NK Cells and T Cells Are Phenotypically and Functionally Defective

The Journal of Immunology

NK Cells and ␥␦ⴙ T Cells Are Phenotypically and Functionally Defective due to Preferential Apoptosis in Patients with Atopic Dermatitis1 Michie Katsuta,2* Yukio Takigawa,* Momoko Kimishima,* Miyuki Inaoka,* Ryo Takahashi,† and Tetsuo Shiohara*† Innate immune cells mediate a first line of defense against pathogens and determine the nature of subsequent acquired immune responses, mainly by producing profound amounts of cytokines. Given these diverse tasks, it is predictable that defective NK and ␥␦ⴙ T cell responses could be the underlying mechanism for the immunological alterations observed in atopic dermatitis (AD). Indeed, the frequencies of circulating NK cells and ␥␦ⴙ T cells were profoundly reduced in AD patients. They also displayed a defective ability to sustain TNF-␣ and IFN-␥, but not IL-4, production after in vitro stimulation, and the defect was restricted to innate immune cells. Surprisingly, on the depletion of CD14ⴙ monocytes, this selective impairment of TNF-␣ and IFN-␥ production was restored to levels comparable to that observed in controls. Release of IL-10 from monocytes was not a major mechanism of the NK and ␥␦ⴙ T cell dysfunction. Apoptosis as revealed by annexin V binding, was preferentially observed in NK and ␥␦ⴙ T cells from AD patients when stimulated in the presence of monocytes, and depletion of monocytes significantly protected these cells from apoptotic cell death. Preferential apoptosis of NK cells by activated monocytes in AD patients was cell-contact-dependent. These results indicate that, once NK and ␥␦ⴙ T cells in AD patients are in immediate contact with activated monocytes, these cells are specifically targeted for apoptosis, leading to the reduced type 1 cytokine production, thereby directing subsequent acquired immune responses toward a type-2 pattern and increasing susceptibility to infection. The Journal of Immunology, 2006, 176: 7736 –7744.

A

predominance of type 2 immune response and increased susceptibility to cutaneous viral and bacterial infections have been generally regarded as specific features reflecting immune dysregulation in atopic dermatitis (AD)3 (1, 2). It remains unknown, however, whether there might be a logical link between these alterations in AD. In this regard, there is mounting evidence to suggest that innate immunity not only provides a rapid antimicrobial host defense that precedes the acquired immune response, but also have a role in determining the type of downstream acquired immune response through their ability to quickly release large quantities of cytokines (3–5). These immunological phenomena led to the notion that innate immune cells, such as NK cells and ␥␦⫹ T cells, particularly those producing the type 1-polarizing cytokine, may be phenotypically and functionally defective in AD, providing a mechanism linking development of type-2-dominated acquired immune responses with a decreased resistance against various pathogens in AD. Nevertheless, the importance of innate immune cells in AD has not been analyzed in great detail yet.

*Department of Dermatology and †Division of Flow Cytometry, Kyorin University School of Medicine, Tokyo, Japan Received for publication April 29, 2005. Accepted for publication March 16, 2006. 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 The study was supported in part by grants from the Ministry of Education, Sports, Science, and Culture of Japan (to R.T. and T.S.), the Ministry of Health, Labor, and Welfare of Japan (to T.S.), and Long-Range Research Initiative by Japan Chemical Industry Association (to T.S.). 2 Address correspondence and reprint requests to Dr. Michie Katsuta, Department of Dermatology, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 181-8611, Japan. E-mail address: [email protected] 3 Abbreviations used in this paper: AD, atopic dermatitis; MFI, mean fluorescence intensity.

Copyright © 2006 by The American Association of Immunologists, Inc.

NK cells were among the first of these cell types to be examined extensively in AD: many earlier studies demonstrated a dysfunction of NK cells in AD, as indicated by a decrease in NK cell numbers (6) and MHC-nonrestricted cytotoxicity against the standard NK-sensitive target cells (7). Nevertheless, conflicting data have been also reported (8). In addition, most recent studies that have addressed innate cell numbers and functions in AD have largely emphasized the role of dendritic cells/macrophages (9, 10), while ignoring the fact that ␥␦ⴙ T cells, another important cell type involved in innate immunity, also have the potential to regulate the functional activities not only of acquired immune cells but also of other innate immune cell, such as NK cells (11). Although human ␥␦ⴙ T cells express a variety of NK receptors, and the phenotypic and functional overlaps between NK cells and ␥␦ⴙ T cells have been suggested (12), the frequency and function of ␥␦ⴙ T cells in AD have not been as well studied (13) as that of monocytes or NK cells, and their relationships to one another have not been defined. Thus, it remains undetermined to what extent NK cells and ␥␦ⴙ T cells are defective in AD, and whether their functions are intrinsically altered. To define, on a cell-specific basis, the frequency and function of NK and ␥␦ⴙ T cells freshly isolated from patients with AD, we have analyzed their frequencies and cytokine expression of proinflammatory (TNF-␣), type 1 (IFN-␥), and type 2 (IL-4) cytokines in NK and ␥␦ⴙ T cells as well as acquired immune cells. The results presented in this study indicate that the frequencies of circulating NK cells and ␥␦ⴙ T cells are profoundly reduced in patients with AD as compared with healthy control subjects. These NK and ␥␦ⴙ T cells in AD displayed a defective ability to sustain TNF-␣ and IFN-␥, but not IL-4, production after an in vitro stimulation. The impairment of early TNF-␣ and IFN-␥ production in AD appears to be restricted to cells of innate immune cell lineage, 0022-1767/06/$02.00

