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Originally published online as doi:10.1189/jlb.0806514 on October 31, 2006

Published online before print October 31, 2006
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(Journal of Leukocyte Biology. 2007;81:458-464.)
© 2007 by Society for Leukocyte Biology

Proteinase 3 and CD177 are expressed on the plasma membrane of the same subset of neutrophils

Susanne Bauer*, Mohamed Abdgawad{dagger}, Lena Gunnarsson{dagger}, Mårten Segelmark{dagger}, Hans Tapper* and Thomas Hellmark{dagger},1

* Departments of Clinical Sciences, Section for Clinical and Experimental Infection Medicine, and
{dagger} Nephrology, Clinical Sciences in Lund, Lund University, Lund, Sweden

1 Correspondence: BMC-C14, Dept. of Nephrology, Clinical Sciences in Lund, Lund University, 221 84 Lund, Sweden. E-mail: thomas.hellmark{at}med.lu.se


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ABSTRACT
 
Proteinase 3 (PR3) is found in granules of all neutrophils but also on the plasma membrane of a subset of neutrophils (mPR3). CD177, another neutrophil protein, also displays a bimodal surface expression. In this study, we have investigated the coexpression of these two molecules, as well as the effect of cell activation on their surface expression. We can show that CD177 is expressed on the same subset of neutrophils as mPR3. Experiments show that the expression of mPR3 and CD177 on the plasma membrane is increased or decreased in parallel during cell stimulation or spontaneous apoptosis. Furthermore, we observed a rapid internalization and recirculation of mPR3 and plasma membrane CD177, where all mPR3 is replaced within 30 min. Our findings suggest that the PR3 found on the plasma membrane has its origin in the same intracellular storage as CD177, i.e., secondary granules and secretory vesicles and not primary granules. PR3- and CD177-expressing neutrophils constitute a subpopulation of neutrophils with an unknown role in the innate immune system, which may play an important role in diseases such as Wegener’s granulomatosis and polycythemia vera.

Key Words: trafficking • colocalization • serine proteases • Wegener’s granulomatosis • polycythemia vera


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INTRODUCTION
 
Proteinase 3 (PR3) was first described in 1973 [1 ] and is a member of the neutrophil serine protease family, which also includes human neutrophil elastase (HNE), cathepsin G, and enzymatically inactive azurocidin. These proteins are stored in primary granules [2 ]. However, PR3 has also been found in secondary granules, in secretory vesicles [3 ], and on the plasma membrane (mPR3) of a subset of neutrophils [4 ]. This nonuniform, bimodal expression of mPR3 on neutrophils is specific to PR3. Other serine proteinases are found on the plasma membrane of activated neutrophils but do not exhibit a bimodal distribution [5 ]. Furthermore, PR3 has been demonstrated to have specific biological functions such as processing of cytokines [6 7 8 ] and induction of IL-8 synthesis by endothelial cells [9 ]. It has been shown that PR3 can promote platelet aggregation [10 ] and that it interferes with the apoptosis machinery [11 12 13 14 ]. Secreted pro-forms of PR3 as well as azurocidin and granzymes A, B, H, K, and M, but not cathepsin G or HNE, have been shown to reduce the fraction of granulopoietic progenitors in S-phase [15 , 16 ]. In summary, PR3 is considered to be essential for the regulation of proliferation, maturation, and apoptosis of granulocytes.

Several investigations have shown that the proportion of neutrophils expressing mPR3 varies between individuals but is remarkably stable over time in a given individual [17 18 19 ]. The percentage of mPR3-positive cells seems to be determined genetically and regulated by two co-dominant alleles [17 , 19 ]. Patients with Wegener’s granulomatosis but also with rheumatoid arthritis have shown an increased proportion of mPR3-positive cells [17 ]. A high percentage of mPR3-positive neutrophils has been linked to an increased risk of developing vasculitis [17 , 19 ], increased relapse rate [18 ], and adverse renal outcome [20 ]. PR3 is important in Wegener’s granulomatosis, as these patients develop autoantibodies against PR3 {PR3-antineutrophil cytoplasmic antibody (ANCA) [21 22 23 24 ]}.

