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Originally published online as doi:10.1189/jlb.0306172 on September 22, 2006

Published online before print September 22, 2006
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(Journal of Leukocyte Biology. 2007;81:92-99.)
© 2007 by Society for Leukocyte Biology

The maturation potential of NK cell clones toward autologous dendritic cells correlates with HMGB1 secretion

Claudia Semino*, Jenny Ceccarelli*, Lavinia V. Lotti{dagger}, Maria R. Torrisi{dagger}, Giovanna Angelini* and Anna Rubartelli*,1

* Laboratory of Experimental Oncology E, Department of Translational Oncology, National Institute for Cancer Research, Genova, Italy; and
{dagger} Department of Experimental Medicine and Pathology, University of Rome "La Sapienza," Rome, Italy

1Correspondence: Laboratory of Experimental Oncology E, Department of Translational Oncology, National Cancer Research Institute, Largo R. Benzi, 10, 16132 Genova, Italy. E-mail: anna.rubartelli{at}istge.it

ABSTRACT

Interaction of NK cells with autologous immature dendritic cells (iDCs) results in reciprocal activation. We have previously reported that NK cells trigger iDC to polarize and secrete IL-18; in turn, DC-activated NK cells secrete the nuclear protein/proinflammatory cytokine high mobility group box protein 1 (HMGB1), which induces DC maturation and prevents DC from lysis. However, activated NK cells can also kill iDC. To investigate whether effector and maturative properties may coexist or segregate in different NK subsets, human NK cell clones were generated and analyzed for their effects on iDC. We found that the ability of different NK cell clones to induce iDC maturation is unlinked to their phenotypic and cytolytic features but correlates with the relocation of HMGB1 from nucleus to cytoplasm. "Maturative" NK cell clones secrete HMGB1 spontaneously. It is interesting that secretion is strongly enhanced by engagement of the surface molecule NKp30 but only slightly induced by triggering of the activating NK receptor CD16. However, culturing freshly isolated NK cells for 1 week with low doses of anti-CD16 triggers the relocation of HMGB1 from nucleus to cytoplasm and its spontaneous secretion, resulting in a stronger maturation potential of the NK cells. Together, our data indicate that NK cells comprise functionally different subsets, endowed with different capacities to secrete HMGB1 and to induce maturation of autologous iDC. Nonetheless, maturation properties can be modulated by different stimuli. This suggests that depending on the environmental stimuli, NK/iDC interaction can lead to different outcomes, thus influencing immune response.

Key Words: innate immunity • cytokine • cytotoxicity

INTRODUCTION

NK cells were defined originally by their ability to lyse tumor cells without prior activation [1 ]. In addition to this effector function, NK cells also display regulatory functions. Following engagement of surface receptors by other immune cells or signaling by soluble factors, NK cells release cytokines, which in turn influence the outcome of the interacting cells [2 ]. A number of studies have focused on the cross-talk between NK cells and autologous myeloid dendritic cells (DC) [3 4 5 ], which takes place after the recruitment of DC and NK cells into inflamed sites in response to tissue damage. DC activate NK cells; in turn, NK cells can kill immature DC (iDC) or induce their maturation. What determines the fate—death or maturation—of DC is still largely unclear. NK cells are able to kill iDC (which underexpress HLA-Class I molecules) but not mature DC, which after antigen uptake, up-regulate Class I expression [6 ]. Although activated polyclonal NK-cell populations display a strong cytolytic activity against iDC, this functional capability is confined to NK cells that lack inhibitory killer Ig-like receptors (KIRs) specific for self-HLA-Class I alleles and express the HLA-E-specific CD94-NKG2A inhibitory receptor [7 ]. Engagement of the activating receptor NKp30 on these NK cells by a so-far unknown ligand expressed by DC has been proposed to play a central role in the process of DC killing by NK cells [8 ]. It is interesting that the same receptor has been found essential in mediating NK-induced DC maturation also [9 ]. We have shown previously that NK cells trigger iDC to secrete IL-18 [10 ]. This cytokine, differently from other NK cell-activating factors, does not enhance the cytolytic activity of NK cells but induces a distinct "helper" pathway of their differentiation [11 ]. IL-18 lacks a secretory signal peptide and accumulates in the soluble cytosol of the producing cells [12 ]. A fraction of cytoplasmic IL-18, however, is found into secretory lysosomes [13 ], a specialized subset of lysosomes that undergo exocytosis induced by extracellular triggering signals [14 ]. Interaction of DC with NK cells is followed by a Ca2+-dependent and tubulin-mediated recruitment of IL-18-contaning secretory lysosomes toward the adhering NK cells [10 ]. Lysosome exocytosis and IL-18 secretion are thus restricted at the immunological synapse, allowing activation of the interacting NK cells without spreading of the cytokine. In turn, DC-activated NK cells secrete the proinflammatory cytokine high mobility group box protein 1 (HMGB1), which induces DC maturation and prevents DC from lysis [10 ]. HMGB1, in spite of its nuclear localization and function, is also a "leaderless" cytokine [15 ], which undergoes regulated secretion by inflammatory cells, including activated monocytes [16 ] and NK cells [10 ]. In these cells, activation results in HMGB1 hyperacetylation [10 , 17 ], which allows its relocation from the nucleus to cytoplasm, followed by lysosome-mediated, regulated secretion [16 ]. Secreted HMGB1 behaves as a powerful, proinflammatory cytokine [18 ] and a DC maturation-inducing factor [10 , 19 , 20 ].

