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Originally published online as doi:10.1189/jlb.0705370 on February 3, 2006

Published online before print February 3, 2006
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(Journal of Leukocyte Biology. 2006;79:828-836.)
© 2006 by Society for Leukocyte Biology

Differential expression of leukocyte-associated Ig-like receptor-1 during neutrophil differentiation and activation

Annelies Verbrugge, Talitha de Ruiter, Christian Geest, Paul J. Coffer and Linde Meyaard1

Department of Immunology, University Medical Center Utrecht, The Netherlands

1Correspondence: Department of Immunology, University Medical Center Utrecht, Room KC02.085.2, Lundlaan 6, 3584 EA Utrecht, The Netherlands. E-mail: l.meyaard{at}azu.nl


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Inhibitory receptors containing immunoreceptor tyrosine-based inhibitory motifs play an important regulatory role in immune cell activation. In addition, several studies suggest that these receptors are involved in the regulation of hematopoietic cell differentiation. Here, we have investigated the expression of leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1), an inhibitory receptor expressed on most peripheral blood leukocytes and on CD34+ hematopoietic progenitor cells, in neutrophil differentiation and activation. We found that although LAIR-1 was expressed on peripheral blood eosinophils, cell-surface expression on mature neutrophils was low, suggesting that LAIR-1 expression is regulated during granulocyte differentiation. Indeed, the promyeloid cell line HL-60 expressed LAIR-1, but the expression decreased during chemical-induced differentiation toward neutrophils. Similarly, in bone marrow-derived neutrophil precursors, the most immature cells expressed LAIR-1, and loss of LAIR-1 expression was associated with neutrophil maturation. LAIR-1 was re-expressed rapidly on the membrane of mature neutrophils upon stimulation with tumor necrosis factor {alpha}, granulocyte macrophage-colony stimulating factor, or N-formyl-methionyl-leucyl-phenylalanine, indicating that LAIR-1 may also regulate neutrophil effector function. Our studies suggest that LAIR-1 may play a regulatory role in differentiation and function of human granulocytes.

Key Words: inhibition • ITIM • progenitor • HL-60 • cytokine


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
For an adequate function of the immune system, a tight balance between activation and inhibitory signals is required. A growing number of inhibitory receptors have been identified, which regulate the function of cells in the immune system [1 , 2 ]. Most of these receptors contain one or more immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Upon engagement of the receptor, the tyrosines in the ITIMs become phosphorylated and serve as docking sites for the Src homology 2 (SH2) domain-containing phosphatase (SHP)-1, SHP-2, and/or the SH2 domain-containing 5' inositol phosphatase (SHIP). These phosphatases dephosphorylate key molecules involved in the signaling from activating receptors and thereby inhibit cellular activation [2 3 4 ]. Most cells of the immune system express several ITIM-bearing receptors [2 , 4 ]. However, although negative regulation by ITIM-bearing receptors has been investigated extensively in other immune cells, little is known about the function of inhibitory receptors in neutrophils, which play an essential role in the innate and adaptive immune response [5 , 6 ].

In addition to their regulatory function in immune cell activation, indirect evidence suggests that ITIM-bearing receptors may control the differentiation of immune cells, including neutrophils. Motheaten and viable motheaten mice, which carry homozygous mutations in the SHP-1 gene, show a dramatic infiltration of neutrophils into the lungs, skin, and extremities [7 ]. The accumulation of neutrophils probably reflects a disturbance in neutrophil production, as it was shown recently that SHP-1 negatively regulates myelopoiesis [8 ]. Also, SHIP-deficient mice and Lyn-deficient mice show increased numbers of myeloid progenitors [9 10 11 ]. As Lyn is important for the phosphorylation of ITIM-bearing receptors and the recruitment of SH2 domain-containing phosphatases in myeloid cells [10 , 12 ], these studies suggest that ITIM-bearing receptors may be involved in the regulation of myelopoiesis. Indeed, ligation of CD33 or p75/adhesion receptor molecule (AIRM)-1 inhibits in vitro proliferation of myeloid progenitors [13 ].

