Immune Netw.  2017 Dec;17(6):410-423. 10.4110/in.2017.17.6.410.

Terminally Differentiating Eosinophils Express Neutrophil Primary Granule Proteins as well as Eosinophil-specific Granule Proteins in a Temporal Manner

• Kim K
• Hwang SM
• Kim SM
• Park SW
• Jung Y
• Chung IY

• KMID: 2400627
• DOI: http://doi.org/10.4110/in.2017.17.6.410

Abstract

Neutrophils and eosinophils, 2 prominent granulocytes, are commonly derived from myelocytic progenitors through successive stages in the bone marrow. Our previous genome-wide transcriptomic data unexpectedly showed that genes encoding a multitude of neutrophil primary granule proteins (NPGPs) were markedly downregulated during the end period of eosinophilic terminal differentiation when cord blood (CB) cluster of differentiation (CD) 34+ cells were induced to differentiate toward the eosinophil lineage during a 24-day culture period. Accordingly, this study aimed to examine whether NPGP genes were expressed on the way to eosinophil terminal differentiation stage and to compare their expression kinetics with that of genes encoding eosinophil-specific granule proteins (ESGPs). Transcripts of all NPGP genes examined, including proteinase 3, myeloperoxidase, cathepsin G (CTSG), and neutrophil elastase, reached a peak at day 12 and sharply declined thereafter, while transcript of ESGP genes including major basic protein 1 (MBP1) attained maximum expression at days 18 or 24. Growth factor independent 1 (GFI1) and CCAAT/enhancer-binding protein α (C/EBPA), transactivators for the NPGP genes, were expressed immediately before the NPGP genes, whereas expression of C/EBPA, GATA1, and GATA2 kinetically paralleled that of eosinophil granule protein genes. The expression kinetics of NPGPs and ESGPs were duplicated upon differentiation of the eosinophilic leukemia cell line (EoL-1) immature eosinophilic cells. Importantly, confocal image analysis showed that CTSG was strongly coexpressed with MBP1 in differentiating CB eosinophils at days 12 and 18 and became barely detectable at day 24 and beyond. Our results suggest for the first time the presence of an immature stage where eosinophils coexpress NPGPs and ESGPs before final maturation.

Keyword

Cathepsin G; Cord blood; Eosinophils; Major basic protein 1; Neutrophils

MeSH Terms

Bone Marrow
Cathepsin G
Cell Line
Eosinophils*
Fetal Blood
Granulocytes
Hypereosinophilic Syndrome
Kinetics
Leukocyte Elastase
Myeloblastin
Neutrophils*
Peroxidase
Trans-Activators
Cathepsin G
Leukocyte Elastase
Myeloblastin
Peroxidase
Trans-Activators

Figure

• 

  Figure 1 mRNA levels of neutrophil- and eosinophil-specific genes during differentiation of CB CD34+ cells toward eosinophils. Expression of 14 genes was determined by real-time PCR. The results represent the mean±standard error of the mean from 3–4 donors in which each measurement was performed in triplicate (*p<0.05 and **p<0.01, compared with day 6 CB cells).

• 

  Figure 2 mRNA levels of neutrophil- and eosinophil-specific genes in EoL-1 eosinophilic cells. EoL-1 cells were induced to differentiate in the absence or presence of dbcAMP (0.1 mM) for 1 and 3 days. Transcript levels are expressed as fold increase relative to expression at day 1 in the absence of dbcAMP. The results represent the mean±standard error of the mean of 3 to 5 independent experiments. Each measurement was performed in triplicate (*p<0.05 and **p<0.01 for comparison between the absence and presence of dbcAMP on each day).

• 

  Figure 3 Immunofluorescence microscopy analysis of MBP1 and CTSG expression. (A) Differentiating CB cells at 4 different time points. Cells were stained with ALEXA488-anti-MBP1 (green) and rhodamine-anti-CTSG (red) antibodies. Fluorescence images were acquired using a ZEISS LSM 800 confocal microscope (ZEISS, Oberkochen, Germany), and their mean fluorescence intensities were analyzed in ten developing CB cells using ImageJ software (National Institutes of Health, Bethesda, MD, USA). These photos are representatives of developing CB cells from 3 donors (*p<0.01, compared with day 6 CB cells). (B, C) PB eosinophils (B) and neutrophils (C) were stained with the same Abs and visualized. The scale bars indicate 20 µm.

• 

  Figure 4 Western blot analysis of neutrophil-specific granule proteins and ESGPs. (A) CTSG, MPO, Olig2, and MBP1 proteins were analyzed in differentiating CB cells at 6, 12, 18, and 24 days. This result is representative of cultures of CB cells from 3 donors. (B) CTSG, MPO, and MBP1 proteins were evaluated in PB eosinophils (E) and neutrophils (N) purified from 3 donors. The anti-MPO Ab used cross-reacted weakly with EPX.

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