Despite the identification of the two distinct "regulatory" versus "effector" functions of NK cells, it remains unclear whether these functions can be induced in all NK cells, possibly at different differentiation/activation stages, or they are restricted to different NK cell subsets. In this study, we generated human NK cell clones to investigate at a clonal level the coexistence or the segregation of the NK cell regulatory and effector functions.

MATERIALS AND METHODS

Generation of polyclonal or clonal NK cell populations
PBMC from healthy donors were isolated by Ficoll (Cedarlane Lab, Hornby, ON, Canada) density centrifugation and depleted of plastic-adherent cells. NK cells were enriched by incubating nonadherent cells 30 min at 4°C with anti-CD3 (UCHT1), anti-CD4 (Leu3a), and anti-CD8 (Leu2a) mAb (all from Becton Dickinson, Milan, Italy), followed by 30 min of incubation at 4°C with goat antimouse Ig-coated magnetic beads (Dynabeads, Dynal, Oslo, Norway) and immunomagnetic depletion as described [10 ]. CD3–CD4–CD8– cells were stimulated with 10 µg/ml PHA (Sigma-Aldrich, Milan, Italy), cultured in RPMI-1640 medium (Sigma-Aldrich), and supplemented with 2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin (Euroclone, Milan, Italy) and 10% heat-inactivated FCS (Sigma-Aldrich) in the presence of 500 IU/mL recombinant human (rhu)IL-2 (Proleukin, Chiron, Emeryville, CA) to obtain polyclonal NK cell populations. NK cell clones were obtained by limiting dilution from CD3–CD4–CD8– cells cultured under the same conditions as described [8 ]. The NK-cell populations were assessed for purity, and only those homogeneously displaying a CD3–CD56+ (BD PharMingen, Milan, Italy) phenotype of NK cells were selected and used. NK cell clones were also characterized for the presence of CD94 (BD PharMingen) or anti-KIR2D mAb (NKvSF1, kind gift of Dr. Alessandro Poggi, National Cancer Research Institute, Genova, Italy; ref. [21 ]), followed by the appropriated secondary reagent labeled with FITC (BD PharMingen) by flow cytometry.

Derivation of iDC from adhering cells
iDC were generated from PBMC as described previously [10 ] by culturing adhering cells for 7 days in RPMI-1640 medium (Sigma-Aldrich), supplemented with L-glutamine, penicillin, streptomycin, 10% heat-inactivated FCS, and 40 ng/ml rhuGM-CSF (Schering-Plough, Milan, Italy) plus 1000 IU/ml rhuIL-4 (Euroclone). The DC populations obtained at the end of the culture were analyzed by flow cytometry using the following mAb: PE-conjugated anti-CD14, FITC-conjugated anti-HLA-DR (Beckman-Coulter, Milan, Italy), PE-conjugated anti-CD86, PE-conjugated anti-CD1{alpha}, FITC-conjugated anti-CD80 and anti-CD83 (Immunotech-Coulter, Marseille, France), and FITC-conjugated anti-CD25 (ImmunoTools GmBH, Friesoythe, Germany).

NK/iDC culture conditions
iDC, prepared as described, were incubated alone or with polyclonal NK cells or NK cell clones at different NK/iDC ratios for different periods of time in RPMI medium, supplemented with 1% Nutridoma-HU (Roche, Milan, Italy) as described [10 ]. When indicated, LPS (1 µg/ml, Sigma-Aldrich) or rHMGB1 (kind gift from Dr. Marco Bianchi, San Rafaele Scientific Institute, Milan, Italy; used 1 µg/mL) was added during the last 24 h of culture to induce DC maturation. HMGB1 preparations did not contain LPS activity sufficient to interfere in the in vitro assays [10 , 20 ]. In some experiments, spent medium deriving from 24 h of incubation of polyclonal NK cells or NK cell clones was added to iDC cultures for the indicated time. At the end of the incubations, iDC were tested by flow cytometry for the expression of the maturation marker CD86 (anti-CD86 PE-conjugated, Immunotech-Coulter).

Cytolytic assay and redirected killing assay
NK cell cytolytic activity against iDC was tested in a 51Cr-release assay as described previously [10 ]. Briefly, iDC were loaded with 51Cr and cocultured for 4 h with polyclonal NK cells or NK clones used as effector cells at different E:T ratios. To assess the presence of activating or inhibiting surface molecules on the surface of NK cell clones, they were tested in a classical, redirected killing assay [9 ]. Briefly, the 51Cr-loaded P815 cell line, previously incubated for 30 min with mAb against anti-CD94 or NKvSF1 at 1 µg/ml, was coincubated for 4 h with the different NK cell clones at a 10:1 E:T ratio. Results are expressed as percentage of cytotoxicity as described [10 ].

