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Originally published online as doi:10.1189/jlb.0407205 on August 3, 2007

Published online before print August 3, 2007
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(Journal of Leukocyte Biology. 2007;82:1095-1105.)
© 2007 by Society for Leukocyte Biology

A phenotypic and functional characterization of NK cells in adenoids

Sa’ar Mizrahi*, Eitan Yefenof*, Menahem Gross{dagger}, Pierre Attal{ddagger}, Avraham Ben Yaakov{dagger}, Debra Goldman-Wohl§, Bella Maly||, Noam Stern*, Gil Katz*, Roi Gazit*, Ronit Vogt Sionov*, Ofer Mandelboim* and Stella Chaushu*,¶,1

* Lautenberg Center of General and Tumor Immunology, Hebrew University–Hadassah School of Medicine and
Hadassah School of Dental Medicine, founded by the Alpha Omega Fraternity, Jerusalem, Israel; Departments of
{dagger} Otolaryngology–Head and Neck Surgery,
§ Obstetrics and Gynecology, and
|| Pathology, Hadassah Medical Center, Jerusalem, Israel; and
{ddagger} Department of Otolaryngology, Shaare Zedek Medical Center, Jerusalem, Israel

1 Correspondence: Lautenberg Center of General and Tumor Immunology, Hebrew University–Hadassah School of Medicine, P.O. Box 12272, Jerusalem, Israel 91120. E-mail: drchaushu{at}gmail.com


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ABSTRACT
 
Adenoids are part of the MALT. In the present study, we analyzed cell surface markers and cytolytic activity of adenoidal NK (A-NK) cells and compared them with NK cells derived from blood of the same donors (B-NK). NK cells comprised 0.67% (0.4–1.2%) of the total lymphoid population isolated from adenoids. The majority (median=92%) of the A-NK cells was CD56brightCD16–. A-NK cells were characterized by the increased expression of activation-induced receptors. NKp44 was detected on >60%, CD25 on >40%, and HLA-DR on >50% of freshly isolated A-NK cells. Functional assays indicated that the cytotoxic machinery of A-NK is intact, and sensitive target cells are killed via natural cytotoxicity receptors, such as NKG2D. Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1; CD66) expression was up-regulated in 23% (median) of the A-NK cells by IL-2 activation but unchanged in B-NK cells. CEACAM1 inhibited the A-NK killing of target cells. CXCR4 was expressed on more than 40% A-NK cells prior to activation. Its ligand, CXCL12, was found in endothelial cells of the capillaries within the adenoid and in cells of the epithelial lining. In addition, A-NK cells migrated in vitro toward a gradient of CXCL12 in a dose-responsive manner, suggesting a role for this chemokine in A-NK cell recruitment and trafficking. We conclude that the A-NK cells are unique in that they display an activated-like phenotype and are different from their CD16– B-NK cell counterparts. This phenotype presumably reflects the chronic interaction of A-NK cells with antigens penetrating the body through the nasal route.

Key Words: human • receptors • chemokines


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INTRODUCTION
 
Adenoids belong to the lymphoepithelial secretory immune system. They constitute part of the Waldeyer’s ring or nasopharynx-associated lymphoid tissue, which includes the palatine, nasopharyngeal (adenoid), and lingual tonsils. The lymphoid tissue of Waldeyer’s ring is located at the gateway of the respiratory and alimentary tract and belongs to the MALT. It is a primary site of encounter with inhaled and ingested microorganisms and thus, considered to be a first line of defense against exogenous pathogens [1 , 2 ].

The generation of humoral immunity in the MALT has been studied extensively [3 4 5 6 ]. Lymphoid tissues of Waldeyer’s ring provide B cells for mucosal effector sites. These lymphoid organs contain IgA-expressing B cells, which migrate to the upper airway mucosa, lacrimal glands, and salivary glands. Vaccination via the nasal route induces regional mucosal immunity but can also enhance systemic immunity [3 ].

Recent studies have shown that immune protection at the MALT is also provided by innate immunity components, most notably, NK cells [7 8 9 ], which afford immediate response against viral infection and tumor cells before adaptive immunity has evolved and use two major effector mechanisms: target cell killing and cytokine production [10 ]. In addition, it has been demonstrated recently that NK cells can function as APCs [11 ].

