TLR-dependent cross talk between human Kupffer cells and NK cells

Abstract

The liver protects the host from gut-derived pathogens yet is tolerant of antigenic challenge from food and commensal sources. Innate responses involving liver macrophages (Kupffer cells) and effector liver natural killer (NK) cells form the first line in this defense. We address the impact of Toll-like receptor (TLR) signaling on the cross talk between these two cells, and reveal how the liver displays a down-regulated inflammatory response to constitutive bacterial elements through the secretion of interleukin (IL) 10 yet retains a vigorous response to viral challenge. The data support the model that (a) human liver Kupffer cells respond to TLR ligands and indirectly activate NK cells; (b) the activation depends on cell-cell contact; (c) the Kupffer cells synthesize NK cell activating signals, among which IL-18 is critical, and NK cell inhibitory factors, including IL-10; (d) ligands that signal via myeloid differentiation factor 88 induce IL-10, giving a blunted response in the NK cells; and (e) ligands that signal via the Toll-IL-1 receptor domain-containing adaptor inducing interferon (IFN) beta-IFN regulatory factor 3 pathway induce less IL-10, and also directly potentiate the stimulatory effect of IL-18 on NK cells, resulting in enhanced activation. Subversion of cellular mechanisms of innate immune response against viruses may be important for hepatotropic viruses (e.g., hepatitis B and C) to develop persistence.

Figure 1.

Kupffer cells from healthy living donors are phenotypically different from PBMCs. (A) There were differences in cell-surface expression of CD1b, DC-SIGN, CD40, and CD83, and most strikingly in the lower expression of HLA class I and greater expression of HLA class II in the Kupffer cells (open). (B) Kupffer cells were negatively selected from liver sinusoidal mononuclear cells before immunomagnetic bead separation. The CD14⁻ cells that were depleted as a by-product of Kupffer cell isolation, and the negatively selected (purified) CD14⁺ cells are shown (top). NK cells (bottom) were purified by positive selection. Contour plots show the whole liver leukocyte population, the discarded CD56⁻ cells, and the purified NK cells (positive selection). SSC, side scatter.

Figure 2.

Kupffer cell cytokine elaboration in response to TLR ligands. (A) TLR ligands stimulate Kupffer cells to secrete IL-10 and IL-18 in a dose-dependent manner. Kupffer cells from a living liver donor were cultured at 10⁶ cells/ml for 16 h in medium supplemented with the various concentrations of the TLR ligands. (B) The time course of IL-10 and IL-18 cytokine secretion in response to TLR ligands. Kupffer cells from a living liver donor were cultured at 10⁶ cells/ml in medium supplemented with the TLR ligands (5 μg/ml LTA, 100 μg/ml poly I:C, and 0.5 μg/ml LPS). Cell-culture supernatants were harvested at various time points. All samples were assayed in triplicate. IL-18 concentration was measured by ELISA; IL-10 was measured by CBA. (C) Consistent pattern of Kupffer cell cytokine secretion across multiple donors. All three TLR ligands (LTA, poly I:C, and LPS) cause IL-12, TNF-α, and IL-10 secretion; however, poly I:C caused less IL-10 secretion than the other two ligands. Only LTA causes IL-1β secretion. Horizontal bars represent means.

Figure 3.

IFN-γ production in co-culture experiments. The data shown are representative examples from six separate living donor experiments. Freshly isolated human LMNCs, LMNCs depleted of CD14⁺ cells, NK cells, and NK cells co-cultured with Kupffer cells together and separated by a transwell were stimulated by TLR agonists for 16 h. Medium and IL-12/15 served as negative and positive control, respectively. Cells were stained with antibodies for cell-surface CD3, CD56, and CD16 and for intracellular IFN-γ. Results show intracellular IFN-γ in CD3⁻, CD56⁺ NK cells. The synthesis of IFN-γ in NK cells was dependent on direct contact with Kupffer cells.

Figure 4.

