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. 2000 Aug;121(2):384–390. doi: 10.1046/j.1365-2249.2000.01310.x

The differential effect of stress on natural killer T (NKT) and NK cell function

H Oya *,†, T Kawamura *, T Shimizu *,†, M Bannai *,†, H Kawamura *, M Minagawa *,†, H Watanabe *, K Hatakeyama †, T Abo *
PMCID: PMC1905713  PMID: 10931157

Abstract

When C57Bl/6 mice were exposed to restraint stress for 12 h or 24 h, lymphocytopenia was induced in the liver, spleen, and thymus. We examined which types of lymphocytes were sensitive or resistant to such stress by a immunofluorescence test. T cells of thymic origin were sensitive while NKT and NK cells were resistant. In contrast to the increase in the proportion of NK cells, NK activity of liver lymphocytes against YAC-1 targets decreased at 24 h after stress. On the other hand, their NKT cytotoxicity against syngeneic thymocytes increased in parallel with an increase in their proportion. In perforin −/– B6 mice and B6-gld/gld (Fas ligand−) mice, NK cells were found to mediate cytotoxicity through perforin while NKT cells mediated self-reactive cytotoxicity through Fas ligand. These results suggest that stress increases the proportion of both NK and NKT cells, but that NK cytotoxicity is suppressed while self-reactive NKT cytotoxicity is not, due to a diversity of their functional mechanisms.

Keywords: restraint stress, NK cells, NKT cells, liver, cytotoxicity

Introduction

In previous studies [1–3] we reported that NK cells and NK T cells increase in number or proportion in the peripheral blood of patients with cancer and of patients with autoimmune diseases (e.g. rheumatoid arthritis). We previously suspected that one of the causes might be sympathetic nerve activation in these patients. Sympathetic nerve activation is always seen in these patients and it might be caused by increased levels of the metabolism in the bodies of these patients (e.g. supply of oxygen and nutrients for tumours or inflammatory sites). Similar to the case of granulocytes, these NK and NKT cells carry adrenergic receptors on the surface [4,5] and they increase in number or proportion by sympathetic nerve stimulation [6–8]. It is therefore conceivable that sympathetic nerve activation increases the number or proportion of NK and NKT cells in these patients.

The functional capability of NK and NKT cells remains controversial. Namely, many patients with cancer show decreased levels of NK function, despite the consistent increase in the number of NK cells and NKT cells [9–12]. This raises the possibility that the number and function of NK and/or NKT cells are regulated by different mechanisms. To investigate this subject, we applied a mouse model in which the mice were exposed to restraint stress for 12 h or 24 h. Restraint stress resulted in an increase in the proportion of both NK and NKT cells. Interestingly, a clear dissociation between the number and function was observed in NK cells, but not in NKT cells. This study also revealed a diversity of the functional mechanisms between NK and NKT cells. The present results may shed light on the discrepancy between the number and function seen in NK and NKT cells.

Materials and methods

Mice

C57Bl/6 (B6) mice, perforin −/– mice (B6 background) [13], and B6-gld/gld mice [14] at the ages of 6–8 weeks were used. They were obtained from Charles River Japan (Kanagawa, Japan) and were maintained in the animal facility of Niigata University under specific pathogen-free conditions.

Restraint stress

Mice were fixed in stainless steel mesh and kept for 12h or 24 h in a cage [15–17]. Briefly, mice were placed in the immobilization mesh cage and were barely able to turn around.

Cell preparations

Mice were anaesthetized with ether and killed after complete exsanguination through incised axillary arteries and veins [18]. To obtain liver mononuclear cells (MNC), the liver obtained from one mouse was cut into small pieces with scissors, pressed through 200-gauge stainless steel mesh, and then suspended in 40 ml Eagle's minimal essential medium (MEM) supplemented with 5 mm HEPES (Nissui Pharmaceutical, Tokyo, Japan) and 2% heat-inactivated newborn calf serum. After being washed once with the medium, the cells were fractionated by centrifugation in 15 ml of 35% Percoll solution containing 100 U/ml heparin for 15 min at 2000 rev/min (450 g) [18]. The resultant cell pellets containing erythrocytes were resuspended and washed with the medium. To deplete erythrocytes, 170 mm Tris–HC1 buffer (pH 7·6) containing 155 mm NH4Cl, 10 mm KHCO3, and 1 mm Na2EDTA was added, and the mixture was kept on ice for 5 min.