Here, we investigated the expression in polymorphonuclear granulocytes of leukocyte-associated immunoglobulin (Ig)-like receptor-1 (LAIR-1), a receptor that is expressed broadly in the immune system [14 ]. LAIR-1 contains two ITIMs, which recruit SHP-1 and SHP-2, and acts as an inhibitory receptor on natural killer, T, and B cells [14 15 16 17 ]. It is interesting that LAIR-1 is also expressed on CD34+ precursor cells, suggesting a role in hematopoiesis [18 ]. In addition, LAIR-1 has been reported to inhibit the differentiation of peripheral blood precursors toward dendritic cells (DC) [19 ]. Thus, LAIR-1 may be involved in the regulation of hematopoietic cell differentiation.

We report that LAIR-1 is expressed on peripheral blood eosinophils but only at low levels on mature neutrophils. The difference in LAIR-1 expression appears to occur during differentiation, as CD34+ precursor cells and immature neutrophils expressed LAIR-1, and decreased LAIR-1 surface expression was associated with differentiation toward mature neutrophils. However, LAIR-1 cell-surface expression on mature neutrophils was up-regulated after stimulation with tumor necrosis factor {alpha} (TNF-{alpha}), granulocyte macrophage-colony stimulating factor (GM-CSF), or N-formyl-methionyl-leucyl-phenylalanine (fMLP). Our findings suggest that LAIR-1 may be involved in the regulation of neutrophil differentiation and function.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Blood and bone marrow donors
Peripheral blood was drawn from healthy volunteers or healthy donors receiving recombinant human (h)G-CSF (Neupogen, 5 µg/kg body weight, twice daily for 5 days; Amgen, Thousand Oaks, CA). Bone marrow samples were obtained from cardiac patients undergoing surgery. Studies were approved by the Medical Ethical Committee of Utrecht University (The Netherlands), in accordance with the Declaration of Helsinki. All donors gave informed consent.

Isolation and cytokine stimulation of granulocytes
Granulocytes were isolated from heparin-anticoagulated peripheral blood samples by standard Ficoll-Histopaque (Sigma Chemical Co., St. Louis, MO) density gradient centrifugation. To remove remaining erythrocytes, the cells were incubated with ice-cold lysis buffer (0.16 M ammonium chloride, 0.01 M potassium bicarbonate, 0.1 mM sodium edetate, pH 7.4) for 5 min and washed. The granulocytes were stained with fluorescein isothiocyanate (FITC)-conjugated anti-human CD16 antibodies (BD Biosciences, San Jose, CA), as neutrophils and eosinophils can be separated based on the level of CD16 expression [20 ]. The granulocytes obtained consisted of 98.7% neutrophils and 1.3% eosinophils.

To investigate LAIR-1 expression on activated neutrophils, the cells were stimulated with 30 ng/ml G-CSF (Amgen), 10 U/ml hGM-CSF (Leucomax®, molgramastin, Novartis, Basel, Switzerland), 125 U/ml TNF-{alpha} (Boehringer Mannheim, Germany), or 50 nM interleukin (IL)-8 (72 amino acids, PeproTech Inc., Rocky Hill, NJ) for the indicated periods. Alternatively, the cells were primed with 10 U/ml hGM-CSF for several periods and subsequently stimulated with 1 µM fMLP (Sigma Chemical Co.) for 2 min. The cells were then analyzed by flow cytometry as described below.

Neutrophil precursors from the bone marrow were isolated as described previously [21 , 22 ], based on the difference in cell density during neutrophil maturation. Briefly, bone marrow samples were incubated in lysis buffer on ice for 5 min to remove erythrocytes. After 1 h of incubation in media, nonadherent cells were harvested, and neutrophil precursors were separated by discontinuous Percoll gradient centrifugation using layers of successive 81%, 62%, 55%, 50%, and 45% Percoll. Layers 2–4 from the top contained immature, intermediate, and mature neutrophils, respectively. The morphology of the neutrophil precursors was analyzed by microscopy of cells stained with Diff-Quick (Dade Behring, Germany).