Different receptors for mPR3 have been proposed, and most of the evidence points toward a ß2 integrin, Cd11b/CD18 [25 ], but it has also been shown that mPR3 colocalizes with CD16 (Fc{gamma}RIIIb) [26 , 27 ]. However, these molecules are expressed on all neutrophils and can thus not explain why only a subset of the cells is mPR3-positive.

CD177 is the only other cell surface molecule known to have a bimodal plasma membrane expression pattern in neutrophils. It is a GPI-anchored glycoprotein, first described in 1971 as the NB1 antigen [28 ] and a member of the leukocyte antigen 6 superfamily [29 ]. The gene coding for CD177 is localized to chromosome 19q13.2 and has at least two known alleles, NB1 and polycythemia rubra vera (PRV)-1 [30 ]. The plasma membrane expression of CD177 can be increased by administration of G-CSF [31 ], regarding the percentage of positive cells and the number of molecules per cell. Furthermore, it is increased during pregnancy and is probably affected by estrogen and progesterone. Several studies have shown that 95–100% of patients with PRV have elevated levels of CD177 mRNA [29 , 32 33 34 ]. A similar increase, but in smaller proportion, has been found in essential thrombocythemia and idiopathic myelofibrosis [32 33 34 ], whereas chronic myelogenous leukemia is not associated with an increased CD177 expression [29 ]. Recently, a dominant gain of function mutation in the JAK2 was found in a high proportion of patients with myeloproliferative disorders [35 ].

In this study, we investigate if CD177 and PR3 are expressed on the plasma membrane of the same subpopulation of neutrophils. We also study the effect of cell activation and spontaneous apoptosis on the plasma membrane expression of these two molecules.


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MATERIALS AND METHODS
 
Neutrophil isolation
Polymorphonuclear leukocytes were isolated from human blood, kindly provided by healthy volunteers. In total, 10 different donors have been used, and in each experiment, we have used neutrophils from at least three donors. As described by the manufacturer, whole blood from EDTA-anticoagulated tubes was layered carefully on PolymorphprepTM (Axis-Shield PoC AS, Oslo, Norway) and centrifuged at 625 g for 1 h at room temperature (RT). The neutrophil layer was recovered and suspended in PBS before pelleting the cells at 250 g for 10 min at RT. Contaminating erythrocytes were removed by hypotonic lysis during 7 min at 37°C using 0.83% NH4Cl in 10 mM Hepes, pH 7.0. The cells were then pelleted at 250 g for 5 min at RT and resuspended in RPMI (PAA Labs, Göteborg, Sweden). The percentage of neutrophils was 95–99%, as determined by Türk staining. Viability, checked using Trypan blue staining, was >95%. All experiments were performed in RPMI medium within 1 h after isolation of the cells.

Colocalization experiments
The neutrophil-containing samples were blocked using human IgG (0.5 mg/ml in PBS containing 5% goat serum and 1% BSA; Sigma Chemical Co., St. Louis, MO) for 20 min on ice, followed by addition of the primary antibodies, rabbit anti-PR3 (3 µg/ml; Wieslab AB, Lund, Sweden), mouse anti-CD177 MCA2045 (1:200–1000; Serotec, Oxford, UK), mouse anti-CD18 (1:1000), or mouse anti-CD16 (1:1000; both from BD PharMingen Biosciences, Palo Alto, CA) for 15 min on ice. The samples were washed and then stained with the secondary antibodies antirabbit ALEXA 488 (1:1000; Molecular Probes, Eugene, OR) and antimouse R-PE-cytochrome 5 (RPE-Cy5; 1:200; Dako Cytomation, Glostrup, Denmark) for flow cytometry (FACS) analysis or antimouse ALEXA 594 (1:1000; Molecular Probes) for fluorescence microscopy for another 15 min on ice. Some samples were stained with cholera toxin subunit B conjugated with Alexa Fluor 594 (CT-B; Molecular Probes). These samples were prefixed with 0.1% paraformaldehyde (PFA), washed, and then incubated in the dark with CT-B for 60 min on ice.