Significant progress has been made in characterizing NK cells of the systemic innate immune system [12 , 13 ]. It is surprising that information regarding NK cells in the MALT, in general, and in adenoids, in particular, is scarce [14 15 16 17 18 ]. A recent study demonstrated that NK cells in palatine tonsils differ from their blood counterparts by phenotype and function [19 ]. It should be stressed that adenoids differ from palatine tonsils, not only by their location in the aerodigestive tract and their morphology but also by their immune response. These tissues display different humoral immune responses, as expressed in the proportion of Ig-secreting cells [20 ] and secretion of antibodies to different antigens [1 , 21 ], and cellular immune responses, as expressed in the secretion panel of various cytokines [22 ]. Differences have also been found in the expression of vascular addressins, homing receptors, and chemokine transcripts [23 ]. However, a literature search revealed no studies aiming to characterize the phenotype and function of adenoid NK (A-NK) cells.

The aim of the present study was to analyze cell surface markers and cytolytic activity of NK cells from adenoids and compare them with blood NK (B-NK) cells of the same donors. Our results show that the A-NK cells are mostly CD56brightCD16– and display an activated-like phenotype, totally distinguishing them from their CD16– B-NK cell counterparts. A-NK cells are characterized with increased expression of several activation-induced receptors, such as NKp44, CD25, HLA-DR, carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), and CXCR4. The activation state of A-NK cells is presumably a result of the chronic interaction with antigens penetrating the body through the nasal route.


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MATERIALS AND METHODS
 
Adenoid and blood cell isolation and culture
Adenoid and blood samples were obtained from 10 children undergoing adenoidectomy as a result of adenoid hypertrophy and snoring. The adenoids were collected immediately after surgery and processed for isolation of lymphocytes as described previously [24 ]. The protocol was approved by the institutional ethical committee, in accordance with the Helsinki guidelines. Briefly, tissues were trimmed into 1 mm pieces and digested enzymatically for 20 min using vigorous shaking with 1.5 mg type I DNase and 24 mg type IV collagenase diluted in 15 ml RPMI-1640 medium. The procedure was repeated three times. After an additional 5 min incubation at room temperature without shaking, the supernatants were collected and incubated overnight in a tissue-culture dish. Nonadherent cells were collected and loaded on Ficoll density gradient to purify the lymphocyte population. PBLs were isolated from the blood using Ficoll gradient. For isolation of the adenoid stromal (AS) cells, the cells that adhered were detached from the culture flask by treatment with 0.25% trypsin (Sigma-Aldrich, St. Louis, MO, USA). The adenoid/PBLs and the AS cells were then processed for flow cytometry analyses or cryopreserved.

To assess the effect of IL-2 activation of NK cells, the lymphocytes were cultured for 72 h in the presence of recombinant IL-2 (Sigma-Aldrich, Rehovot, IL, USA), used at a concentration of 5 IU/ml.

Preparation of NK clones
Isolation of NK cells was performed using NK Isolation Kit II (Miltenyi Biotec Inc., Auburn, CA, USA), according to the manufacturer’s instructions. The isolated cells were cultured on feeder cells in media supplemented with human IL-2 and PHA, as described previously [25 ].

Flow cytometry
The following mouse anti-human mAb were used:

Single staining
Single staining included anti-MHC class I-related chain A (MICA), anti-MICB, anti-MICA/B, anti-MHC class I W6/32, anti-UL16-binding protein 1 (ULBP1), anti-ULBP2, and anti-ULBP3 (all from R&D Systems, Minneapolis, MN, USA). FITC-labeled goat anti-mouse Ig (BD Pharmingen, San Diego, CA, USA) was added after the first mAb for indirect labeling.

Double staining
Double staining included FITC-conjugated anti-CD34 and APC-conjugated anti-CD45 (BD Pharmingen).

Triple staining
Triple staining included PE-conjugated anti-CD56, FITC-conjugated anti-CD16, CyChrome-conjugated anti-CD3, PE-conjugated anti-NKp30, PE-conjugated anti-NKp44, PE-conjugated anti-NKp46, PE-conjugated anti-NKG2D, PE-conjugated anti-killer inhibitory receptors (KIRs), PE-conjugated anti-CCR4, PE-conjugated anti-CXCR3, PE-conjugated anti-CXCR4, PE-conjugated anti-CCR7, and FITC-conjugated anti-HLA-DR (all from BD Pharmingen). FITC-conjugated anti-CD66 and FITC-conjugated anti-CD25 were from Dako (Glostrup, Denmark); FITC-conjugated anti-CD56 was from Sigma-Aldrich (St. Louis, MO, USA).

Quadruple staining
As a fourth color, biotinylated anti-CD16 mAb (Serotec, Oxford, UK) was used, followed by Cy5-streptavidin (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA). The quadruple staining allowed for analysis of cell surface markers on the two distinct CD16+ and CD16– subpopulations found in the PBLs.