Chemical-induced differentiation of HL-60 cells
The human promyeloid tumor cell line HL-60 has previously been shown to differentiate into neutrophil-like cells upon culture in the presence of dimethyl sulfoxide (DMSO) [23 ]. HL-60 cells were grown in RPMI-1640 media (Gibco, Paisley, UK) supplemented with 10% fetal calf serum (FCS; Integro, Dieren, The Netherlands), 50 µM ß-mercaptoethanol (ß-ME), and antibiotics. To induce differentiation, 1.25% DMSO was added to the media. Cells were grown for 1–5 days and were analyzed by microscopy after staining of the cells with Diff-Quick and by flow cytometry as described below.

For eosinophilic differentiation of HL-60 cells, the cells were cultured in media containing 25 mM N-[2-hydroxyethyl]piperazine-N'-3-propane-sulfonic acid (EPPS; Sigma Chemical Co.) at pH 7.7 for 2 months. To induce further differentiation, 0.5 mM butyrate (Sigma Chemical Co.) was added as described before [24 ]. The cells were cultured for up to 9 days and analyzed by flow cytometry.

In vitro differentiation of CD34+ progenitors toward neutrophils
CD34+ cells were isolated from bone marrow and differentiated in vitro as described previously [25 ]. Briefly, mononuclear cells were isolated from bone marrow by Ficoll-paque density gradient centrifugation. CD34+ progenitor cells were isolated by immunomagnetic cell sorting using the CD34 isolation kit (Miltenyl Biotec, Auburn, CA) and cultured in Iscove’s modified Dulbecco’s medium (IMDM; Gibco) supplemented with 10% FCS, 50 µM ß-ME, 2 mM glutamine, and antibiotics. Differentiation was induced by adding stem cell factor (50 ng/ml), fetal liver tyrosine kinase 3 ligand (50 ng/ml), GM-CSF (0.1 nM), and G-CSF (30 ng/ml). After 3 days, cells were counted and resuspended in IMDM containing G-CSF only. Cells were maintained at a density of 0.5 x 106 cells, which were grown for 14 days in media containing G-CSF and were subsequently grown in media, with or without G-CSF, for another 3 days. To analyze cell differentiation, cytospins were made of differentiating cells and fixed in methanol. The cells were stained by subsequent incubation with 50% eosin methylene blue solution according to May-Grunwald (Sigma-Aldrich, Seelze, Germany) for 15 min and 10% Giemsa solution (Merck, Darmstadt, Germany) for 20 min.

Flow cytometry
To analyze cell-surface expression of LAIR-1 and neutrophil differentiation markers, isolated neutrophils and HL-60 cells were incubated with 10% normal mouse serum to block Fc receptors (FcRs). Subsequently, the cells were incubated with FITC-conjugated, anti-human CD64 monoclonal antibody (mAb; Serotec, Oxford, UK), allophycocyanin (APC)-conjugated, anti-human CD11b mAb, phycoerythrin (PE)-conjugated, anti-CD89 mAb, PE-conjugated, anti-LAIR-1 mAb, PE-conjugated, anti-IL-5 receptor {alpha} (IL-5R{alpha}), or isotype-matched, conjugated antibodies (BD Biosciences) in the presence of 10% normal mouse serum and analyzed by flow cytometry.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
LAIR-1 is expressed on eosinophils but not on neutrophils
To investigate the expression of LAIR-1 on granulocytes, we isolated cells from peripheral blood samples of healthy donors. As eosinophils have lower CD16 expression than neutrophils, reactivity of anti-CD16 antibodies was used to distinguish between these two types of granulocytes [20 ]. We found that CD16-dim eosinophils expressed LAIR-1 at the cell surface (Fig. 1 , left panel), whereas LAIR-1 expression was barely detectable on CD16-bright neutrophils (Fig. 1 , right panel). The low reactivity of neutrophils, which form the vast majority of blood granulocytes with anti-LAIR-1 antibodies, confirmed our previous observation that granulocytes do not express cell-surface LAIR-1 [14 ]. Thus, in contrast to eosinophils, neutrophils in peripheral blood of healthy donors express only low levels of LAIR-1 at the cell surface.