Spleen cells and thymocytes were obtained by forcing each organ through 200-gauge stainless steel mesh. These lymphocytes were used after exposure to the erythrocyte lysing buffer.

Immunofluorescence test

The surface phenotype of cells was analysed using MoAbs in conjunction with a two-colour immunofluorescence test [18]. The MoAbs used here included FITC-, PE-, or biotin-conjugated anti-CD3 (145-2C11) (PharMingen, San Diego, CA), anti-IL-2R β-chain (IL-2Rβ) (TM-β1), and anti-NK1.1 (PK136) MoAbs. FITC- or PE-conjugated anti-CD4 (L3T4), and anti-CD8 (Lyt-2) MoAbs were obtained from Becton Dickinson (Mountain View, CA). Biotin-conjugated reagents were developed with PE-conjugated streptavidin (Becton Dickinson). The fluorescence-positive cells were analysed with a FACScan using LYSIS II software (Becton Dickinson).

Cytotoxicity assay

Cytotoxicity was examined by a specific 51Cr-release assay with an incubation time of 4 h [14]. NK cytotoxicity was examined using YAC-1 targets, whereas self-reactive cytotoxicity mediated by NKT cells was examined by using syngeneic (B6 mice) thymocytes. YAC-1 cells or thymocytes labelled with sodium chromate (51Cr) (Amersham Int., Arlington Heights, IL) were used, and effector cells were either hepatic or splenic lymphocytes. Percent cytotoxicity was determined using 104 YAC-1 cells or 2 × 104 thymocytes at the indicated target-to-effector ratios in triplicate cultures. During a 4-h incubation assay, spontaneous chromium release of both targets ranged from 10% to 15%. This release was eliminated by calculation.

Administration of adrenaline or hydrocortisone

Normal B6 mice were intraperitoneally administered with either adrenalin (20 and 100 μg/mouse) or hydrocortisone (1 mg/mouse) and were used 24 h after the injection. Control mice were injected with only saline (0·2 ml). As shown previously, these doses of adrenalin [6] and hydrocortisone [19] induced acute thymic atrophy (> 40% decrease in the number of thymocytes), similar to the case of restraint stress.

Cell sorting

MNC obtained from the liver were stained with FITC-conjugated anti-CD3 MoAb and PE-conjugated NK1.1 MoAb, and sorted into NK and NKT cells by FACS Vantage (Becton Dickinson) [20]. Cell fractions were harvested, washed, and used for NK or NKT cytotoxicity assay.

Statistical analysis

Difference was determined by Student's t-test.

Results

Stress resistance of NK1.1+CD3int cells in the liver of mice exposed to restraint stress

To investigate the effect of stress on an immunological system, mice were exposed to restraint stress and the numbers of lymphocytes were enumerated in various immune organs (Fig. 1). The numbers of lymphocytes in the liver, spleen, and thymus were found to decrease at a level of < 50% by 24 h after the exposure. It was speculated that severe stress induced apoptotic death of lymphocytes or the perturbation of their distribution.

Fig. 1.

Fig. 1

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A comparison of the number of lymphocytes yielded by the liver, spleen, and thymus in mice before and after restraint stress. Four mice were used to produce the mean and 1 s.d. (/mouse) at each point. *P < 0·05.

It was examined whether some specific types of lymphocyte subsets decreased due to the stress. Two-colour staining for CD3 and IL-2Rβ, that for CD3 and NK1.1, and that for CD4 and CD8 were conducted (Fig. 2). As shown previously [18], the staining for CD3 and IL-2Rβ (Fig. 2, left column) simultaneously identified NK cells (CD3−IL-2Rβ+), primordial T cells (CD3intIL-2Rβ+), and conventional T cells (CD3hiIL-2Rβ−). This staining demonstrated the proportional change of lymphocyte subsets in the liver after exposure to stress. A change was an increase in the proportion of CD3intIL-2Rβ+ cells in this organ at both 12 h and 24 h after stress. The proportion of CD3−IL-2Rβ+ NK cells slightly increased, whereas that of CD3hiIL-2Rβ− T cells decreased. The changes in the spleen were minimal. In the thymus, the proportion of CD3hi cells increased in a time-dependent manner after stress (12·0→14·2→31·3%). This might have been due to the fact that the apoptotic death of immature thymocytes (i.e. CD3− and CD3dull cells) was greater than that of mature thymocytes (i.e. CD3hi cells).

