passages: PMC-2193592-sec-11__text
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passages: Stimulation/Inhibition of iDC Functions by Activated NK Cells.
  Two previous studies have shown that both resting and activated NK cells partially kill autologous DCs, as measured by 3-4-h chromium release assays using high NK/DC ratios (11, 12). However, in our long-term cultures (24-48 h) the NK cells stimulated the iDCs rather than lysing them. Based on these conflicting results, we examined in more detail the apparent stimulation of iDCs after contact with NK cells. We reasoned that activated NK cells would be more useful for these experiments because (i) an encounter in vivo between NK cells that normally traffic in the blood (4) and iDCs which reside in the tissues (1, 3), should only occur when both cells are activated; (ii) activated NK cells mediate more potent lysis of iDCs (11-13), thereby allowing for a more rigorous examination of a potential cytotoxic effect; and (iii) the number of NK cells obtained after short-term in vitro culture would be far greater providing more cells for analysis (9).
  The DC maturation induced by resting NK cells we observed was dependent on endogenous TNF-alpha production. Furthermore, TNF-alpha and other proinflammatory cytokines are important amplifiers of immune responses (14). Therefore, we used TNF-alpha production as a readout for DC stimulation in our culture system. Culturing activated NK cells with DCs at low NK/DC ratios (1:5) led to increases in TNF-alpha production, which were augmented dramatically by the addition of suboptimal doses (10 ng/ml) of LPS (Fig. 2 A). The increased TNF-alpha production was dependent on cell-to-cell contact because when the two cell types were separated in trans-wells, no stimulation was observed (Fig. 2 B). In separate experiments we observed this NK-stimulatory effect on TNF-alpha production at NK/DC ratios as low as 1:20 and 1:40 (data not shown). Raising the NK/DC ratio to 5:1 changes the interaction from one of potent DC stimulation to that of complete inhibition in the presence or absence of LPS (Fig. 2 C). An identical pattern is seen for IL-12 p40 production (Fig. 2 D). No IL-10 production in the cultures was detected under any of the conditions tested (data not shown). The massive production of TNF-alpha in the cultures at the lowest ratios was mediated primarily by DCs in the culture due to the low number of NK cells (2 x 104 cells per well) used in these experiments. In addition, efforts to stimulate NK cells directly with optimal doses of anti-CD16, rIL-2, rIL-12, and PMA/ionomycin alone or in combination induced only moderate (<500 pg/ml) TNF-alpha production by the NK cells alone which were present at 2 x 105 cells per well (data not shown).
  Figure 2.
  Activated/cultured NK cell-mediated amplification/inhibition of DC cytokine production. Results are presented on log scales. Cultured iDCs were incubated alone or with cultured/purified NK cells for 24 h (A and B) or 48 h (C-E) in the presence or absence of the indicated concentrations of LPS. After culture the supernatants were removed and analyzed by ELISA for TNF-alpha or IL-12 p40. (A) TNF-alpha production was measured in the supernatants from cultures of: DCs alone (gray bars); NK+DC (1:5) (black bars). LPS concentrations used were: 0, 10, and 1,000 ng/ml (DCs alone only). (B) Contact-dependent stimulation of iDC TNF-alpha production by NK cells was tested under the following conditions: DCs alone (gray bars); NK+DC (1:5) (black bars); NK/DC (1:5) trans-wells (striped bars). LPS concentrations used were: 10 ng/ml (donor 1) and 20 ng/ml (donor 2). (C and D) Effect of increasing the NK/DC ratio (DCs alone; NK:DC-1:5; 1:1; 5:1) on (C) TNF-alpha and (D) IL-12 p40 production. LPS concentrations used were: 0 ng/ml (*); 20 ng/ml (*); 50 ng/ml (^); and 1,000 ng/ml-LPS (*) (DCs alone only). (E) The effect of neutralizing IFN-gamma production was tested under the following conditions: DCs alone (gray bars); NK+DC (1:5) (black bars); NK+DC (1:5) and 10 mug/ml of blocking anti-IFN-gamma mAb (stippled bars). LPS concentrations used were: 0 ng/ml (NO LPS) and 20 ng/ml (LPS 20).
  Blocking reagents for CD80, CD86, CD154, CD95, CD11a, CD50, or IL-12, had no effect on the NK cell-induced TNF-alpha or IL-12 p40 production, whereas a neutralizing antibody for IFN-gamma while not affecting TNF-alpha production partially blocked (<20%) IL-12 p40 production induced by the NK cells (Fig. 2 E). This partial inhibition is consistent with numerous previous reports demonstrating a central role for an IL-12/IFN-gamma positive feedback loop active between NK cells and IL-12-producing cells (15-17). Clearly, however, this well-described cytokine cross-talk mechanism is secondary to the contact-dependent activation of DCs by NK cells described here. Taken together these results indicate that novel mechanisms control cognate interactions between NK cells and DCs and that NK-DC contact has the potential to serve as an important control switch for both amplifying and attenuating innate immune responses in vivo.
