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The CD16^–/CD56^bright Subset of NK Cells Is Resistant to Oxidant-Induced Cell Death¹

Department of Infectious Medicine, Sahlgrenska Academy at Göteborg University, Göteborg, Sweden

	Abstract

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Phagocyte-derived reactive oxygen species ("oxygen radicals") have been ascribed a suppressive role in immunoregulation by inducing dysfunction and apoptotic cell death in lymphocytes. Earlier studies show that human NK cells are exceptionally sensitive to oxygen radical-induced apoptosis and functional inhibition. Two subsets of human CD56⁺ NK cells have been identified: the highly cytotoxic CD56^dim cells which constitute >90% of NK cells in peripheral blood, and the less cytotoxic but efficiently cytokine-producing CD56^bright cells. In this study, we demonstrate that the CD56^bright subset of NK cells, in contrast to CD56^dim cells, remains viable and functionally intact after exposure to phagocyte-derived or exogenously added oxygen radicals. The resistance of CD56^bright cells to oxidative stress was accompanied by a high capacity of neutralizing exogenous hydrogen peroxide, and by a high cell-surface expression of antioxidative thiols. Our results imply that CD56^bright NK cells are endowed with an efficient antioxidative defense system that protects them from oxygen radical-induced inactivation.

	Introduction

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Natural killer cells are large granular lymphocytes that comprise up to 15% of the lymphocyte population in human peripheral blood (1). As innate effectors, NK cells are considered to play a critical role in the early phase of the immune response to pathogens and malignant cells before an adaptive immune response has developed (2, 3). Upon encounter with a tumor cell or a virus-infected cell, NK cells can become activated and kill the target cell by releasing preformed granules containing perforin and granzymes. When activated, NK cells modulate the adaptive immune responses by secreting cytokines, primarily IFN- (4, 5). Approximately 90–95% of human NK cells in peripheral blood display a relatively low density of the CD56 Ag on the cell surface (CD56^dim) and also express the low-affinity IgG FcR, FcRIII (CD16). The CD56^dim NK cells produce low amounts of cytokines but express high levels of perforin and granzymes and thus exert high natural and Ab-dependent cellular cytotoxicity against susceptible target cells (6).

A smaller subset of peripheral blood NK cells (5–10%) expresses the CD56 Ag at a higher density and also typically lacks the CD16 Ag (CD56^bright16^dim/neg phenotype). CD56^bright NK cells differ from CD56^dim cells by more efficiently producing cytokines and by exerting lower natural cytotoxicity (7, 8). Rather than forming part of the cytotoxic armaments of innate immunity, the CD56^bright cells appear to play a role at the innate/adaptive immunity interface, as reflected by their expression of CD62L and CCR7, which are adhesion molecules needed for homing to secondary lymphoid organs (9, 10). CD56^bright cells are thus localized in T cell areas of human lymph nodes where they interact with homing dendritic cells (11, 12, 13). By releasing IFN-, the CD56^bright NK cells are assumed to shape the adaptive immune response, promoting Th1 polarization (14).

Oxidative stress, defined as cellular toxicity inflicted by reactive oxygen species ("oxygen radicals"), has been ascribed a role in immunomodulation. First, oxygen radicals have been proposed to contribute to the dysfunction of cytotoxic lymphocytes characteristic of malignant disorders and chronic infections (15, 16, 17, 18, 19, 20). Second, oxygen radicals reportedly suppress autoimmunity and arthritis development by controlling the cell-surface level of antioxidative thiols on lymphocytes (21, 22). Although these functional consequences of oxygen radical-related immunoregulation have been studied in some detail, relatively little is known about the sensitivity of individual lymphocyte subsets to oxidative stress. Earlier studies show that NK cells, in contrast to, e.g., CD4⁺ T cells, are rapidly inactivated and highly prone to acquire features of apoptosis upon exposure to oxygen radicals, delivered either as exogenous hydrogen peroxide or produced by adjacent phagocytic cells (23, 24, 25, 26). In this study, we report that CD56^bright NK cells are unexpectedly resistant to oxidative stress, based on the finding that CD56^bright cells, in contrast to CD56^dim cells, remain viable and functional after exposure to oxygen radicals. Our results imply that CD56^bright NK cells are endowed with a strong antioxidative defense system that protects them from oxidant-induced inactivation.