Negative controls included labeled, isotype-matched IgG mAb (BD Pharmingen). Cells (1–5x105) were incubated with saturating amounts of antibodies (0.2–0.5 µg) for 30 min at 4°C. FACS analysis was performed using FACScan flow cytometers (BD Biosciences, Heidelberg, Germany). First, all viable cells were selected by a widely set gate on a two-parameter plot of side-scatter versus forward-angle-scatter. Among these cells, a second gate was set to include all CD3– or CD56+ CD3– cells.

Incubation of B-NK cells with AS cells
B-NK cells were isolated, washed twice in serum-free RPMI 1640, and incubated at a final concentration of 106 cells/ml in RPMI-1640 with 1 µM CFSE (Molecular Probes, Eugene, OR, USA) for 30 min at 37°C, 5% CO2. Labeled cells were washed twice in RPMI 1640 containing 10% FCS and were then plated at a concentration of 106 cells/ml in the presence of the same number of AS cells for 72 h. The cells were then processed for flow cytometry analysis. Staining for CEACAM1 was performed after gating on CFSE-labeled cells, thus excluding possible contaminating A-NK cells expressing the receptor.

NK cell cytotoxicity assay
The cytotoxic activity of A-NK and B-NK cells against various targets was measured after labeling target cells overnight with 35S-methionine. Target cells were washed, and 5000 of them were incubated with the NK cells at various E:T ratios (from 20:1 to 2.5:1). Cytotoxicity was calculated according to the formula: Percent 35S-methionine release = (cpm sample–cpm spontaneous release)/(cpm total–cpm spontaneous release) x 100. Total 35S-methionine release was measured after incubation of the cells with 0.1 M NaOH. The spontaneous release was less than 25% of maximal release. In the experiments where rabbit polyclonal anti-CEACAM (DakoCytomation, Carpinteria, CA, USA) was included, the final antibody concentration was 10 µg/ml.

Immunohistochemical staining
Adenoid tissues were fixed with formalin and embedded in paraffin. Immunohistochemical staining was performed as described previously [26 ]. Briefly, sections were incubated with 10 µg/ml anti-human CXCL12 mAb (R&D Systems) overnight at 4°C, followed by detection using the avidin-biotin Histostain Plus kit, according to the manufacturer’s instructions (Zymed Laboratories, San Francisco, CA, USA). Color development was with 3-amino-9-ethylcarbazole, and sections were counterstained with hematoxylin.

In vitro cell migration assay
Adenoidal lymphocytes (2x106) in 100 µL were loaded into each Transwell filter (5 µm pore filter Transwell, 24-well cell clusters, Corning, Corning, NY, USA). Filters were then plated in each well containing 600 µL medium supplemented with effective concentrations of CXCL12 described previously [27 ] (R&D Systems). At least three wells were used for each chemokine concentration. To determine nonspecific or background migration, six wells, which did not contain any chemokine, were used as controls. After 3 h of incubation at 37°C, 5% CO2, the upper chambers were removed, and cells in the bottom chamber were collected and analyzed by flow cytometry (cell counting and immunophenotyping). The percentage of CD56+A-NK-migrated cells was calculated by subtracting the value of spontaneously CD56+A-NK-migrating cells from the number of CD56+A-NK-migrating cells at a given chemokine concentration, and afterward, the result was divided by the total number of CD56+A-NK cells (out of the adenoidal lymphocytes), which were initially loaded on the transwell filter.


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RESULTS
 
A-NK cells are different from B-NK cells
We first compared the percentage of NK cells obtained from adenoids and blood of the same donor. Initially, we observed that NK cells are a minor fraction (median 0.67%; range 0.4–1.2%) of the total lymphoid cells isolated from adenoids, and B-NK cells comprised 14.8% (range 10–19.7%) of the PBLs isolated from the blood using Ficoll gradient (Fig. 1A ). We next activated A-NK and B-NK cells with IL-2 for 72 h and observed a significant enrichment of the percentage of NK cells in all the adenoids studied (median 3.1%; range 2.5–4.2%, Fig. 1B ). By contrast, the percentage of NK cells in blood consistently decreased after stimulation (median 3.3%; range 2.1–3.5%). In addition, IL-2 stimulation caused a significant decrease in the number of CD56dim cells with a concomitant increase in the number of CD56bright cells (Fig. 1B) . CD56+ T cells accounted for a small percentage of adenoid or PBLs before stimulation but expanded significantly in response to IL-2 (Fig. 1A and B) .


Figure 1
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  		Figure 1. Characterization of A-NK cells. Double FACS staining of (A) freshly isolated and (B) IL-2-cultured adenoid and PBLs for CD56 expression (x-axis) and CD3 expression (y-axis). NK cells are characterized by the CD3–CD56+ phenotype. The analysis was performed after gating on CD3– cells, thus excluding contaminating, activated T cells expressing CD56. (C) CD16 expression pattern of A-NK and B-NK cells. The dot-plots were gated on CD3–CD56+ cells. The data are representative of at least five separate analyses.