Measurement of TNF-{alpha} and IL-12 production
Cell-free supernatants derived from 5 x 105 cell iDC, untreated or treated with LPS or HMGB1 or cocultured with NK cells, were collected after 24 h of incubation. IL-12 and TNF-{alpha} concentrations in culture supernatants were measured by ELISA, using the huIL-12 (IL-12p70; Biotrak ELISA system kit, Amersham Biosciences, Milan, Italy) and the huTNF-{alpha} (TNF-{alpha} Duo set, R & D Systems, Minneapolis, MN), following the manufacturer’s instructions. Results were expressed as pg/ml.

Secretion of HMGB1 by polyclonal NK cells or NK cell clones
Polyclonal NK cells or NK cell clones were tested for the ability to secrete HMGB1, spontaneous or triggered by mAb, interacting with relevant surface molecules. For this purpose, mAb (anti-NKp30, Alexis, Vinci, Italy; anti-CD56 or anti-CD16, BD PharMingen; 5 µg/mL) were precoated in plastic plates for one night at 4°C. NK cells were added to the plates for the time indicated, and at the end of the incubation, cells and supernatants were processed by Western blotting. To test the involvement of cytosolic phospholipase A2 (cPLA2), the cPLA2 inhibitor pyrrolidine-2 (analogous to pyrrolidine-1; ref. [22 ] and kind gift of Michael H. Gelb, University of Washington, Seattle) was added at 20 µM during the last 6 h of incubation.

Activation of NK cells by cytokines or mAb
Purified, resting NK cells were cultured 7 days with suboptimal doses (50 IU/mL) of rhuIL-2, with rhuIL-2 (50 IU/mL) plus rhuIL-18 (1 ng/ml, R & D Systems), with rhuIL-2 (50 IU/mL) plus low doses of anti-CD16 mAb (1 ng/ml), or with PHA (10 µg/ml, Sigma-Aldrich). At the end of the culture period, NK cells were washed and cultured for an additional 6 h in RPMI-1% Nutridoma-HU (Sigma-Aldrich). Supernatants and cells were then collected for Western blot analyses.

Isolation and purification of exosomes
NK cells or DC were washed by centrifugation and recultured in RPMI medium supplemented with 1% Nutridoma-HU (Sigma-Aldrich) for 24 h. Cell culture media (35 ml) containing 30 x 106 cells were centrifuged for 10 min at 300 g to remove the cells. After a second centrifugation at 300 g, the medium was centrifuged for 10 min at 1200 g (2x), 30 min at 10,000 g, 60 min at 70,000 g, and 60 min at 100,000 g, sequentially, using a rotor (SW27, Beckman Instruments, Inc., Fullerton, CA) as described [23 ]. The pellets were solubilized in nonreducing SDS sample buffer, and the soluble supernatants were prepared for Western blotting analyses.

Western blot analysis
At the end of the various incubation times, supernatants were collected, centrifuged at 13,000 g for 5 min, and concentrated by 10% trichloroacetic acid precipitation [10 ]. Cells were lysed in 1% Triton X-100 lysis buffer as described [16 ]. Aliquots of cell lysates and the correspondent trichloroacetic acid-concentrated supernatants and exosomes were boiled in reducing Laemmli sample buffer, resolved on 12% SDS-PAGE, and electrotransferred onto a polyvinylidene difluoride membrane (Amersham Biosciences) as described [12 , 13 ]. Filters were hybridized with rabbit anti-huHMGB1 antibody (BD PharMingen) or with antiactin mAb (Clone AC74, Sigma-Aldrich) as a loading control, followed by the appropriated HRP-conjugated secondary reagent (Dako, Milan, Italy). Mouse mAb to lysosome-associated membrane protein-2 (Lamp-2; H4B4) and CD63 (H5C69) were obtained from Developmental Studies Hybridoma Bank, University of Iowa (Iowa City) and used to characterize exosomes [23 ]. Western blots were developed by ECL Plus (Amersham Biosciences), according to the manufacturer’s instructions.

Immunofluorescence
NK cells were fixed and permeabilized with 3% paraformaldehyde and 0.5% Triton X-100. After washings and saturation with 2% BSA (Sigma-Aldrich), cells were labeled with rabbit anti-HMGB1 antibody (BD PharMingen) followed by incubation with the appropriate secondary reagents labeled with cyanine 3 (Jackson ImmunoResearch, Soham, UK) as described [10 , 16 ] and with anti-{alpha}-tubulin. Finally, cells were examined under an Olympus AX70 microscope (Milan, Italy) by using a x100/1.30 oil objective lens [10 , 16 ]. Images were acquired with a Hamamatsu digital camera (Milan, Italy).