Fig. 2.

Fig. 2

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Phenotypic characterization of lymphocytes by immunofluorescence tests. Two-colour staining for CD3 and IL-2Rβ, for CD3 and NK1.1, and for CD4 and CD8 were conducted. The data shown here are representative of three experiments. Numbers in the figure represent the percentages of fluorescence-positive cells in corresponding areas.

There are two subsets of CD3int cells in the liver and other immune organs of normal mice [21]. We examined which subsets of CD3int cells increased in proportion after restraint stress. Two-colour staining for CD3 and NK1.1 was conducted (Fig. 2, middle column). The majority of CD3int cells which increased in proportion after stress were NK1.1+ (approximately 90%). As shown in control mice, 60–70% of CD3int cells in the liver were NK1.1+. In contrast to the liver, a change was not seen in the spleen and thymus of mice exposed to stress.

Two-colour staining for CD4 and CD8 was conducted (Fig. 2, right column). The population expanding in the liver was estimated to be CD4+. At this time, the proportion of CD8+ cells decreased to some degree. A similar change (i.e. an increase in the proportion of CD4+ cells) was seen in the spleen and thymus.

Comparison of the change in absolute number among various lymphocyte subsets in the liver

To determine the change in the absolute number of various lymphocyte subsets in the liver after exposure to stress, repeated experiments (n = 6) were conducted (Fig. 3). The data of the mean and 1 s.d. are represented. Although the absolute numbers of all tested lymphocyte subsets decreased after stress, such decrease was prominent in CD3hi cells but not prominent in NK cells and CD3int cells. In the case of CD3hi cells, the decrease was up to 70%. However, the decrease was only up to 35% in CD3int cells.

Fig. 3.

Fig. 3

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Variation in the proportion of lymphocyte subsets in the liver of mice after restraint stress. Six mice were used to produce the mean and 1 s.d. at each point. NK1.1+CD3int cells were found to be most resistant to stress. *P < 0·05.

When attention was focused on the subsets of CD3int cells, the decrease in the absolute number was much more prominent in the NK1.1− subset than in the NK1.1+ subset. In other words, the number of cells in the NK1.1+ subset of CD3int cells (i.e. NKT cells) was quite resistant to stress.

Parallel expansion of CD4−CD8− T cells after stress

Since one-third of CD3int cells or approximately 50% of NKT cells comprise an unusual T cell population with double-negative (DN) CD4−CD8− phenotype [21], it was confirmed whether such population expanded after exposure to stress (Fig. 4). In this experiment, two-colour staining for CD3 (green) and a mixture of CD4 and CD8 (red) was conducted. It was clearly demonstrated that, in parallel with the expansion of CD3int cells and NKT cells, DN CD4−CD8− cells did also expand.

Fig. 4.

Fig. 4

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A prominent increase in the proportion of DN CD4−8− cells in the liver of mice exposed to restraint stress. Two-colour staining of liver lymphocytes for CD3 and a mixture of CD4 and CD8 was conducted. The data shown here are representative of three experiments. Numbers in the figure represent the percentages of fluorescence-positive cells in corresponding areas.

Stress suppressed NK cytotoxicity but not self-reactive NKT cytotoxicity

We investigated how restraint stress modulated these cytotoxicities by using hepatic and splenic lymphocytes (Fig. 5a). NK cytotoxicity was identified by YAC-1 targets, whereas self-reactive NKT cytotoxicity was identified using syngeneic thymocytes. Both cytotoxicities were demonstrated in hepatic lymphocytes isolated from control mice and stress-exposed mice. However, NK cytotoxicity was prominently suppressed at 24 h after restraint stress (P < 0·05). Such suppression was not observed in NKT cytotoxicity after stress.

Fig. 5.

