Olfactomedin 4 defines a subset of human neutrophils

Abstract

OLFM4 was identified initially as a gene highly induced in myeloid stem cells by G-CSF treatment. A bioinformatics method using a global meta-analysis of microarray data predicted that OLFM4 would be associated with specific granules in human neutrophils. Subcellular fractionation of peripheral blood neutrophils demonstrated complete colocalization of OLFM4 with the specific granule protein NGAL, and stimulation of neutrophils with PMA resulted in corelease of NGAL and OLFM4, proving that OLFM4 is a genuine constituent of neutrophil-specific granules. In accordance with this, OLFM4 mRNA peaked at the MY/MM stage of maturation. OLFM4 was, however, present in only 20-25% of peripheral blood neutrophils, as determined by immunocytochemistry and flow cytometry, whereas mRNA for OLFM4 was present in all MY/MM, indicating post-transcriptional regulation as a basis for the heterogeneous expression of OLFM4 protein.

Figure 1.. OLFM4 is a constituent of neutrophil-specific granules and is maximally transcribed at the MY/MM stage of myeloid differentiation.

(A) Relative expression levels of mRNA for OLFM4, MPO, LCN2 (NGAL), and gelatinase in human bone marrow cells separated into three populations: MB/PM (MB/PM), MY/MM, bm-PMN, and on PMN isolated from peripheral blood (Pb-PMN). Human ACTB was used as a normalizer; performed in triplicates. Bars represent sd. (B) Immunoblot of subcellular neutrophil fractions showing OLFM4 with a MW of ∼64 kDa present in fraction 9-18. (C) Distribution profiles of MPO (marker of azurophil granules), NGAL (marker of specific granules), gelatinase (marker of gelatinase granules), and HSA (marker of secretory vesicles), all measured by ELISA. Distribution profiles for OLFM4 and cytochrome c are based on intensity measurements of bands on immunoblot. y-Axis represents an arbitrary scale where the highest measured value of each protein is given the value 1. (D) Distribution profiles of OLFM4, NGAL, MPO, and cytochrome c from unstimulated neutrophils and neutrophils stimulated with 100 ng/mL PMA prior to nitrogen cavitation and subcellular fractionation. Stimulation of cells with PMA causes mobilization of ∼60% of specific granules and leads to a similar reduction in OLFM4 and in the specific granule protein marker NGAL. The content of the azurophil granule protein MPO and the mitochondrial marker cytochrome c is virtually unchanged by stimulation. Results for MPO and NGAL were measured by ELISA (y-axis represents ng/mL). Results for OLFM4 and cytochrome c are based on density measurements of bands on immunoblots [Supplemental Fig. 1 A and B; y-axis represents intensity (INT)/mm²].

Figure 2.. OLFM4 protein is differentially expressed in neutrophils and myeloid precursors.

(A) Immunocytochemistry of OLFM4 and NGAL in mature PMNs isolated from peripheral blood with antibody raised against synthetic OLFM4 peptide (antibody 3569). Original scale bars represent 20 μm. Preabsorbtion of the antibody with synthetic OLFM4 peptide completely abrogated the immunostaining (data not shown). (B) Confocal microscopy of isolated neutrophils stained for OLFM4 [green fluorescence (Alexa488)], in combination with the azurophil granule marker MPO, the specific granule marker NGAL, or the tertiary granule marker gelatinase [all marked with red fluorescence (Alexa594)]. In merged pictures, colocalization (yellow) with NGAL and gelatinase is seen together with DAPI staining (blue) of nuclei. (C) Immunocytochemistry of OLFM4 and NGAL in bone marrow populations enriched in MB/PM, MY/MM, and in bm-PMN shows that OLFM4 appears at the myelocyte stage of maturation. OLFM4 is differentially expressed, whereas NGAL is present in all neutrophils. (D) Isolated neutrophils from healthy donors. Fraction of OLFM4 high cells measured by immunocytochemistry (ICC; n=15) or flow cytometry (FC; n=11). Circles represent female donor; triangles represent male donor; bars represent mean value.

Figure 3.. FACS of neutrophils confirms the existence of two subsets: OLFM4 high and OLFM4 low.

(A) Isolated neutrophils from a healthy donor unstained or stained with a FITC-conjugated isotype control or with a FITC-conjugated anti-OLFM4 antibody [GC-1(N-20)], clearly showing two distinct populations of OLFM4, whereas staining for gelatinase (B) reveals only one population. (C) Sorting strategy for FACS of OLFM4 high and OLFM4 low populations depicting the gating of the two populations. FITC-A, FITC-area; FSC-A, forward-scatter-area. (D) Western blot of sorted populations. Samples representing 4 × 10⁵ cells were applied in each well. Whereas the OLFM4 low population has no immunoreactivity for OLFM4, the amount of NGAL is equal in the two populations. (E) Cytospins of the sorted populations acquired in a LSM 700 (Zeiss) microscope.

Figure 4.. OLFM4 mRNA is present in all cells at the MY/MM stage of differentiation.

Fluorescent in situ hybridization of MY/MM isolated from human bone marrow using no probe, a probe for OLFM4 mRNA, a probe for NGAL mRNA, or a probe for hCAP18 mRNA. mRNA for OLFM4 is present in all cells at the MY/MM stage of differentiation and shows no differential expression compared with NGAL and hCAP18.