The Journal of Immunology probably resulting in a sustained impairment of type 1-acquired immune function. We further demonstrate that the selective impairment of TNF-␣ and IFN-␥ production by ␥␦ⴙ T cells and NK cells in AD can be restored to levels comparable to that observed in controls on the depletion of CD14⫹ monocytes. Importantly, a preferential apoptosis of the type 1 cytokine producing NK and ␥␦⫹ T cells in the presence of monocytes was found to be a major mechanism responsible for a sustained impairment of type 1 innate immune function in AD. Our results highlight the importance of functional interactions between cells of the innate immune system for the elucidation of pathogenetic mechanisms of AD.

Materials and Methods Subjects Twenty patients (16 men and 4 women; age, 24 – 40; median age, 31.4 ⫾ 2.2 years) with adult type AD, according to the criteria of Hanifin and Rajka (14), were enrolled in this study. Ten healthy volunteers (7 men and 3 women; age, 25–35 years; median age, 28.7 ⫾ 1.0 years) who had no history of asthma, allergic rhinitis, or AD were selected as age-matched normal controls. Informed consent was obtained from all subjects before entry into the study. All patients had long-standing mild-to-severe disease with typical eczematous skin lesions, elevated serum IgE levels (⬍1000 IU/ml), and specific serum IgE Abs to aeroallergens. The severity of the disease was clinically assessed as mild (a body involvement of ⬍20% of the surface, and IgE levels lower than 1000 U/ml), moderate (a body involvement of 20 –50% of the surface, and IgE levels higher than 1000 IU/ml), or severe (a body involvement of ⬎50% of the surface, and IgE levels higher than 1000 IU/ml). The severity of skin involvement was evaluated according to the method of Rajka and Langeland (15): mild (score 3– 4), moderate (4.5–7.5), and severe (⬍8). None of the patients or control subjects received medications such as corticosteroids or cyclosporin A, or any systemic therapy, including UV light therapy, within at least 4 wk before study entry. Topical corticosteroids were withheld for at least 2 days before blood collection. All blood samples were taken between 9:00 am and 11:00 am to avoid problems associated with circadian rhythm. In some patients with AD and controls, heparinized venous blood samples were taken on several occasions for estimation of the fluctuation of the results depending on the severity of the disease: only those patients who were defined as fully relapsed or fully in remission were included in this study. PBMC were isolated from heparinized venous blood from study subjects by density gradient centrifugation on Ficoll-Paque (Pharmacia) for phenotypic and cytokine studies.

Flow cytometry PBMCs were stained with a combination of labeled mAb and measured by flow cytometry as described previously (15). Expression of surface Ags on gated lymphocytes was assessed by flow cytometry on a FACSCalibur (BD Biosciences) with a Paint-a-Gate program (BD Biosciences), with the following Abs: FITC-labeled anti-TCR␣␤ and anti-TCR␥␦, PE-labeled antiIFN-␥, anti-TNF-␣, and anti-IL-4, PerCP-labeled anti-CD8, and allophycocyanin-labeled anti-CD3 and anti-CD4 were purchased from BD Biosciences. Anti-CD19-FITC and anti-CD56-allophycocyanin were purchased from Beckman Coulter. The absolute numbers of circulating lymphocyte subpopulations were calculated from the results of standard white blood cell counts and differential analysis from blood smears. The flow rate was adjusted to ⬍200 cells/s, and, at the least, 5 ⫻ 103 cells were analyzed for each sample. Simultaneous flow-cytometric assessment of surface phenotype and intracellular cytokine synthesis was performed as described previously (16). Briefly, isolated PBMCs (106/ml) were stimulated for various times, as indicated, with 25 ng/ml PMA plus 1 ␮g/ml ionomycin in the presence of 10 ␮g/ml Brefeldin A, an inhibitor of protein translocation from the endoplasmic reticulum to the Golgi apparatus: intracellular cytokines, TNF-␣, IFN-␥, and IL-4, produced during the 2- or 4-h period (in some experiments, 2-, 4-, 6-, 8-, 12-, or 24-h) after stimulation with PMA and ionomycin, were detected in cells by adding Brefeldin A 2 h before cell harvest. In some experiments, in which intracellular cytokines were produced during the 4-h period after stimulation was detected, Brefeldin A was added 4 h before cell harvest. For intracellular staining, cells harvested from stimulation cultures were washed twice with PBS/BSA, incubated in 0.5 ml of lysing solution and 0.5 ml of permeabilizing solution (BD Biosciences) for 10 min at room temperature, washed with PBS/BSA, and then incubated for 25 min with appropriately titered PE-labeled anti-IFN-␥, antiTNF-␣, or anti-IL-4 mAb. The samples were analyzed on a FACSCalibur

7737 flow cytometer (BD Biosciences). For control, mAbs were replaced with nonreactive isotype-matched mAbs. The percentage of cytokine-producing cells upon stimulation for the indicated period was calculated by subtracting the percentage of background production in parallel nonstimulated samples. In some experiments, mean fluorescence intensity (MFI) was used as a parameter to assess levels of cytokine synthesis in the indicated populations. MFI in linear scale was obtained by adjusting the fluorescence gain so that 5% of the cell sample with the greatest fluorescence were positive in the highest fluorescence channel.