Immunoelectron microscopy
The NK cell population was processed for immunoelectron microscopy as described previously [16 ]. As fixation in 1.0% glutaraldehyde resulted in loss of immunoreactivity to anti-HMGB1 antibody, cells were fixed with 2% paraformaldehyde in 0.1 M phosphate buffer for 2 h at 25°C, washed, and embedded in 10% gelatin (Sigma-Aldrich) in 0.1 M phosphate buffer, which was solidified on ice. Gelatin blocks were infused with 2.3 M sucrose overnight at 4°C, frozen in liquid nitrogen, and cryosectioned using an Ultracut EM FC6 (Leica, UK). Ultrathin cryosections were collected with sucrose and methyl cellulose and incubated with rabbit anti-HMGB1 antibody followed by 10 nm diameter protein A-colloidal gold conjugates (British BioCell International, Cardiff, UK). Control experiments were performed by omission of the primary antibody from the immunolabeling procedure. After labeling, all ultrathin cryosections were fixed in 1% glutaraldehyde and finally stained with a solution of 2% methyl cellulose and 0.4% uranyl acetate.

RESULTS

NK cell clones display different cytolytic and maturative properties
NK clones were generated by limiting dilution from NK cells isolated from peripheral blood of three healthy donors and analyzed for their phenotypic and cytolytic characteristics. All NK clones analyzed (n=50) were CD3–, CD94+, CD56+ (90% bright, 10% dim), whereas 50% were NKvSF1+. Using a classical, redirected killing assay [9 ], we found that CD94 was expressed in the activatory form by 25% and in the inhibitory form by 75% of the clones. Among NKvSF1+ clones, the ratio was inverted (25% expressed the inhibitory form and 75% the activatory form of the receptor). Five representative clones are shown in Table 1 . The cytolytic capacity of the different clones was also assayed toward autologous iDC. The results indicate that 30% of the clones were not or were weakly cytotoxic (<15% of lysis at a 10:1 E:T ratio), whereas 70% of the clones, including Clones 2/1, 33/1, 37/2, and 13/3, displayed a stronger cytolytic activity, resulting in 25–37% lysis of iDC at a 10:1 E:T ratio. The ability of these cytotoxic clones to induce autologous iDC maturation was then investigated. Twenty-four hours of coculture of NK cell Clones 37/2 and 13/3 (Fig. 1A ) with autologous iDC at a NK:DC ratio of 5:1 resulted in an increase of CD86 expression; a CD86 increase, although less pronounced, was also observed after incubation of iDC with supernatants from 24 h of culture of the same NK clones. It is different that other clones, including Clones 2/1 and 33/1, which also displayed cytotoxic activity, were not able to induce DC maturation, neither when tested in coculture nor when iDC were incubated with their conditioned medium (Fig. 1A) . Different maturative potential was also observed among noncytolytic clones (not shown). In keeping with previous observations [3 , 10 ], polyclonal NK cell populations, obtained by culturing with IL-2 NK cells purified from peripheral blood of the same donors, displayed cytolytic (35% of lysis at a 10:1 E:T ratio) and maturative properties (Fig. 1A) with respect to autologous iDC. As a positive control, LPS strongly up-regulated CD86 expression (Fig. 1A) . These data indicate that the capacity of the different clones to induce maturation of autologous iDC is independent of their cytolytic potential. The increase in CD86 expression induced by "maturative" clones was consistent but not impressive. We then analyzed the expression of other maturation markers expressed by DC following coculture with the NK cell clones, as well as the production of IL-12 and TNF-{alpha}. Figure 1B shows the results obtained with the cytotoxic Clone 39/1, derived from a different donor. All the DC maturation markers analyzed (CD86, CD83, CD80, CD25, and HLA-Class II) were up-regulated significantly after 24 h of coculture with the NK cell clone. In addition, secretion of IL-12 and TNF-{alpha} was detected in the supernatants of the cocultures but not of iDC cultured alone (Fig. 1C) . Although IL-12 is a DC-specific cytokine, expressed on DC maturation [24 ], TNF-{alpha} can be produced by DC [19 , 20 ] and NK cells [5 ]. In keeping with previous reports indicating that HMGB1 is a DC maturation factor [10 , 19 , 20 ], exposure of DC to rHMGB1 resulted in increased surface expression of the different maturation markers analyzed [10 , 19 , 20 ]. It is more important that HMGB1 induced DC to secrete IL-12 and TNF-{alpha}, indicating that the TNF-{alpha} found in supernatants of NK/DC cocultures may derive at least in part from DC.


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  		Table 1. Phenotypic and Cytolytic Properties of Selected NK Cell Clones

 

Figure 1
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  		Figure 1. Maturation properties of polyclonal NK cells and NK cell clones. (A) The expression of CD86 by autologous iDC was analyzed by flow cytometry after 24 h of culture without (–) or with NK cells (at a NK:DC ratio of 5:1, solid bars) or NK cell supernatants (sups; from 24 h of culture, open bars) from the indicated NK cell clones (cl 2/1, cl 33/1, cl 37/2, cl 13/3) or from activated polyclonal NK cells (bulk). The effect of LPS (1 µg/ml) on CD86 expression is shown as a control. Data are expressed as mean fluorescence intensity (MFI). (B) The expression of maturation markers (CD86, CD83, CD80, CD25, and HLA-Class II) by autologous DC incubated for 24 h with LPS (1 µg/ml), HMGB1 (1 µg/ml), or with the NK cells from Clone 39/1 was analyzed by flow cytometry. Data are expressed as MFI. (C) TNF-{alpha} (open bars) or IL-12 (solid bars) secretion by DC cultured 24 h alone (–) or with NK cells from Clone 39/1, with HMGB1 (1 µg/ml), or with LPS (1 µg/ml) as indicated. Data obtained by ELISA are expressed by pg/ml of the secreted cytokine.