  We next tested directly whether NK cell killing was active in our system using both short-term (4-h) chromium release assays (data not shown) and a flow cytometric assay which allowed us to follow the survival of the autologous iDCs in the presence of NK cells over longer periods (Fig. 3). The results demonstrate that NK cells kill autologous iDCs (Fig. 3 A) at all NK/DC ratios tested after 4 h and the level of killing is similar to the lysis of K562 cells over the same time (Fig. 3 B). After 24 h, however, the presence of NK cells led to completely divergent effects on the DCs. At the 5:1 (NK/DC) ratio, NK cell-mediated destruction of the iDCs was the dominant feature, whereas at the low ratio (1:5) the same NK cells promoted iDCs survival compared with iDCs alone over the same period (Fig. 3 A). The equal ratio of NK/DC (1:1) fell between these two extremes. The NK cell-mediated lysis of the K562 targets increased at all ratios tested after the 24-h incubation. These striking differences between death and survival of DCs upon encountering NK cells correlate precisely with our previous results on TNF-alpha and IL-12 p40 production where increasing numbers of NK cells in the culture shifts the NK effect from potent stimulation to complete inhibition (Fig. 2).
  Figure 3.
  Activated/cultured NK cell-mediated death/survival of DCs and NK-DC conjugate formation. NK cell mediated killing/survival of CFDA.SE-loaded iDCs (A) and K562 cells (B) was measured by flow cytometry over 4 h (*) or 24 h (*) at the indicated NK/DC ratios as described in Materials and Methods. Results are presented as percentage of specific lysis +/- SEM (n = 3 independent experiments). NK-DC (C-E) or NK-K562 (F-H) conjugate formation was measured by flow cytometry, at the following ratios (NK/DC or NK/K562): 1:5 (C and F); 1:1 (D and G); and 5:1 (E and H). Snarf-1 (FL3)-labeled iDCs or K562 cells were incubated (30 min) with CFDA.SE (FL1)-labeled NK cells at the different ratios. The percentage of conjugates formed (top right quadrant) was calculated by determining the percentage of FL3+ (Snarf-1) events which were also positive for FL1 (CFDA.SE). The results are representative of four experiments performed with activated NK cells. Resting NK cells also readily formed conjugates with autologous iDCs (data not shown).
  NK cells can both efficiently kill and potently stimulate DCs in our model system and each of these opposing effects appear dependent on cell-to-cell contact. To quantify these apparent cognate interactions we measured the formation of NK-DC conjugates and compared them to conjugates formed by NK cells and tumor targets. Our analysis revealed that NK cells bound DC (Fig. 3 C-E) with an efficiency almost equal to that of NK cell binding to the classical NK-target, K562 (Fig. 3 F-H). Binding was detectable even at the lowest NK/DC ratio tested (1:5) indicating that NK-DC conjugates are formed in our other experiments measuring cytokine production and cell killing.
  Our initial experiments using resting NK cells (Fig. 1) demonstrated a contact-dependent NK effect driving iDCs to mature. Therefore, we tested what effects activated NK cells mediated on DC maturation (Fig. 4). Similar to our previous results with activated NK cells (Figs. 2 and 3), addition of NK cells to the culture leads to opposing effects on the iDC population depending on the NK/DC ratio. Compared with the untreated DCs (Fig. 4 A-C), low NK/DC ratios (1:5 or 1:1) consistently augmented expression of the maturation markers: CD86 (Fig. 4 D-I), CD83, and HLA-DR (data not shown). At a 5:1 ratio (NK/DC) most of the DCs are nonviable (Fig. 4 J-L). Addition of suboptimal doses of LPS (Fig. 4 C, F, I, and L) augmented the NK-induced maturation at NK/DC ratios of 1:5 and 1:1, but had no effect at the 5:1 ratio. Again, DC maturation was dependent on NK cell contact and endogenously produced TNF-alpha, because it did not occur when the two cell types were separated in trans-wells or upon the addition of a neutralizing antibody for TNF-alpha (Fig. 5). Moreover, addition of blocking reagents for CD80, CD86, CD154, CD95, CD11a, CD50, IL-12, IFN-gamma, IFN-alpha, or IFN-beta had no effect on the NK-induced maturation (data not shown).
  Figure 4.
  Activated/cultured NK cell-mediated maturation/death of DCs. Cultured iDCs were incubated for 48 h, alone (A-C), or with NK cells at the following ratios (NK/DC), 1:5 (D-F), 1:1 (G-I), and 5:1 (J-L). Forward versus side scatter plots (A, D, G, and J) show the gating on the DC subset and demonstrate its disappearance at the 5:1 (NK/DC) ratio (J). Propidium iodide staining of the NK/DC (5:1) cultures confirmed the almost complete absence of viable DCs (data not shown). Staining for CD86 expression was performed as described in Fig. 1 and the histograms are shown for DCs cultured in the presence (C, F, I, and L) or absence (B, E, H, and K) of 1 ng/ml of LPS. Results are representative of six experiments performed.
  Figure 5.
  Activated/cultured NK cell-induced DC maturation is dependent on cell-to-cell contact and endogenous TNF-alpha production. Cultured iDCs were incubated alone (A, D, and G); with activated NK cells at an NK/DC ratio of 1:5 (B, E, and H); with activated NK cells (NK/DC; 1:5) separated in trans-wells (C and F); or with activated NK cells and 10 mug/ml of a neutralizing antibody for TNF-alpha (I). LPS concentrations used were: 0 ng/ml (A-C); 10 ng/ml (D-F) and 50 ng/ml (G-I). CD86 staining of the DC subset was performed as in Figs. 1 and 4. Results are representative of four experiments performed.
  