Depletion of CD14⫹ monocytes from PBMC CD14-depletd PBMC were obtained by negative selection with anti-CD14 mAb using the magnetic cell separation system (MACS; Miltenyi Biotec). Briefly, 1 ⫻ 107 PBMC were incubated with anti-CD14 mAb-conjugated MACS beads, and the labeled cells (CD14⫹) were removed with a magnetic column. By this procedure, the CD14-depleted PBMC contained ⬍0.5% contaminating CD14⫹ monocytes. Phenotypically, the CD14⫺ population was composed of 49.0 ⫾ 1.8% CD4⫹, 20.1 ⫾ 1.0% CD8⫹, 17.6 ⫾ 1.1% CD56⫹, and 6.4 ⫾ 1.5% TCR-␥␦⫹ cells as determined by flow cytometry. There was no significant phenotypic difference before and after the removal of CD14⫹ cells from the cultures, because PBMC before the removal of CD14⫹ cells were composed of 51.2 ⫾ 1.3% CD4⫹, 20.3 ⫾ 2.7% CD8⫹, 16.9 ⫾ 0.9% CD56⫹, and 6.0 ⫾ 1.6% TCR-␥␦⫹ cells.

Apoptosis in NK cells and ␥␦⫹ T cells Apoptotic cells were stained with annexin V-FITC (Medical and Biological Laboratories) and propidium iodide (BD Pharmingen) according to the manufacturer’s directions and analyzed by flow cytometry. Cell debris was excluded from analysis by appropriate forward light scatter threshold setting. Cells staining with annexin V-FITC, but not propidium iodide, were regarded as early apoptotic cells. This method was used to avoid contamination of the data with debris or late apoptotic cells.

Coculture of CD14⫹ monocytes and CD14-depleted PBMC using Transwell plates In some experiments, CD14⫹ monocytes and CD14-depleted PBMC prepared from AD patients and controls, as described above, were cultured in 24-well plates equipped with a Transwell insert (Corning), to determine whether preferential apoptosis of innate immune cells by monocytes in AD was cell-contact-dependent. The Transwell insert consisted of a 200-␮l upper well separated from a 800-␮l lower well by a 0.4-␮m microporous polycarbonate membrane, which permits the circulation of soluble factors between both wells but prevents cell contact between cells seeded in the upper and lower wells. Two different concentrations of CD14⫹ monocytes (1 ⫻ 105 and 3 ⫻ 105 cells/200 ␮l) were seeded in the upper well and cocultured with two different concentrations of CD14-depleted PBMC (9 ⫻ 105 and 7 ⫻ 105 cells/800 ␮l) seeded in the lower well, at ratios of 1:9 and 3:7, respectively. These cells were cultured in the presence of PMA and ionomycin for 4 h in the Transwell plates. As controls, CD14-depleted PBMC were cocultured either with or without CD14⫹ monocytes in the lower well in the same plate at identical ratios.

Statistical analysis Data were analyzed using an unpaired Student t test to determine significant differences between patient and control groups. All findings were considered significant at a p value of ⬍0.05. Nonparametric Spearman rank correlation was used to test for correlation between clinical variables and number of NK and ␥␦⫹ T cells.

Results NK and ␥␦

⫹

T cells in AD vs control

We first compared the percentages of NK cells and ␥␦⫹ T cells as well as acquired immune cells such as ␣␤⫹ T cells in PBMC of AD patients and healthy controls, because previous phenotypic and functional studies on NK cells in AD have yielded conflicting results: many authors demonstrated significantly lower percentages of CD56⫹NK cells in PBMC and significant reduction in NK cell activity (6, 7), whereas others reported no significant differences in AD as compared with controls (8). Because NK cells are major components of the innate immune system, particularly in antiviral defense (3, 17), subclinical viral infections often associated with the exacerbations of AD lesions may have obscured the large differences in number and function of NK cells between the two

7738 groups. In this study, therefore, blood samples were usually obtained from patients not associated with exacerbations of AD lesions; and in some patients, on several occasions they were obtained at active and quiescent phases. As shown in Fig. 1, the frequencies of CD56⫹NK cells and ␥␦⫹ T cells were significantly lower in the patients with AD (median ⫽ 8.9% in NK and 4.1% in ␥␦, respectively) than controls (median ⫽ 27.1% in NK and 8.4% in ␥␦, respectively). In contrast, there were no significant differences in the percentages of acquired immune cells such as ␣␤⫹ T cells, CD4⫹ T cells, and CD8⫹ T cells, with the exception of CD19⫹ B cells, which were found to be significantly increased in AD. When patients with AD were subdivided into the two groups according to the clinical severity, severe and mild/moderate, there was no significant difference in the frequencies of these cells between the two (Table I), although the percentages of NK cells were somewhat lower in the mild/moderate group than in the severe group. Because exacerbations associated with viral infections were much more often observed in the severe group than the mild/moderate group, repeated viral infections in the severe group may have caused a compensatory increase in the percentage of circulating NK cells; in support of this possibility, the percentages of CD56⫹NK cells were higher during the exacerbation than the quiescent phase in each individual, when the same patients were analyzed on two occasions, fully relapsed or fully in remission (data not shown). However, because we considered that no fundamental difference was found with respect to innate immune cells between the two subgroups, the two were analyzed together without dividing for all additional experiments. Impairment of IFN-␥ and TNF-␣ production by NK and ␥␦⫹ T cells in AD We next asked whether NK cells and ␥␦⫹ T cells in AD were impaired in their ability to produce proinflammatory (TNF-␣) and type 1 (IFN-␥) cytokines, as well as their frequencies in PBMC. An important but poorly studied aspect of cytokine production by

