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The Journal of Immunology, 2008, 181, 6711 -6719
Copyright © 2008 by The American Association of Immunologists, Inc.
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Interaction of Monocytes with NK Cells upon Toll-Like Receptor-Induced Expression of the NKG2D Ligand MICA1

Mercedes Kloss2,*, Patrice Decker2,{dagger}, Katrin M. Baltz*, Tina Baessler*, Gundram Jung{dagger}, Hans-Georg Rammensee{dagger}, Alexander Steinle{dagger}, Matthias Krusch3,* and Helmut R. Salih3,4,*

* Department of Hematology and {dagger} Department of Immunology, Eberhard Karls University of Tuebingen, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Reciprocal interactions between NK cells and dendritic cells have been shown to influence activation of NK cells, maturation, or lysis of dendritic cells and subsequent adaptive immune responses. However, little is known about the crosstalk between monocytes and NK cells and the receptors involved in this interaction. We report in this study that human monocytes, upon TLR triggering, up-regulate MHC class I-Related Chain (MIC) A, but not other ligands for the activating immunoreceptor NKG2D like MICB or UL-16 binding proteins 1–3. MICA expression was associated with CD80, MHC class I and MHC class II up-regulation, secretion of proinflammatory cytokines, and apoptosis inhibition, but was not accompanied by release of MIC molecules in soluble form. TLR-induced MICA on the monocyte cell surface was detected by autologous NK cells as revealed by NKG2D down-regulation. Although MICA expression did not render monocytes susceptible for NK cell cytotoxicity, LPS-treated monocytes stimulated IFN-{gamma} production of activated NK cells which was substantially dependent on MICA-NKG2D interaction. No enhanced NK cell proliferation or cytotoxicity against third-party target cells was observed after stimulation of NK cells with LPS-activated monocytes. Our data indicate that MICA-NKG2D interaction constitutes a mechanism by which monocytes and NK cells as an early source of IFN-{gamma} may communicate directly during an innate immune response to infections in humans.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
NK cells are components of the innate immune system initially identified as lymphoid cells capable of lysing target cells without prior sensitization (1, 2). They exert their cytotoxic effector functions mainly after sensing "missing self", i.e., the absence of MHC class I, and/or "induced self" by triggering activating receptors upon interaction with specific ligands leading to antiviral and antitumor immune responses (3). Although the cytolytic potential of NK cells is widely appreciated, recently the capability of NK cells to initiate and shape adaptive immune responses by providing an early source of IFN-{gamma} receives increasing attention (4). The immunomodulatory function of NK cells is further exemplified by numerous studies addressing the crosstalk between NK cells and dendritic cells (DC).5 This interaction can result in activation, potentiation of the cytolytic capacity, and cytokine production of NK cells and in maturation, lymphokine release, but also in lysis of certain autologous DC (5, 6, 7). However, surprisingly few studies addressed the crosstalk of NK cells with other components of the myeloid lineage and the receptors involved in this interaction, especially in humans (8, 9, 10, 11, 12, 13, 14, 15). Very recently it was shown that TLR stimulation induces expression of activation-induced C-type lectin (AICL) on myeloid cells, which, by binding to the human NK cell receptor NKp80, stimulates the release of proinflammatory cytokines from both NK cells and monocytes (16). While the encounter of DC and macrophages with NK cells occurs, according to their tissue distribution, across various lymphoid and nonlymphoid tissues, a major part of the human NK cell compartment is contained in peripheral blood and thus in direct proximity of monocytes.

NK cell functions result of an integrative response emerging from multiple activating and inhibitory receptors far beyond the well-characterized receptors that recognize MHC class I allelic variants (3). The unidentified nature of the corresponding cellular ligands of many activating NK cell receptors has hampered the analysis of their involvement in NK cell-mediated immunoregulation and immunosurveillance. An exception is the stress-inducible ligands of the C-type lectin-like immunoreceptor NKG2D (3, 17, 18). The NKG2D ligands (NKG2DL) are proteins that are structurally similar to MHC class I molecules and comprise members of the MHC class I-related chain (MIC) family (MICA and MICB) and the UL16-binding protein family (ULBP1–4, RAET1G, RAET1L) (17, 19, 20). The various NKG2DL exhibit only moderate sequence similarities among each other: MICA, for example, only shares 20–25% sequence identity with ULBP molecules, but all NKG2DL share an MHC class I-like {alpha}1{alpha}2 domain that binds to NKG2D (17). NKG2DL can be found on many cancer cells and on cells infected with bacteria or viruses (12, 21, 22, 23, 24, 25). After recognition of its ligands, NKG2D potently stimulates NK cell functions.