	Materials and Methods

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Separation of leukocytes

Peripheral venous blood was obtained as freshly prepared acid citrate dextrose-containing leukopacks from healthy blood donors at the Blood Centre (Sahlgren’s University Hospital, Göteborg, Sweden). To remove catalase-containing erythrocytes, the blood (65 ml/donor) was mixed with 92.5 ml of Iscove’s DMEM, 35 ml of 6% dextran, and 7.5 ml of acid citrate dextrose. After incubation for 15 min at room temperature, the supernatant was carefully layered on top of a Ficoll-Hypaque (Lymphoprep) density gradient. After centrifugation at 380 x g for 15 min, mononuclear cells were collected at the interface, while the pellet contained erythrocytes and polymorphonuclear phagocytes (PMN)³ (25). Mononuclear cells were further separated into lymphocytes and mononuclear phagocytes (MP) using a countercurrent centrifugal elutriation technique in which the sedimentation rate of cells in a spinning rotor is balanced by a counterdirected flow through the chamber. To this end, the mononuclear cells were resuspended in elutriation buffer, i.e., buffered NaCl supplemented with 0.5% BSA and 0.1% EDTA, and fed into a Beckman J2-21 ultracentrifuge with a JE-6B rotor (Beckman Coulter) at 2100 rpm. By slowly increasing the flow rate through the chamber, fractions of cells of well-defined sizes were collected. A fraction with >90% mononuclear phagocytes was obtained at a flow rate of 19 ml/min. The procedure was repeated for the pellet recovered after the Ficoll-Hypaque gradient centrifugation, and a fraction with >95% PMN (with >98% neutrophilic granulocytes) was obtained at flow rates of >22 ml/min. Lymphocyte fractions recovered at flow rates between 14 and 16 ml/min were enriched for NK cells and T cells and contained <3% phagocytes. These lymphocyte fractions were pooled and subjected to further separation.

The elutriated lymphocytes were depleted of CD3⁺ cells (NK cell-enriched lymphocytes) using IMag CD3 magnetic particles (BD Biosciences) according to the instructions provided by the manufacturer. In some experiments, the NK cell-enriched lymphocytes were stained with anti-human mAbs to CD3, CD16, CD56, and CD8, and >98% pure populations of CD3^–/16⁺/56^dim and CD3^–/16^–/56^bright were sorted out using a BD FACSAria equipped with three laser lines (405, 488, and 633 nm) and FACSDiva version 5 software (BD Biosciences). Separated cells were resuspended in Iscove’s DMEM supplemented with 10% human AB⁺ serum and this medium was used in all experiments below unless otherwise stated.

Lymphocyte cell death

Lymphocytes, enriched NK cells, or FACS-sorted NK cells were incubated overnight in 96-well round-bottom plates with medium, autologous mononuclear phagocytes, hydrogen peroxide (H₂O₂), or autologous PMN. After 18 h, end-stage oxidant-induced cell death in NK cells was assayed using flow cytometry, based on the altered light-scattering characteristics displayed by end-stage apoptotic cells, i.e., a reduced forward scatter and an increased right angle scatter (23). Apoptosis was confirmed using annexin V and To-Pro-3 staining as described elsewhere (26). Apoptotic CD56^dim NK cells characteristically lose the expression of CD16 and CD56 (27). Therefore, in mixed lymphocyte preparations, the percentage of apoptotic CD56^dim cells was determined by comparing the fraction of live CD56^dim cells after exposure to oxidants with the fraction in the untreated control: percentage of apoptotic cells = 100 x (percentage of live CD56^dim of total lymphocytes in control – percentage of live CD56^dim of total lymphocytes in experiment)/(percentage of live CD56^dim of total lymphocytes in control). To investigate PJ34-mediated protection of NK cells, the H₂O₂ concentration that induced at least 30% apoptosis in the CD56^bright subset in the absence of PJ34 was used for analysis. That concentrations varied between experiments (range: 125–250 µM). The presented data indicate the fraction of dead NK cells in the control that were protected by addition of PJ34.