Figure 1
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  		Figure 1. Expression of LAIR-1 on granulocytes from peripheral blood. Granulocytes were isolated from peripheral blood and incubated with FITC-conjugated, anti-CD16 antibody (upper panel). LAIR-1 expression on CD16-dim eosinophils (left panel) and CD16-bright neutrophils (right panel) was evaluated by flow cytometry after incubation with PE-conjugated, anti-LAIR-1 mAb (solid histograms) or PE-conjugated, isotype-matched, control antibody (open histograms). The data shown are representative of three independent experiments. FSC, Forward-scatter.

 
Loss of LAIR-1 expression is associated with neutrophil differentiation in HL-60 cells
The difference in LAIR-1 expression between eosinophils and neutrophils suggests that LAIR-1 expression could be regulated during granulocyte differentiation. We have reported previously that the promyeloid tumor cell line HL-60 expresses LAIR-1 [14 ]. HL-60 cells can be induced to differentiate toward neutrophils by culturing in the presence of 1.25% DMSO for several days [23 ], and we used this system to investigate whether neutrophil differentiation is associated with a loss of surface LAIR-1 expression.

Undifferentiated HL-60 cells showed a high LAIR-1 expression. In addition, they expressed the high-affinity FcR for IgG (Fc{gamma}R)I receptor CD64 but were negative for CD11b and the receptor for the Fc portion of IgA (Fc{alpha}R) CD89 (Fig. 2A , top panels), correlating with the phenotype of neutrophil progenitors [26 ]. Upon culture in the presence of DMSO, the cells differentiated toward neutrophil-like cells, based on the morphology of the cells: whereas undifferentiated HL-60 cells have round nuclei, DMSO-differentiated cells were smaller and had banded (3 days) or segmented (5 days) nuclei, characteristic of polymorphonuclear neutrophils (Fig. 2B) . This was associated with increased expression of SHP-1, as described previously (data not shown and refs. [27 , 28 ]). In addition, the cells increased CD11b and CD89 expression during DMSO-induced differentiation, and CD64 expression was lost (Fig. 2A) , in accordance with normal neutrophil development [22 , 26 ]. No change in morphology or cell-surface expression was observed upon longer culture in the presence of DMSO (data not shown).


Figure 2
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  		Figure 2. Differentiation of HL-60 cells toward neutrophils is associated with decreased LAIR-1 expression. (A) HL-60 cells were grown in the presence of DMSO for up to 5 days and analyzed for the expression of cell-surface markers by flow cytometry using PE-conjugated, anti-LAIR-1 mAb, APC-labeled, anti-CD11b mAb, FITC-labeled, anti-CD64 mAb, PE-conjugated, anti-CD89 mAb (solid histograms), or isotype-matched, control antibodies (open histograms). (B) The morphology of differentiating HL-60 cells was determined at Days 0, 3, and 5 by staining cytospins of the cells with the Diff-Quick reagent. The results are representative of three independent experiments. (C) HL-60 cells were cultured in media containing EPPS, pH 7.7, for 2 months. 0.5 mM butyrate was added at Day 0, and the cells were cultured for indicated periods. The cells were analyzed by flow cytometry using PE-conjugated, anti-LAIR-1 antibody (upper panels, solid histograms), anti-IL-5R{alpha} antibody (lower panels, solid histograms), or isotype-matched, control antibody (open histograms).

