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Evaluation of the expression of human CAP18 gene during neutrophil maturation in the bone marrow
I Nagaoka1, M Hirata, K Sugimoto
1Department of Biochemistry, Juntendo University, School of Medicine, Tokyo, Japan. nagaokai@med.juntendo.ac.jp
Abstract:
To understand the gene expression of CAP18 (18-kDa cationic antibacterial protein), a member of cathelicidins, we evaluated mRNA and protein expression of CAP18 using human bone marrow cells and mature neutrophils. Northern blot analysis revealed that CAP18 mRNA was expressed more abundantly in bone marrow cells than mature neutrophils, whereas Western blot analysis indicated that CAP18 protein was more abundant in mature neutrophils than bone marrow cells. Consistent with this, in situ hybridization using bone marrow cells demonstrated that the expression of CAP18 mRNA was neutrophil lineage-specific and was observed primarily in myelocytes (>95%) with limited expression in more immature cells (promyelocytes) and mature cells (metamyelocytes, band cells, and segmented neutrophils). Furthermore, immunohistochemical study indicated that, coincident with the increase of CAP18 mRNA levels, CAP18-positive cells increased markedly at myelocyte stage, and the increased levels remained almost constant (>95%) in metamyelocytes, band cells, and segmented neutrophils, although the mRNA levels were remarkably reduced in these cells. Together these observations indicate that CAP18 gene transcription likely occurs lineage- and stage-specifically at the myelocyte stage of neutrophil maturation in the bone marrow and results in the synthesis and cytoplasmic accumulation of CAP18, which is present in the subsequent stages of neutrophil maturation.
Insights
Gene expression of CAP18 (18-kDa cationic antibacterial protein) is specific to neutrophil maturation. CAP18 mRNA and protein levels indicate transcription occurs at the myelocyte stage, with protein accumulating in mature neutrophils.
Area of Science:
- Immunology
- Molecular Biology
- Hematology
Background:
- Cathelicidins are a family of antimicrobial peptides.
- CAP18 (18-kDa cationic antibacterial protein) is a cathelicidin with known antimicrobial properties.
- Understanding the regulation of CAP18 expression is crucial for comprehending innate immunity.
Purpose of the Study:
- To investigate the gene expression profile of CAP18 during neutrophil differentiation.
- To correlate mRNA and protein expression levels of CAP18 in human bone marrow cells and neutrophils.
- To determine the specific stages of neutrophil maturation involved in CAP18 synthesis.
Main Methods:
- Northern blot analysis to assess CAP18 mRNA levels.
- Western blot analysis to quantify CAP18 protein abundance.
- In situ hybridization and immunohistochemistry to localize CAP18 mRNA and protein expression in bone marrow cells.
Main Results:
- CAP18 mRNA expression was higher in bone marrow cells than mature neutrophils.
- CAP18 protein was more abundant in mature neutrophils compared to bone marrow cells.
- CAP18 mRNA expression is neutrophil lineage-specific, primarily in myelocytes, with protein accumulating throughout later maturation stages.
Conclusions:
- CAP18 gene transcription is lineage- and stage-specific, occurring during the myelocyte stage of neutrophil development.
- Post-transcriptional regulation leads to the accumulation of CAP18 protein in mature neutrophils.
- These findings elucidate the regulatory mechanisms governing CAP18 expression in neutrophils.