Intracellular staining for IFN-

NK cell-enriched lymphocytes were incubated overnight with autologous phagocytes MP/NK ratio 0.5 and 1) or hydrogen peroxide. After 18 h, cells were stimulated with 100 ng/ml PMA and 1 ng/ml ionomycin as described previously (28). After 1 h, brefeldin A (GolgiPlug; BD Biosciences) was added to retain IFN- intracellularly. After another 4 h, cells were harvested, washed, and stained for cell-surface Ags. Lymphocytes were then fixed and permeabilized with Cytofix/Cytoperm (BD Biosciences) and stained for intracellular IFN- using a PE-conjugated mAb.

Staining for cell-surface thiols

The relative number of cell-surface thiols on lymphocytes was determined using Alexa-633 C5-maleimide (ALM-633; Invitrogen Life Technologies) (22, 29). Freshly isolated PBMCs were incubated with 5 µM ALM-633 for 15 min on ice. After extensive washing, cells were stained with Abs directed against appropriate surface Ags and data were acquired and analyzed using a BD FACSCanto II with FACSDiva version 5 software. Results are presented as median fluorescence intensity (MFI) values of ALM-633 staining.

Hydrogen peroxide consumption assay

FACS-sorted preparations of CD56^dim and CD56^bright NK cell subsets were resuspended in Kreb’s Ringer glucose buffer (500,000 cells/ml) and incubated with 30 µM hydrogen peroxide for 30 min at 37°C. The remaining hydrogen peroxide was detected using an isoluminol-ECL technique (30). Isoluminol (10 µg/ml) and HRP (4 U/ml) were added and the chemiluminescence activity was monitored for 30 s using a Mithras LB 940 plate reader (Berthold Technologies).

Abs and reagents

The following anti-human mAbs were purchased from BD Biosciences: anti-CD3 (PerCP, allophycocyanin), anti-CD8 (AmCyan, PerCP, allophycocyanin), anti-CD16 (FITC, allophycocyanin-Cy7), anti-CD56 (PE, PE-Cy7, allophycocyanin), anti-CD107a (PE-Cy5). Anti-CD3 (Pacific blue) and anti-IFN- (PE) were obtained from Invitrogen Life Technologies. FITC- and PE-conjugated annexin V were from BD Biosciences. The following compounds were used: N-ethyl maleimide, isoluminol, PJ34 (Sigma-Aldrich); dextran (Kabi Pharmacia); acid citrate dextrose (Baxter); BSA (ICN Biomedicals); EDTA and hydrogen peroxide (VWR); Ficoll-Hypaque, Lymphoprep (Nycomed); To-Pro-3, ALM-633 (Invitrogen Life Technologies); HRP (Boehringer Mannheim).

Statistics

Paired samples t tests were used throughout the study. For MFI values, data were transformed logarithmically before statistical calculation. All reported p values are two-sided.

	Results

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The CD56^bright NK cell subset is resistant to oxidant-induced cell death

We investigated the susceptibility of CD56^dim and CD56^bright cells to the toxicity of reactive oxygen species, derived from autologous phagocytes or added exogenously as hydrogen peroxide. NK cell-enriched lymphocytes were incubated with hydrogen peroxide, MP, or PMN overnight and assayed for end-stage apoptosis. CD56^dim NK cells were highly sensitive to exogenously added hydrogen peroxide and to phagocyte-derived oxygen radicals. In contrast, CD56^bright cells were almost completely resistant to oxidant-induced apoptosis when incubated with phagocytes or at lower H₂O₂ concentrations. At higher concentrations of hydrogen peroxide, CD56^bright cells were significantly less prone to acquire features of apoptosis than CD56^dim cells (Fig. 1). The difference in viability between CD56^bright and CD56^dim NK cells after exposure to oxidants also remained after 72 h of culture, indicating that the observed difference was not due to delayed cell death in the CD56^bright subset (data not shown).

