Subcellular localization of OLFM4.

<p><b>A</b>) Neutrophils were immunofluorescently stained for OLFM4 (green) together with specific granule marker NGAL or azurophil granule marker CD63 (red), and DNA was labeled with DAPI (blue). Colocalization was analyzed by imaging flow cytometry and images show representative cells from the double positive populations (BF = brightfield). The diagram shows the mean colocalization score for the two fluorophores +SD from at least three experiments. The technical positive control was FITC-conjugated mouse anti-human CD16 antibody followed by Alexa Fluor 647-coupled goat anti-mouse secondary antibody and the technical negative control was FITC-conjugated mouse anti-human CD16 antibody together with DAPI, showing the minimum and maximum values that can be obtained by this analysis. The biological positive control was specific granule marker lactoferrin (LF) together with NGAL, and the biological negative control was lactoferrin together with CD63, showing the resolution of the analysis. <b>B</b>) Pooled neutrophils from three donors were subjected to subcellular fractionation, and the fractions containing peak content of azurophil granule marker (MPO; fraction 1), specific granule marker (lactoferrin; fractions 10-12), gelatinase granule marker (gelatinase; fractions 13-15) and secretory vesicle marker (latent alkaline phosphatase; fraction 22) were subjected to Western blot with anti-OLFM4 antibody using rOLFM4 as a positive control. One representative blot out of three is shown. Lactoferrin (specific granule marker) and gelatinase (gelatinase granule marker) blots are also shown for fractions 10-15.</p

Effect of rOLFM4 protein on neutrophils.

<p><b>A</b>) Neutrophils were allowed to enter apoptosis spontaneously (untreated) or in the presence of rOLFM4 at a range of concentrations, after which they were stained using Annexin V-Fluos (for apoptosis) and 7-AAD (for necrosis). The dot plots show Annexin V and 7-AAD staining in untreated and rOLFM4-treated cells in one representative donor after 20 h incubation, and the histogram shows the percentage of neutrophils that have undergone each type of cell death in untreated and rOLFM4-treated samples, respectively. The mean +SD of three experiments is shown. <b>B</b>) Neutrophils were treated with rOLFM4 (1 µg/ml) , TNFɑ (10 µg/ml; positive control) or left untreated, for 20 min at 37^oC, and analyzed for surface expression of L-selectin. The shedding of L-selectin from the cells is indicative of neutrophil activation. The histogram shows L-selectin staining in untreated cells and cells treated with rOLFM4 or TNFɑ in one representative experiment. The bar graph shows the mean fluorescence intensity (MFI) of L-selectin +SD from three independent experiments.</p

Conventional degranulation of OLFM4.

<p>Control neutrophils and neutrophils treated with fMLF or cytochalasin B/ionomycin were subjected to immunofluorescent staining of OLFM4 and NGAL, and analyzed by flow cytometry. The histograms show OLFM4 or NGAL staining in control (Con, yellow), fMLF-stimulated (blue), and cytochalasin B/ionomycin-stimulated (CB+io, red) neutrophils from one representative experiment out of four.</p

Presence of OLFM4 in NETs.

<p><b>A</b>) Neutrophils isolated from heparinized blood were adhered to glass coverslips and either stimulated with PMA to form NETs (+PMA) or left unstimulated (-PMA). They were then left unpermeabilized (-Perm.) or permeabilized using acetone/methanol (+Perm.), after which they were immunostained for OLFM4 (red), plasma membranes (PM) were stained using FITC-conjugated WGA (green), and DNA was stained with DAPI (blue). Confocal images show representative cells or NETs from at least three individual experiments. <b>B</b>) Adherent neutrophils were induced to form NETs as in A, and unpermeabilized samples were immunostained for OLFM4 (green) and MPO (red). DNA was stained with DAPI (blue). Confocal images show representative NETs from three individual experiments. <b>A</b>–<b>B</b>: Arrows indicate OLFM4-containing cells or NETs, while arrow heads indicate cells or NETs without OLFM4. The fluorophore conjugates used for each staining are indicated (AF = Alexa Fluor). The scale bars represent 5 µm.</p

OLFM4 expression in transmigrated tissue neutrophils.

