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Originally published online as doi:10.1189/jlb.0508278 on August 7, 2008

Published online before print August 7, 2008
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(Journal of Leukocyte Biology. 2008;84:1298-1305.)
© 2008 by Society for Leukocyte Biology

Trogocytosis and killing of IL-4-polarized monocytes by autologous NK cells

Mary Poupot, Jean-Jacques Fournié and Rémy Poupot1

INSERM, U.563, Centre de Physiopathologie de Toulouse-Purpan, Toulouse, France; and Université Paul-Sabatier, Toulouse, France

1 Correspondence: INSERM 563, Centre de Physiopathologie de Toulouse-Purpan, CHU Purpan, place Baylac, 31024 Toulouse Cedex, France. E-mail: remy.poupot{at}toulouse.inserm.fr


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ABSTRACT
 
Cross-regulations between innate immune cells have been given more and more emphasis. Here, we address the question of bidirectional interactions between activated monocytes and autologous NK cells. Classically activated monocytes (class-monocytes), obtained by priming with IFN-{gamma}, drive an inflammatory immune response. On the contrary, alternatively activated monocytes (alt-monocytes), obtained by stimulation with IL-4 or IL-13, engage an anti-inflammatory immune response. We show that alt-monocytes inhibit proliferation and production of IFN-{gamma} by autologous, IL-2-activated NK cells, whereas class-monocytes do not inhibit these NK cell functions. Reciprocally, IL-2-activated NK cells interact and undertake intensive synaptic transfer with alt-monocytes, whereas interactions with class-monocytes are weaker. This strong trogocytosis correlates with an efficient killing of alt-monocytes, mediated by natural cytotoxicity receptors and a lowered killing of class-monocytes. These results suggest that interactions between NK cells and autologous-activated monocytes modulate inflammatory responses. This might be extended further in the elimination of tumor-associated macrophages, which actively promote solid tumor progression and metastasis.

Key Words: cytotoxicity • inflammation • membrane capture • activation


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INTRODUCTION
 
Human NK cells are essentially cytotoxic cells representing a pivotal population of innate immunity. They play a critical role in antiviral, antibacterial, antiparasitic, and anticancer immunity. Their cytotoxicity is regulated by integration of antagonist signals delivered by activating and inhibitory receptors [1 ]. The different activating or inhibitory cellular ligands recognized by these receptors enable discrimination between target and nontarget cells for NK cell cytotoxicity. In recent years, it has been shown that NK cells can engage in a cross-talk with other innate immune cells, thus regulating the extent of immune responses. Many reports emphasize the reciprocal activation of NK cells and dendritic cells (DCs) in initial stages of the innate immune responses. With mouse cells, it was shown that DCs are able to trigger cytotoxicity and production of IFN-{gamma} by NK cells in a cell-to-cell, contact-dependent manner [2 ]. Later on, it was demonstrated that a bidirectional cross-talk between human DCs and NK cells results in the activation of both partners. Mature or immature DCs in the presence of maturation stimuli activate fresh NK cells and strongly augment their cytotoxicity and production of IFN-{gamma}. Moreover, NK cells activated by IL-2 or by mature DCs directly induce maturation of DCs [3 ]. These IL-2-activated or DC-activated NK cells efficiently kill immature DCs, involving the NKp30 natural cytotoxicity receptor (NCR) [4 ]. It has then been specified that killing of immature DCs is restricted to the CD94/NKG2A-expressing subset of NK cells [5 ]. Other reports have depicted interactions between NK cells and monocytes or macrophages. One of the earliest reports in the field shows that monocytes have a role in the IL-2-induced proliferation of human NK cells. This effect is dependent on cell-to-cell contact [6 ]. In the same idea, Carson et al. [7 ] showed that NK cells can produce IFN-{gamma} after coculture with autologous monocytes preliminarily activated by LPS. More recently, it has been shown that NK cells and monocytes can engage in a reciprocal program of activation depending on cell-to-cell contact. This activating cross-talk may result in a sustained, inflammatory response [8 ]. Finally, Caumartin et al. [9 ] described the acquisition by a tumor NK cell line of HLA-G1 molecules from monocytes after IFN-{gamma} treatment or from transfected tumor cells. This molecular transfer impacts the functional properties of the NK cells.