Stimulation experiments
Neutrophils were stimulated with TNF-{alpha} (20 ng/ml, 15 min), PMA (100 nM, 15 min), fMLP (1 µM, 5 min), or cytochalasin B (CyB; 10 µM, 15 min), followed by addition of fMLP (1 µM, 5 min; all from Sigma Chemical Co.). All incubations were performed at 37°C. The samples were prepared as described in the colocalization experiment section and stained with antibodies against PR3 and CD177.

Apoptosis and necrosis investigation
Apoptotic and necrotic cells were demonstrated by double-staining using 1 µl AnnexinV-Alexa Fluor 488 (Molecular Probes) and 20 µl BD Via-Probe (BD PharMingen Biosciences) for 5 min in the dark. Samples were then investigated by FACS.

Internalization experiments
Neutrophil samples were stained with primary antibodies, the monoclonal anti-PR3 4A5 (1:100; Wieslab AB), anti-CD177 (1:1000), or anti-CD18 (1:1500) for 15 min on ice. After washing, the secondary antibody ALEXA 488 antimouse (1:1000) was added for another 15 min on ice. The samples were then put on ice or incubated for 15, 30, or 60 min, rotating at 37°C, 8 rpm. Incubation with the primary antibodies, as described above, was repeated followed by staining with the secondary antibody ALEXA 594 antimouse (1:1000) for 15 min on ice. The samples were fixed using 2% PFA and mounted for fluorescence microscopy evaluation.

Blockage of protein synthesis
Neutrophils were incubated with cycloheximide (100 µg/ml) for 0, 30, 60, 90, and 120 min. After washing, the cells were stained with mAb against PR3 (1:100) and CD177 (1:300) for 15 min on ice and then washed again. The secondary antimouse PE-Cy5 antibody was added for 15 min on ice (1:200). Finally, the cells were stained with Annexin V-Alexa Fluor 488 for 5 min in the dark to monitor the viability of the cells. The samples were evaluated by FACS.

Time-course study
Staining with the primary antibodies, 4A5 (1:300), anti-CD177 (1:200), or anti-CD18 (1:1500), was performed for 15 min on ice followed by antimouse PE-Cy5 (1:200) for another 15 min on ice. Fluorescent Annexin V was then added for 5 min in the dark at RT. Viability was also checked by double-staining with Annexin V and BD Via-Probe. During incubation, the samples were rotated at 37°C, 8 rpm. The samples were evaluated by FACS analysis.

FACS analysis
Flow cytometry analysis was performed using a FACSCalibur flow cytometer, equipped with a 15-mW argon laser tuned at 488 nm (Becton Dickinson, Franklin Lakes, NJ). The neutrophil population was selected by gating with appropriate settings for forward scatter and sideward scatter. The FL1 fluorescence channel was used to record the emitted fluorescence from ALEXA 488, and the FL3 fluorescence channel was used for PE-Cy5. For each condition, 20,000 gated events were acquired. Acquisition and analysis of the data were performed using the CellQuest Pro software.

Fluorescence microscopy
Samples were fixed using 2% PFA for 15 min at 4°C, followed by overnight incubation at 8°C. Glass coverslips were washed with methanol and overlaid with 0.25 ml poly L-lysine (0.2 mg ml–1 in water; Sigma Chemical Co.). After evaporating the added fluid at 50–65°C, the coverslips were washed twice with distilled water. Following a final wash with PBS, the fixed and stained cells were adhered to the poly L-lysine-coated coverslips, and the samples were mounted using Dako mounting medium (Dako, Carpinteria, CA).

Visual inspection and recording of images were performed using a Nikon Eclipse TE300 inverted fluorescence microscope equipped with a Hamamatsu C4742-95 cooled charged-coupled device camera, using a Plan Apochromat 100x oil immersion objective. Images were acquired and handled using Image Pro Plus and Adobe Photoshop 7.0.


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RESULTS
 
Cells were costained with antibodies against CD18/CD16/CD177 and PR3. The samples were evaluated by FACS and fluorescence microscopy. A strong correlation between PR3 and CD177 surface expression was found, as shown in Figure 1 . Anti-CD18, anti-CD16, and CT-B stained virtually all cells, and no correlation to mPR3 staining was seen (data not shown). Representative fluorescence microscopy data are displayed in Figure 2 . CD18 (Fig. 2B) and CD16 (Fig. 2E) are found on the majority of the neutrophils and not only on cells expressing mPR3. However, the expression of CD177 (Fig. 2H) is heterogeneous and coincides with the expression of PR3 (Fig. 2I) . Cells that are mPR3-positive are also positive for CD177, and cells that are mPR3-negative are also negative for CD177. When examining 100 cells, 96% were double-positive or double-negative, and only 4% displayed an expression of one of the markers and not the other.