IL-10 production in co-cultures. Freshly isolated human LMNCs, LMNCs depleted of CD14⁺ cells, NK cells, and NK cells co-cultured with Kupffer cells together and separated by a transwell were stimulated by TLR agonists for 16 h. Antibodies to cell-surface CD14 and intracellular IL-10 were used to detect IL-10 expression in Kupffer cells. The results shown are from one representative donor out of six separate experiments. In the co-cultures, cytoplasmic IL-10 staining was weak in Kupffer cells stimulated with poly I:C.

Figure 5.

IFN-γ production in co-culture experiments treated with blocking antibodies to mediators of NK cell function. Freshly isolated NK cells were co-cultured with Kupffer cells and stimulated by TLR agonists for 16 h in the presence or absence of anticytokine antibody. Medium and IL-12/15 served as negative and positive controls, respectively. Shown is a co-culture experiment of one representative donor showing IFN-γ production in CD3⁻, CD56⁺ NK cells. The most striking inhibitory effect was obtained using anti–IL-18, with a more modest effect of blocking IL-12, a minimal effect of blocking with anti-NKG2D, and a dramatic increase in IFN-γ after IL-10 blockade in the case of LTA- and LPS-stimulated cells. The data are representative examples taken from one out of six experiments.

Figure 6.

IFN-γ production in co-culture experiments treated with IL-10 and IL-18 blockade. Freshly isolated human liver sinusoidal NK cells and Kupffer cells from adult live donors were co-cultured and stimulated by TLR agonists for 16 h in the presence or absence of anti–IL-10 (A) or anti– IL-18 (B) antibodies. Media and IL-12/15 served as negative and positive controls, respectively. The percentages of IFN-γ–positive cells were measured by flow cytometry (A and B, top). Supernatants were collected, and cytokine protein content was measured by CBA (A and B, bottom). The dramatic effects of IL-10 blockade are seen in LTA- and LPS-stimulated cells (A). The effects on poly I:C–stimulated cells were not significant in terms of the percentage of NK cells synthesizing IFN-γ, whereas they were significant in terms of IFN-γ secretion. On the other hand, IL-18 blockade significantly inhibited IFN-γ synthesis induced by all three ligands, and such inhibition was also significant in terms of secreted IFN-γ for LTA and poly I:C (B).

Figure 7.

IL-18 secretion by Kupffer cells alone and in co-culture. IL-18 secretion was assessed by ELISA in culture supernatants from six separate experiments. IL-18 increased in response to all three TLR ligands and was similar whether the Kupffer cells were cultured in isolation or in co-culture without or with the intervening transwell membrane. Note that IL-18 secretion was in the range of 50–200 pg/ml. Error bars are means ± SEM.

Figure 8.

Recombinant human IL-18 induced IFN-γ expression of purified NK cells in the presence of TLR agonists at nanogram concentrations. Freshly isolated human liver sinusoidal NK cells from adult live donors were stimulated by TLR agonists for 16 h in the presence of different concentrations of IL-18. Intracellular IFN-γ expression was assessed in CD3⁻, CD56⁺ NK cells by flow cytometry. Only poly I:C synergistically induced an NK cell IFN-γ response together with IL-18. However, note that the effective concentration of IL-18 was in the range of 100 ng/ml.

Figure 9.

Detection of TLR expression intracellularly and on the cell surface of NK and Kupffer cells. Isolated NK and Kupffer cells were stained (open) to detect TLR on the cell surface (Perm −) or permeabilized to detect intracellular TLR. Results revealed the intracellular expression of TLR3 in both NK and Kupffer cells, whereas the expression of TLR2 and TLR4 was limited to the Kupffer cells.

Figure 10.

Model of TLR ligand activation of Kupffer cell–NK cell cross talk in co-culture. Salient features of this model, consistent with the experimental data, are the dependence of NK activation by Kupffer cells on cell-to-cell contact, IL-18 as the critical activating signal, and IL-10 as the modulating inhibitory signal induced mainly through the MyD88 signaling cascade. The model predicts that bacteria-associated TLR agonists signaling through TLR2 and TLR4 result in down-modulated NK cell activation caused by IL-10, whereas a virus-associated TLR agonist (double-stranded RNA) acting through the MyD88-independent pathway results in stronger activation of NK cells. The model also recognizes the intracellular location of TLR3 in NK cells, as well as Kupffer cells, with the possibility of direct NK cell activation through this channel.