A-NK cells express a different phenotype than B-NK cells
Previous studies demonstrate that NK cells from lymph nodes and palatine tonsils express a different phenotype compared with NK cells from peripheral blood [19 ]. However, no information is available regarding NK cells in adenoids. Hence, we examined the phenotype of A-NK cells and compared it to B-NK cells. To begin with, we studied the CD16 expression, as CD56dim and CD56bright cells in the blood differ in the expression of CD16 [28 ]. As expected, the vast majority of the A-NK cells was CD56bright CD16– (median 92%; range 90–94%), and the opposite was observed in the blood (median 23%; range 22–25%, Fig. 1C ).

Our next goal was to analyze a plethora of receptors and compared their expression between A-NK and B-NK cells. It is important that we always compared the expression of the various receptors between A-NK and B-NK cells of the same individual.

Activating NK receptors
NK cell cytotoxicity is controlled by activating receptors, coreceptors, and inhibitory receptors. Three activating NK receptors, termed natural cytotoxicity receptors (NCRs), were discovered by Moretta et al. [29 ]. Thus far, only viral ligands were identified for these receptors, such as haemagglutinin for the NKp44 and NKp46 receptors [25 , 30 ] and pp65 protein of cytomegalovirus for the NKp30 receptor [31 ]. The identity of tumor cell ligands for these receptors is still unknown. NKG2D is another major activating receptor expressed on NK cells and activated T cells [32 ]. Analysis of the expression pattern of the NCR on A-NK cells revealed significant differences with regard to NKp44, a receptor that is normally expressed on activated NK cells only [29 , 33 ]. It is surprising that this receptor was detected on freshly isolated A-NK cells, and no expression was observed in the other B-NK cell populations (Fig. 2A and Tables 1 and 2 ). IL-2 activation induced the expression of NKp44 on all of the NK populations examined, in agreement with previous publications (Tables 1 and 2 ) [29 , 33 ]. In contrast, the NKp30, NKp46 expression was not significantly different between A-NK and B-NK cells (Fig. 2A and Tables 1 and 2 ). NKG2D expression was slightly lower on A-NK cells before and after activation (Fig. 2A and Tables 1 and 2 ).


Figure 2
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  		Figure 2. Expression of surface receptors on A-NK and B-NK cells. Triple FACS staining was performed for the expression of CD56, CD3, and various receptors. (A) Activating receptors; (B) inhibitory receptors; (C) chemokine receptors. The analysis was performed after gating on CD3– cells, thus excluding contaminating, activated T cells expressing CD56. The data are representative of at least five independent experiments.


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  		Table 1. Phenotype of A-NK Versus CD16+ and CD16– B-NK Cells


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  		Table 2. Phenotype of A-NK Versus CD16+ and CD16– B-NK Cells

Inhibitory NK receptors
The activity of NK cells is negatively regulated by NK inhibitory receptors interacting with HLA class I proteins. The HLA class I-specific KIRs exist in two forms: an inhibitory receptor, such as the KIR2DL1 (CD158a), and a shorter version of the receptors, such as KIR2DS4 (KAR p50.3), which transmits an activating signal and activates the killing capability. In addition, the NKG2A inhibitory receptor interacts with the HLA-E protein when it is found in complex with the CD94 protein [34 ]. In agreement with previous publications [19 ] and similar to the palatine tonsil NK population, A-NK cells showed almost no expression of KIR2DL1 and KIR2DS4 (Fig. 2B and Tables 1 and 2 ). NKG2A, conversely, was expressed significantly on A-NK and B-NK cells in similar levels (Fig. 2B and Tables 1 and 2 ). After IL-2 activation, the expression of the KIR2DL1 and NKG2A receptors was increased in A-NK cells to levels similar to B-NK cells (Tables 1 and 2) .

Chemokine receptors
Several studies [35 , 36 ] demonstrated that CD56bright and CD56dim NK cells have unique repertoires of chemokine receptors. In an earlier study, we observed that the differential expression of chemokine receptors on the CD16– cells leads to the recruitment of this particular subset to the deciduas during pregnancy [27 ]. We therefore aimed to examine whether expression of these receptors is unique in A-NK vis-à-vis B-NK cells.

A significant difference was found in the expression CXCR4. It was hardly detectable on B-NK cells, whereas a significant number of A-NK cells were CXCR4-positive (Fig. 2C and Tables 1 and 2 ).

A-NK and B-NK cells expressed the CXCR3; however, no significant differences were observed between adenoids and blood in the expression of this receptor (Fig. 2C) .

