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Selective synthesis of mRNA and proteins by human peripheral blood neutrophils
1Department of Medicine, Harvard Medical School, Boston, MA 02115.
Abstract:
Human peripheral blood polymorphonuclear neutrophils (PMN) have been considered to be capable of little if any protein biosynthesis. We evaluated the ability of PMN to synthesize both mRNA and proteins. Using in vitro [35S]methionine pulse-chase labeling of purified PMN, followed by immunoprecipitation of cell lysates with immobilized mAb and analysis by gel electrophoresis, PMN were shown to synthesize CR1, FcR, CR3 alpha-chain, MHC class I, and actin. In contrast, incorporation of [35S]methionine into either CR3 beta-chain or the secondary granule protein lactoferrin was not detected. Purification of mRNA from PMN and analysis by Northern blots demonstrated the presence in PMN of CR1, actin, and MHC class I transcripts. However, despite the apparent lack of CR3 beta-chain biosynthesis, specific beta-chain message was detectable in PMN RNA. Inhibition of mRNA synthesis in PMN with actinomycin D resulted in decreased synthesis of nascent CR1, FcR, MHC class I, and actin compared with control cells. Thus, PMN continue to transcribe and translate the genes for certain membrane and cytoskeletal proteins. In contrast, the lack of detectable synthesis of either lactoferrin or CR3 beta-chain suggested that biosynthesis in circulating PMN is selective.
Insights
Human neutrophils can synthesize certain proteins and mRNA, including CR1 and actin, but not lactoferrin or the CR3 beta-chain. This protein biosynthesis in polymorphonuclear neutrophils (PMN) is selective.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- Human peripheral blood polymorphonuclear neutrophils (PMN) were traditionally thought to have minimal protein synthesis capabilities.
- Recent studies suggest a need to re-evaluate the protein biosynthesis potential of PMN.
Purpose of the Study:
- To investigate the capacity of human peripheral blood PMN to synthesize both mRNA and proteins.
- To identify specific proteins and their corresponding transcripts synthesized by PMN.
Main Methods:
- In vitro [35S]methionine pulse-chase labeling of purified PMN.
- Immunoprecipitation of cell lysates using immobilized monoclonal antibodies (mAb).
- Gel electrophoresis and Northern blot analysis for mRNA detection.
Main Results:
- PMN synthesize proteins including CR1, FcR, CR3 alpha-chain, MHC class I, and actin.
- Incorporation of [35S]methionine into CR3 beta-chain and lactoferrin was not detected.
- CR1, actin, and MHC class I transcripts were present in PMN mRNA, with detectable CR3 beta-chain message despite lack of protein synthesis.
Conclusions:
- Circulating PMN actively transcribe and translate genes for specific membrane and cytoskeletal proteins.
- The observed lack of lactoferrin or CR3 beta-chain synthesis indicates selective protein biosynthesis in PMN.
- PMN protein synthesis is regulated, with distinct pathways for different proteins.