FIGURE 1. The percentages of CD4, CD8, ␥␦, NK, and B cells in PBMC from AD patients and controls. Twenty patients and 10 healthy controls were analyzed as described in Materials and Methods. Results are expressed as the mean percentage of cells ⫾ SEM in whole PBMC. ⴱ, Significantly different from the mean value of healthy controls; p ⬍ 0.05. ⴱⴱ, p ⬍ 0.01.

DEFECTIVE NK AND ␥␦⫹ T CELLS IN AD these innate cells in allergic diseases is the time frame after stimulation, because in previous studies intracellular cytokine determinations by PBMC were exclusively performed on those during a certain time period (usually 0 – 4 h) for the sake of simplicity: this indicates the need for a more extensive kinetic analysis of cytokines production for various time spans after stimulation. Our initial kinetic analysis of the intracellular cytokine response of NK cells to PMA and ionomycin for up to 24 h showed that IFN-␥ synthesis by NK cells of healthy controls increased up to 4 h of stimulation and gradually declined thereafter, whereas that of AD patients peaked at 2 h and rapidly declined (Fig. 2). Based on these findings, the ability of NK cells and ␥␦⫹ T cells to produce TNF-␣, IFN-␥, and IL-4 was analyzed at the two time points (0 –2 and 2– 4 h) after starting stimulation. IFN-␥ production by NK and ␥␦⫹ T cells from AD patients showed initially (at 2 h) the same frequencies as that from controls (Fig. 3). However, at later time points (2– 4 h), whereas a significant fraction of NK and ␥␦⫹ T cells in controls were found to produce IFN-␥, the frequency of IFN-␥-producing NK and ␥␦⫹ T cells in AD decreased dramatically (Fig. 3, A and B). A loss of IFN-␥-producing capability of innate immune cells from AD patients was not attributable to the exhaustion of the capability as the result of repeated stimuli received in vivo, because the dramatic decrease in IFN-␥ production with time was observed regardless of whether the patients were analyzed during relapses or while being in remission (data not shown). High levels of TNF-␣ expression were detected at 2 h in all subsets tested (Fig. 3C). Proportionally more ␥␦⫹ and ␣␤⫹ (CD4⫹) T cells produced TNF-␣ than did NK cells at any time point examined. There was no significant difference in frequency of TNF-␣-producing NK and ␥␦⫹ T cells between AD patients and controls at 2 h. However, TNF-␣ production by ␥␦⫹ T cells and NK cells isolated from AD patients rapidly decreased to levels much lower than those observed in controls at later time points (4 h). The difference between AD patients and controls was not obvious in acquired immune cells such as ␣␤⫹ T cells. The frequency of IL-4-producing cells was very low compared with those of TNF-␣- and IFN-␥-producing cells (Fig. 3D). IL-4 production by innate immune cells in controls increased up to 4 h of stimulation, whereas the increase in innate immune cells from AD patients was less obvious. In contrast, the increased levels of IL-4 production by ␣␤⫹ T cells from AD patients were persistently observed throughout the time we followed: the frequency of IL4-producing ␣␤⫹ T cells was significantly higher in AD patients than in controls, a finding consistent with previous studies (18). Interestingly, a selective impairment of TNF-␣ and IFN-␥ production by NK and ␥␦⫹ T cells in AD patients was not obvious when intracellular flow cytometric analyses for cytokine expression were performed at a conventional fixed time point (0 – 4 h) (data not shown). We also compared the levels of cytokine expression by innate immune cells in AD patients and controls as revealed by MFI (Fig. 4, A and B). With regard to TNF-␣ and IFN-␥ expression, no significant difference was detectable between the two groups, despite the observed differences in frequency of cytokine-producing cells. These results suggest that the significant decrease in cytokine production by innate immune cells from AD patients at later time points (2– 4 h) would result from a preferential loss of the cytokine-producing cell population during the culture period. Recovery of TNF-␣- and IFN-␥-producing ability by innate immune cells on the depletion of CD14⫹ monocytes Because earlier studies have shown that NK cells are functionally suppressed by autologous monocytes (19, 20), we were interested


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Table I. Comparison of profile and immunological parameters between the two groups of AD patientsa Group

Severe Mild to Moderate Control a b c

No. of Patients

Age (year)