Figure 1
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  		Figure 1. Coexpression of CD177 and PR3. Neutrophils were stained with anti-CD177 and anti-PR3 antibodies. Figure 1a 1b 1c 1d , represents four different individuals with different proportions of mPR3/CD177 expressing neutrophils (in total, 10 different individuals were studied). As can be seen, PR3 and CD177 are expressed on the plasma membrane of the same subset of neutrophils.


Figure 2
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  		Figure 2. Plasma membrane expression of CD18, CD16, or CD177 and mPR3. Neutrophils were stained using antibodies directed against CD18 (B), CD16 (E), or CD177 (H) and PR3 (C, F, I). CD18 and CD16 are expressed on the majority of neutrophils; however, the CD177 surface staining is bimodal and coincides with the mPR3 expression. The images are a representative of five experiments. Original size bar = 10 µm.

Next, we investigated how the surface expression was affected by stimulation of the neutrophils and whether mPR3- and CD177-negative cells could be stimulated into becoming mPR3- and CD177-positive. Cells were treated with TNF-{alpha}, PMA, fMLP, or fMLP in combination with CyB and analyzed by FACS. Stimulation with TNF-{alpha} or fMLP caused a moderate increase (a factor 1.7 and 2.0, respectively) in fluorescence intensity for mPR3- and CD177-positive cells (Fig. 3 ). However, stimulating the neutrophils with PMA caused a major shift in fluorescence intensity. The mPR3- and CD177-positive cell population was shifted further to the right; i.e., the expression of mPR3 and CD177 increased by a factor 4.9. Furthermore, the mPR3- and CD177-negative population was converted into mPR3- and CD177-expressing cells. The distribution remained bimodal, but instead of a positive/negative population, the cells divided into a mPR3 and CD177 high and low subgroup. The proportion of mPR3 high-expressing cells was constant and independent of stimulation. When stimulating with the combination of fMLP and CyB, a similar effect was observed. Fluorescence microscopy verified a plasma membrane localization of the staining (not shown). Our results indicate that PR3 and CD177 are coregulated, as their surface expression is increased in parallel. Moreover, PR3 and CD177 surface-negative cells have an intracellular pool of these markers, which can be transported to the plasma membrane upon stimulation.


Figure 3
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  		Figure 3. Up-regulation of mPR3 and plasma membrane CD177. The histograms show the fluorescence intensity of unstimulated neutrophils (–––) and neutrophils stimulated with TNF-{alpha} (–·–·–), fMLP (– – –), PMA (–··–··–), or CyB in combination with fMLP (·······) at 37°C. (A and B) The mPR3 is shown; (C and D) CD177 staining is shown. This demonstrates one experiment out of four.

mPR3 has been suggested to be a preapoptotic marker for aging neutrophils. To study this, we incubated purified neutrophils at 37°C for various times, up to 22 h. The neutrophils were analyzed every 3 h for mPR3 expression (Fig. 4A ) and for viability (Fig. 4C) . The percentage of mPR3-positive cells remained constant over time, whereas the MFI slowly decreased with increasing age of the live neutrophils (Annexin V-negative cells). When analyzing the total neutrophil population, similar results were obtained (data not shown). CD177 plasma membrane expression was also evaluated at time-point 0 and after 22 h incubation in three experiments (Fig. 4B) . The results show that the percentage of positive cells tended to diminish (not significant); however, the MFI was reduced, and after 22 h, only 40% of the plasma mPR3 remained. This loss of CD177 and mPR3 could be a result of shedding into the surrounding media or internalization.