View larger version (37K): [in this window] [in a new window]   FIGURE 1. CD56bright NK cells are resistant to oxidant-induced cell death. NK cell-enriched lymphocytes were incubated overnight in the absence (A) or presence (B) of mononuclear phagocytes at a 1:1 ratio. Dot plots show viable CD3– lymphocytes based on a live scatter gate. In B, a selective loss of live CD56dim NK cells is observed, while the population of CD56bright NK cells remains intact, as indicated by the increased fraction of CD56bright cells among viable lymphocytes after exposure to phagocytes. Similar results were observed in six different experiments. In C and D, NK cell-enriched lymphocytes were exposed to mononuclear phagocytes at indicated ratios or concentrations of hydrogen peroxide overnight and assayed for end-stage apoptosis. CD56dim cells were significantly more sensitive to phagocyte-derived radicals as well as to hydrogen peroxide than CD56bright cells. Data are mean ± SEM. (MP/NK ratio 0.5, p < 0.01, n = 6; MP/NK ratio 1, p < 0.01, n = 6; 125 µM H2O2, p < 0.001, n = 8; 250 µM H2O2, p < 0.001, n = 4; **, p < 0.01; ***, p < 0.001).

View larger version (11K): [in this window] [in a new window]   FIGURE 2. Phagocytes induce cell death in CD56dim cells. FACS-sorted CD56bright and CD56dim cells were incubated overnight with mononuclear (A) or polymorphonuclear (B) phagocytes at indicated phagocyte to lymphocyte ratios. CD56bright cells were significantly more resistant to phagocyte-induced cell death than CD56dim cells (MP/NK ratio 0.5, p < 0.01, n = 8; ratio 1, p < 0.0001, n = 12; PMN/NK ratio 1, p < 0.01, n = 5; **, p < 0.01; ***, p < 0.001).

We have recently shown that oxidant-induced cell death in NK cells is mediated by the PARP/apoptosis-inducing factor (AIF) axis. Thus, exposure of NK cells to oxidants activates nuclear PARP, which in turn triggers translocation of mitochondrial AIF to the nucleus with ensuing DNA fragmentation and cell death (26). To investigate the role of the PARP/AIF axis for the oxidant-induced cell death in CD56^bright NK cells, we pretreated NK cells with a PARP inhibitor, PJ34, before exposing them to hydrogen peroxide. As shown in Fig. 3, the pronounced degree of apoptosis in CD56^dim cells as well as the lower degree of apoptosis in CD56^bright cells were efficiently prevented by PJ34, thus suggesting that the mechanisms of oxidant-induced cell death is similar in these NK cell subsets.

View larger version (10K): [in this window] [in a new window]   FIGURE 3. A PARP-1 inhibitor, PJ34, protects both NK cell subsets from oxidant-induced cell death. NK-enriched lymphocytes were preincubated with 0.5 µM PJ34 before exposure to hydrogen peroxide. Data are from the H2O2 concentration that induced at least 30% apoptosis in the CD56bright subset (range: 125–250 µM). Data are normalized and displayed as a percentage of the corresponding control without PJ34. Thus, for both NK cell subsets, PJ34 protected 70–75% of the otherwise apoptosing NK cells (CD56bright, p < 0.01, CD56dim, p < 0.01, n = 4; **, p < 0.01).

Next, we sought to determine whether CD56^bright cells remained functionally intact after exposure to phagocytes or hydrogen peroxide. For this purpose, we investigated the capacity of NK cell subsets to express intracellular IFN- after activation. After incubation with oxidants, NK cell-enriched lymphocytes were stimulated with PMA and ionomycin and stained for intracellular IFN-. In line with the previous findings that CD56^bright cells are the principal cytokine-producing NK cells (7), a significantly higher percentage of CD56^bright cells stained positively for IFN- and these cells were to a significantly larger extent IFN-^bright as compared with CD56^dim cells. After addition of hydrogen peroxide or incubation with mononuclear phagocytes, the responsiveness to PMA/ionomycin stimulation dropped in CD56^dim cells. In contrast, CD56^bright cells preserved their capacity to produce IFN- after exposure to oxygen radicals (Fig. 4). In line with results from earlier studies in T cells, higher concentrations of hydrogen peroxide were required to induce NK cell death than those required to suppress NK cell function (31).