Additional NK receptors
We have demonstrated that the activity of NK cells could be negatively regulated by inhibitory receptors, whose ligands are not MHC class I proteins such as CD66a (CEACAM1) [37 ]. CEACAM1 is a member of the CEACAM receptor family and is expressed on a broad spectrum of cells after IL-2 activation [38 ]. Previous studies showed that CEACAM1 homotypic and heterophilic interactions between NK cells and target cells inhibit NK cytotoxicity [39 , 40 ]. In addition, it has been shown that CEACAM1 plays a pivotal role in inhibiting functions of activated, decidual lymphocytes, such as lysis, proliferation, and cytokine secretion [24 ].

We demonstrated so far that the adenoid lymphocyte composition resembles that of the deciduas in that A-NK cells are mostly CD56brightCD16–. Accordingly, we anticipated a role for the CEACAM1 protein in regulating A-NK cell functions and therefore examined the expression of this protein on A-NK cells. In agreement with previous results [24 ], immunofluorescence staining revealed low-level expression of CEACAM1 in A-NK and B-NK cells. However, following IL-2 activation, A-NK cells expressed high levels of CEACAM1, and B-NK cells did not (Fig. 3 and Tables 1 and 2 ).


Figure 3
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  		Figure 3. Activation markers on A-NK and B-NK cells. Triple FACS staining was performed for the expression of CD56, CD3, and various receptors indicated in the figure, which shows staining of freshly isolated CD56+ cells and on the same cells, culture for 72 h in the presence of IL-2. The analysis was performed after gating on CD3– cells, thus excluding contaminating activated T cells expressing CD56. The figure shows one representative experiment out of at least five performed.

Another consistent difference between A-NK and B-NK cells was the expression of CD25 (high-affinity IL-2R). CD25 was expressed on a large percentage of A-NK cells. IL-2 stimulation did not increase this expression (Fig. 3 and Tables 1 and 2 ). In contrast, B-NK cells and CD16– and CD16+ cells were CD25-negative. However, following activation with IL-2, the expression of CD25 was increased on B-NK cells (Fig. 3 and Tables 1 and 2 ).

Another consequence of NK activation is the expression of MHC class II molecules (HLA-DR). Our previous study showed unexpectedly that activated NK cells expressing MHC class II can serve as APCs [11 ]. We therefore examined the HLA-DR expression on A-NK cells. HLA-DR was detected on more than 50% of the A-NK cells even before IL-2 activation. In contrast, no HLA-DR expression was found in resting B-NK cells, and IL-2 stimulation enhanced the expression of HLA-DR in B-NK cells to a similar extent as in A-NK cells (Fig. 3 and Tables 1 and 2 ).

It is noteworthy to clarify that the variations seen in CD56bright staining between A-NK cells and B-NK cells in some of the figures are only technical and result from slightly different FL2-PE amplification voltage settings.

In summary, it seems that A-NK cells appear in a certain state of activation, marked by the expression of the activation markers NKp44, MHC class II, and CD25. A-NK cells seem to be phenotipycally different from other NK subsets found in the blood but are similar to those found in palatine tonsils.

CEACAM1 on activated A-NK cell clones inhibits cytotoxicity
One of the notable differences between A-NK and B-NK cells was the expression of CEACAM1 on A-NK cells after activation. To test the possible role of CEACAM1 in controlling A-NK cell function, IL-2-activated A-NK cells clones were tested in killing assays against 721.221-expressing CEACAM1 (0.221/CEACAM1) as described previously [24 ]. A-NK clones expressing high levels of CEACAM1 (seen in Fig. 4 ) were effective killers of control 0.221 cells but not 0.221/CEACAM1 cells (Fig. 4) . 221/CEACAM1 cells were protected of lysis by virtue of CEACAM1 inhibition, as anti-human CEACAM antibodies abolished the inhibition of killing (Fig. 4) . Statistical analysis of the results from five experiments (Fig. 4) proved that the differences in the levels of cytotoxicity were highly, statistically significant (P<0.001). We showed that CEACAM1 was induced only on A-NK cells by IL-2. A possible scenario is that other cells in the adenoid stroma prime A-NK cells differently to those in blood. Therefore, we tested whether incubation of B-NK cells with self-AS cells might induce the expression of CEACAM1 on their surface. FACS staining of CFSE-labeled B-NK at the end of the incubation period showed that AS cells had no effect on the expression of CEACAM1 on B-NK cells (data not shown).