In this study, we analyzed the role of NKG2D in the immunoregulatory crosstalk between NK cells and monocytes derived from human peripheral blood. We report that monocytes up-regulate the NKG2DL MICA upon TLR activation and studied the role of MICA expression by monocytes in the modulation of NK cell responses in humans.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Preparation of monocytes, DC, NK cells, and transfectants

PBMC were obtained from healthy volunteers by Ficoll-Hypaque density gradient centrifugation. Monocytes and NK cells were isolated from PBMC by negative selection using the Monocyte Isolation Kit II and the NK Cell Isolation Kit II, respectively, and MACS columns (Miltenyi Biotec) yielding monocyte/NK populations of at least 95% purity. In some cases, monocytes were alternatively isolated from PBMC by a second density centrifugation through a 46% Percoll cushion as described previously (26), yielding a cell population of ~70% purity. In all cases, monocyte and NK cell purity was determined using FACS analysis by staining for CD14+ and CD56+CD3–, respectively. DC were generated from plastic adhering monocytes by culture in RPMI 1640 medium supplemented with GM-CSF (100 ng/ml, Leucomax; Novartis) and IL-4 (20 ng/ml; R&D Systems). Cytokines were added to differentiate DCs every 2 to 3 days. C1R-MICA, C1R-MICB, and C1R-mock transfectants were previously described (27).

Reagents and mAbs

Highly purified LPS (Salmonella typhimurium, prepared by phenol extraction, purified by gel filtration chromatography and cell culture tested), poly I:C, and Pam3Cys were from Sigma-Aldrich. mAbs recognizing MICA (AMO1), MICB (BMO1), ULBP1–3 (AUMO3, BUMO1, CUMO3, respectively), and NKp80 (5D12) were previously described (16, 27). Mouse IgG1 was from BD Pharmingen. F(ab')2 of the blocking MICA/B mAb BAMO1 and of the NKp80 Ab were generated by pepsin digestion and purified from endotoxin by Triton X-114 extraction (28). Endotoxin levels in mAb preparations were tested with a limulus amebocyte lysate assay (QCL-1000; Cambrex) and were below 0.1 EU/µg of Ab. Control IgG1 F(ab')2 were from Southern Biotechnology Associates. The goat anti-mouse-PE and goat anti-mouse IgG2a-HRP conjugates were from Jackson ImmunoResearch Laboratories and Southern Biotechnology Associates. Anti-CD56-FITC, anti-CD80-PE, anti-CD14-FITC and -PE, anti-HLA-A/B/C-PE, anti-HLA-DR-PE, anti-CD107a-PE mAb, and the corresponding isotype controls, as well as Annexin V-FITC and 7-amino-actinomycin D (7-AAD) and the anti-human IL-12p40/p70 mAb were from BD Pharmingen. Anti-NKG2D mAb, NKG2D-Ig fusionprotein, human IgG1, IFN-{alpha}, and IL-15 were from R&D Systems and PromoKine.

Flow cytometry

Cells were incubated with the indicated specific mAb or isotype control (all 10 µg/ml) followed by goat anti-mouse-PE conjugate (1/100) as secondary reagent and then analyzed on a FACSCalibur (BD Biosciences). Conjugated mAb and the respective isotype controls were used at 2 µl/100,000 cells. Annexin V-FITC and 7-AAD were used according to manufacturer’s recommendations. Where indicated, specific fluorescence indices of MICA staining were calculated by dividing median fluorescences obtained with the specific mAb by median fluorescences obtained with isotype control.

Real-time PCR

Real-time PCR analysis was performed as described previously (27). Samples were normalized to GAPDH RNA to account for the variability in the initial concentration of the total RNA and conversion efficiency of the reverse transcription reaction. Primers for GAPDH RNA were 5-GGGTGTGAACCATGAGAAG-3 and 5-GGCAGGGATGATGTTCTGG-3; Primers for MICA were 5-CCTTGGCCATGAACGTCAGG-3 and 5-CCTCTGAGGCCTCGCTGCG-3.

ELISA

Detection of soluble MICA and MICB in cell culture supernatants was performed using our previously described sandwich-ELISA with a detection limit of 80pg/ml (27). IFN-{gamma} production by NK cells as well as IL-6, IL-8, IL-12p40, IL-12p70, and TNF secretion by monocytes was analyzed using OptEIA sets or Ab pairs and streptavidin-peroxidase conjugate from BD Pharmingen or R&D Systems according to the manufacturer’s instructions. MIC and cytokine concentrations in supernatants are depicted as mean ± SEM of triplicates.

Chromium release assays

Target cells were labeled with 1.85MBq of Na251CrO4 (Amersham Biosciences) for 1 h at 37°C. Cells were washed and effector cells were titrated on the target cells and incubated for 4 at 37°C. Maximum release was determined from target cells lysed in 1% Triton X-100. Percentage of lysis was calculated as follows: 100 x (experimental release – spontaneous release): (maximum release – spontaneous release).