10 10 10

33.8 ⫾ 2.1 29.4 ⫾ 2.3c 28.7 ⫾ 1.0 c

Mean Score

IgE

8.6 ⫾ 0.2 5.6 ⫾ 0.4b

23096 ⫾ 7791 5879 ⫾ 2044b ⬍170

b

␥␦ (%)

NK (%) b

10.7 ⫾ 1.8 7.1 ⫾ 1.2c 27.1 ⫾ 3.3 c

B (%)

4.2 ⫾ 0.9 4.1 ⫾ 1.9c 8.4 ⫾ 1.6 c

16.1 ⫾ 1.3c 21.3 ⫾ 2.6c 9.7 ⫾ 1.7

Results are expressed as the mean ⫾ SEM. Data from the two groups of AD patients were considered significantly different when p ⬍ 0.05. No differences were found when the two groups of AD patients were compared.

in investigating whether depletion of CD14⫹ monocytes could result in the recovery of TNF-␣- and IFN-␥-producing ability by innate immune cells. We compared the cytokine-producing ability of these cells in PBMC with that of corresponding cells in monocytes-depleted PBMC. As shown in Fig. 5, A and B, unlike our initial expectation, removal of CD14⫹ monocytes did not significantly enhance the ability of these cells from controls to produce the cytokines. Surprisingly, however, depletion of CD14⫹ monocytes restored the cytokine-producing ability of NK (Fig. 5, A and B) and ␥␦⫹ T cells (data not shown) from AD patients to the same levels as controls. However, the cytokine-producing capability of ␣␤⫹ T cells from AD patients was not significantly enhanced by the depletion of CD14⫹ monocytes (Fig. 5C). The finding that CD14⫹ monocytes from AD patients significantly suppressed the cytokine-producing capability of innate immune cells suggested that IL-10 endogenously produced by the CD14⫹ monocytes may have significantly contributed to the inhibition. Therefore, we investigated whether treatment with anti-IL10R mAb could reverse the inhibitory effect of CD14⫹ monocytes on the cytokine-producing capacity of innate immune cells from AD patients. Although the IFN-␥-producing ability of innate immune cells from AD patients was marginally enhanced by treatment with anti-IL-10R mAb, the observed enhancement was neither drastic nor confined to innate immune cells from AD patients, as compared with an enhancement occurring after depletion of CD14⫹ monocytes, because IFN-␥ production by ␣␤⫹ T cells from either AD patients or controls was also enhanced by treatment with anti-IL-10R mAb (Fig. 6): thus, anti-IL-10R mAb did not reproduce the effect of depletion of CD14⫹ monocytes in AD patients. These results indicate that the release of IL-10 from CD14⫹ monocytes was not a major mechanism responsible for monocytes from AD patients to inhibit the cytokine-producing capacity of innate immune cells, and that factors other than IL-10 may contribute to the inhibition mediated by the monocytes.

FIGURE 2. Kinetics of intracellular expression of IFN-␥ by NK cells from AD patients and healthy controls up to 24 h after stimulation with PMA and ionomycin. Six patients and six healthy controls were analyzed as described in Materials and Methods. Results are expressed as the mean percentage of IFN-␥-positive cells ⫾ SEM in NK cells. ⴱ, Significantly different from the mean value of healthy controls; p ⬍ 0.05.

Innate immune cells from AD patients differ on the sensitivity to monocyte-induced apoptosis Because previous studies demonstrated that NK cells acquire features characteristic of apoptosis after incubation with autologous monocytes (21), we next examined whether innate immune cells from AD patients were more prone to undergo apoptosis upon stimulation in the presence of autologous monocytes than corresponding cells from controls. To test this hypothesis, we compared the degree of apoptosis of innate immune cells and acquired immune cells in PBMC left undepleted and CD14-depleted PBMC from AD patients and controls after stimulation. To confirm apoptosis, flow cytometric detection of extracellular annexin V binding was used. As shown in Fig. 7, apoptosis as revealed by annexin V binding was preferentially observed in innate immune cell populations from AD patients, and depletion of CD14⫹ monocytes significantly protected these cells from apoptotic cell death. These results indicate that innate immune cells from AD patients may have an intrinsically lower capacity for survival after stimulation in the presence of monocytes, and that this impairment could result from their increased sensitivity to apoptosis upon contact with autologous monocytes as compared with the corresponding populations from controls. Preferential apoptosis of NK cells by monocytes is contactdependent To determine whether preferential apoptosis of innate immune cells in the presence of activated monocytes was contact-dependent, CD14⫹ monocytes seeded in the upper well were cocultured with CD14-depleted PBMC seeded in the lower well at two different ratios, 1:9 and 3:7, respectively. These two ratios used were determined so as to closely reflect the concentrations of CD14⫹ monocytes in PBMC of healthy controls and AD patients, respectively, because the monocyte/NK cell interaction may dramatically

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FIGURE 3. Intracellular expression of IFN-␥ (A and B), TNF-␣ (C), and IL-4 (D) by NK cells, ␥␦⫹ T cells, and ␣␤⫹ T cells from AD patients (f) and healthy controls (䡺) at different time points (0 –2 and 2– 4 h) after stimulation with PMA and ionomycin (n ⫽ 9). A, Representative experiment showing that intracellular IFN-␥ expression in NK cells from AD patients decreases with time. The percentage of IFN-␥-positive NK cells in each sample is indicated in the upper right quadrant. Results are expressed as the mean percentage of cytokine-positive cells ⫾ SEM in each subset. ⴱ, Significantly different from the mean value of healthy controls; p ⬍ 0.05. ⴱⴱ, p ⬍ 0.01; ⴱⴱⴱ, p ⬍ 0.005

