Depressed and anxious mood and T-cell cytokine expressing populations in ovarian cancer patients

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Abstract

The adaptive immune response of ovarian cancer patients has been linked to survival, and is known to be impaired in the tumor microenvironment. Little is known about relationships between biobehavioral factors such as depressed mood and anxiety and the adaptive immune response in ovarian cancer. Thirty-seven patients with epithelial ovarian cancer and 14 patients with benign ovarian neoplasms completed psychosocial questionnaires pre-surgery. Lymphocytes from peripheral blood, tumor, and ascites (fluid around the tumor), were obtained on the day of surgery. Expression of the Type-1 cytokine interferon-gamma (IFNγ), and the Type-2 cytokine interleukin-4 (IL-4) by T-helper (CD4+) and T-cytotoxic (CD8+) cells was measured under autologous tumor-stimulated, polyclonally-stimulated, or unstimulated conditions. Links with mood were examined. Among cancer patients, marked elevations in unstimulated and tumor-stimulated Type-2 responses were seen, particularly in ascites and tumor-infiltrating lymphocytes (P values < 0.01). With polyclonal stimulation, lymphocytes from all compartments expressed elevated Type-1 cytokines (P values < 0.014). Depressed and anxious mood were both associated with significantly lower ratios of polyclonally-stimulated CD4+ cells producing IFNγ (TH1 cells) vs. IL-4 (TH2 cells) in all compartments (depressed mood: P = 0.012; anxiety: P = 0.038) and depressed mood was also related to lower ratios of polyclonally-stimulated CD8+ cells producing IFNγ (TC1) vs. IL-4 (TC2) (P = 0.035). Although effects of polyclonal stimulation should be generalized with caution to the in vivo immune response, findings suggest that depressed and anxious mood are associated with greater impairment of adaptive immunity in peripheral blood and in the tumor microenvironment among ovarian cancer patients.

Introduction

Although ovarian cancer has the second highest incidence among gynecologic cancers, more women die annually from ovarian cancer than from cancers of all other gynecologic sites combined (American Cancer Society, 2007). Presence of tumor-infiltrating CD3+ T-lymphocytes is linked to an approximately threefold increase in overall survival, highlighting the importance of the adaptive immune response in ovarian cancer (Zhang et al., 2003). In a second study, tumor-infiltrating T-cells were associated with disease-free interval, survival, and improved response to chemotherapy (Raspollini et al., 2005). The poor prognosis in ovarian cancer is thought in part to reflect local immunosuppression coupled with a well-developed capacity for immune escape (Santin et al., 2004). Considerable impairment of local anti-tumor effector mechanisms has been observed in lymphocytes derived from malignant ascites (fluid surrounding the tumor, largely composed of secretions by tumor cells) and in tumor-infiltrating lymphocytes (TIL) as compared to peripheral blood mononuclear cells (PBMC) (Badger et al., 1981, Berek et al., 1984, Rabinowich et al., 1996, Santin et al., 2004). T-cells from ovarian ascites show decreased expression and function of signaling molecules, defects in the T-cell receptor and transmembrane receptors, decreased ability to produce interleukin-2 (IL-2) and interferon-gamma (IFNγ) (Lai et al., 1996), and decreased activation markers, even in the presence of stimulatory cytokines (Chen et al., 1999). Although tumor-specific cytotoxic T-lymphocytes (CTL) have been observed in ascites and tumor of ovarian cancer patients (Yamada et al., 1999) and are capable of recognizing multiple antigens on ovarian tumors, their functional capacity is largely impaired (Ioannides et al., 1991).

The elimination of tumor cells by immune mechanisms is generally thought to involve cytokine-mediated upregulation of immune effector cells (Olver et al., 2006). This relies more on a Type-1 cytokine response, including cytokines that stimulate the cellular immune response such as IFNγ, as opposed to a Type-2 response, which includes cytokines such as interleukin-4 (IL-4) that predominantly stimulate the humoral immune response (Kemp and Ronchese, 2001, Takashi et al., 1999). The Type-1 and Type-2 response, traditionally measured in helper T-cells (called TH1 and TH2, respectively), has been shown to also be relevant in cytotoxic T-cells (called TC1 and TC2, respectively) (Kemp and Ronchese, 2001). Low levels of intratumoral IFNγ expression have been related to poorer prognosis in ovarian cancer (Marth et al., 2004). In contrast, IL-4 in the tumor microenvironment has been linked to poorer CTL function and poorer tumor clearance (Olver et al., 2006), although not all findings are consistent (Mattes et al., 2003, Rodolfo et al., 1999).

Differences in cytokine profiles have been noted between peripheral blood lymphocytes and TIL. CTL cell lines generated from peripheral blood lymphocytes of ovarian cancer patients preferentially produced Type-1 cytokines such as IL-2 and IFNγ when cultured with autologous tumor cells (Goedegebuure et al., 1997). In contrast, TIL from ovarian tumors show a predominance of Type-2 cytokine gene expression (Rabinowich et al., 1996), although not all reports are consistent (Santin et al., 2004). Ovarian tumor cells have also been shown to inhibit IFNγ production by TIL (Kooi et al., 1993). Taken together, these findings show a general downregulation of the Type-1 response in the tumor microenvironment. However, little is known about host factors that may influence the polarity (relative Type-1 vs. Type 2 predominance) of the adaptive immune response in ovarian cancer.