Figure 4
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  		Figure 4. CEACAM1 inhibits the killing activity of A-NK cells. NK clones were isolated from adenoids and cultured in the presence of IL-2 as described previously [24 ]. Shown are five representative A-NK cells clones, which expressed a high level of the CEACAM1 protein (right histogram in each killing assay). CEACAM1 expression was monitored using the anti-CEACAM1 mAb (DakoCytomation; empty histogram). The control, filled histogram is the staining of the second antibody only. Killing assays were performed as described in Materials and Methods. The figure shows one representative experiment out of three performed. The E:T ratio presented is 10:1. The error bars represent the SD of the triplicate. **, P < 0.05; ***, P < 0.001.

NKG2D interactions up-regulate cytotoxicity of A-NK cells
The above results demonstrate that the killing machinery of A-NK cells is intact, as 721.221 cells, which are killed via the NCR [29 ], were killed effectively by A-NK cells, and also, they demonstrate that this killing could be inhibited, for example, by CEACAM1. The 721.221 cells, however, do not express ligands for the NKG2D receptor. To test whether NKG2D functions in A-NK cells, IL-2-activated A-NK as well as CD16– B-NK cell clones were tested for killing against C1R/Neo cells (parental C1R, which lack MICA, the ligand of NKG2D, transfected with the empty vector) versus C1R/MicA cells (transfected with MICA). As shown in Figure 5 , A-NK cells lysed C1R/MICA cells effectively and less efficiently, the C1R/Neo cells. Thus, NKG2D is functional in A-NK cells. NKG2D down-regulation can be elicited by sustained NKG2D ligand expression on normal cells [41 ]. A-NK cells had a slightly lower MFI for NKG2D than B-NK cells. Therefore, we examined the expression of NKG2D ligands on the AS cells and observed no expression of MHC class I chain-related molecules A/B (MICA/MICB) and ULBP1, -2, and -3 (Fig. 6 ).


Figure 5
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  		Figure 5. NKG2D is functional in A-NK cells. Killing assays were performed with CD16–NK cells from PBL (lower) or with A-NK cell clones (upper), as described in Materials and Methods. Killing assays were performed against the C1R cells transfected with a control, empty vector (designated as Neo) or against C1R cells transfected with the MICA protein (designated as MicA). The figure shows one representative experiment out of three performed. The E:T ratio presented is 10:1. The error bars represent the SD of the triplicate.


Figure 6
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  		Figure 6. NKG2D ligand expression on AS cells. FACS staining was performed for the expression of MICA, MICB, and ULBP1–3. Dashed line, AS cells stained with an isotype control. Black, solid line, AS cells stained with anti-MICA, -MICB, and -ULBP1–3 antibodies. The secondary reagent was a FITC-conjugated anti-mouse antibody. The data are representative of five independent experiments.

The source of A-NK cells
Our results so far demonstrate that NK cells of the CD56bright CD16– phenotype can be found in adenoids. What is the source of this special subset of NK cells? It was demonstrated recently that a significant number of human CD56bright NK progeny, CD34dimCD45RA+β7bright hematopoietic progenitors are found within lymph nodes, implicated the peripheral lymphoid tissue as a site of early, human NK cell differentiation [42 ]. However, Figure 7 shows that CD34+CD45RA+ cells could be detected only in a small percentage (0.02%) in adenoidal tissues. Thus, one alternative is that the source of the A-NK cells is derived from the NK precursors. It is also possible, however, that NK cells migrate to the adenoids from the periphery. Peripheral CD16– NK cells have been shown previously to bind in vitro various CXCR4 and CXCR3 ligands [38 , 43 ]. The enhanced expression of CXCR4 in A-NK cells (Fig. 2) suggested that this receptor might be involved in the recruitment of the CD16– NK population from the peripheral blood to adenoids. We therefore looked for the presence of the CXCR4 ligand (CXCL12) in adenoid tissues using immunochemistry staining. The presence of CXCL12 was clearly confirmed in endothelial cells of adenoid blood vessels (Fig. 8A ) and adenoid respiratory epithelial cells (Fig. 8B) . Immunostaining of palatine tonsillar tissue, which was performed for comparison, also illustrated the presence of CXCL12 in blood vessel endothelium (Fig. 8C) . However, the nonkeratinizing epithelium lining showed no significant CXCL12 staining (Fig. 8D) . To test whether the expression of CXCR4 on A-NK is functional, we performed migration assays. Figure 9 shows that the significant expression levels of the CXCR4 receptor on A-NK cells resulted in migration to CXCL12 in a dose-responsive manner. A-NK cells responded to CXCL12 concentrations as low as 12.5 ng/ml. On average, 20% of the CD56+A-NK cells migrated to CXCL12 at the concentration of 100 ng/ml.


Figure 7
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  		Figure 7. FACS staining of adenoidal lymphocytes for expression of CD34 and CD45RA receptors. The analysis is representative of three separate experiments.