 
It is interesting that LAIR-1 expression started to decline after 2 days of DMSO-induced differentiation and was decreased significantly after 5 days (Fig. 2A , left panels). Similar results were obtained when HL-60 cells were differentiated in the presence of 1 µM all-trans retinoic acid (data not shown).

To determine whether the loss of LAIR-1 expression is specific for neutrophil differentiation of HL-60 cells, we also investigated the effect of eosinophil differentiation on LAIR-1 expression. HL-60 cells can be differentiated toward eosinophils by culturing them at increased pH for 2 months and subsequent culture in the presence of butyrate [24 ]. Three days after the addition of butyrate, HL-60 cells expressed the IL-5R{alpha} (Fig. 2C , lower panels), which was identified recently as a marker of cells committed toward the eosinophilic lineage [29 ]. In accordance with the observation that eosinophils in blood express LAIR-1, the differentiated eosinophilic HL-60 cells retained LAIR-1 expression (Fig. 2C , upper panels). Thus, in HL-60 cells, neutrophil differentiation is associated with decreased cell-surface expression of LAIR-1, whereas differentiation toward eosinophils does not affect LAIR-1 expression.

Maturation of neutrophils is associated with decreased LAIR-1 expression in vivo
The expression of LAIR-1 on undifferentiated HL-60 cells suggests that LAIR-1 is expressed on myeloid progenitors. Indeed, CD34+ precursor cells express high levels of LAIR-1 (see Fig. 5 , left panel (Day 0), and ref. [18 ]). We therefore investigated whether differentiation of myeloid progenitors toward neutrophils is also associated with loss of LAIR-1 expression. First, we compared neutrophils from G-CSF-treated, healthy individuals with those from untreated donors. G-CSF treatment results in a large increase of neutrophil numbers in the peripheral blood, which is also associated with cells of a more immature phenotype [30 , 31 ]. Neutrophils from G-CSF-treated individuals had greatly increased LAIR-1 expression levels compared with neutrophils from untreated donors, suggesting that immature neutrophils still express LAIR-1 (Fig. 3A and 3B ).


Figure 5
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  		Figure 5. CD34+ progenitor cells decrease LAIR-1 expression during neutrophil differentiation. CD34+ cells were isolated from bone marrow and differentiated toward neutrophils in the presence of G-CSF. On indicated days, cell samples were taken and analyzed by flow cytometry after incubation with PE-conjugated, anti-LAIR-1 mAb (solid histograms) or isotype-matched control (open histograms). After 14 days, the cells were split and grown until Day 17 in the presence (right, upper histogram) or absence (right, lower histogram) of G-CSF. To confirm neutrophil differentiation, cytospins were made and analyzed using the May-Grunwald Giemsa staining (lower panels).

 

Figure 3
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  		Figure 3. Neutrophils from G-CSF-treated donors express high levels of LAIR-1. (A) Representative analysis of LAIR-1 expression in an untreated donor (Control) and a G-CSF-treated donor. The cells were incubated with PE-conjugated, anti-LAIR-1 mAb and APC-conjugated, anti-CD11b antibody (solid histograms) or isotype-matched, control antibody (open histograms) and analyzed by flow cytometry. (B) Mean fluorescence intensity of neutrophils stained with PE-conjugated, anti-LAIR-1 mAb in untreated donors (Control, n=11) or G-CSF-treated donors (n=6). –, Mean of the mean fluorescence intensity.

 
To investigate the relation between neutrophil maturation and LAIR-1 expression further, we isolated neutrophil precursors from the bone marrow of healthy individuals. Neutrophils at different stages of maturation can be separated based on a difference in cell density by centrifugation on a discontinuous Percoll density gradient [21 ]. Using this method, we isolated three neutrophil fractions, which are designated as "immature", "intermediate", and "mature", based on the morphology of the cells and their nuclei (Fig. 4 , right panels, and ref. [22 ]).