NK cell/monocyte interaction also promotes NK cell-mediated cytolysis of malignant cells. During this mutual activation, activation-induced C-type lectin/NKp80 (a C-type, lectin-like homodimeric receptor on NK cells) interaction is involved [10 ]. This report suggests that NK cells can kill autologous monocytes. Human NK cells and macrophages also regulate one another. Macrophages activate NK cell proliferation and secretion of cytokines and prime NK cell cytotoxicity against target cells. Reciprocally, NK cells regulate macrophage activity by killing highly stimulated macrophages [11 ].

Monocyte activation can take several aspects [12 ]. Besides the classical activation pathway described earlier, an alternative activation mechanism emerged in 1992 in the mouse model [13 ]. The classical activation of monocytes is mediated by IFN-{gamma} as primer, whereas alternative activation is mediated by IL-4, IL-13, or glucocorticoids [12 , 14 ]. The classical activation pathway of monocytes evolves toward an inflammatory immune response (secretion of high levels of IL-1, IL-6, IL-12, and TNF-{alpha}), and the alternative pathway evolves toward an anti-inflammatory response (secretion of high amounts of IL-10 and IL-1R antagonist) [14 ].

Hematopoietic effector cells need to establish intercellular contacts with partners to achieve immunological surveillance and to initiate their immunological responses if necessary [15 ]. These contacts take place via an immunological synapse, which is an interactive zone, well-defined in time and space. During these contacts, the effector cell interacts with the surface of its partner and takes membrane patches (this phenomenon is also known as trogocytosis). The partner can be an APC for {alpha}β T lymphocytes [16 ] or B lymphocytes [17 ] or a putative target cell (cancer cell or infected cell) for cytotoxic effectors such as CTL [18 ], {gamma}{delta} T cells [19 ], or NK cells [20 ]. At the immunological synapse, synaptic transfer of molecular materials from the target cell to the effector cell occurs through a physical bridge formed by membrane fusion between the two cells [21 ]. All lymphoid effectors can make trogocytosis and transfer cell-surface components [22 ]. In this way, NK cells establish an immunological synapse upon exposure to putative targets. In this immunological synapse, inhibitory and activating signals are integrated and converted or not in a cytolytic response. It has been shown that the balance between the triggering of activating and inhibitory receptors involved in the immunological NK cell synapse determines trogocytosis and killing of target cells [20 ]. A flow cytometry technique was proposed recently to monitor trogocytosis between cells for quantifying cell–cell interactions [23 ]. This assay enables the selective monitoring of different subsets among PBMC without physically separating them. Target cells are labeled with a membranous fluorescent molecular probe, and after 4 h incubation between target cells and effector cells, fluorescence is quantified on effector cells.

Following conjunction of a NK cell with an appropriate target cell, lytic granules are directed to the point of contact with this target cell, and the granule contents are released into the immune synapse established between the two partners [21 , 24 ]. The granule core is surrounded by a lipid bilayer containing lysosomal-associated membrane glycoproteins (LAMPs), including CD107a (LAMP-1), CD107b (LAMP-2), and CD63 (LAMP-3). These proteins become transiently expressed on the NK cell surface upon granule exocytosis. Transient CD107a expression at the membrane surface can be an assay to evaluate cell-mediated cytotoxicity [25 , 26 ].

Here, we compare the cross-talk between NK cells and monocytes according to the type of monocyte activation. Monocytes were primed toward classical activation with IFN-{gamma} (class-monocytes) or were alternatively activated with IL-4 (alt-monocytes). We show that IL-2-activated NK cells interact with autologous monocytes and perform trogocytosis, which is associated with functional consequences on monocytes and NK cells, depending on the type of activation of the former.


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MATERIALS AND METHODS
 
Cell preparation and culture
Fresh blood samples were collected from healthy donors, and PBMC were prepared on a Ficoll-Paque density gradient (Amersham Biosciences AB, Uppsala, Sweden) by centrifugation (800 g, 30 min at room temperature).

Highly pure CD14+ monocytes (>98%, as checked by flow cytometry) were positively selected from PBMC by MACS on an LS separation column (CD14 Microbeads, Miltenyi Biotec, Auburn, CA, USA), according to the manufacturer’s instruction manual. The CD14– fraction was collected as PBL.