View larger version (19K): [in this window] [in a new window]   FIGURE 4. CD56bright NK cells remain responsive to activation after exposure to oxidants. NK-enriched lymphocytes were exposed to mononuclear phagocytes or hydrogen peroxide overnight, and assayed for intracellular IFN- after stimulation with PMA/ionomycin. A, CD56dim cells lost their ability to produce IFN- after exposure to radicals, while a significantly higher fraction of CD56bright cells remained functional (MP/NK ratio 0.5, p<0.01; ratio 1, p < 0.0001; H2O2 30 µM, p < 0.01; 60 µM, p < 0.0001, n = 7). In B, a representative experiment with PMA/ionomycin-stimulated NK cells shows that CD56bright cells expressed more IFN- on a per cell basis and were thus stained more intensely (higher MFI) than CD56dim cells (p = 0.01, n = 6; **, p < 0.01; ***, p < 0.001).

Cells are endowed with elaborate systems for protection against the detrimental effect of oxidants. Central in these antioxidative systems are thiols, such as glutathione and thioredoxin (32). Exofacial surface thiols are needed for proper activation of lymphocytes (33, 34), but are also considered to function as a first line of defense against oxidative challenge by neutralizing oxygen radicals. To investigate thiol expression in CD56^dim and CD56^bright cells, we stained freshly isolated PBMCs for cell-surface thiols using fluorochrome-conjugated maleimide. As shown in Fig. 5, A and B, the MFI value (which reflects the median number of thiols expressed per cell) for CD56^bright cells was approximately twice that of CD56^dim cells. Pretreatment with N-ethyl maleimide (at 2.5 µM), which blocks cell-surface thiols (29) was found to sensitize CD56^bright NK cells to H₂O₂ (data not shown), thus supporting that thiol expression contributed to the resistance of this subset to oxidants.

View larger version (16K): [in this window] [in a new window]   FIGURE 5. CD56bright cells express significantly more surface thiols than CD56dim cells and consume hydrogen peroxide. Data in A are the mean ± SEM of MFI values (p < 0.001, n = 4). B, A representative experiment showing thiol-specific binding of Alexa 633 maleimide (ALM-633). C, CD56bright cells neutralize exogenous hydrogen peroxide more efficiently than CD56dim cells. Pure populations of the two NK cell subsets were incubated with 30 µM H2O2 for 30 min. Remaining hydrogen peroxide was assayed using chemiluminescence. Data are mean ± SEM, p = 0.03, n = 5. *, p < 0.05; ***, p < 0.001.

	Discussion

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In recent years, it has become clear that the human NK cell population comprises at least two functionally distinct subsets, the highly cytotoxic CD56^dim cells and the less cytotoxic but more efficiently cytokine-producing CD56^bright cells (7). A main finding in the present report is that CD56^bright NK cells remain viable when exposed to phagocyte-derived or exogenously added hydrogen peroxide, while CD56^dim cells rapidly undergo apoptosis. In addition, we show that 1) CD56^bright cells express higher levels of cell-surface thiols than CD56^dim cells, 2) CD56^bright cells are significantly more efficient at neutralizing hydrogen peroxide than CD56^dim cells, and 3) CD56^bright cells, in contrast to CD56^dim cells, retain their capacity to synthesize IFN- in conditions of oxidative stress.