Figure 4
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  		Figure 4. Loss of LAIR-1 expression is associated with maturation in bone marrow-derived neutrophil progenitors, which were separated in an immature (top panels), intermediate (middle panels), and mature (bottom panels) fraction using Percoll density gradient centrifugation. The cells were incubated with PE-conjugated, anti-LAIR-1 mAb (solid histograms) or PE-conjugated, isotype-matched control antibody (open histograms) and analyzed by flow cytometry. Cell morphology was determined by staining cytospins of the cells with the Diff-Quick reagent. The results shown are representative of four donors.

 
The immature neutrophils showed a relatively high level of LAIR-1 surface expression, and LAIR-1 expression was reduced on more mature neutrophils (Fig. 4 , left panels). Thus, also in vivo LAIR-1 expression level decreases during neutrophil maturation.

In vitro differentiation of CD34+ precursors toward neutrophils is associated with decreased LAIR-1 expression
The decreased LAIR-1 expression on mature neutrophils compared with the LAIR-1 expression on immature neutrophils and CD34+ precursor cells suggests that LAIR-1 expression is lost during differentiation of CD34+ cells. To investigate this, we used an in vitro differentiation assay in which CD34+ precursor cells isolated from bone marrow were differentiated toward neutrophils in the presence of G-CSF [25 ]. As expected, LAIR-1 expression on CD34+ cells was down-regulated during the first days of the differentiation (Fig. 5 , compare Day 0 with Day 7). It is surprising, however, that the expression of LAIR-1 did not decline during further maturation, and cells still expressed LAIR-1, even when matured completely (Fig. 5 , lower panels).

To investigate whether the expression of LAIR-1 was caused by the presence of G-CSF in the culture medium, we cultured the cells in the absence of G-CSF for 3 days. Indeed, deprivation of G-CSF resulted in a down-regulation of LAIR-1 cell-surface expression (Fig. 5 , right panels), without affecting neutrophil maturation (data not shown). Thus, in the absence of G-CSF, mature neutrophils do not express LAIR-1 at the cell surface.

Induction of surface expression of LAIR-1 by stimulation of mature neutrophils
The observation that neutrophils retain LAIR-1 expression when matured in vitro in the presence of G-CSF suggests that cytokine stimulation of neutrophils might induce LAIR-1 expression. We therefore treated mature neutrophils with several cytokines. When left in medium for several hours, neutrophils showed a slight increase in LAIR-1 expression on the membrane (Fig. 6A , Control panels). Treatment of mature neutrophils with G-CSF for up to 4 h did not have a significant effect on surface LAIR-1 expression as compared with culture in medium alone (Fig. 6A) . Similarly, treatment with IL-8 did not affect surface LAIR-1 expression. However, in all donors tested, treatment of neutrophils with TNF-{alpha} or GM-CSF resulted in LAIR-1 expression (Fig. 6A) , indicating that neutrophils express surface LAIR-1 upon stimulation with these cytokines.


Figure 6
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  		Figure 6. Mature neutrophils express LAIR-1 on the membrane upon stimulation with cytokines. (A) Neutrophils were isolated from peripheral blood and were left in medium or stimulated with 30 ng/ml G-CSF, 10 U/ml hGM-CSF, 125 U/ml TNF-{alpha}, or 50 nM IL-8 for 30 min, 1 h, 2 h, or 4 h (solid histograms). The open histograms represent cells directly after isolation. The cells were incubated with PE-conjugated, anti-LAIR-1 mAb and analyzed by flow cytometry. A PE-conjugated, isotype-matched, control antibody did not stain the cells. The results shown are representative of three independent experiments. (B) Neutrophils were cultured for 0, 10, 30, 60, or 90 min in medium alone (left panels) or in the presence of 10 U/ml GM-CSF (middle and right panels). Subsequently, fMLP was added (1 µM final concentration, left and right panels), and the cells were stimulated for 2 min (solid histograms). The open histograms represent cells treated with medium only. The cells were incubated with PE-conjugated, anti-LAIR-1 mAb and analyzed by flow cytometry. A PE-conjugated, isotype-matched, irrelevant antibody was used as a negative control. The results are representative of three experiments.