Highly pure CD3–CD56+ NK cells (>91%, as checked by flow cytometry) were negatively selected by MACS on an LS separation column (NK cell isolation kit II, Miltenyi Biotec) from PBL, prepared as described above.

NK cells and PBL were cultured at 37°C in a humidified incubator with 5% CO2 in air for 2 days in RPMI-1640 culture medium supplemented with penicillin, streptomycin (Cambrex Bio Science, Verviers, Belgium), sodium pyruvate, and 10% of heat-inactivated FCS (Invitrogen Corp., Paisley, UK). During the cultures, NK cells and PBL were activated with 100 U/mL IL-2 (Sanofi-Aventis, Labège, France).

Monocyte activation
Alt-monocytes were obtained by culture of freshly purified monocytes stimulated by 300 U/mL IL-4 (PeproTech, Rocky Hill, NJ, USA) during 2 days at 106 cells per mL in complete RPMI-1640 culture medium. Class-monocytes were obtained by culture of freshly purified monocytes stimulated by 1000 U/mL IFN-{gamma} (R&D Systems Europe, Abingdon, UK) during 2 days at 106 cells per mL in complete RPMI-1640 culture medium.

Phenotype analyses by flow cytometry
Staining for flow cytometry was performed using FITC-, PE-, PE-Cyanine 5 tandem (PE-Cy5)-, or allophycocyanin-Cy7-conjugated mAb against human CD14, HLA-A, -B, and -C, HLA-DR, CD86, CD64, CD206, CD36 (Beckman Coulter-Immunotech, Marseilles, France), or appropriate isotype-matched fluorescent mAb as controls. Monocytes (105) were diluted in 30 µL PBS containing 5% FCS with 20 µg/mL mAb and incubated 15 min at 4°C.

NK cells were labeled for NKp30, NKp44, NKp46, or NKG2D using the corresponding mAb (10 µg/mL, R&D Systems Europe, Abingdon, UK) or appropriate isotype-matched mAb as controls, followed by staining with FITC-conjugated goat anti-mouse IgG.

For the detection of NKG2D ligands on monocytes, staining was performed using mAb (20 µg/mL) against human UL16-binding protein 1 (ULBP1; clone 170818), ULBP2 (clone 165903), ULBP3 (clone166510; R&D Systems Europe), MHC class I polypeptide-related sequence A (MICA; clone AM01), or MICB (clone BM02; Immatics Biotechnology, Coger, Paris, France), followed by incubation with FITC-conjugated goat anti-mouse IgG.

Cells were then washed and resuspended in PBS, and 104 cells were analyzed on a LSR-II cytometer using Diva software (both from BD Biosciences, San Jose, CA, USA).

Trogocytosis and CD107a expression
Activated monocytes were stained with the lipophilic green-emitting dye PKH67 (Sigma-Aldrich, St. Louis, MO, USA), according to the manufacturer’s instruction manual. Then, they were coincubated 4 h with autologous IL-2-activated PBL or with IL-2-activated NK in a 96-well U-bottom culture plates at a cell ratio of 2:1. A final concentration of 6 x 105 cells in 120 µL complete RPMI-1640 culture medium was used for labeling with anti-CD107a-PE-Cy5 or with isotype-matched PE-Cy5 conjugate as control at 4.8 µg/mL (BD Biosciences). Cells were pelleted by gentle centrifugation (110 g for 1 min) and left in coincubation for 3 min or 4 h and then washed with PBS/EDTA 0.5 mM. Cells were then stained for 15 min at 4°C with 5 µg/mL anti-CD3-PE/anti-CD56-PE-Cy7/anti-CD8-allophycocyanin-Cy7 or anti-TCR{gamma}9-PE/anti-CD4-PE-Cy5/anti-CD3-PE-Cy7 (Beckman Coulter-Immunotech for all mAb, except for anti-TCR{gamma}9-PE, which is from BD Bioscience). Synaptic transfer was measured as the acquisition of PKH67 fluorescence from the PKH67+-labeled activated monocytes by the different lymphoid populations. This acquisition was characterized through the increase of the mean fluorescence intensity (mfi) of PKH67 by a flow cytometry analysis. The mfi values (see Go Go Fig. 3 ) were visualized as described previously [23 ].