<p>Skin chamber neutrophils (<b>A</b>) were obtained from healthy volunteers and synovial fluid neutrophils (<b>B</b>) were collected from inflammatory arthritis patients. Blood samples were also drawn from all subjects. After fixation of tissue and blood neutrophils, OLFM4 was immunolabelled. The histograms show OLFM4 staining in blood and skin chamber (Skin ch.) or synovial fluid neutrophils from one donor each. The diagrams show the percentage of OLFM4-positive neutrophils in blood and transmigrated neutrophils from three different donors.</p

The Human Neutrophil Subsets Defined by the Presence or Absence of OLFM4 Both Transmigrate into Tissue <i>In Vivo</i> and Give Rise to Distinct NETs <i>In Vitro</i>

<div><p>Neutrophil heterogeneity was described decades ago, but it could not be elucidated at the time whether the existence of different neutrophil subsets had any biological relevance. It has been corroborated in recent years that neutrophil subsets, defined by differential expression of various markers, are indeed present in human blood, calling for renewed attention to this question. The expression of the granule protein olfactomedin 4 (OLFM4) has been suggested to define two such neutrophil subsets. We confirm the simultaneous presence of one OLFM4-positive and one OLFM4-negative neutrophil subpopulation as well as the localization of the protein to specific granules. <i>In vitro</i>, these neutrophil subsets displayed equal tendency to undergo apoptosis and phagocytose bacteria. In addition, the subpopulations were recruited equally to inflammatory sites <i>in vivo</i>, and this was true both in an experimental model of acute inflammation and in naturally occurring pathological joint inflammation. In line with its subcellular localization, only limited OLFM4 release was seen upon <i>in vivo</i> transmigration, and release through conventional degranulation required strong secretagogues. However, extracellular release of OLFM4 could be achieved upon formation of neutrophil extracellular traps (NETs) where it was detected only in a subset of the NETs. Although we were unable to demonstrate any functional differences between the OLFM4-defined subsets, our data show that different neutrophil subsets are present in inflamed tissue <i>in vivo</i>. Furthermore, we demonstrate NETs characterized by different markers for the first time, and our results open up for functions of OLFM4 itself in the extracellular space through exposure in NETs.</p> </div

Functional analyses of the neutrophil subpopulations.

<p><b>A</b>) Neutrophils were either fixed immediately after separation (Fresh) or allowed to enter apoptosis spontaneously (-FasL) or through the Fas pathway (+FasL) for 4 or 20 h, after which they were fixed and stained using a TUNEL assay in combination with immunostaining for OLFM4. The histogram shows fresh (blue) and apoptotic (20 h +FasL, red) neutrophils stained using TUNEL. The dot plot shows double staining of fragmented DNA (TUNEL) and OLFM4, with the quadrants set using fresh neutrophils for TUNEL and control (omitted primary antibody) for OLFM4. The bar graph shows the mean percentage of apoptosis +SD, based on TUNEL-positivity, in the OLFM4-positive (black) and -negative (grey) subpopulations of three donors. <b>B</b>) Freshly isolated and apoptotic neutrophils (20 h incubation without or with FasL) were subjected to immunostaining of OLFM4 (black) and NGAL (grey). The diagram shows the mean percentage of positive cells +SD for each protein from three independent experiments. <b>C</b>) Neutrophils were allowed to phagocytose <i>M. tuberculosis</i> H37Ra strain expressing GFP (<i>M</i>.<i>tb</i>-GFP), either unopsonized or serum-opsonized, at an MOI of 5 for 30 min, fixed, and immunostained for OLFM4. The histogram shows neutrophils incubated without (No prey) or with (+<i>M</i>.<i>tb</i>-GFP ops) serum-opsinized <i>M. tuberculosis</i>. The dot plot shows fluorescence intensity in the GFP and OLFM4 channels after phagocytosis of opsonized bacteria, with the quadrants set using neutrophils with no prey added for <i>M</i>.<i>tb</i>-GFP and control (omitted primary antibody) for OLFM4. The bar graph shows the mean percentage of phagocytosing neutrophils +SD in the OLFM4-positive (black) and -negative (grey) subpopulations from three independent experiments.</p