 
To investigate the induction of surface expression of LAIR-1 further, we treated the cells with fMLP, a potent activator of neutrophils [32 ]. Two minutes of fMLP stimulation of unprimed cells (0 min priming) resulted in a significant membrane expression of LAIR-1 (Fig. 6B , top panels). The expression of LAIR-1 was increased further when the cells were first primed with GM-CSF (Fig. 6B , right panels), even at time-points where GM-CSF alone had no effect (Fig. 6B , middle panels). GM-CSF enhances the neutrophil response to fMLP [33 ], and the enhanced up-regulation of LAIR-1 upon fMLP treatment correlated with the kinetics of priming by GM-CSF.

Together, these results indicate that mature neutrophils can re-express LAIR-1 at the cell surface, suggesting that LAIR-1 may regulate the function of mature neutrophils.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we have demonstrated that the expression of LAIR-1 is regulated during neutrophil differentiation: LAIR-1 is expressed on CD34+ precursor cells and immature neutrophils, and peripheral blood neutrophils have only low cell-surface LAIR-1 expression. Similarly, the promyeloid tumor cell line HL-60 showed decreased LAIR-1 expression upon differentiation toward neutrophils.

ITIM-bearing receptors play an important role in the control of immune cell activation [1 , 2 ]. However, correlative evidence suggests that these receptors may also be involved in regulating the production of immune cells: SHP-1 and SHIP, known effectors of ITIM-bearing receptors [2 3 4 ], have been shown to be important negative regulators of myelopoiesis [7 8 9 ]. In addition, mice deficient for Lyn, a Src family kinase that phosphorylates ITIM-bearing receptors, also have increased numbers of myeloid progenitors [10 , 11 ]. In contrast, the role of SHP-2, another phosphatase that associates with ITIM-bearing receptors, is less straightforward. SHP-2 has been suggested to be a positive regulator of hematopoiesis, and gain-of-function mutants of SHP-2 increase GM-CSF-induced colony formation in hematopoietic progenitors [34 35 36 37 ]. However, SHP-2 negatively affects macrophage maturation [37 ]. In addition, overexpression of SHP-2 inhibits IL-3-induced survival and proliferation of hematopoietic progenitors [38 ]. Thus, SHP-2 appears to act as a positive and negative regulator of immune cell differentiation.

Several ITIM-bearing receptors have been suggested to regulate differentiation of myeloid cells. CD33 and AIRM-1 become expressed during myeloid cell differentiation, but the expression is lost in mature neutrophils. Both receptors have been shown to inhibit proliferation of myeloid precursors in vitro [13 ]. Also, the expression of CD31/platelet endothelial cell adhesion molecule (PECAM)-1 is high on early myeloid cells but down-regulated during neutrophil maturation [39 ]. Signal regulatory protein (SIRP) is expressed on CD34+ hematopoietic progenitor cells and mature granulocytes, but the expression is reduced in several myeloid leukemias, suggesting a role in the control of myeloid cell proliferation [40 ].

Previously, it has been described that LAIR-1 expression is decreased during maturation of peripheral blood precursors toward DC, and cross-linking of LAIR-1 on the surface of the precursor cells inhibited differentiation of the cells [19 ]. It is tempting to speculate that LAIR-1 regulates the differentiation of neutrophil precursors in a similar manner. LAIR-1 engagement inhibits GM-CSF-induced proliferation in acute myeloid leukemia blasts and induces apoptosis in myeloid leukemic cell lines [41 , 42 ], suggesting that LAIR-1 regulates the proliferation of myeloid cells. Thus, expression of LAIR-1 on CD34+ precursor cells and neutrophil precursors may be required for the controlled proliferation and/or differentiation of these cells, and maturation of neutrophils may require down-regulation of LAIR-1 expression.