Proliferation assay

Proliferation was determined by culture of a total of 105 autologous NK cells with or without 103 monocytes in 96-well plates. Thymidine incorporation was determined on day 5 by 16 h pulse with [3H]-thymidine (1µCi/well; Amersham Biosciences) Results shown are means ± SEM of quadruplicates.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
TLR stimulation induces MICA expression on monocytes

As a first step, we incubated monocytes for 16 h in the presence or absence of the TLR ligands (TLRL) LPS (500 ng/ml), Pam3Cys (1 µg/ml), or poly I:C (10 µg/ml) and subsequently determined the expression of the NKG2DL MICA, MICB, and ULBP1–3 by flow cytometry. TLR stimulation substantially induced surface expression of MICA, but surprisingly not of MICB or ULBP molecules (Fig. 1A). Next, concentrations of the three stimuli were titrated and the kinetics of MICA up-regulation was estimated. As shown in Fig. 1B, freshly isolated monocytes did not express MICA. After 16 and 39 h without stimuli, monocytes of some but not all donors acquired marginal MICA expression, possibly due to unspecific activation by the isolation procedure or in vitro culture. In contrast, the three stimuli induced substantial MICA expression already after 16 h, which was observed with all investigated donors. While MICA was already substantially induced by LPS concentrations as low as 4 ng/ml, higher concentrations of poly I:C and Pam3Cys were required (Fig. 1B). In contrast to available data obtained with DC (29, 30), we did not detect MICA up-regulation upon treatment of monocytes with 30 ng/ml IL-15, 3,000 U/ml IFN-{alpha}2a or 3,000 U/ml IFN-{alpha}2b (Fig. 1C).


Figure 1
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  		FIGURE 1. Stimulation of monocytes with TLRL up-regulates MICA but not MICB or ULBP expression. Monocytes were isolated using MACS columns and cultured in the presence or absence of the indicated stimuli before flow cytometry. Dead cells were excluded by 7-AAD staining. A, Open peaks and shaded peaks represent isotype control and the indicated NKG2DL-specific Ab stainings, respectively, after 16 h. B, Concentrations of LPS, poly I:C, and Pam3Cys were titrated, and the kinetics of MICA up-regulation were determined (bar graphs). A representative histogram plot of MICA up-regulation (Isotype, isotype control; ex vivo, freshly isolated; Medium, 39 h without LPS; LPS high and low: 39 h with 500 and 4 ng/ml LPS, respectively) is also depicted. C, Effects of the indicated stimuli on MICA expression are depicted. D, Monocytes were isolated using MACS columns and cultured for the indicated times in the presence or absence of LPS (500 ng/ml). Subsequently RNA was isolated and reverse transcribed. MICA levels were determined by real-time PCR and normalized with GAPDH expression. {diamondsuit}, untreated monocytes; {blacksquare}, LPS-treated monocytes. E, Monocytes were cultured as in A and cell culture supernatants were analyzed after 24 h for the presence of sMICA and sMICB by ELISA. Supernatants from C1R-MICA and C1R-MICB transfectants served as controls. ND, not detectable. Shown is one representative experiment each out of at least four experiments with similar results. SFI, Specific fluorescence indices.

 
To confirm the effect of TLR ligation on MICA expression, we utilized quantitative PCR analysis. Monocytes were cultured for the indicated times alone or in the presence of LPS, and subsequently MICA transcripts in treated and untreated monocytes were quantified by standardization with GAPDH RNA levels. In line with the results obtained by flow cytometry, we detected a time-dependent increase of MICA mRNA levels in LPS-activated monocytes compared with untreated cells (Fig. 1D).

To exclude that our results regarding MICA/B surface expression on monocytes after TLR stimulation were affected by, or the consequence of, release of MICA and MICB as soluble forms (31, 32), we analyzed cell culture supernatants of untreated and TLR-stimulated monocytes by ELISA with C1R-MICA and C1R-MICB transfectants (300,000 cells/well each for 24 h) as positive controls. While the C1R transfectants released high levels of sMICA and sMICB into the culture supernatants, no sMICA or sMICB was detected in any of the investigated monocyte culture supernatants, indicating that differences in MIC surface expression are not due to a differential release in soluble form (Fig. 1E). Likewise, no sMICA or sMICB was detectable when monocytes were activated with LPS at different concentrations and analyzed at different timepoints (data not shown). Together, these results demonstrate that monocytes exclusively up-regulate MICA, but none of the other investigated NKG2DL, upon activation via TLR.

Functional characterization of MICA up-regulation in monocytes

Because conflicting data regarding the ability of various APCs to express NKG2DL have been reported (10, 11, 12, 29, 30, 33, 34), we aimed to characterize the functional response of TLR-stimulated monocytes leading to increased MICA expression in greater detail. No additive or synergistic effect on MICA expression was observed when monocytes were treated with both LPS and poly I:C as compared with LPS or poly I:C alone (Fig. 2A), either at high concentrations (500 ng/ml LPS, 10 µg/ml poly I:C, upper panel) or at low concentrations (middle panel). However, at the level of IL-6 secretion, an at least additive effect of the two stimuli was observed at low concentrations (lower panel). MICA induction after 16 h was associated with CD80 and MHC class II up-regulation on live, CD14-positive cells after stimulation with all the TLRL tested (Fig. 2B). Moreover, TLR-stimulated monocytes secreted higher levels of IL-6, IL-8, TNF, and IL-12p40, but no bioactive IL-12p70 (Fig. 2C), confirming that monocytes upregulating MICA were fully activated and functional. No substantial presence of the cytokines IL-15, IL-18, or type I IFNs was detected in supernatants of monocytes cultured with or without LPS for 24 or 48h under our experimental conditions (data not shown). Because LPS was a potent inducer of MICA, we aimed to characterize the LPS-induced MICA up-regulation in greater detail. MICA up-regulation on monocytes by LPS was accompanied by MHC class I up-regulation (increase mean expression from 129 to 236). Levels of MICA/B on activated DC as well as C1R-MICA and C1R-mock transfectants, which are susceptible to NK cell lysis, were at least the same or even by far higher than on activated monocytes, while these cells expressed lower levels of MHC class I (Fig. 3A). Monocyte IL-6 secretion was detected after incubation with all the LPS concentrations tested both after 16 and 39h, while no IL-6 production was observed with untreated monocytes (Fig. 3B). Importantly, MICA up-regulation upon LPS stimulation was accompanied by a decrease in early (Annexin V-positive/7-AAD-negative cells) and late (Annexin V-positive/7-AAD-positive cells) apoptotic monocytes compared with untreated monocytes after 39 h (Fig. 3C) excluding that MICA up-regulation was a consequence of apoptosis induction. Moreover, pronounced MICA up-regulation was observed already after 16 h and thus before a substantial percentage of monocytes became apoptotic, and MICA up-regulation was specifically observed on nonapoptotic (Annexin V-negative) LPS-stimulated monocytes (data not shown). It should be noted that the observed extent of apoptosis in monocytes cultured in medium only as well as the protective effect of LPS has already been reported (35).