 
Maturative but not nonmaturative NK cell clones displays cytoplasmic localization and secretion of HMGB1
We then compared the spontaneous secretion of HMGB1 by NK cell clones displaying a different ability to induce iDC maturation and by their polyclonal parental NK cell line. As shown in Figure 2 , NK cells from Clone 13/3 secrete HMGB1, as indicated by the presence of a 30 KD band on supernatants from 12 h culture (Fig. 2A , upper panel, lane 2). On the contrary, HMGB1 was absent from supernatants of NK cells from Clone 2/1 (Fig. 2A , upper panel, lane 4). The parental NK cell line also displayed HMGB1 secretion (Fig. 2A , upper panel, lane 6). In keeping with the different secretory capacity of the two clones, the amount of HMGB1 in Triton X-100 cell lysates was consistently higher in HMGB1-secreting (lane 1) than in nonsecreting clones (lane 3), in spite of equal amounts of loaded proteins, confirmed by hybridization of the same blot with anti-actin antibody (lower panel, lanes 1 and 3). The cytoplasmic HMGB1 band detected in the nonsecreting Clone 2/1 is probably a result of, in part, the nuclear-cytoplasmic shuttle characteristic of HMGB1 [17 ] and in part, the leakage from the nucleus occurring during the lysis procedure. Indeed, when mechanic separation of nucleus and cytoplasmic fraction was carried out [10 ], the HMGB1 present in cell lysates of nonsecreting NK cells was consistently lower (ref. [10 ] and not shown). However, as a result of the large number of cells required for mechanic subcellular fractionation [10 , 13 , 16 , 25 ] and to the low number of NK cells obtained from the clones, this procedure could be performed only in pilot experiments. Support to this interpretation came from immunofluorescence analysis, which revealed the presence of abundant cytoplasmic HMGB1 in Clone 13/3 but not in Clone 2/1, where HMGB1 is restricted to the nucleus (Fig. 2B) . Similar results were obtained with several different maturative and nonmaturative NK cell clones, which consistently displayed cytoplasmic and secreted HMGB1 or only nuclear HMGB1, respectively (data not shown). To further explore the localization of HMGB1 within maturative NK cells, immunoelectron microscopy analyses were performed. As shown in Figure 2C , cytoplasmic HMGB1 was found associated in part with membranous structures displaying features of multivesicular bodies. Cytoplasmic proteins associated with multivesicular bodies have been reported to be secreted in exosomes [23 ]. To investigate whether HMGB1 is also released through this pathway, exosomes were isolated from 24 h culture fluid of a polyclonal NK cell culture, and the presence of HMGB1 in the exosomal fraction and in the soluble supernatants was compared. As a control, the same fractions were isolated from supernatants of DC, which are known to generate abundant exosomes [23 ], found in NK and iDC supernatants (Fig. 2D) , as indicated by the presence of exosomal markers such as the lysosomal protein Lamp-2 and the tetraspanin CD63 [23 ]. Little if any HMGB1 was found in exosomes from NK cells (Fig. 2D) , whereas HMGB1 was abundantly detected in the soluble supernatants (Fig. 2E) . It is different that HMGB1 was absent in the exosomal fraction and the supernatants from DC cultures (Fig. 2D and 2E) .


Figure 2
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  		Figure 2. Localization and secretion of HMGB1 in polyclonal NK cells or NK cell clones. (A, Upper panel) Western blot analysis of HMGB1 in cell lysates (CL; lanes 1, 3, and 5) and supernatants (sup; lanes 2, 4, and 6) from 12 h of cultures of the polyclonal, parental NK cells (lanes 5 and 6) or of the NK cell Clones 2/1 (lanes 3 and 4) and 13/3 (lanes 1 and 2). (Lower panel) The same blots were hybridized with anti-actin as a loading control. (B) Immunofluorescence analysis of the NK cell Clones 13/3 and 2/1. Cells were stained for HMGB1 (red) and tubulin (green). Note that in the maturative Clone 13/3, HMGB1 is present in the nucleus and the cytoplasm, where it colocalizes in part with the cytoplasmic marker tubulin. It is different that in the nonmaturative Clone 2/1, HMGB1 is abundant in the nucleus and absent in the cytoplasm, where tubulin is highly stained. (C) Immunoelectron microscopy analysis. Activated polyclonal NK cells were stained with rabbit anti-HMGB1 antibody followed by 10 nm goat antirabbit IgG gold-conjugates. (Lower panel) Two adjacent NK cells are shown. Note the presence of gold particles in the nucleus of one NK cell (arrows) and in structures enriched with internal membrane (multivesicular bodies) in the other. (Upper panel) A structure with multivesicular body features, heavily labeled by gold particles, is shown at higher magnification (arrow). PM, Plasma membrane; N, nucleus; M, mitochondria. (D) Western blot analysis of exosomes purified from 24 h of culture of 30 x 106 iDC (left panel) or polyclonal-activated NK cells (right panel). Blots were hybridized with HMGB1, Lamp-2, or CD63 as indicated. (E) Western blot analysis of HMGB1 in soluble supernatant from 24 h of culture of 30 x 106 iDC or polyclonal-activated NK cells.

