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Published on: April 10, 2012
Biosynthesis of complement factor P (properdin) by the human pre-monocyte cell line (U-937)
1Department of Pathology, University of Toronto, Ontario, Canada.
Abstract:
The human monocyte-like cell line, U-937, is known to differentiate into macrophage-like cells following stimulation with phorbol myristate acetate (PMA) or interferon-gamma (IFN-gamma). The activated cells have been reported to have enhanced capacity to synthesize C2, C3, Factors B and H. Here, U-937 cells were used as a model system to investigate the effects of immunomodulatory agents on the biosynthesis of Factor P by monocytoid cells. Non-stimulated U-937 cells progressively secreted increasing amounts of Factor P over a 72-hr culture period. The secreted Factor P was hemolytically active. The daily production of Factor P was nearly linear (approx. 2.1 +/- 0.2 ng/10(6) cells; mean +/- SEM). Factor P synthesis was reversibly inhibited by cycloheximide indicating de novo synthesis. Both secreted Factor P and Factor P in normal plasma contained Factor P of heterogeneous molecular sizes and eluted from Sephacryl S-300 gel filtration column as a broad peak (mol. wt 250-800 kDa). The synthesis of Factor P by U-937 cells was augmented 1.8-, 2.1- and 2.5-fold respectively following induction with PMA (30 ng/ml), IFN-gamma (100 U/ml) and LPS (0.1 microgram/ml). Metabolic labeling of U-937 cells and autoradiograms of SDS-PAGE analysis of Factor P immunoprecipitates demonstrated a 54 kDa band in the culture supernate, co-migrating with purified 125I Factor P. Intracellular Factor P however had an apparent mol. wt that was 4000 kDa smaller than secreted Factor P. Thus U-937 cells synthesize a precursor Factor P subunit polypeptide chain which undergoes post-synthetic glycosylation and polymerization to give rise to the oligomers characteristic of native Factor P in fresh plasma. Our data also demonstrate that Factor P synthesis by monocytic cells can be enhanced by immunomodulatory factors or mediators that are generally found at sites of inflammation and immune response.
Insights
Monocytoid cells, like U-937, synthesize Factor P, a complement protein. Immunomodulatory agents such as PMA and IFN-gamma significantly enhance Factor P production in these cells.
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.
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