DEFECTIVE NK AND ␥␦⫹ T CELLS IN AD

FIGURE 4. Intracellular expression of IFN-␥ by NK cells and ␥␦⫹ T cells from AD patients and healthy controls, as revealed by MFI. NK cells and ␥␦⫹ T cells stimulated for the indicated period of time with PMA and ionomycin were analyzed for intracellular expression of IFN-␥ (MFI) using three-color techniques and FACSCalibur (BD Biosciences). A, Representative examples of intracellular expression of IFN-␥ by NK cells and ␥␦⫹ T cells from an AD patient and a healthy control during the 0- to 2-h (solid lines) and 2- to 4-h (broken line) periods are displayed in the histograms. MFI for each histogram is expressed by the number indicated by arrows. B, Intracellular expression of IFN-␥ by NK cells and ␥␦⫹ T cells from AD patients (f) and healthy controls (䡺), as revealed by the average MFI (n ⫽ 4). No significant differences were found when the two groups were compared.

alter depending on the ratios. As shown in Fig. 8, CD14⫹ monocytes from AD patients failed to induce apoptosis of autologous NK cells when both populations were physically separated by a Transwell membrane during culture, as compared with those cocultured in the lower well at the identical ratio. The effect of


7741

FIGURE 5. Influence of depletion of CD14⫹ monocytes from stimulation cultures on intracellular expression of IFN-␥ or TNF-␣ by NK cells or ␣␤⫹ T cells from AD patients and healthy controls. A, Flow cytometry dot plots of IFN-␥ expression by NK cells in PBMC left undepleted and CD14-depleted PBMC from healthy controls and AD patients. Proportions of cells in each quadrant are expressed as the percentage of the total population. The number in the upper right quadrant represents the percentage of NK cells expressing IFN-␥ in the PBMC on stimulation with PMA and ionomycin for the indicated culture period; the number in parentheses represents the percentage of IFN-␥-positive NK cells in total NK cells; and the number in the lower right quadrant represents the percentage of IFN-␥-negative NK cells in the PBMC. B, The percentages of NK cells expressing IFN-␥ or TNF-␣ in PBMC left undepleted and CD14-depleted PBMC from healthy controls (䡺) and AD patients (f) after stimulation with PMA and ionomycin for the indicated culture period (n ⫽ 4). Results are expressed as the mean percentage of IFN-␥ or TNF-␣-positive cells ⫾ SEM in NK cells. ⴱ, Significantly different from the mean value of healthy controls; p ⬍ 0.01. C, The percentages of ␣␤⫹ T cells expressing IFN-␥ or TNF-␣ in PBMC left undepleted and CD14-depleted PBMC from healthy controls (䡺) and AD patients (f) after stimulation with PMA and ionomycin for the indicated culture period (n ⫽ 4). Results are expressed as the mean percentage of IFN-␥- or TNF-␣-positive cells ⫾ SEM in ␣␤⫹ T cells. No significant differences were found when the two groups were compared.

7742

FIGURE 6. Marginal enhancement of IFN-␥ expression by NK cells and ␣␤⫹ T cells in anti-IL-10R mAb-treated cultures. PBMC from healthy controls and AD patients were stimulated with PMA and ionomycin in the presence of anti-IL-10 mAb at 5 ␮g/ml (3F9, rat IgG 2a, ␬; BD Pharmingen) or isotype-matched control mAb (JES3-19F1, rat IgG 2a, ␬; BD Pharmingen) at the same concentration, for the indicated culture period. Proportions of cells in each quadrant are expressed as the percentage of the total population: the number in the upper right quadrant represents the percentage of IFN-␥-positive NK cells (A) and ␣␤⫹ T cells (B) in the PBMC, and the number in the lower right quadrant represents the percentage of IFN-␥negative NK cells (A) and ␣␤⫹ T cells (B) in the PBMC. Results are from one representative experiment of four independent experiments using samples from four additional individuals each (four controls and four AD patients), in which similar results were obtained.

monocytes to induce apoptosis of NK cells was observed only when the two populations (CD14⫹ monocytes and CD14-depleted PBMC) from AD patients were cocultured in the lower well. In contrast, CD14⫹ monocytes from healthy controls did not induce a significant degree of apoptosis of autologous NK cells, regardless of whether they were separated by a Transwell membrane or cocultured in the lower well. As compared with NK cells, CD3⫹ T cells from the same AD patients were not susceptible to the effect of monocytes, regardless of whether they were separated from or cocultured with CD14⫹ monocytes. Thus, preventing cell contact between NK cells and monocytes clearly diminished the capacity of monocytes to induce apoptosis of NK cells. These results show that apoptosis of NK cells by activated monocytes in AD patients is a cell-contact-dependent process.