Figure 1
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  		Figure 1. Analysis of surface phenotype of alt-monocytes and class-monocytes by flow cytometry for the expression of the indicated markers by using mAb (gray histograms) and isotype-matched mAb as controls (white histograms) before (Day 0) and after a 2-day activation with IL-4 (alt-mo) or IFN-{gamma} (class-mo). Data shown come from one representative experiment out of five.


Figure 2
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  		Figure 2. Alt-monocytes interfere with proliferation and production of IFN-{gamma} by autologous NK cells. (A) Effect of alt-monocytes and class-monocytes on the IL-2-induced proliferation of NK cells measured by CFSE dilution after 6 days of coculture. (B) IL-2–activated NK cells were cocultured or not (white bars) with alt-monocytes (black bars) or class-monocytes (hatched bars) at different ratios. The percentage of NK cell proliferation represents the proportion of NK cells that have divided at least once. Error bars show SD of eight independent experiments. ns, Not significant. (C) Expression of IFN-{gamma} by NK cells stimulated by IL-2, IL-15, and IL-18 in cocultures without or with alt-monocytes or class-monocytes. Numbers in the upper-right quadrants indicate percentages of IFN-{gamma}+ NK cells among CD3–CD56+ cells. (D) IL-2-activated NK cells were cocultured or not (white bars) with alt-monocytes (black bars) or class-monocytes (hatched bars) with or without the TranswellTM system at the 1:1 ratio. Error bars show SD of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.05.


Figure 3
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  		Figure 3. NK cells engage much more trogocytosis with autologous alt-monocytes than with class-monocytes. (A) Profiling interactions of four subsets of IL-2-activated PBL with autologous alt-monocytes or class-monocytes preliminarily stained with PKH67 and after 3 min or 4 h of coincubation. Each mfi value corresponding to the acquisition of PKH67 was converted to a gray color scale from one representative experiment [23 ]. Gray intensity is correlated with interaction intensity. t, Time. (B) Acquisition of PKH67 fluorescence by IL-2-activated NK cells after 3 min or 4 h of coincubation with alt-monocytes or class-monocytes. Percentage represented the trogocytic NK cells. (C) PKH67 mfi resulting from trogocytosis by IL-2-activated NK cells or by the CD56+ subset of IL-2-activated PBL after 4 h of coincubation with autologous alt-monocytes (black bars) or class-monocytes (hatched bars) preliminarily stained with PKH67; white bars represent 3 min coincubation as negative control. Data shown come from one representative experiment out of five.

Lytic reactivity of IL-2-activated NK cells was evaluated by analysis of cell-surface CD107a expression as described in literature [25 , 26 ]. The mfi values were computed from 3 x 104 gated cells in all experiments.

Cytotoxicity assays
Standard assays of 51Cr release were used with differently activated monocytes as target and IL-2-activated NK cells as effectors. Target cells were labeled with 100 mCi 51Cr-sodium bichromate (Amersham Biosciences AB). When specified, purified NK cells were incubated for 15 min at 4°C with mAb against NKp30, NKp44, NKp46, or NKG2D at 10 µg/mL (R&D Systems Europe) and washed, and then, the Fc{gamma} of these mAb were blocked by incubation during 15 min at 4°C with a F(ab')2 goat anti-mouse Fc{gamma} (used at 52 µg/mL, Jackson ImmunoResearch Laboratories, Baltimore, MD, USA) before contact with targets. After 4 h of contact, 50 µL supernatant was taken and transferred on the LumaPlateTM 96; then after drying, 51Cr release was measured with a MicroBeta TriLux (Perkin Elmer, Wellesley, MA, USA). The percentage of specific lysis was calculated as follows: {[(51Cr release)–(spontaneous release)]/[(maximum release)–(spontaneous release)]} x 100.

Proliferation assays
Freshly purified NK cells were stained with 1.5 µM CFSE (Molecular Probes-Invitrogen Life Technology, Paisley, UK) for 8 min at 37°C and then washed three times in RPMI-1640 culture medium.

Activated monocytes were added to CFSE-labeled NK cells at ratios 0:1, 1:2, or 1:1 in 96-well culture plates. All cells were resuspended in complete RPMI-1640 culture medium supplemented with 100 U/mL IL-2. At Day 6 of coculture, the CFSE dilution in NK cells was measured by flow cytometry, as described above after staining for 15 min at 4°C with 5 µg/mL anti-CD3-PE-Cy7/anti-CD56-PE-Cy5.

