New Investigator Award
Distress and expression of natural killer receptors on lymphocytes

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Abstract

Chronic distress has been associated with alterations in natural killer (NK) cell and T cell percentages and function. NK cells express inhibitory and stimulatory receptors that regulate cytotoxicity and cytokine secretion. T cells and T cells expressing CD56 (NKT cells) also express these NK-associated receptors, which are thought to serve the same function. The objective of this study was to examine the relationship between distress and expression of NK-associated receptors on NK cells, T cells, and NKT cells. Using multicolor flow cytometry and validated questionnaires, we studied twenty-nine healthy adults with a bimodal age spread. Whereas distress was related to significantly lower percentages of CD3+ T cells, it was related to significantly higher percentages of NKT cells. Distress was associated with significantly higher percentages of T cells expressing NK-associated receptors including CD94 and KIR. In contrast, distress was associated with significantly lower percentages of NK cells bearing KIR (GL183) receptors. Our findings suggest a possible role for NK-associated receptors in distress-related alterations in lymphocyte maturation, trafficking, or activity.

Introduction

Chronic distress has been associated with modulation of the activity of natural killer (NK) cells and T lymphocytes. Depression (Irwin et al., 1986, Irwin et al., 1992), bereavement (Irwin et al., 1988), and life stress (Ben-Eliyahu et al., 1999, De Gucht et al., 1999, Esterling et al., 1994) have all been associated with impaired NK cell activity, and multiple studies have shown associations of depression (Irwin, 2001) and life stress (Herbert and Cohen, 1993b) with impairments in functional activity of lymphocytes. Moreover, daily hassles (Brosschot et al., 1994), life stress (Castle et al., 1995, Pariante et al., 1997), and depression have been associated with decrements in numbers or percentages of NK cells and T cells (Herbert and Cohen, 1993a, Herbert and Cohen, 1993b), although not all findings have been consistent (Schleifer et al., 1989). This modulation is thought to be mediated in part by adrenergic activity (Gan et al., 2002) and by neuroendocrine hormones such as cortisol (Gatti et al., 1987). Distress-induced modulation of trafficking and functional activity of lymphocytes via beta adrenergic receptors (Gan et al., 2002, Sanders et al., 2001) and glucocorticoid receptors (Dhabhar and McEwen, 2001, Plaut, 1987) has been demonstrated. However, both T cells and NK cells possess a number of other surface receptors that regulate their activity. Little is known regarding the relationships of psychosocial distress with the expression of other surface receptors, such as NK-associated receptors, on lymphocytes.

NK cells are commonly defined by the expression of a CD3 CD56+ surface phenotype (Borrego et al., 2002). In addition, NK cells express a variety of surface receptors that regulate their activity. One function of this regulation is to prevent expression of cytotoxic activity against cells identified as self (Backstrom et al., 2004). Several families of regulatory NK receptors have been described. One type is a heterodimer of CD94 and NKG2 of the C-type II lectin superfamily. NKG2 exists in several forms—NKG2A and B isoforms are inhibitory whereas NKG2C and E isoforms are stimulatory. A second family of receptors includes “killer immunoglobulin-like receptors” or KIR. KIR isoforms have 2 or 3 immunoglobulin-like domains (called 2D or 3D, respectively). KIR expression is genetically determined, and KIR molecules may be either stimulatory or inhibitory (Long et al., 2001).

The cytotoxic activity of NK cells is thought to be determined by a balance between stimulatory and inhibitory signals, with activation of NK cells occurring when stimulatory signals exceed inhibitory signals (Backstrom et al., 2004). Ligation of stimulatory receptors results in downstream signaling that initiates NK effector activities. Engagement of inhibitory receptors results in interruption of intracellular signals that would otherwise result in activation (Backstrom et al., 2004). Inhibitory KIR have also been shown to impair NK cell adhesion to target cells, thereby allowing NK cells to release from cells expressing “self” major histocompatibility complex (MHC) class I ligands. This frees them to attach to more appropriate targets and protects normal cells from NK killing (Long et al., 2001).