Figure 2
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  		FIGURE 2. Monocytes upregulating MICA are fully activated and functional. Monocytes were either isolated from PBMC by MACS columns (A and C) or by density gradient centrifugation (B) and cultured for 16 h in the presence or absence of LPS, poly I:C, or Pam3Cys. A, The effect of a combination of LPS and Poly I:C on MICA expression in monocytes was investigated using high (upper panel, LPS 500 ng/ml, poly I:C 10 ng/ml) and low (middle panel) concentrations. In addition, the influence on IL-6 secretion was studied by ELISA (lower panel). B, Cells were stained with CD80- or MHC class II-specific mAb (shaded peaks) or the corresponding isotype controls (open peaks) and analyzed by FACS gating on live (7-AAD exclusion) CD14-positive monocytes. To highlight MHC class II up-regulation, mean fluorescence intensities are included. C, LPS, poly I:C, and Pam3Cys were used at 0.5, 10, and 1 µg/ml, respectively, and cell culture supernatants were analyzed by ELISA for the presence of IL-6, IL-8, TNF, IL-12p40, and IL-12p70.

 

Figure 3
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  		FIGURE 3. Characterization of LPS-induced MICA-up-regulation. Monocytes were either isolated from PBMC by MACS columns (A and B) or by density gradient centrifugation (C) and cultured in the presence or absence of LPS. A, MICA, MICB, and MHC class I expression was determined after 24 h by FACS on resting and LPS-activated (500 ng/ml) monocytes and DC as well as C1R-MICA and C1R-mock transfectants. Open peaks and shaded peaks represent isotype control and the indicated specific Ab stainings, respectively. After treatment with the indicated concentrations of LPS, (B) cell culture supernatants were analyzed after 16 and 39h by ELISA for the presence of IL-6. C, Early apoptosis (Annexin V-positive/7-AAD-negative) and late apoptosis (Annexin V-positive/7-AAD-positive) among CD14-positive monocytes was estimated after 39 h by flow cytometry. Shown is one representative experiment each out of at least four.

 
NK cell recognition of monocyte-expressed MICA

To determine whether MICA expressed on activated monocytes was sufficient to be detected by NKG2D on NK cells, we took advantage of the fact that NKG2D is down-regulated on NK cells after interacting with its ligands (36). Monocytes were cultured for 24 h in the presence or absence of LPS and subsequently autologous NK cells were added and cultured for additional 24 h before the analysis of NKG2D surface expression (Fig. 4A). Presence of untreated monocytes caused a minor down-regulation of NKG2D expression which may be explained by a weak MICA expression on untreated monocytes due to in vitro culture. Presence of LPS-treated monocytes, in contrast, induced a marked down-regulation of NKG2D expression on cocultured NK cells, which did not occur when LPS-treated monocytes and NK cells were separated by a transwell insert. NKG2D expression was also largely restored when interaction with MICA was blocked by anti-MICA/B F(ab')2 using IgG1 F(ab')2 as control, which was almost as effective as transwell separation of monocytes and NK cells. Thus, in fact MICA expression and not soluble factors produced by the activated monocytes is largely responsible for NKG2D down-regulation, and accordingly MICA-expressing monocytes are capable of interacting with autologous NK cells via NKG2D.