Figure 4
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  		Figure 4. Apoptosis and plasma membrane expression of CD177 and mPR3. Neutrophils were incubated for 22 h, and mPR3 was measured every 3 h. Plasma membrane CD177 was measured at time-point 0 and after 22 h. (A) The relative percentage of mPR3-positive cells compared with the baseline is indicated ({square}), and the mean fluorescence intensity (MFI) of the positive cells ({blacksquare}). (B) The relative expression of mPR3 and CD177 is shown. (C) The percentage of live ({blacksquare}), apoptotic ({circ}), and necrotic ({blacktriangleup}) cells is shown. All values are given as mean ± SEM. (A and C) Based on eight experiments; (B) based on three experiments.

The dynamics of the membrane traffic of mPR3 and CD177 was studied by internalization experiments. CD177 was transported into the cells from the surface, and after 15 min of incubation, intracellular CD177 was observed (Fig. 5E 5II ), with only weak staining on the cell surface. After 30 min, the majority of the surface-bound CD177 had been internalized (Fig. 5F 5II) . For PR3, the majority of the surface staining had been internalized already after 15 min (Fig. 5H 5II) . CD18, used as a control, displayed a cell surface staining throughout the study, and only a smaller proportion was transported intracellularly (Fig. 5A 5B 5C II) . After this incubation, the cells were stained again using the same primary antibodies but a different secondary antibody, labeled with another fluorochrome. All cells displayed a strong surface staining (Fig. 5 III) , indicating that new proteins had been transported to the plasma membrane. Samples incubated for 60 min were similar to those incubated for 30 min. FACS analysis of the cells showed that the MFI for the green and red probes was unchanged throughout the experiment (data not shown). This shows that the plasma membrane-bound proteins were internalized and that the amount of proteins on the plasma membrane was constant. Our results demonstrate an active uptake of surface PR3 and CD177 and that new PR3 and CD177 molecules are transported to the surface. These proteins could originate from granules, a recycling compartment, or from de novo synthesis.


Figure 5
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  		Figure 5. Time-course study of membranes CD18 and CD177 and mPR3. Neutrophils were stained with anti-CD18 (A–C), anti-CD177 (D–F), or anti-PR3 (G–I) at time-point 0. After incubation (at 37°C for 0, 15, and 30 min), the cells were stained again with the same primary antibodies but using another secondary antibody labeled with a different fluorochrome. II denotes staining before incubation and III, surface staining done after incubation was completed. CD18, Cell surface staining is present during the entire study. CD177 and PR3, The majority of cell surface staining is internalized already after 15 min. The images are a representative of five experiments. Original size bar = 10 µm.

To investigate whether de novo synthesis is a major factor contributing to the mPR3 and CD177, which reappeared at the cell surface, as seen in Figure 5 III , we inhibited protein synthesis with cycloheximide, and the cycloheximide treatment did not cause any major difference in the number or the staining of the positive cells.


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DISCUSSION
 
It is well established that circulating human neutrophils have a bimodal distribution of PR3 and CD177 on their plasma membrane. In this study, we show that the two markers are expressed on the same subset of neutrophils. Furthermore, both proteins can be up-regulated in parallel, indicating that they share mechanisms of trafficking and that they originate from the same intracellular storage. When stimulating the cells with PMA or with CyB in combination with fMLP, we show that mPR3- and CD177-negative cells become positive. The increased expression of plasma membrane CD177 follows that of mPR3, and levels of both proteins are increased about five times. The majority of the intracellular PR3 is stored in primary granules, and only smaller amounts are found in other granule types. CD177 is not present in primary granules but is found mainly in secondary granules. Still, both proteins are up-regulated to the same degree after potent stimulation using fMLP in combination with CyB, causing a total degranulation. This suggests that only PR3 stored in secretory vesicles and secondary granules can be expressed on the plasma membrane.

Recently, it has been shown that mPR3 can be coprecipitated with CD18 and CD16 and that mPR3 is localized in lipid rafts on neutrophils [25 , 26 ]. Our data do not exclude that mPR3 colocalizes with CD16 and CD18 and that it, to some degree, is found in lipid rafts (colocalization experiments with CT-B, data not shown). However, mPR3 is also expressed in other areas of the plasma membrane, indicating that there may be other proteins linking PR3 to the plasma membrane. Binding of mPR3 to CD16 and CD18 cannot explain the bimodal expression of mPR3, as they are present on all neutrophils. This leaves us with two possible explanations to the bimodal expression. The first is adaptor proteins, which transport proteins, possibly CD177, and are expressed primarily in mPR3-positive cells. The adaptor proteins are the limiting factor, and the amount of PR3 stored intracellularly is the same in all cells. In the second explanation, the positive cells have stored much more PR3 and CD177 in their secondary and secretory vesicles during the granulopoiesis compared with the negative ones. This indicates common signals regulating the gene expression of the two genes during the later stages of the granulopoiesis. The 4% of the cells expressing only one of the two molecules favors this latter explanation.