 
Secretion of HMGB1 by maturative NK clones is enhanced upon engagement of the surface molecule NKp30
Several evidences indicate that DC maturation induced by NK cells requires a direct cell-to-cell contact, implying that a receptor-ligand interaction is crucial for this event [26 ]. To investigate which surface molecules are involved, we studied NKp30, which has been found implicated in the NK-DC cross-talk [8 , 9 ]. As HMGB1 is contained into structures resembling multivesicular bodies, we also studied the effect on secretion of anti-CD16 antibody, which is known to induce NK cell degranulation [27 , 28 ]. Activated NK cells were incubated 4 h (a time too short to allow detection of spontaneous HMGB1 secretion) on anti-NKp30 or anti-CD16 mAb-coated plates, and supernatants were analyzed for the presence of HMGB1. As shown in Figure 3A , supernatants derived from NK cells incubated with anti-NKp30 mAb contained abundant HMGB1 (Fig. 3A , upper panel, lane 1), whereas supernatants from NK cells incubated with anti-CD16 mAb displayed little or no HMGB1 (Fig. 3A , upper panel, lane 2). In keeping with these data, when maturative NK cell clones were tested in the same assay (Fig. 3B , shows Clones 13/3 and 37/2), secretion of HMGB1 was strongly increased by anti-NKp30 antibody (Fig. 3B , lanes 1 and 2) but not or slightly by anti-CD16 mAb (Fig. 3B , lanes 3 and 4). This effect was specific, as the incubation with an isotype-matched control mAb (anti-CD56) failed to induce HMGB1 secretion (not shown). It is different that nonmaturative NK cell clones failed to secrete HMGB1 after engagement of NKp30 (not shown). Treatment of NK cells with anti-CD16 antibody for 5 min elicited a sustained rise in intracellular-free Ca2+ concentration (not shown and ref. [29 ]), indicating that CD16 triggering is not a good inducer of HMGB1 secretion in spite of its ability to generate different physiologic responses in NK cells. The release of the cytosolic enzyme lactate dehydrogenase (LDH), used as a marker of cell lysis, ranged in the different experiments from 2% to 5%. As the percent of HMGB1 release induced by NKp30 triggering varied from 20% to 50%, this ruled out a role for cell death in the antibody-triggered release of HMGB1 by NK cells.


Figure 3
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  		Figure 3. Induction of HMGB1 secretion by engagement of surface receptors. (A) Western blot analysis of HMGB1 in supernatants (upper panel, sup) and cell lysates (lower panel, cell lys) of polyclonal-activated NK cells cultured 4 h on plates precoated with anti-NKp30 (lane 1) or anti-CD16 (lane 2) mAb. (B) Western blot analysis of HMGB1 in supernatants (upper panel) and cell lysates (lower panel) of the maturative NK cell Clones 13/3 (lanes 1 and 3) and 37/2 (lanes 2 and 4) cultured 4 h on plates precoated with mAb anti-NKp30 (lanes 1 and 2) or anti-CD16 (lanes 3 and 4). (C) Western blot analysis of HMGB1 in supernatants (upper panel) and cell lysates (lower panel) of polyclonal NK cells incubated 4 h on plates precoated with anti-NKp30 alone (–, lane 1) or with the cPLA2 inhibitor pyrrolidine-2 (20 µm, Pyrrol, lane 2). (D) Western blot analysis of HMGB1 in supernatants (upper panel) and cell lysates (lower panel) from the last 6 h of culture of resting polyclonal NK cells cultured 1 week with a suboptimal dose of rhuIL-2 (50 IU/ml, lane 1), suboptimal dose of rhuIL-2 plus 1 ng/ml anti-CD16 (lane 2), PHA (10 µg/ml, lane 3), or 50 IU/ml rhuIL-2 plus rhuIL-18 (1 ng/ml, lane 4). The blots shown in A–D were hybridized with anti-actin as a loading control. Results are shown in the lower panels of cell lysates.