Recent findings highlight that oxygen radical-induced inactivation of lymphocytes may constitute a significant mechanism of immunosuppression, with implications for the development of autoimmunity as well as for the function of cytotoxic lymphocytes in malignant diseases. Rodents with mutations in a gene encoding a pivotal component of the phagocyte NADPH oxidase, with an ensuing deficiency of oxygen radical synthesis, display increased susceptibility to arthritis. T cells from these animals show lower threshold for reactivity, suggesting that oxygen radicals participate in controlling autoimmunity and arthritis development (22, 35). Although oxygen radical-induced inhibition of lymphocyte function thus may contribute to protection against autoimmunity, a reverse situation appears to be at hand in malignant diseases. Thus, oxygen radicals, formed by phagocytic cells at the site of malignant expansion, are believed to significantly contribute to the characteristic state of anergy of cytotoxic lymphocytes in, e.g., colorectal cancer, malignant melanoma, renal cell carcinoma, and certain hemopoietic cancers, in addition to chronic viral infections such as HIV and hepatitis C (15, 16, 17, 18, 19, 20, 25, 36).

It may be hypothesized that the sensitivity of the highly cytotoxic CD56^dim NK cells to oxygen radicals has evolved as an emergency brake for potentially dangerous immune responses, complementing the inhibitory signals transduced by the CD94/NKG2A heterodimer and the killer Ig-like receptor group of receptors on CD56^dim cells, and that oxygen radical-mediated inactivation of CD56^dim cell may be used by microbes and transformed cells to evade NK cell-mediated cytotoxicity. Regarding CD56^bright cells, oxidant-mediated inactivation might not be urgently needed as 1) these cells display low cytotoxicity, and 2) recent data, albeit a subject of discussion, suggest that CD56^bright cells are precursors of CD56^dim cells (13). The resistance of CD56^bright cells to oxygen radical-inflicted toxicity is coherent with earlier reports demonstrating that activating NK cell receptors, such as NKp46 and NKG2D, are retained on the surface of CD56^bright cells but down-regulated on CD56^dim cells after oxygen radical exposure (37).

A tentative conclusion from the present report is that CD56^bright cells, in contrast to CD56^dim cells, remain viable and functional also in tissues exposed to oxidative stress, such as acute and chronic bacterial infections, chronic viral infections, chronic inflammation, and in the malignant microenvironment. Although further studies are needed to clarify the functional consequences of the differential regulation of these NK cell subsets, recent reports regarding the distribution of CD56^bright and CD56^dim cells in such tissues may reflect their differential sensitivity to radical-induced toxicity. Thus, the CD56^dim/CD56^bright ratio of NK cells is reportedly shifted in favor of the CD56^bright subset in pathologies such as head and neck cancer, breast cancer, hepatitis C, tuberculosis, and chronic inflammatory diseases (38, 39, 40, 41). It is tempting to speculate that these clinical findings may mirror the scope reported in this study: in the oxidatively challenging environment in these pathologies, the oxygen radical-sensitive CD56^dim NK cells succumb, while the resistant CD56^bright NK cells survive.

In conclusion, our results demonstrate an unexpected dissimilarity between the two dominant NK cell subsets in that NK cells with CD56^bright phenotype are strikingly resistant to oxygen radicals, whereas CD56^dim cells are highly prone to undergo apoptosis. The resistance to oxidants of CD56^bright cells appears to be explained by a high capacity of neutralizing oxygen radicals, which in turn may be related to a high cell-surface expression of antioxidative thiol groups.

	Acknowledgment

 
We thank Marie-Louise Landelius for excellent technical assistance.

	Disclosures

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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 Swedish Cancer Society, Swedish Research Council, Inga-Britt and Arne Lundberg Research Foundation, and Sahlgrenska Academy at Göteborg University.

² Address correspondence and reprint requests to Dr. Kristoffer Hellstrand, Department of Infectious Medicine, Sahlgrenska Academy at Göteborg University, Guldhedsgatan 10b, S-413 46 Göteborg, Sweden. E-mail address: kristoffer.hellstrand{at}microbio.gu.se

³ Abbreviations used in this paper: PMN, polymorphonuclear phagocyte; MP, mononuclear phagocyte; MFI, median fluorescence intensity; ALM-633, Alexa-633 C5-maleimide; PARP, poly(ADP-ribose)polymerase; AIF, apoptosis-inducing factor.

Received for publication January 26, 2007. Accepted for publication May 13, 2007.

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