Some T-cells also express NK-associated receptors, including those of the CD94/NKG2 and KIR families. It has been proposed that KIR may act to “fine-tune” specific aspects of the immune response by altering the ability of lymphocytes to be activated by the T cell receptor-CD3 complex (Besostri et al., 2000). Stimulatory receptors amplify signals from the T cell receptor, prolong the immune response, and protect cells from activation induced cell death. However, if this stimulation goes unchecked, autoimmunity may result (Snyder et al., 2004). NK-associated receptors appear to regulate terminally differentiated effector and memory T cells with high levels of clonal expansion (Snyder et al., 2004) and have been identified on clonally expanded CTL populations such as those found in chronic inflammation as well as on T lymphocytes in older adults (Tarazona et al., 2000). T cells with NK-associated receptors have also been found in higher frequencies among patients with autoimmune diseases (Markovic-Plese et al., 2001, Moosig et al., 1998, Warrington et al., 2001), atherosclerotic plaques (Nakamima, 2003), and chronic infections (Dalod et al., 1999, Trimble et al., 2000). Inhibitory KIR have been shown to inhibit the activation of tumor-specific CTL, thus impairing T-cell function, including cytolytic activity and cytokine production (Mingari et al., 1996, Mingari et al., 1998). On both T and NK cells, inhibitory receptors generally outnumber stimulatory receptors (Valiante et al., 1997).

In addition to classically defined NK (CD3CD56+) and T (CD3+CD56) lymphocytes, a subset of T lymphocytes expressing the NK marker CD56 has been described; these have been called NKT cells (Smyth and Godfrey, 2000). NKT cells are heterogeneous and may represent recently activated effector T cells (Pittet et al., 2000), senescent T cells (Tarazona et al., 2000), or a distinct T cell lineage (Godfrey et al., 2004). These latter NKT cells utilize a unique T cell receptor (TCR) to recognize glycolipid antigen in the context of CD1d, a non-classical MHC class I molecule. These cells are cytotoxic and show rapid production of high levels of both TH1 and TH2 cytokines, particularly IFNγ and IL-4 (Smyth and Godfrey, 2000, Tarazona et al., 2000). NKT cells are thought to have an immunoregulatory function, and have been shown to both promote (Kawano et al., 1998, Smyth et al., 2000) and suppress anti-tumor immunity (Pardoll, 1996, Terabe et al., 2000) and to suppress delayed type hypersensitivity (Moodycliffe et al., 2000). NKT cells display KIR and CD94 family receptors as well.

Expression of NK-associated receptors on lymphocytes has been shown to be modulated by cytokines including TGF-β and interleukin-2 (Guerra et al., 1999, Shin et al., 2004) which, in turn, are known to be influenced by behavioral factors (Glaser et al., 1998, Kohut, 2002, Smith et al., 2002). We hypothesized that KIR and CD94/NKG2 receptors may be part of regulatory pathways by which stress effects on lymphocytes are mediated. The development of monoclonal antibodies to these receptors allowed us to address this question. As expression of both families of NK-associated receptors is more likely to be inhibitory than stimulatory (Valiante et al., 1997), we hypothesized that individuals with higher levels of distress would have higher percentages of both KIR and CD94 receptors on conventional NK cells (CD3CD56+), T cells (CD3+CD56) and NKT cells (CD3+CD56+). Furthermore, as few psychoneuroimmunology studies include a wide spectrum of age (Segerstrom and Miller, 2004) we included both younger and older age groups in the sample.

Section snippets

Participants

Participants were recruited via community advertisements and from the local senior citizen center as part of a larger study investigating effects of age on NK-associated receptors. To maximize inclusion of both young and older participants, inclusion criteria included age between 21 and 30 or over 65. Exclusion criteria included potential immunologic confounds such as history of illness affecting the immune response (e.g., multiple sclerosis, lupus), current use of immunomodulatory medications

Descriptive statistics and covariates

Descriptive statistics for cells expressing NK-associated receptors are shown in Table 1. Whereas over half of NK cells expressed receptors of the CD94 (64.4%) or KIR (52.9%) families, rather few T cells expressed CD94 (3.97%) or KIR (1.56 %) receptor families. Slightly under half (48.9%) of NK cells expressed the inhibitory receptor NKG2A whereas only about 2.39 percent of T cells expressed this receptor. NKT cells expressed intermediate percentages of CD94 (40.0%), NKG2A (19.64%), and KIR

Discussion

This exploratory study examined relationships of distress with NK associated receptors on lymphocytes. The main findings of this study were that individuals with greater distress had higher percentages of T lymphocytes expressing surface receptors characteristic of NK cells, including CD94 and KIR, and higher percentages of CD3+CD56+ NKT cells. This was in the context of an association of distress with lower percentages of CD3+CD56 T cells overall. Distress was associated with lower

Acknowledgments

This work was supported in part by NIH R01 DE11139, the Carver Foundation, NIH R21 CA88293, and Grant #RR00059 from the General Clinical Research Centers Program, National Center for Research Resources, National Institutes of Health. We thank Linda Snetselaar for help with study design, Colleen Fullenkamp and Phyllis Stumbo for help with recruitment and data collection, and Bridget Zimmerman for statistical assistance.

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