Figure 4
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  		FIGURE 4. Monocyte-expressed MICA is detected, but does not stimulate lysis by autologous NK cells. Monocytes were isolated using MACS columns, cultured for 24 h in the presence or absence of LPS and subsequently freshly isolated autologous NK cells were added. A, Where indicated, LPS-treated monocytes and NK cells were separated by a transwell insert (TW), or experiments were performed in the presence of anti-MICA/B F(ab')2 or isotype control. After 24 h of coculture NK cells were double-stained with anti-CD56 and anti-NKG2D or isotype control and analyzed by flow cytometry. Numbers in dot plots represent NKG2D/CD56 positive cells. B, Cytotoxicity of NK cells against untreated and LPS-activated monocytes was evaluated in the absence (left panel) or presence (right panel) of 50U/ml IL-2 by chromium release assays using C1R-mock and C1R-MICA transfectants as controls. Triangles and circles represent untreated and LPS-treated MICA-expressing monocytes, respectively. Diamonds and squares represent C1R-mock and C1R-MICA transfectants, respectively. Data shown are means of triplicates with SDs. C, After 3 h of coculture with monocytes (M) in the absence (upper panels) or presence (lower panels) of 50 U/ml IL-2, NK cells were double-stained with anti-CD56 and anti-CD107a or isotype controls and analyzed by FACS. Cocultures with K562 cells served as positive controls. Numbers in dot plots represent CD107a/CD56 positive cells. One representative experiment each from a total of at least four is shown.

 
Because NK cells can lyse NKG2DL-expressing cells despite the expression of substantial levels of MHC class I, we next analyzed whether NK cells exerted cytotoxicity against activated autologous monocytes which display MICA expression. Monocytes were again cultured for 24 h in the presence or absence of LPS, and up-regulation of MICA expression on activated monocytes was confirmed by FACS. Subsequently, cytotoxicity of autologous NK cells against monocytes in the presence or absence of IL-2 was analyzed by chromium release assays. Neither untreated nor LPS-treated monocytes were substantially lysed by autologous NK cells under our experimental conditions (Fig. 4B). Efficacy of 51Cr-labeling of monocytes was validated by comparison of spontaneous and maximal release, and the spontaneous release was <20% in all experiments (data not shown). Furthermore, no late apoptosis was detectable in resting monocytes after 28 h, the time corresponding to the setting of the chromium release assays, which further excludes that results were largely affected by differences in survival and spontaneous chromium release of untreated vs LPS-stimulated monocytes (data not shown). It is noteworthy that C1R-mock transfectants were substantially lysed by the NK cells, and a significantly enhanced lysis was observed when C1R-MICA transfectants were used as target cells (Fig. 4B). This confirmed both the cytolytic potential of the NK cells and the stimulatory effect of MICA expression on NK cell reactivity. To confirm further that LPS-induced MICA expression does not sensitize autologous monocytes for NK cell lysis we determined CD107a expression on NK cells as a surrogate marker for granule mobilization (Fig. 4C). Neither in the presence nor in the absence of IL-2 an enhanced CD107a expression on NK cells was observed in cocultures with LPS-treated compared with untreated monocytes, while high levels of CD107a expression were observed in cocultures with K562 cells. Together, these data demonstrate that autologous NK cells do not display cytotoxicity against MICA-expressing monocytes, at least under our experimental conditions.

Modulation of NK cell reactivity by activated monocytes via NKG2D

Next, we wanted to analyze how MICA expression on monocytes influenced NK cell reactivity. As a first step, we cultured untreated and LPS-treated monocytes with autologous NK cells for 24 h in the presence or absence of IL-2 and analyzed cytotoxicity of NK cells against bystander K562 and Daudi cells. Although K562 are readily killed by both resting and activated NK cells, Daudi cells are largely resistant to resting NK cells and rather susceptible to lysis by activated NK cells. Accordingly, in the absence of IL-2, only weak killing of Daudi cells was detected while in the presence of IL-2 Daudi cells were lysed sufficiently. A higher percentage of lysis, which was not markedly affected by IL-2, was observed with K562 cells. Importantly, the presence of LPS-activated compared with resting monocytes did neither with K562 nor with Daudi cells cause relevant differences in target cell lysis (Fig. 5A).


Figure 5
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  		FIGURE 5. MICA expression on activated monocytes stimulates NK cell IFN-{gamma} production, but not cytotoxicity or proliferation. Monocytes were isolated using MACS columns and incubated for 24 h in the absence or presence of LPS. A, Subsequently MACS-isolated autologous NK cells were added and cocultured with untreated (M untreated) or LPS activated (M LPS) monocytes with or without IL-2 (50 U/ml) before chromium-release assays with the indicated target cells (NK cell-target ratio 10:1). B–D, Autologous NK cells and IL-2 were added to monocytes for additional 24 h in the presence or absence of NKG2D-Ig fusion protein (B), blocking anti-MICA/B F(ab')2 (C), or anti-MICA/B F(ab')2 together with anti-NKp80 F(ab')2 (D) and the respective isotype controls. Subsequently IFN-{gamma} in culture supernatants was determined by ELISA. E, NK cells were cultured with IL-2 in the presence or absence of immobilized anti-NKG2D mAb or isotype control. Where indicated, supernatants of untreated (S untreated) and LPS-activated (S LPS) monocytes (1:1), 10 µg/ml blocking anti-IL12 mAb or isotype control were added. After 24 h, IFN-{gamma} in culture supernatants was determined by ELISA. F, Autologous NK cells were cocultured with resting and activated monocytes (M) with or without IL-2 for 5 days followed by a 16 h pulse with thymidin. Afterwards, thymidin incorporation was determined. Untreated and LPS activated monocytes as well as NK cells cultured alone served as controls. Statistically significant differences (p < 0.05, Student’s t test) are indicated by *. One representative experiment each from a total of at least four is shown.