Intracellular detection of IFN-{gamma} in NK cells
IL-2-activated NK cells were coincubated in complete culture medium with or without differently activated monocytes (at ratio 1:1) and with or without TranswellTM inserts (BD Biosciences). IL-2-activated NK cells (6x105 cells in 400 µL) were plated in 24-well culture plates in the bottom part of the TranswellTM, and the insert was filled with 400 µL complete culture medium containing or not 6 x 105 of differently activated monocytes with 100 U/mL IL-2, 10 ng/mL IL-15, and 10 ng/mL IL-18 (R&D Systems Europe) for 18 h at 37°C. Brefeldin A (Sigma-Aldrich) was then added at a final concentration of 10 µg/mL, and 6 h later, cells were washed, stained for CD56 and CD3 surface markers, fixed in 4% paraformaldehyde, permeabilized with PBS + 1% saponin, and then stained for cytoplasmic IFN-{gamma} with 4 µg/mL anti-IFN-{gamma}-PE conjugate or with 4 µg/mL PE-labeled, isotype-matched mAb as control (both from BD Biosciences). After washing, cells were resuspended in PBS, and NK cells were analyzed by flow cytometry after CD3–/CD56+ gating.

Statistical analysis and graphic representation
Data (see Figs. 2 B and D , 4 B and D , and 5B ) were analyzed using two-tailed Student’s t-tests. The results shown are representative of at least three separate experiments, and values are expressed as the mean ± SEM with * for P < 0.05, ** for P < 0.01, and *** for P < 0.005 when indicated.


Figure 4
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  		Figure 4. NK cells preferentially kill autologous alt-monocytes compared with class-monocytes. (A) Analysis by flow cytometry of the expression of CD107a by the CD56+ subset of IL-2-activated PBL after coincubation with alt-monocytes or class-monocytes. Numbers in the upper-right quadrants indicate percentages of CD107a+ cells among CD56+ cells. (B) Percentage of CD107a+ cells for different IL-2-activated PBL subsets and for IL-2-activated NK cells after coincubation with alt-monocytes (black bars) or class-monocytes (hatched bars). Error bars show SD of three independent experiments. (C) Analysis of coexpression of CD107a and PKH67 of IL-2-activated NK cultivated with alt-monocytes during 3 min or 4 h in the presence of isotype control IgG or CD107a antibody. Numbers in upper-right quadrant indicate percentages of cells. (D) Specific lysis of alt-monocytes ({blacksquare}) or class-monocytes ({square}) by NK cells in 51Cr release. Percentages of specific lysis are shown for three E:T ratios. Error bars show SD of five independent experiments.


Figure 5
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  		Figure 5. NK cells kill autologous alt-monocytes through NCRs. (A) Analysis of NK cells by flow cytometry for the expression of the NK cell receptors NKp30, NKp44, NKp46, and NKG2D by using mAb (gray histograms) and isotype-matched mAb as controls (white histograms). (B) Percentage of specific lysis is reported for the NK cell:alt-monocyte ratio of 30:1. Before coincubation of 51Cr-labeled alt-monocytes with NK cells for 51Cr-release assay, IL-2-activated NK cells were preincubated or not with anti-human NK receptor mAb, separately or mixed (as indicated under the histograms). Error bars show SD of three independent experiments, and significance is established with assays without mAb.


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RESULTS
 
Alt-monocytes but not class-monocytes inhibit NK cell proliferation and production of IFN-{gamma}
After magnetic cell sorting of monocytes, class-monocytes were generated by IFN-{gamma} priming, and alt-monocytes were generated by IL-4 activation. Their respective phenotypes were verified by a flow cytometry analysis of the specific expression of surface markers (Fig. 1 ). On the one hand, an increase of the expression of Fc{gamma}RI (CD64) [27 ], CD86 [12 ], HLA-class I (HLA-A, -B, and -C), and HLA-class II (HLA-DR) molecules [28 ] and on the other hand, the strong decrease of the expression of the scavenger receptor (CD36) [29 ] confirmed the phenotype of class-monocytes. An increase of the expression of the mannose receptor (CD206) [13 ] and partial increase of the expression of CD86 and HLA-class II molecules [12 ] corresponded to the phenotype of alt-monocytes. As expected, CD36 was expressed more intensively on alt-monocytes than on class-monocytes [12 ]. In addition, we found that the expression of CD64 and HLA-class I molecules was decreased on alt-monocytes when compared with class-monocytes, whereas expression of CD206 was decreased on class-monocytes and increased on alt-monocytes.