Discussion

Our results demonstrate that not only NK cells, but also ␥␦⫹ T cells, are profoundly decreased in number in AD regardless of the severity of the disease. Contrary to our initial expectation, the fre-

DEFECTIVE NK AND ␥␦⫹ T CELLS IN AD quency of NK cells was higher in the severe group than in the mild/moderate group, although the difference was not significant. Two possible explanations could be considered for the seemingly contradictory finding of the decrease in NK cell number in AD and the increase in the severe group and in patients during the exacerbation. First, compensation for the dysfunction through increased mobilization might account, at least partially, for the overall increase in NK cell number in the severe group and patients during the exacerbation, which may reflect the associated viral infections. If there is a true positive association between clinical activity and NK cell number in AD, one might also expect that a selection bias toward patients with a tendency toward repeated viral infections may have occurred in previous studies demonstrating no significant decrease in NK cell number. Second, prolonged treatment with topical corticosteroids, which was usually twice daily to affected areas until 2 days before the enrollment, may have caused the reduction in NK cell number, because previous studies demonstrated that topical corticosteroids causes a decrease in the circulating NK cell activity of normal subjects (22). This possibility is unlikely, however, because no significant reduction in NK cell number and function was seen in patients with psoriasis who used much higher amounts of topical corticosteroids for the longer period (M. Katsuta and T. Shiohara, unpublished data). In a sequential analysis in a single patient with AD, the amount of topical corticosteroids was not inversely related to the percentage of NK cells (data not shown). Thus, a decrease in NK cell and ␥␦⫹ T cell numbers seen in AD patients is unlikely a simple consequence of disease activity and prior treatment. Much effort has been devoted to NK cell cytotoxic activity against NK-sensitive tumor cells in AD, but their capacity to produce cytokines has remained largely undefined, although the potential relevance of these in vitro findings to the in vivo situation is important. The results of this study reveal that TNF-␣ and IFN-␥ production by NK cells and ␥␦⫹ T cells at single-cell level was significantly reduced in AD and that the impairment was evident, when cytokine production was individually assessed at various time intervals after stimulation, particularly at 2– 4 h. Because the kinetics of IFN-␥ production by acquired immune cells such as ␣␤⫹ cells were delayed compared with NK cells and ␥␦⫹ T cells, NK and ␥␦⫹ T cells are the major producers of IFN-␥ at earlier time points (2– 6 h) after stimulation. In considering the crucial role of IFN-␥ rapidly produced by these innate cells early after stimulation in mediating a first line of defense against pathogens and determining the nature of subsequent acquired immune responses, this early impairment of NK and ␥␦⫹ T cell functions in AD is likely to contribute to the overall response in a type-2 direction and to increased susceptibility to infection. The defective production of IFN-␥ and TNF-␣ by these innate immune cells in vitro in AD could be interpreted in several mechanisms, which may not be mutually exclusive: 1) the intrinsic defect in the capacity to produce type 1 cytokine; 2) an “exhaustion” of these innate immune cells that have been continuously activated in vivo; 3) overproduction of immunosuppressive cytokines such as TGF-␤ and IL-10 that may down-regulate immune responses mediated by these innate cells; and 4) preferential apoptosis of cytokine-producing NK and ␥␦⫹ T cells upon contact with monocytes. Because we have shown in this study that the capacity to produce IFN-␥ and TNF-␣ by these innate cells in AD was restored to levels comparable to that in controls upon depletion of monocytes before stimulation, it is unlikely that circulating NK and ␥␦⫹ T cells from AD patients are intrinsically defective in promptly producing IFN-␥ and TNF-␣ upon stimulation in vitro. A second possible explanation is that a reduced capacity of NK and ␥␦⫹ T cells to sustain IFN-␥ and TNF-␣ production represents an


7743

FIGURE 7. Preferential apoptosis of NK cells and ␥␦⫹ T cells from AD patients stimulated with PMA and ionomycin in the presence of CD14⫹ monocytes. Annexin V binding cells in each subset was evaluated using flow cytometry as described in Materials and Methods. Results are combined from four independent experiments and are expressed as the mean percentage of annexin V binding cells ⫾ SEM in each subset in undepleted PBMC before (䡺) and after stimulation with PMA and ionomycin (f), and CD14-depleted PBMC after stimulation (dotted bars). ⴱ, Significantly different from the mean value in undepleted PBMC stimulated with PMA and ionomycin; p ⬍ 0.05. ⴱⴱ, p ⬍ 0.01. †, Significantly different from the mean value of healthy control; p ⬍ 0.05.

effect resulting from a transient exhaustion of cytokine production in these cells, and therefore only a fraction of these cells may have been receptive or been able to receive the activation signal: many of these cells may be “burned out” in vivo in AD and become transiently refractory to reactivation by stimulation in vitro. Because our data indicate that the defect can be rapidly restored upon removal of CD14⫹ monocytes and that the defect can be demonstrated independently of disease severity, it is difficult to assign the defect in a reversible manner to a transient exhaustion of these innate cells. Because the addition of anti-IL-10R mAb did not restore the impaired capacity of monocyte-exposed NK cells in AD to produce IFN-␥, IL-10 released from monocytes appear to play at most a minor role. Importantly, our data indicate that the reduced capacity of NK and ␥␦⫹ T cells to produce IFN-␥ and TNF-␣ was particularly evident at longer times (2– 4 h) of stimulation, but not at 2 h, and that the levels of cytokine expression by NK and ␥␦⫹ T cells in