Fig. 5

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NK and NKT cytotoxicities in the liver and spleen. (a) Analysis of the cytotoxicities before and after restraint stress. ○, Control liver; Δ, control spleen; •, stress liver; ▴, stress spleen. (b) Analysis in the liver of perforin −/– mice and gld mice. ○, Control; •, stress. Cytotoxicity mediated by NK cells was examined against YAC-1 targets, whereas that mediated by NKT cells (i.e. self-reactive cytotoxicity) was examined against syngeneic thymocytes. Effector lymphocytes were obtained from the liver and spleen in B6 mice with or without exposure to stress (12 h and 24 h), and were used at the indicated target-to-effector cell ratios of 4 h incubation. Effector lymphocytes were also obtained for the liver of normal B6 mice, perforin −/– B6 mice, and B6-gld/gld mice before and after restraint stress (24 h). The mean and 1 s.d. were produced by triplicate cultures. *P < 0·05.

We have reported that NKT cells have fewer cytoplasmic granules which contain perforin than do NK cells [21]. They express the Fas ligand and mediate self-reactive cytotoxicity via the Fas ligand/Fas system rather than the perforin system [14]. This possibility was examined using perforin knockout (perforin −/–) B6 mice and B6-gld/gld (Fas ligand−) mice (Fig. 5b). These mice were exposed to restraint stress for 24 h, and hepatic lymphocytes were isolated for use as effector cells in the cytotoxicity assays. Almost all NK cytotoxicity against YAC-1 targets disappeared when effector cells were obtained from perforin −/–mice. On the other hand, such a phenomenon was not observed when effector cells were obtained from Fas ligand− mice. In contrast, all cytotoxicity against syngeneic thymocytes disappeared when effector cells were obtained from Fas ligand− mice. This was true when effector cells were obtained from stress-exposed mice, but not observed in perforin −/–mice.

Administration of adrenalin or glucocorticoid induced the similar phenomenon in vivo to that of stress

When mice are exposed to restraint stress, the major humoral events include the secretion of catecholamines and glucocorticoids [15]. We investigated whether the administration of either adrenalin or hydrocortisone induced the similar phenomenon to that of restraint stress (Fig. 6). A single injection of adrenalin or hydrocortisone at the indicated doses was conducted and the NK (against YAC-1) and NKT (against syngeneic thymocytes) cytotoxicities were analysed 24 h after the injection. Both injections were found to induce the suppression of NK cytotoxicity (P < 0·05) and the enhancement of NKT cytotoxicity (P < 0·05), simultaneously. Repeated experiments (n = 3) confirmed this result.

Fig. 6.

Fig. 6

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Variation of NK and NKT cytotoxicities in liver mononuclear cells (MNC) of mice when administered with either adrenalin or hydrocortisone. NK (against YAC-1) and NKT (against syngeneic thymocytes) cytotoxicities of liver MNC were examined 24 h after the injection of adrenalin or hydrocortisone. The cytotoxicities were measured at the indicated target-to-effector cell ratios of 4 h incubation. The data shown here are representative of three experiments. *P < 0·05. ○, Control; •, adrenalin 20 μg; ▴, adrenalin 100 μg; ▪, steroid 1 mg.

Determination of effector cells for NK and NKT cytotoxicities

In a final portion of the present experiments, we directly confirmed the effector cells for NK and NKT cytotoxicity. NK cells with the CD3−NK1.1+ phenotype and NKT cells with the CD3intNK1.1+ phenotype were purified from liver MNC in B6, perforin −/–, and gld mice by a cell sorter (Fig. 7). As shown previously [20] and in this study, NK cells were the major effector cells for NK cytotoxicity against YAC-1 cells, while NKT cells were the restricted effector cells for NKT cytotoxicity against syngeneic thymocytes in control B6 mice. It is noteworthy that purified NKT cells mediated a significant level of NK cytotoxicity in control mice, similar to the results in our previous study [20]. However, NK cells isolated from perforin −/–mice lacked NK cytotoxicity, NK cells isolated from gld mice showed a normal level of NK cytotoxicity, and NKT cells of perforin −/–mice showed a normal level of NKT cytotoxicity.

Fig. 7.

Fig. 7

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NK (□) and NKT (▪) cytotoxicities mediated by purified NK and NKT cells in the liver of B6, perforin −/–, and gld mice. Liver mononuclear cells (MNC) were isolated from B6, perforin −/–, and gld mice. After the staining for CD3 and NK1.1, CD3−NK1.1+ NK cells and CD3intNK1.1+ NKT cells were purified by a cell sorter. NK cytotoxicity was determined by YAC-1 targets while NKT cytotoxicity was determined by syngeneic thymocytes. Triplicate cultures were conducted at an effector-to-target ratio of 20:1. The mean and 1 s.d. are represented.