Many reports emphasize reciprocal interactions between monocytes and NK cells [6 7 8 9 ], such as NK cell proliferation promoted by freshly purified monocytes [6 ]. We thus sought after an impact of differently activated monocytes on autologous NK cell proliferation induced by IL-2. NK cell proliferation was measured by CFSE dilution after 6 days in cocultures with alt-monocytes or class-monocytes (Fig. 2A ). Alt-monocytes significantly reduced NK cell proliferation induced by IL-2 at the 1:1 ratio, whereas class-monocytes did not (Fig. 2 A and B) .

Then, we studied the influence of differently activated monocytes on the ability of autologous NK cells to produce proinflammatory cytokines, in particular, IFN-{gamma}. NK cells were cultured with autologous alt-monocytes or class-monocytes in the presence of IL-2, IL-15, and IL-18, and intracellular IFN-{gamma} was quantified. Without monocytes, 71% of NK cells stimulated by the cytokines produced IFN-{gamma}. When added, alt-monocytes inhibited production of IFN-{gamma} by NK cells, whereas class-monocytes did not (Fig. 2C) . Using TranswellTM cocultures, we showed that this inhibition of the production of IFN-{gamma} by NK cells was dependent on cell-to-cell contact (Fig. 2D) .

Profiling blood lymphocyte interactions with autologous monocytes
As alt-monocytes inhibited the production of IFN-{gamma} by NK cells in a cell-to-cell, contact-dependent manner, we analyzed the interactions between NK cells and autologous monocytes activated by IL-4 or by IFN-{gamma}.

First, using a flow cytometry technique developed to identify the lymphoid subsets that interacted with cancer cell lines in bulks of PBMC [23 ], we sought after interactions between PBL and autologous monocytes. After activation, class- and alt-monocytes were stained with a green fluorescent membranous probe (PKH67) and mixed with autologous IL-2-activated PBL. Interaction between partners was quantified by measuring the green fluorescence acquisition of membrane patches from monocytes by different lymphoid subsets after 4 h of coincubation (trogocytosis) [23 ]. This experiment shows that each PBL subset took much more membrane patches from autologous alt-monocyte targets than from class-monocyte targets, rendered by a higher PKH67 mfi (Fig. 3A ). In details, among the four lymphoid subsets that have been tested, CD56+ cells showed the highest potential to make trogocytosis with autologous monocytes and particularly strongly with alt-monocytes. CD4+ and CD8+ T lymphocytes also interacted, but weakly, with alt-monocytes, whereas the {gamma}{delta} T cell subset poorly recognized monocytes.

As the main population encountered in the CD56+ subset of PBL was most probably NK cells, we then searched for interactions between purified NK cells and autologous monocytes. As expected, pure IL-2-activated NK cells interacted more with alt-monocytes than with class-monocytes, rendered by a higher percentage of PKH67-positive NK cells after 4 h of contact with alt-monocytes (Fig. 3B) . Then, we showed that synaptic transfers between pure IL-2-activated NK cells and activated monocytes were similar with what was observed with the CD56+ subset of IL-2-activated PBL (Fig. 3C) . Therefore, the global-scanning results (Fig. 3A) were confirmed with pure, IL-2-activated NK cells, indicating that trogocytosis of monocytes by NK cells was not dependent on a bystander cellular population and that NK cells were the main effectors of trogocytosis toward autologous monocytes in a bulk of IL-2-activated PBL.