AD as revealed by the average MFI were not different from those in controls. The most likely interpretation of these observations we favor is that a reduced IFN-␥ and TNF-␣ production by NK and ␥␦⫹ T cells in AD at longer times would be due to a preferential apoptosis of cytokine-producing NK and ␥␦⫹ T cell subsets after stimulation in vitro in the presence of monocytes. Indeed, this preferential apoptosis of NK cells in the presence of monocytes is reflected in substantial increases in the percentages of NK cells by depletion of CD14⫹ monocytes in the 2- to 4-h treatment group (from 12.5 to 18.6%) in Fig. 5A. Moreover, a sizable fraction of NK and ␥␦⫹ T cells did undergo apoptosis after stimulation in vitro, and removal of CD14⫹ monocytes almost completely rescued these cells from undergoing apoptosis. Although our data show that NK and ␥␦⫹ T cells from AD are more prone to undergo apoptosis than ␣␤⫹ T cells upon cell-to-cell contact with monocytes, the mechanisms responsible for the difference in apoptotic propensity between innate immune cells and acquired immune

FIGURE 8. Contact-dependent apoptosis of NK cells, but not CD3⫹ T cells, by activated monocytes in AD patients. CD14⫹ monocytes seeded in the upper well were cultured with autologous CD14-depleted PBMC seeded in the lower well in the presence of PMA and ionomycin for 4 h. As controls, CD14-depleted PBMC were cocultured either with or without CD14⫹ monocytes in the lower well, and undepleted PBMC were cultured in the same conditions. Annexin V binding cells in each subset was evaluated using flow cytometry as described in Materials and Methods. Data represent the mean percentage of annexin V binding cells ⫾ SEM in cells gated on NK cells or CD3⫹ T cells under different culture conditions indicated, from three independent experiments performed at two different (1:9 and 3:7) CD14⫹/CD14⫺ ratios using samples from three individuals each, in which similar results were obtained. ⴱ, Significantly different from the mean value in CD14-depleted PBMC-cocultured CD14⫹ monocytes in the lower well; p ⬍ 0.05.

7744 cells remain unknown. Although earlier studies showed that an interaction between NK cells and monocytes has profound effects on NK cell function (18, 19), it remains unknown why NK and ␥␦⫹ T cells in AD are solely susceptible to the inhibitory signal from monocytes. Because one might assume that the functional result of the interaction is determined by the sum of the opposing signal, an alternative explanation for the differential effects of monocytes on NK and ␥␦⫹ T cells between AD patients and controls is that monocytes from AD patients may be specifically defective in supporting survival of these innate immune cells. In view of the preferential action of IL-15 on innate immune cells for mediating their development, homeostasis, and activation (23–26), the most likely candidate responsible for the observed difference in the capacity of monocytes between AD patients and controls would be IL-15. In support of this possibility, a recent study reported by Ong et al. (10) has demonstrated that the membranebound IL-15 expression is significantly lower in the monocytes of AD patients compared with that in normal controls and psoriatic patients. If IL-15 is an important physiological agent in preventing NK cells and ␥␦⫹ T cells from undergoing apoptosis, the net balance between antiapoptotic actions manifested by IL-15 and apoptotic actions probably manifested by reactive oxygen species (27) may be the critical set of signals delivered by monocytes that determine whether NK and ␥␦⫹ T cells are targeted for apoptosis upon contact with monocytes. Nevertheless, it would be premature to conclude that the immunological abnormality in AD primarily resides within the monocytes rather than the NK and ␥␦⫹ T cells. In this regard, future mix/match experiments using monocytes and NK cells from AD patients and controls will be required to definitely show that the defect in AD resides primarily in monocytes or NK cells. In view of the reported ability of IFN-␥ produced by NK cells to elicit production of IL-15 by activated monocytes (28), which may in turn stimulate NK cells to produce IFN-␥, the communication between NK or ␥␦⫹ T cells and monocytes seems to be a dialogue rather than a monologue, in which the monocytes respond to NK and ␥␦⫹ T cells as well. Similar reciprocal activating interactions between NK cells and CD14⫹ monocytes have been also reported in patients with inflammatory arthritis (29). NK cells enriched at inflammatory sites have been shown to engage with CD14⫹ monocytes in a reciprocal activatory fashion by promoting TNF-␣ production by CD14⫹ monocytes. The outcome of such communication, which is bidirectional regulation, would be reflected in the immunological alterations observed in AD. Such reciprocal functional interaction between monocytes and either NK cells or ␥␦⫹ T cells would play an important physiological role in the regulation of both innate and acquired immune responses. In conclusion, our observations raise the hypothesis that once NK cells and ␥␦⫹ T cells in AD patients are in immediate contact with activated monocytes in vivo, these cells are specifically compromised in their capacity to produce type 1 and proinflammatory cytokines, thereby directing subsequent acquired immune responses toward a type-2 pattern and increasing susceptibility to infection. On the basis of these results, we propose that the design of successful therapy for AD should target a regulatory pathway linking NK cells, ␥␦⫹ T cells, and monocytes.

Disclosures The authors have no financial conflict of interest.

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