Discussion

In the present study, we exposed mice to restraint stress and found that the number of NK and NKT cells in the liver did not decrease prominently after stress. This was striking when compared with a change in the number of conventional T cells. Namely, the number of conventional T cells (i.e. NK1.1−CD3hi cells) decreased prominently after stress (after both 12 h and 24 h). It is conceivable that conventional T cells are much more sensitive to stress than NK and NKT cells. At present, we do not know the actual reason why the number of conventional T cells decreased in the liver. However, it is speculated that conventional T cells might undergo apoptosis in the liver or migrate from the liver to other sites. Other evidence was the differential effect of stress on NK and NKT cell function.

We reveal here a diversity of functional mechanisms between NK and NKT cells. Cytotoxicity mediated by NK cells was suppressed 24 h after stress, whereas self-reactive NKT cytotoxicity was rather elevated. This might be due to the fact that NK cytotoxicity was mediated through the perforin system but that NKT cytotoxicity functioned through the Fas ligand/Fas system. These results seem to be able to explain our clinical observation that many patients with tumours or with autoimmune diseases had elevated levels of NK and NKT cells in the blood [1–3] but showed a decreased level of NK cytotoxicity [9–12].

A direct comparison of the mechanisms involved in NK cytotoxicity with those in NKT cytotoxicity had remained to be investigated, although some independent studies on each lymphocyte subset have been reported [22–25]. We show here a clear difference in their cytotoxicity mechanisms: NK cytotoxicity decreased after exposure to stress. The underlying mechanisms remain unclear. One speculation is that the perforin system employed by NK cells requires the excretion of cytoplasmic granules and that stress-induced sympathetic nerve stimulation suppressed this excretion. It is known that the sympathetic nerve system is responsible for the suppression of secretion at the level of the cell and of the body [26]. On the other hand, the Fas ligand/Fas system may not require a secretion mechanism due to direct molecular interaction.

The relationship between NK cytotoxicity and the perforin system has already been reported by various other investigators [22–25]. We previously reported that NKT cells mediated the cytotoxicity against targets (e.g. syngeneic thymocytes) different from those of NK cells [14]. The major effector mechanism of NKT cytotoxicity seems to be the Fas ligand/Fas molecules. In the final part of these experiments, we confirmed that the major effector cells for NK cytotoxicity were CD3−NK1.1+ NK cells through the perforin system, while those for NKT cytotoxicity were CD3−NK1.1+ NK cells through the Fas ligand/Fas system. On the other hand, cytotoxic T cells (i.e. conventional T cells) have the ability to use both the perforin system and the Fas ligand/Fas system [27].

Other evidence in this study was that the NK1.1− subset of CD3int cells was considerably stress-sensitive in number, as were conventional T cells. There were two subsets among CD3int cells (i.e. extrathymic T cells in the liver): 60–70% of CD3int cells being NK1.1+ (i.e. NKT cells), while the other being NK1.1− [21]. NKT cells comprise DN CD4−8− and CD4+ cells, whereas the NK1.1− subset comprises CD8+ cells. Many investigators have emphasized only the function of NKT cells [28–31]. However, the biological function of the NK1.1− subset of CD3int cells cannot be neglected. Thus, point mutations of polymorphic MHC antigens (e.g. bm1 or bm12 disparity) were recognized by the NK1.1− cells of CD3int cells [32,33]. Such a NK1.1− subset (of bm1 or bm12 origin) showed cytotoxicity against hepatocytes of B6 origin after activation. Similarly, thymus-derived T cells acquire potent cytotoxicity (i.e. non-specific killer activity) after mitogenic stimulation [34–37].

We previously reported that the secretion of catecholamines and glucocorticoids is induced when mice are exposed to restraint stress [15]. Therefore, we investigated whether the in vivo administration of adrenalin or hydrocortisone induced a similar response to that of restraint stress. Both of them could simultaneously induce the suppression of NK cytotoxicity and the augmentation of NKT cytotoxicity. It is conceivable that the present phenomenon induced by stress was eventually mediated by these humoral factors. In any case, the present results add additional information about NKT cells as well as NK cells in mice.

Acknowledgments

We wish to thank Mrs Yuko Kaneko for preparation of the manuscript.

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