A high level of LAIR-1 expression was found in neutrophils isolated from G-CSF-treated donors. This may reflect the fact that the mobilized neutrophils are more immature than in untreated donors [30 , 31 ], but it may also be a consequence of the treatment. We found that prolonged culture in the presence of G-CSF during in vitro differentiation of CD34+ progenitors toward neutrophils was associated with incomplete down-regulation of LAIR-1 expression, and G-CSF removal resulted in further down-regulation, suggesting that G-CSF treatment may affect LAIR-1 expression directly. Treatment of mature neutrophils with G-CSF, however, did not result in induction of surface expression of LAIR-1. Thus, G-CSF may have different effects on immature neutrophils compared with fully matured neutrophils. Alternatively, G-CSF treatment may not be sufficient for re-expression of LAIR-1 once surface expression has been lost.

Mature neutrophils showed LAIR-1 cell-surface expression upon treatment with several but not all cytokines. This suggests that neutrophils can re-express LAIR-1 upon stimulation, depending on the mode and/or extent of activation. The largest increase in LAIR-1 cell-surface expression was observed upon treatment with fMLP of neutrophils primed with GM-CSF. The rapid re-expression of LAIR-1 on neutrophils activated with fMLP suggests that there is an intracellular pool of LAIR-1, which can be externalized upon activation of the cells. Thus, LAIR-1 may also control neutrophil effector functions.

Although mature human neutrophils express several ITIM-bearing receptors, little is known about inhibitory functions mediated by these receptors. The expression levels of PECAM-1, SIRP{alpha}, and CD66a are regulated during neutrophil activation, but research, so far, has focused on their function as adhesion molecules [43 44 45 46 ] rather than possible inhibitory functions. In fact, engagement of PECAM-1 and CD66a induces up-regulation of CD11b, a marker of neutrophil activation [43 , 46 ]. Similarly, the expression of Siglec-5 increases upon neutrophil activation, and engagement of the receptor with mAb primes the cells for a fMLP-induced oxidative burst. Another ITIM-bearing receptor, C-type lectin superfamily 6 (CLECSF6; also known as DCIR), has been suggested to be a negative regulator of neutrophil function [28 ], but this has not yet been confirmed by functional studies.

However, there are several studies that show that ITIM-bearing receptors regulate neutrophil function in mice. Mouse neutrophils express gp49B1, and mice lacking this receptor show increased neutrophil tissue infiltration upon lipopolysaccharide injection [47 ]. Another murine receptor, paired Ig-like receptor B, has been shown to be an important regulator of integrin and chemokine receptor signaling in neutrophils [48 , 49 ]. Human neutrophils are likely regulated in a similar manner by ITIM-bearing receptors.

It is interesting that the expression of CLECSF6 and LAIR-1 on mature neutrophils is regulated in an opposite manner: Although expression of LAIR-1 is up-regulated, CLECSF6 expression is down-regulated upon neutrophil activation [28 , 50 ]. Thus, whereas CLECSF6 may inhibit initial neutrophil activation, LAIR-1 is more likely to regulate neutrophils once they are activated. This suggests that neutrophil function may be regulated at distinct stages by different ITIM-bearing receptors.

In conclusion, our studies show for the first time that the expression of LAIR-1 is regulated tightly in human neutrophils and suggest that LAIR-1 may be involved in the regulation of neutrophil differentiation and function.


   ACKNOWLEDGEMENTS
 
This work was supported by the Netherlands Organization for Scientific Research (NWO, Grants 901-07-230 and 016-026-008). C. G. was supported by the Dutch Cancer Society (KWF, Grant 2003-2920). A. V. and T. d. R. contributed equally to this work. We thank M. A. Otten and E. Rudolph for technical assistance and Drs. E. J. Petersen and A. Brutel for providing blood and bone marrow samples.

Received July 8, 2005; revised November 25, 2005; accepted December 6, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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