 
To study whether NK cell cytokine production was affected via NKG2D in cocultures with untreated vs LPS-treated monocytes, we determined IFN-{gamma} levels in culture supernatants by ELISA (Fig. 5, B–D). Incubation of NK cells alone with LPS did not induce IFN-{gamma} production (data not shown). Presence of untreated monocytes did not affect NK cell IFN-{gamma} release. In contrast, LPS-activated monocytes significantly stimulated IFN-{gamma} production in the presence, but not in the absence, of IL-2. Saturating concentrations of NKG2D-Ig fusionprotein (Fig. 5B) or blocking anti-MICA/B F(ab')2 (Fig. 5C) significantly (Student’s t test, p < 0.05) reduced IFN-{gamma} levels, while the respective isotype controls had no effect confirming that in fact MICA contributed substantially to NK cell cytokine production. The observation that IFN-{gamma} production in cocultures with activated monocytes in the presence of either blocking agent was still significantly (Student’s t test, p < 0.05) higher compared with cultures with resting monocytes indicates that, besides IL-2 and MICA, other factors contribute to the induction of NK cell cytokine production in our experimental setting.

Because AICL-NKp80 interaction stimulates monocyte-NK cell crosstalk (16), we determined whether this molecular system contributed to NK cell IFN-{gamma} production in our experimental setting (Fig. 5D). In fact, NK cell IFN-{gamma} production caused by activated monocytes was found to be dependent, in addition to MICA, on AICL-NKp80 interaction, since the addition of anti-NKp80 F(ab')2 significantly further (p < 0.05, Student’s t test) diminished the levels of IFN-{gamma} detectable in the presence of anti-MICA/B F(ab')2.

To determine whether and how soluble factors produced by activated monocytes contributed to NK cell IFN-{gamma} production in our experimental setting, we crosslinked NKG2D using an immobilized agonistic anti-NKG2D mAb, which, in the presence but not the absence of IL-2, substantially induced release of IFN-{gamma} by NK cells (Fig. 5E and data not shown). Although addition of LPS alone did not alter IFN-{gamma} levels (data not shown), supernatant of LPS-activated, but not of resting monocytes slightly but significantly (Student’s t test p < 0.05) increased the production of IFN-{gamma} by NK cells. Because the supernatants of our activated monocytes contained elevated levels of IL-12 we studied whether IFN-{gamma} levels were altered by the presence of a blocking anti-IL-12p40/p70 mAb. However, addition of the mAb did not reduce IFN-{gamma} production indicating that other soluble factors are responsible for the effect of the supernatant of activated monocytes.

Next, we studied whether proliferation of NK cells was affected by activated monocytes. Thus, monocytes were again cultured for 24 h in the presence or absence of LPS and subsequently their capacity to stimulate NK cell proliferation was determined. Although no proliferation was observed in the absence of IL-2, presence of IL-2 alone induced proliferation of NK cells, and this was further enhanced by the presence of monocytes in the cultures. However, this effect was not dependent on activation, since no difference in NK cell proliferation with resting compared with LPS-stimulated monocytes was observed (Fig. 5F). Together, our data demonstrate that TLR-induced expression of MICA provides monocytes with a mechanism to enhance NK cell cytokine production via NKG2D.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
NKG2DL potently stimulate functions of cytotoxic lymphocytes. Therefore it is the current conception that NKG2DL are usually not expressed on healthy tissue but rather are induced upon cellular stress like viral infection or genotoxic stress during malignant transformation (37, 38). Expression of one or more NKG2DL has also been reported in several types of APC, but the available data are at least partially conflicting (10, 11, 12, 29, 30, 33, 34). In this study, we comprehensively studied the induction of NKG2DL on monocytes upon TLR activation using various stimuli and different doses. We report that monocytes rapidly up-regulate expression of MICA upon activation by all investigated TLR stimuli. No additive effect of combining different TLR stimuli was observed at the level of MICA expression, whereas such an at least additive effect was observed regarding monocyte IL-6 secretion. Importantly, using a panel of various NKG2DL-specific mAb, we did neither observe substantial constitutive nor TLR-inducible expression of other NKG2DL besides MICA like the closely related MICB or ULBP molecules. The discrepant results on activation-induced NKG2DL expression in APC prompted us to characterize the activation of monocytes leading to MICA expression in greater detail. We demonstrate that monocytes upregulating MICA are fully activated and functional since they revealed enhanced expression of CD80, MHC class I and class II, increased secretion of the immunomodulatory cytokines TNF, IL-6, IL-8, and IL-12, and were protected from apoptosis. In addition, we excluded that MIC molecules were released as soluble forms. This was of importance since NKG2DL can proteolytically be cleaved from the cell surface leading to altered cell surface expression (31, 32).

Although monocyte-derived macrophages have recently been reported to express NKG2DL upon TLR stimulation (11), we believe that a detailed analysis of NKG2DL induction in monocytes is of major importance: Firstly, monocytes are components of the peripheral blood, and macrophages reside in several tissues. Secondly, monocytes represent a pluripotent lineage that can differentiate into macrophages, but also into DC. Thirdly, the differentiation process itself may influence the reactivity of the cells to different stimuli.

