDa polypeptide subunit for secreted Factor P, with intracellular Factor P being smaller, suggesting post-synthetic modifications.

Conclusions:

• U-937 cells synthesize and secrete active Factor P.
• Factor P biosynthesis involves the production of a precursor subunit followed by post-synthetic glycosylation and polymerization.
• Immunomodulatory agents found at inflammatory sites can enhance Factor P synthesis by monocytic cells.