NK cells differentially kill autologous alt- and class-monocytes
To test whether cell–cell contacts between IL-2-activated NK cells and monocytes led to the NK cell-mediated lysis of the latter, cell-surface expression of CD107a on NK cells was measured after contact with monocytes. During trogocytosis experiments, cells were incubated with an anti-CD107a mAb to detect CD107a expression on the four lymphoid subsets of PBL previously tested for trogocytosis. Expression of CD107a was measured by the percentage of positive cells for this marker. Interaction with autologous alt-monocytes induced a pronounced degranulation of the CD56+ subset when compared with the lowest degranulation obtained with autologous class-monocytes (Fig. 4A ). Among the different subsets of PBL, only the CD56+ subset was capable of engaging activated, autologous monocytes and to express CD107a at the same level than pure, IL-2-activated NK cells (Fig. 4B) . The analysis of the costaining PKH67/CD107a of IL-2-activated NK after 4 h of contact with alt-monocytes showed 33% of NK cells making trogocytosis only and 13% of NK cells that made trogocytosis and expressed CD107a (Fig. 4C) . Moreover, 5% of NK cells expressed CD107a without a real acquisition of PKH67 with regard to the negative control at 3 min. This suggests that autologous monocytes could be susceptible to NK cell-mediated lysis. This possibility was assessed in a standard, 4 h 51Cr-release assay. IL-2-activated NK cells were used as effectors against autologous alt- or class-monocytes. For each E:T ratio tested (from 30:1 to 1:1), alt-monocytes were killed more efficiently than class-monocytes (Fig. 4D) . As HLA-A, -B, and -C molecules on target cells are known as inhibitory signals for NK cell cytotoxicity [1 ], the test of a specific lysis was also performed in the presence of mAb masking HLA-A, -B, and -C molecules on the class-monocytes. Interestingly, in this setting, class-monocytes became more sensible to the killing of IL-2-activated NK cells (data not shown).

NK cells kill autologous alt-monocytes through NCRs
Then, the relative contribution of the different NK receptors in the strong lysis of alt-monocytes was determined. Before coincubation with alt-monocytes, IL-2-activated NK cells were preincubated with blocking mAb against NKp30, NKp44, or NKp46 (NCRs) or against NKG2D (C-type lectin), as these receptors were expressed on IL-2-activated NK cells (Fig. 5A ). The percentage of specific lysis of alt-monocytes was reduced significantly by the blockade of the NKp30 (P<0.01), NKp44 (P<0.005), and NKp46 (P<0.01; Fig. 5B ). Statistical analysis of the reduction of specific lysis by blockade of the NCRs did not show evidence of the pre-eminence of one of these NCRs in the lysis of alt-monocytes by autologous, IL-2-activated NK cells (Fig. 5B) . Moreover, the dramatic reduction of lysis with the mix of the three anti-NCR mAb showed the synergistic effect of NCRs for the lysis of alt-monocytes. Besides, neutralization of NKG2D did not reduce this lysis; this result correlated with the absence of the expression of the NKG2D ligands ULBP1, ULBP2, ULBP3, MICA, and MICB by alt-monocytes (data not shown). On the contrary, we found that MICB was expressed on class-monocytes (data not shown).


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DISCUSSION
 
Altogether, our data depict new insights in interactions between human NK cells and autologous monocytes. In 1992, Miller et al. [6 ] reported that proliferation of NK cells induced by IL-2 is enhanced by freshly purified monocytes. In this study, monocytes had not been activated prior to their incubation with purified NK cells. More recently, Dalbeth et al. [8 ] reported that human NK cells and monocytes from patients with inflammatory disease can engage in a reciprocal program of activation leading to enhanced production of cytokines and thus, amplification of the inflammatory response. Others showed that the cross-talk between NK cells and autologous LPS-activated monocytes or macrophages leads to an increase of the production of cytokines by NK cells and of the proliferation of NK cells [7 , 10 , 11 ]. These reports also show that LPS-activated monocytes or macrophages are killed by autologous NK cells. It is noteworthy that NK cells can also kill autologous, immature DCs [4 ] or mesenchymal stem cells [30 , 31 ].

Here, we show that alt-monocytes, but not class-monocytes, can inhibit the IL-2-induced proliferation of autologous NK cells and production of IFN-{gamma} by autologous NK cells in a cell-to-cell dependent manner for the last property. In the same time, we show evidence that IL-2-activated NK cells strongly engage trogocytosis with autologous alt-monocytes and probably kill them in a simultaneous manner, as demonstrated in a similar model between human {gamma}{delta} T lymphocytes and tumor cells [32 ].