In macrophages, treatment with LPS up-regulates surface expression of MICA and ULBP1–3 resulting in lysis by autologous NK cells. It is noteworthy that in this study induction of NKG2DL expression only occurred on macrophages after stimulation with high LPS doses (200 ng/ml), and no other TLR stimuli were tested (11). With monocytes, we observed induction of MICA expression in a dose-dependent manner with a substantial effect observed with as low as 4 ng/ml LPS. Vankayalapati and coworkers (12) reported that specifically ULBP1, but not MICA/B, ULBP2, or ULBP3 was responsible for NK cell lysis of autologous alveolar macrophages and monocytes infected with Mycobacterium tuberculosis. The authors described that infection with this intracellular bacterium led, via TLR2, to up-regulation of ULBP1, but not of any other NKG2DL. It needs to be considered that no other TLR stimuli were tested in that study. In contrast, upon infection of DC with mycobacteria a pronounced up-regulation of MICA has been reported, but alterations of the further NKG2DL and the effect of other TLR stimuli were not determined in this study (33). Also in DC, Jinushi and coworkers (29, 30) analyzed the effect of various stimuli and reported that specifically IFN-{alpha} and IL-15, but not LPS, poly I:C, CD40L-stimulation, or cytokines like TNF, IL-12, or IL-18 induced MICA/B expression. Alterations in ULBP expression were not studied. With monocytes, we did not observe MICA induction following treatment with IFN-{alpha} or IL-15. Nowbakht and coworkers (34), again partially in contrast to the aforementioned results, described a selective up-regulation of ULBP1 on monocytes by myeloid growth factors while no up-regulation of any ULBP molecule was observed upon treatment with IL-1β, TNF, LPS, and IFN-{alpha}. In this study, MICA/B expression was not analyzed.

This discrepancy of the presently available results may be due to actual characteristic properties of the different types of APC. However, it seems very likely that the differences in previous reports among each other and to our findings may, at least in part, be explained by differing technical approaches and reagents applied. Firstly, various and differing stimuli have been used by several investigators. Secondly, different reagents and methods for NKG2DL detection have been applied. Thirdly, positive selection of monocytes with CD14 beads like in some reported experiments may per se influence NKG2DL expression and TLR signaling. Finally, differing culture conditions may have contributed to conflicting results. The latter seems even more likely since we observed an unspecific induction of MICA expression on monocytes upon in vitro culture even with "untouched" and nonstimulated monocytes. Our detailed characterization of monocyte activation in the course of MICA induction clearly suggests that cell surface expression of MICA, but not of other NKG2DL, is a feature of TLR-stimulated monocytes. In line, TLR stimulation of mouse macrophages induced expression of retinoic acid early inducible-1 proteins, but not of the other murine NKG2DL H-60 or UL16-binding protein-like transcript-1 (10). In addition, there is some evidence that expression of certain NKG2DL like MICB is more tightly controlled than that of others, e.g., MICA (23, 39). Thus, it may well be that NKG2DL expression on activated monocytes is confined to MICA.

Next, we set out to determine the functional role of MICA expression on activated monocytes. We demonstrate that MICA expression was sufficient to be detected by NKG2D as determined by NKG2D down-regulation on autologous NK cells. The possibility that NK cells and autologous monocytes may interact via NKG2D is of special interest in light of the multitude of studies reporting on the important role of the crosstalk between NK cells and DC (reviewed in Ref. 7, 40). NK cells have been shown to induce DC maturation, and immature DC can be lysed by NK cells, while mature DC are protected. By inducing DC activation, NK cells can indirectly promote T cell responses. In turn, activated DC have been shown to enhance NK-cell functions including production of IFN-{gamma} (7, 40). A variety of immunomodulatory cytokines, but also cell-cell contacts via various receptors have been reported to modulate this reciprocal interaction. In contrast, less attention has been given to communication between NK cells and other cells of the myeloid lineage. In both mice and humans, macrophages have been shown to interact with NK cells following activation, and various soluble factors and cell-bound ligands including 2B4, NKG2D, and IL-18 receptor have been shown to modulate this interaction (8, 10, 11, 12). Our finding that TLR-activated monocytes are capable to stimulate NK cells as an early source of IFN-{gamma} to release high levels of this potent immunomodulatory cytokine via MICA-NKG2D interaction suggests that the communication between these two cell types may be of direct physiological relevance, e.g., during infection. It should however be noted that the stimulatory effect of MICA-NKG2D interaction on NK cell cytokine production was only observed in the presence of IL-2, while triggering of NKG2D alone was not sufficient to induce NK cell IFN-{gamma} production. This is in agreement with the recently proposed concept that various receptors/stimuli need to cooperate to stimulate resting NK cells (41). In line, we also found that other soluble and cell bound factors contribute to NK cell cytokine production upon interaction with activated monocytes. Our findings extend the results of previous studies which reported that both humoral factors and cell contact enables monocytes to stimulate NK cells (12). Moreover, monocyte-NK cell interactions can lead to a reciprocal activation of both cell types, which is, at least in part, dependent on cell-cell contact, for example via CD40-CD40L interaction or by binding of AICL to NKp80 on NK cells, which we also found to be involved in the stimulation of NK cell IFN-{gamma} production by activated monocytes (9, 13, 16). The fact that NKG2D-Ig fusion protein caused a slightly more pronounced inhibitory effect than anti-MICA/B F(ab')2 in cocultures of TLR-activated monocytes and NK cells may indicate that ULBP4, RAET1G, or other yet unknown NKG2DL may contribute to stimulation of NK cell IFN-{gamma} production via NKG2D. In line, inhibition of NKG2D down-regulation on NK cells by anti-MICA/B F(ab')2 was slightly less efficient than transwell separation of NK cells and LPS activated monocytes, which may also be indicative for the involvement of another NKG2DL. In contrast to stimulation of IFN-{gamma} production, NK cell proliferation or cytotoxicity against classical target cells was not enhanced by activated, MICA-expressing monocytes. Although it may seem surprising that various functions of NK cells are differentially affected by MICA-NKG2D interaction, it needs to be considered that the different aspects of NK cell reactivity are mediated by tightly regulated and at least partially independent signaling pathways involving various kinases, phosphatases, and transcription factors. Although activation of ERK is crucial for NK cell mediated lytic functions (42), the production of IFN-{gamma} is influenced by diverse pathways leading to activation of transcription factors like NF-{kappa}B or members of the STAT family (43). It may thus well be that activated monocytes serve to stimulate cytokine production, while other effector functions of NK cells are not affected.