 
Secretion of HMGB1 by NK cells is prevented by inhibition of cPLA2
cPLA2 has been found involved in secretory lysosome-mediated release of IL-1ß by monocytes [25 ]. As HMGB1, like IL-1ß, is associated in part with a vesicular compartment, we investigated whether cPLA2 inhibition has any effect on HMGB1 secretion by NK cells, which were exposed to NKp30 in the absence or presence of the cPLA2 inhibitor pyrrolidine-2 [22 ], and the secretion of HMGB1 was evaluated. As shown in Figure 3C , NKp30-induced HMGB1 secretion was strongly impaired by 20 µM pyrrolidine-2 in the absence of any sign of cell toxicity, confirmed by the equal amount of HMGB1 present in the cell lysates of untreated or pyrrolidine-2-treated NK cells. A dose-response assay revealed that 5 µM pyrrolidine-2 was able to inhibit the release of HMGB1 by 50%.

Differentiation of resting NK cells toward HMGB1-secreting phenotype: role of CD16
Resting NK cells neither express cytoplasmic HMGB1 nor secrete this cytokine [10 ]. Triggering of CD16 not only mediates NK cell degranulation [27 , 28 ] but also induces expression of activation markers such as IL-2 and CD25 [30 ] and influences the in vitro proliferation of NK cell subsets [31 ]. We then investigated whether low doses of anti-CD16 mAb, unable to induce degranulation, have any effect on HMGB1 relocation and secretion. Our results show that 1 week of culture of resting NK cells with 1 ng/ml anti-CD16 in the presence of a suboptimal doses of IL-2 induced secretion of HMGB1 more efficiently than other NK cell stimuli able to drive HMGB1 release, such as the polyclonal activator PHA or the combination of the two cytokines IL-2 plus IL-18 (Fig. 3D) . The number and vitality of NK cells (assessed by trypan blue exclusion) after 1 week of culture were similar in the different culture conditions analyzed. Again, the percent of LDH released was far less than the percent of HMGB1 secreted in the different culture conditions, excluding that HMGB1 release in NK cell supernatants was the result of a lytic death (not shown).

DISCUSSION

In this study, we have explored at the clonal level the coexistence of cytolytic and maturative function in NK cells as well as their regulation by the microenvironment. Our data show that the capacity of the different clones to induce maturation of autologous iDC is independent of their cytolytic potential but correlates with the cellular localization of the nuclear protein HMGB1. Indeed, HMGB1 is largely cytoplasmic and abundantly secreted by those NK cell clones able to induce DC maturation. HMGB1 is a proinflammatory cytokine [15 , 18 ] with DC maturation properties [10 , 19 , 20 ]. Exposure to HMGB1 prevents DC from killing by NK cells [10 ], in keeping with the resistance to NK cell-mediated lysis displayed by mature DC. Thus, our findings strongly suggest that HMGB1, secreted by NK cells, plays a major role on NK cell-induced DC maturation and that the high or low availability of this cytokine in the DC/NK microenvironment may switch the fate of DC toward death or maturation.

Several evidences indicate that DC maturation induced by NK cells requires or at least is increased strongly by a direct cell-to-cell contact. This implies that receptor-ligand interactions are crucial for this event [26 ]. Recent data [9 ] reported that triggering of NKp30 on NK cells drives secretion of TNF-{alpha}, which in turn, promotes DC maturation. Our present findings indicate that engagement of NKp30 also induces secretion of HMGB1 by NK cells. Moreover, we show that TNF-{alpha} secretion by DC is triggered by rHMGB1, in agreement with previous studies [19 , 20 ]. It is now accepted that TNF-{alpha} may form a proinflammatory loop with HMGB1, as they can induce the release of each other [32 33 34 ]. Indeed, on the one hand, HMGB1 secretion is induced by TNF-{alpha}, and conversely, rHMGB1 up-regulates TNF-{alpha} expression dose-dependently in many cell types [34 ].

On these bases, we propose that engagement of NKp30 stimulates HMGB1 secretion, which in turn, triggers TNF-{alpha} production by NK or by NK cells and DC when cocultured; the two cytokines then sustain the production of each other. Support for this interpretation comes from a preliminary observation that clones that secrete HMGB1 in the absence of stimulation but not nonsecreting clones also release low amounts of TNF-{alpha} spontaneously (unpublished results). This reciprocal stimulation of HMGB1 and TNF-{alpha} production, which can be relatively long-lasting, may also explain why the maturation effects on autologous iDC are much stronger when iDC are cultured in contact with maturative NK cell clones than when exposed to the supernatants from the same NK clones.

Nonmaturative clones, expressing most HMGB1 in the nucleus, fail to secrete HMGB1 spontaneously (Fig. 2) or in response to NKp30 triggering (not shown). This finding is not unexpected, as the relocation of HMGB1 from the nucleus to cytoplasm, necessary for secretion, has been found to require at least 8 h of activation in monocytes, which similarly to NK cells, undergo regulated secretion of HMGB1 [16 ]. It is possible that longer NKp30 triggering may switch a nonmaturative clone to a maturative one, but this long stimulation in vitro results in NK cell death. Rather, NKp30 triggering induces secretion of HMGB1 already stored into the cytoplasm of maturative NK cell clones. This observation can clarify the apparently discrepant findings that NKp30 is implicated in NK-mediated iDC maturation [9 ] and killing [8 ]. Indeed, one can speculate that DC triggering of NKp30 on the NK cell subsets expressing high amounts of cytoplasmic HMGB1 would result in secretion of HMGB1 and TNF-{alpha} and hence, in DC maturation, and it would lead to DC lysis in the case of the NK cell subsets devoid of cytoplasmic HMGB1. However, many of the maturative NK cell clones are also cytotoxic against autologous DC, suggesting that the pattern of receptors expressed by NK cells and the synergy among them are crucial in determining activation of cytotoxicity or cytokine secretion, as suggested [35 ].