It is known that this active trans-synaptic capture of membrane fragments is down-regulated by signals emanating from inhibitory NK receptors that recognize protective HLA-class I alleles [20 ]. Accordingly, the strong trogocytosis and the efficient killing of alt-monocytes by IL-2-activated NK cells, which we report here, can be explained in part by a low level of HLA-class I molecules at the surface of alt-monocytes. On the contrary, class-monocytes express higher levels of HLA-class I molecules, which account for the low percentage of specific lysis by NK cells. Moreover, we show that specific lysis of alt-monocytes by autologous, IL-2-activated NK cells is engaged by NCRs. Interestingly, whereas NKp30 and NKp44 ligands remain unknown, it has been shown that Mycobacterium tuberculosis-infected monocytes can be killed by autologous NK cells through the engagement of NKp46 with vimentin [33 ], a cytoskeletal protein expressed at the monocyte surface and up-regulated in these infected monocytes [33 , 34 ]. Therefore, IL-4 activation of monocytes could up-regulate some unknown ligands of NCRs, which could be implicated in the recognition and the lysis of these monocytes by autologous, IL-2-activated NK cells.

Besides, we have detected MICB at the surface of class-monocytes, suggesting that NK cells could be activated by autologous class-monocytes through the engagement of NKG2D. However, this activation might be thwarted by the high levels of inhibitory HLA-class I molecules expressed by class-monocytes, leading to a weak, specific lysis. This could explain why IL-2-activated NK cells and IFN-{gamma}-polarized monocytes interact weakly together. Our results can be brought together with recent reports from Wang et al. [35 ] and Dalbeth et al. [8 ]. The first shows that the IFN-{gamma}-induced MIC molecules on monocytes played an essential role in triggering the activation of NK cells, especially for IFN-{gamma} production. The second shows that monocyte-NK cell reciprocal interactions at inflammatory sites enable a sustained inflammatory response. By contrast, Caumartin et al. [9 ] show a strong trogocytosis between a IL-2-dependent NK cell line and IFN-{gamma}-polarized monocytes with acquisition of HLA-G1 by the former. First and contrarily to our study, this report describes an allogenic context using a tumoral NK cell line but not NK cells purified from healthy donors. Second, it is known that a NKG2D-mediated, activating signal may be bypassed by a HLA-G1 inhibitory signal [36 ].

Taken together, our results suggest a cellular regulation of anti-inflammatory responses: Alt-monocytes inhibit the proliferation of autologous NK cells and their production of IFN-{gamma}, but NK cells can thwart this anti-inflammatory direction by killing alt-monocytes efficiently. On the contrary, class-monocytes do not inhibit the inflammatory response of autologous NK cells and are killed weakly.

A majority of malignant solid tumors contains numbers of macrophages as a major component of the host leukocytic infiltrate. These macrophages are referred to as tumor-associated macrophages (TAMs) and are derived from peripheral blood monocytes recruited into the tumor mass from the bloodstream. This recruitment is followed by a local proliferation. As evidenced by clinical and experimental studies, TAMs promote angiogenesis, progression of solid tumors, and metastasis through the release of various cytokines, chemokines, and growth factors [37 ]. TAMs can have, in an inflammatory environment, anti-inflammatory phenotype and properties, such as alt-monocytes [38 ]. Several studies propose a novel, anticancer strategy based on the targeting of TAMs [39 , 40 ]. For instance, Hagemann et al. [41 ] showed recently that the inhibition of NF-{kappa}B signaling in TAMs promotes a direct tumoricidal activity through NO production and reverses their immunosuppressive phenotype by promoting NK cell-mediated antitumor immunity through a release of IL-12.

In our study, we show that anti-inflammatory alt-monocytes inhibit autologous, IL-2-activated NK cells, but the former efficiently kills the latter. The related phenotype of TAMs makes them a good target for autologous NK cells. Therefore, in addition to the lysis of tumor cells (the graft vs. tumor effect), NK cell-based immunotherapies might eliminate anti-inflammatory TAMs. In conclusion, our results strengthened previous reports indicating cross-control of innate immune populations [7 , 8 , 10 , 11 , 41 42 43 ]. Unbalance in this cross-control can be implicated in inflammatory, autoimmune disorders. Understanding cellular and molecular mechanisms in this cross-control could open new therapeutic strategies for inflammatory diseases or for the treatment of solid cancer.


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ACKNOWLEDGEMENTS
 
This work was supported by grants awarded by Région Midi-Pyrénées ("Biothérapies" program), MENSR ("Action de Soutien à l’Innovation"), INSERM, and Paul Sabatier University.

Received May 6, 2008; revised July 8, 2008; accepted July 19, 2008.


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