Importantly, we did not observe killing of MICA-expressing monocytes by autologous NK cells, while Nedvetzki and coworkers (11) reported that NK cells killed macrophages stimulated by high doses of LPS, and this cytolysis was triggered by NKG2D. Moreover, Steinle and coworkers (16), studying the role of NKp80 and AICL in the interaction of monocytes and NK cells, reported that LPS-treated monocytes were lysed by autologous NK cells. However, lysis rates were at a rather low level and observed only for two out of four investigated donors. Thus, the discrepancy between our results and the findings of the other groups may be due to inherent differences between monocytes and macrophages (11) and/or experimental conditions such as differences in isolation procedures, cultivation of monocytes or NK cells and donor variability (16). We further confirmed the results obtained in chromium release assays in our setting by analysis of CD107a-expression on NK cells as a marker for mobilization of cytolytic granules. Neither with resting nor with LPS-activated monocytes an induction of CD107a on NK cells was observed. The phenotype of activated monocytes compared with C1R transfectants and also activated DC observed under our experimental conditions may partly explain the lack of killing: Activated monocytes expressed moderate levels of MICA but high levels of MHC class I molecules. On the contrary, C1R transfectants, which were efficiently killed by NK cells, expressed higher levels of MIC molecules, but only moderate levels of MHC class I and a similar expression pattern was observed with activated DC. Together, our findings regarding the capacity of monocytes to stimulate NK cell IFN-{gamma} production by expression of MICA suggest that NKG2DL may have specialized functions at certain immune interfaces and may not always function to generate cytotoxic NK cell responses against the NKG2DL-expressing cells. It may well be that activated monocytes are protected from NK cell lysis due to sufficient expression of inhibitory molecules or MICA expression below the critical threshold, and thus recognition of activation-induced MICA by NK cells does not result in killing of the monocytes. In this context, it is noteworthy that cells of the bone marrow in both humans and mice express some but not all NKG2DL, and MICA is expressed in the trophoblast during normal pregnancy (17). It is unlikely that NKG2DL are expressed in these cells to trigger cytotoxic responses. Although certainly more work in this area is required, the emerging data suggest that NKG2DL diversity may have allowed for the evolution of individual ligands with functional specialities that are specific for different cell types and tissues (17). In response to pathogenic TLR stimuli, monocytes may act as "danger detectors" providing activating signals for NK cells which, by enhancing IFN-{gamma} production, contributes to both innate and adaptive immune responses. MICA-NKG2D interaction delineates a mechanism by which NK cells and monocytes can interact directly emphasizing the role of NK cell/monocyte crosstalk in the initiation and maintenance of immune responses during inflammation.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from Deutsche Krebshilfe (10-2004-Sa2), the German Research Foundation (SA 1360/2-2), and the IZKF-Nachwuchsgruppe program of the Medical Faculty at Eberhard Karls University Tuebingen (1466-0-0 and 1604-0-0). Back

2 Me.K. and P.D. contributed equally to this work. Back

3 Ma.K. and H.R.S. share senior authorship. Back

4 Address correspondence and reprint requests to Dr. Helmut R. Salih, Department of Hematology and Oncology, Eberhard Karls University, Tuebingen, Germany. E-mail address: Helmut.Salih{at}med.uni-tuebingen.de Back

5 Abbreviations used in this paper: DC, dendritic cell; AICL, activation-induced C-type lectin; NKG2DL, NKG2D ligand; MIC, MHC class I-related chain; 7-AAD, 7-amino-actinomycin D; TLRL, TLR ligand; ULBP, UL-16 binding protein family. Back

Received for publication January 7, 2008. Accepted for publication September 5, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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