Our results show a rapid internalization of PR3 from the surface (Fig. 5) , and similar results were obtained in 1985, when it was shown that ANCA was internalized [24 ]. We now show that mPR3 is replaced constantly and that the amount of mPR3 is constant. As a result of limitations in our methodology, we cannot conclude whether the mPR3 that appears on the cell surface after internalization is recirculated or replaced by PR3 stored in granular structures, e.g., secondary granules. The internalization of mPR3 and CD177 could be antibody-dependent. However, the different kinetics of the internalization of CD18 strongly argues against this. One hour after incubation with antibody, most of the CD18 is still present on the surface. The dynamic trafficking of PR3 in combination with the finding that the MFI of mPR3 is decreasing slowly with the age of the neutrophil (Fig. 4) and the lack of de novo synthesis lead us to propose that mPR3 is recirculating constantly. Part of the recirculating pool of PR3 is lost by storage in granules or by degradation.

The rapid internalization of mPR3 could explain the different results seen when trying to investigate whether PR3-ANCA can bind to mPR3 [36 37 38 ]. Even if ANCA does bind to the neutrophils in the circulation, it will not be found on the surface when analyzing the cells but rather, inside the cells. In fact, if the primary and secondary antibodies are not added simultaneously, even a short incubation in RT, e.g., a centrifugation step, will hide the primary antibody inside the cell. Another interesting question that we have not addressed in this investigation is how ANCA affects the function of the neutrophil once internalized. Previous experiments indicate that internalized PR3-ANCA induces apoptosis [39 ], and further studies about this subject may be crucial to fully understand the pathogenic mechanisms of ANCA on neutrophils.

Elevated levels of the percentage of PR3-positive cells and the MFI of PR3 have been found for neutrophils in patients with ANCA-associated vasculitis. Other results have shown an up-regulation of CD177 in severe bacterial infections [40 ] and diseases such as PRV. G-CSF and/or GM-CSF are a potential link between PR3 and CD177 expression, as they have been shown to increase the level of expressed mRNA and protein on the plasma membrane for PR3 and CD177 [31 , 41 ], and CD177 is considered to be a marker of increased granulopoiesis, and we speculate that the increased percentage of mPR3-positive cells found in patients with ANCA-associated, systemic vasculitis could be a result of similar mechanisms, i.e., an increased granulopoiesis. Potentially, an increased apoptosis rate could be compensated for by increased G/GM-CSF levels, resulting in normal neutrophil counts but increased levels of mPR3 and CD177 and a larger percentage of positive cells. Another explanation could be a defective signaling pathway in analogy with the JAK2 mutation found in patients with PRV [35 ].

In this study, we show that PR3 and CD177 are coexpressed on a subset of neutrophils. Furthermore, both proteins are increased in parallel and exhibit a dynamic plasma membrane expression with rapid internalization and re-expression. Elevated levels of plasma mPR3 are linked to ANCA-associated small vessel vasculitis, whereas elevated levels of CD177 are found in diseases with disturbed myelopoiesis. We believe that future studies in this area will lead to a better understanding of the mechanisms underlying ANCA-associated systemic vasculitis and PRV.


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ACKNOWLEDGEMENTS
 
This work was supported by the Swedish Research Council (Grants 2002-6479, 71X-15152, and 73X-09487), the Crafoord Foundation, the Greta and Johan Kock Foundation, the Kungliga Fysiografiska Sällskapet, the Thelma Zoéga Foundation, the Magn Bergvalls Foundation, the Åke Wibergs Foundation, and the Alfred Österlund Foundation. The authors thank Wieslab AB for providing monoclonal and polyclonal anti-PR3 antibodies.

Received August 14, 2006; revised October 6, 2006; accepted October 9, 2006.


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