Figure 8
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  		Figure 8. In vivo expression of CXCL12. Immunohistochemical staining was performed on paraformaldehyde-fixed adenoid and palatine tonsillar tissue. The various tissues were stained for CXCL12 expression using the anti-human CXCL12 mAb as described in Materials and Methods. CXCL12 expression was detected (marked by arrows) in endothelial cells of adenoid blood venules (A), in the adenoid respiratory epithelial cells (B), and in the endothelial cells of palatine tonsils’ blood vessels (C). The nonkeratinizing palatine tonsillar epithelial lining showed no significant staining (D). Figure shows one representative experiment out of four performed. Original magnification is x200 for all panels.


Figure 9
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  		Figure 9. Dose-dependent migration of A-NK cells to recombinant human CXCL12. Migration assays were performed as described in Materials and Methods. The amount and identity of the migrated NK cells were analyzed by flow cytometry. The percentages of migrated cells are calculated from the total cell population after the subtraction of the spontaneous migration. One representative experiment is shown of three performed. Error bars indicate SD.


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DISCUSSION
 
The present study compares NK cells from adenoids and blood from the same donors and points to unique aspects of the former. The primary physiological role of NK cells is to yield early defense against pathogenic organisms [44 ]. The adenoid tissues studied here were removed because hyperthrophy and snoring provide unique material to study the role of NK cells in the mucosal immune system at sites exposed to airborne antigens. The few studies, which have been conducted in this area, have focused on NK cells from palatine tonsils [19 ]. Although both of these secondary lymphoid organs belong to the same nasopharynx-associated lymphoid tissues and share many common features [45 ], several studies have demonstrated differences in their humoral and cellular adaptive immune responses to various antigens and mitogens [1 , 20 21 22 23 ]. Therefore, the innate immune response in adenoids, as reflected in the phenotype and function of A-NK cells, cannot be automatically assumed to be similar to that in the palatine tonsils and should be studied separately.

Overall, our data demonstrate that A-NK cells are different from B-NK cells in their phenotype, as determined by comparative analysis of several receptors.

Most CD56bright NK cells do not express CD16. By contrast, >95% of CD56dim NK cells are CD16+ [46 ]. As shown here and by previous studies, ~90% of the NK cells in peripheral blood are CD56dimCD16+ [47 , 48 ]. In contrast, >90% of A-NK cells were CD56brightCD16–. This pattern correlates with the phenotype of NK cells from lymph nodes and palatine tonsils [19 ]. It is interesting that IL-2 stimulation for only 72 h resulted in marked expansion of the A-NK cells, and B-NK cells of the same donor were not affected. Ferlazzo et al. [19 ] reported that palatine tonsil and lymph node NK cells expanded more vigorously than T cells after IL-2 stimulation, and this may account for the relative increase in the percentage of NK cells out of the total number of lymphocytes. A possible reason for this expansion might be the presence of the high-affinity IL-2R (CD25) on A-NK cells.

In blood, IL-2 treatment reduces the number of CD56dimand enriches CD56bright cells. This is not surprising in light of previous studies showing that CD56bright NK cells are better responders to cytokine stimulation [49 , 50 ]. Dunne et al. [50 ] reported that after incubation with IL-2, most of the NK cells (71.8%) are CD56bright, which are predominant in human lymph nodes, where endogenous, T cell-derived IL-2 costimulates CD56bright NK cells to secrete IFN-{gamma} by virtue of its binding to the CD25 [51 ]. Furthermore, clinical studies showed that in vivo infusion of IL-2 generates CD56bright NK cells [52 ].

It is important that IL-2 stimulation provoked expansion of CD56+ T cells in adenoids and blood, confirming previous reports [50 ]. These cells exhibit adaptive and innate immune functions. They lyse tumor targets cells spontaneously and upon engagement of their TCRs, secrete IFN-{gamma} and GM-CSF, thus expressing a "hybrid" phenotype [53 ].

Analysis of the activating receptors revealed that a significant percentage of resting A-NK cells but not B-NK cells expresses NKp44 prior to IL-2 stimulation. NKp44 is not expressed on resting B-NK cells but can be induced by IL-2 activation [54 , 55 ]. Previous studies reported that NK cells from palatine tonsils express a high level of NKp44 [19 , 56 ]. Ferlazzo et al. [19 ] attributed this enhanced expression to the inflammatory response for which the palatine tonsils were removed. It is pertinent to note that none of the adenoids used in the present study was removed because of inflammation. Our data suggest that NK cells in adenoids are in an activated-like state, apparently acting as guardians against the potential intrusion of invading pathogens. Moreover, it also shows that the A-NK cells comprise a unique population different from their CD16– counterpart in blood.