Immunoelectron microscopy analysis of the subcellular localization of HMGB1 in NK cells endowed with maturation properties revealed that HMGB1 is associated in part with structures displaying some features of multivesicular bodies. This observation raised the hypothesis that HMGB1 is secreted in exosomes. These are small vesicles that accumulate in multivesicular bodies and are released following fusion of the external membrane of these structures with the plasma membrane [23 ]. However, our results show that HMGB1 is abundantly present in the soluble supernatants of HMGB1-secreting NK cells, after 24 h of culture, but it is virtually absent in the exosomal fraction. This suggests that rather than multivesicular bodies, the membranous structures containing HMGB1 are secretory lysosomes that release soluble HMGB1 following exocytosis, such as the endolysosomal vesicles that mediate secretion of HMGB1 in activated monocytes [16 ]. Subsets of secretory lysosomes also mediate secretion of IL-18 by DC [13 ] and of IL-1ß by monocytes [36 ]. The latter requires active cPLA [25 ]. Similarly, the mechanism underlying HMGB1 externalization in NK cells is likely to involve cPLA2 activation, as specific inhibitors of this enzyme, such as pyrrolidine-2, a new compound analogous to pyrrolidine-1 [22 ], prevent HMGB1 release by NK cells.

In contrast to NKp30 engagement, cross-linking of CD16, which induces granule exocytosis in NK cells [27 , 28 ], fails to trigger abundant HMGB1 secretion. It is conceivable that the two receptors activate different signaling pathways leading to exocytosis of different granules [28 ]. In support of this interpretation, although cPLA2 is activated rapidly upon CD16 cross-linking, active cPLA2 is not required for CD16-triggered granule exocytosis in NK cells [27 ].

Although a short trigger (4 h) of CD16 has little effect on HMGB1 secretion, culturing resting NK cells with anti-CD16 mAb at low doses, unable to induce degranulation, results in differentiation of NK cells toward a maturative phenotype, with abundant secretion of HMGB1. It is remarkable that long culture with low doses of anti-CD16 mAb activates NK cells to secrete HMGB1 more efficiently than other stimuli, including the polyclonal activator PHA or IL-2 plus IL-18 [10 ]. Although a major effect of CD16 ligation is activation of NK cell cytotoxicity [27 , 28 ], CD16 engagement has also been reported to drive the expression of activation markers such as IL-2 and CD25 [30 ] and to induce NK cell apoptosis or proliferation, depending on the presence of costimulatory cells [31 ]. In this context, our data suggest that not only the presence of costimulatory signals but also the strength of CD16 triggering (low vs. high amount of anti-CD16 mAb) may lead to different functional responses of NK cells. In the experimental system used in this study, prolonged stimulation with low doses of ant-CD16 leads to differentiation toward a maturative phenotype. In vivo, it is possible that the availability of CD16 ligands in the microenvironment, depending on the amount, may direct NK cells to degranulation or to differentiation toward a phenotype with DC maturation activity.

In conclusion, our data indicate that NK cells comprise functionally distinct subsets, displaying different maturative capacities. However, maturation properties can be modulated by external stimuli. This suggests that depending on the signals provided by the environment, NK/iDC interaction can lead to generation of cytotoxic or maturative NK cells, with opposite outcome on the interacting DCs. Moreover, NK cells can also differentiate to cells endowed with maturative and cytolytic properties toward DC; in this case, DC maturation or death can depend on the relative amount of released HMGB1, which inducing maturation, prevents DC from NK cell-mediated lysis.

ACKNOWLEDGEMENTS

This work was supported in part by grants from Associazione Italiana per la Ricerca sul Cancro, Ministero della Salute (Ricerca Finalizzata Oncologia, programma oncologico Italia-USA), and CIPE (02/07/2004, CBA project). We thank Dr. M. H. Gelb (University of Washington, Seattle) for the kind gift of pyrrolidine-2, Dr. A. Poggi (National Cancer Research Institute, Genova, Italy) for the gift of NKvFS1 mAb and for helpful discussion, and the Blood Centers of Galliera and San Martino Hospitals (Genova, Italy) for the kind gift of buffy coats. The mAb Lamp-2 and CD63, developed by J. Thomas August and James E. K. Hildreth, were obtained from the Developmental Studies Hybridoma Bank, developed under the auspices of the National Institute of Child Health and Human Development and maintained by the University of Iowa, Department of Biological Sciences (Iowa City).

Received March 5, 2006; revised July 14, 2006; accepted August 6, 2006.

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