Regarding the chemokine receptors, the most significant difference was observed with CXCR4, which was found on more than 40% of the freshly isolated A-NK cells prior to activation. These results parallel the high expression of CXCR4 in decidual cells [27 ].

Similar to the decidual NK cells, A-NK cells migrated in vitro toward a gradient of the CXCR4 ligand (CXCL12). Thus, CXCR4 seems to play an important role in the preferential enrichment of CD16– NK cells in adenoids and deciduas. This result reinforces the conclusion that A-NK cells display an activated-like phenotype, presumably required to allow a prompt, innate immunity response to antigens entering the mucosal tissue through the nasal route. Immunohistochemistry analysis proved that the ligand for CXCR4 is found in endothelial cells of adenoid blood vessels and in the epithelial lining of the adenoids. Therefore, it is likely that CXCR4 is involved in the recruitment of A-NK cells as they traffic within the adenoids, through interactions with its specific ligand, CXCL12. It is tempting to speculate that the respiratory epithelial cells might attract A-NK cells toward the surface of the adenoids, where their encounter with the invading pathogens takes place. This interaction would induce the CD56bright A-NK cells to secrete immunoregulatory cytokines, such as IFN-{gamma}, TNF-{alpha}, and IL-5, which in turn, regulate the function of T and B lymphocytes and other cells of the adaptive immune system. Then, endothelial cells of adenoid blood vessels might attract the already activated NK cells from the adenoids toward the systemic circulation in response to different stimuli. This assumption is in line with previous studies, suggesting that late stages in human CD56bright NK maturation occur in peripheral lymphoid tissue [19 ]. It is interesting that endothelial cells in palatine tonsils also express CXCL12, whereas their epithelial lining lacks it, indicating a different role for the latter in these two secondary lymphoid organs.

The percentage of CXCR4-, CD25-, and HLA-DR-positive cells was only increased amongst B-NK cells following IL-2. These findings support again the assumption that A-NK cells are already in an activated state; therefore, the percentage of A-NK cells presenting CXCR4 is probably at the maximum, and therefore, IL-2 has no further effect. However, B-NK cells are not activated, and therefore, IL-2 activation increases the expression of these receptors. As IL-2 was used at saturation concentrations, far beyond its physiologic levels, their expression is probably maximal.

In contrast to the significant enrichment of human CD56bright NK progeny within lymph nodes [42 ], CD34+CD45RA+ cells were found only in low numbers in the adenoids. This result suggests that the adenoids are not a major site for early NK cell development.

Our results show that NKG2D is functional in A-NK cells, in spite of its slightly lower expression in comparison with B-NK cells. This down-regulation of NKG2D could not be explained by binding to NKG2D ligands, as they were not expressed on AS cells.

In concordance with previous studies about CD56bright NK cells [36 ], the A-NK cells had a low level of KIRs but a high level of the CD94–NKG2A inhibitory receptors. Apparently, the differential expression of these inhibitory receptors by subsets of human NK cells enables unique regulation of cytotoxic properties by these cells [46 ]. The increased expression of these receptors on A-NK cells after IL-2 stimulation supports previous findings in palatine tonsils and lymph nodes [19 ] and the assumption that NK cells in secondary lymphoid organs might differentiate and change their phenotype to become more similar to cytotoxic B-NK cells, in response to invading antigens [19 ].

Previous studies have reported an important role for the CEACAM1 protein in the inhibition of activated, decidual lymphocyte functions [24 ]. In the present study, CEACAM1 (CD66) expression was absent on NK cells from adenoids and blood. Following IL-2 activation, its expression was up-regulated in 23% (median) of the A-NK cells but unchanged in B-NK cells, and CEACAM1 was able to inhibit the A-NK cell activity. The up-regulation of CEACAM1 in response to IL-2 was a distinct feature of A-NK cells and was not the result of priming of A-NK cells by other cells in the adenoid stroma, as incubation of AS cells with B-NK cells did not elicit an increase in the expression of the receptor on the latter. These results parallel a previous study done on decidual, IL-2-activated NK clones [24 ] and suggest that the CEACAM1 proteins play a modulatory role in the inhibition of killing by IL-2-activated NK cells from adenoids.

Another unique property of A-NK cells is that the HLA-DR receptor was expressed on a higher percentage of cells and with stronger intensity on A-NK cells as compared with B-NK cells. The concomitant expression of HLA-DR and CD86 in A-NK cells (data not shown) suggests that such cells may function as APCs similar to NKT cells [57 ].

Received April 4, 2007; revised June 28, 2007; accepted June 29, 2007.


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