Many of the immunologic activities relevant to cancer are known to be modulated by or reflective of psychological states such as depressed or anxious mood. Several meta-analytic studies and reviews have demonstrated consistent correlations between depressed mood, emotional distress and decrements in cellular immune parameters such as the T-cell response to mitogen stimulation and the cytotoxic activity of natural killer (NK) cells (Irwin, 1999, Irwin, 2002, Irwin and Miller, 2007, Reiche et al., 2004, Segerstrom and Miller, 2004). Anxious mood has also been associated with impairments in the cellular immune response (Koga et al., 2001, Thornton et al., 2007), and interventions that decrease anxiety have shown increases in T-cell response to mitogens in women with breast cancer (Andersen et al., 2004) and long-term increases in CD3+CD8+ lymphocytes in men with HIV (Antoni et al., 2000a).

T-cells and NK cells express adrenergic and glucocorticoid receptors, enabling them to be responsive to distress-induced neuroendocrine hormones (Chrousos, 1992, Heilig et al., 1993). Glucocorticoids, catecholamines, and distressed mood have all been associated with a shift from a predominantly Type-1 response to a Type-2 cytokine response (Elenkov and Chrousos, 2000, Glaser et al., 2001). In cancer patients, elevated distress in early stage breast cancer patients at four months post-surgery has been associated with poorer T-cell response to mitogen stimulation in peripheral blood (Andersen et al., 1998, Thornton et al., 2007), whereas stress-reducing behavioral interventions were effective in increasing the T-cell lymphoproliferative response to mitogens in 2 trials in breast cancer patients (Andersen et al., 2004, McGregor et al., 2004).

Most psychoneuroimmunology studies in cancer patients have examined behavioral–immune relationships in the periphery. However because of tumor-induced immune downregulation, it is not known to what extent extrapolations from peripheral blood to the tumor microenvironment are justified. Thus the examination of behavioral–immune relationships in the vicinity of the tumor is important in understanding the in vivo setting in cancer. Little is known about relationships between depressed or anxious mood and adaptive immunity in ovarian cancer patients, and to our knowledge this has not been previously examined in the tumor microenvironment.

With respect to innate immunity, we have previously reported that among epithelial ovarian cancer patients at surgery, depressed mood was related to lower NK cell activity in the tumor microenvironment (Lutgendorf et al., 2005). This finding has clinical relevance as NK cell activity among patients with advanced ovarian cancer has been related to disease progression both prospectively and at the time of recurrence (Garzetti et al., 1993). As many of the same adrenergic and glucocorticoid mechanisms that mediate relationships of psychosocial factors with NK cell activity also modulate T-cell production of cytokines (Elenkov, 2004, Elenkov and Chrousos, 1999, Elenkov et al., 1996) and T-cells have relevance for survival in ovarian cancer (Zhang et al., 2003), we investigated whether depressed and anxious mood were associated with further downregulation of adaptive immunity in ovarian cancer. The adaptive immune response was assessed by relative expression of Type-1 vs. Type 2 cytokines by both CD4+ and CD8+ T-cells in peripheral blood, ascites, and tumor. Cytokine expression stimulated by autologous tumor, by a polyclonal mitogen, or by no stimulation was examined. Stimulation by autologous tumor was performed to examine the T-cell response in the context of tumor-induced modulation of the immune response. In contrast, polyclonal mitogen stimulation allows for observation of the maximum cytokine response available to T-cells.

Based on previous associations of stress hormones and distress with lower levels of Type-1 cytokines and elevations in Type-2 cytokines, (Elenkov, 2004, Elenkov and Chrousos, 1999, Elenkov et al., 1996) we hypothesized that higher levels of depressed and anxious mood would be associated with lower levels of Type-1 cytokines as compared to Type-2 cytokines in T-cells in ovarian cancer patients. Based on our previous findings of behavioral–immune links in NK cells isolated from peripheral blood and tumor but not in ascites, we predicted that these relationships would be seen in peripheral blood and in TIL but not in ascites.

Section snippets

Inclusion and exclusion criteria

This study was approved by the University of Iowa Institutional Review Board. Inclusion as an ovarian cancer patient required confirmation by histological diagnosis of a primary invasive epithelial ovarian, primary papillary peritoneal, or fallopian tube malignant tumor. Patients found to have benign ovarian neoplasms with no inflammatory or other confounding conditions (e.g., endometriosis) were included as a comparison group. The benign comparison group was used to provide a standard of

Patient characteristics

Clinical and demographic characteristics of patients are shown in Table 1. There were no significant differences between benign and ovarian cancer patients in age, ethnicity, education, alcohol use, hours of sleep during the last week or the last night, percentage of current smokers, body mass index, beta blockers or hormone replacement therapy (all P values > 0.18). There were no significant relationships between use of beta blockers or hormone replacement therapy and any of the immune outcome

Discussion

The present findings extend existing literature by demonstrating associations between higher levels of depressed mood and a shift from a Type-1 to a Type-2 pattern of cytokine expression in polyclonally-stimulated cells (both CD4+ and CD8+) among ovarian cancer patients. Similar patterns were observed in lymphocytes isolated from peripheral blood, ascites, and tumor. The presence of an inverse relationship between depressed mood and polyclonally-stimulated Type-1/Type-2 ratios in all

Conclusion

Depressed and anxious mood were associated with a shift from a Type-1 to a Type-2 pattern of cytokine expression in polyclonally-stimulated lymphocytes among ovarian cancer patients. It is particularly noteworthy that these findings were observed in all compartments. Extrapolation to the in vivo immune response of ovarian cancer patients should be done with caution because findings reflect behavior of polyclonally-stimulated cells. Nevertheless, these findings suggest that depressed and anxious

Acknowledgments

This research was funded in part by Grants #R21CA88293 and #RO1CA104825 to Susan Lutgendorf from the National Cancer Institute. We thank Andrew Misfeldt, Joshua Lukenbill, Hannah Chang, Daniel Pederson, and Elizabeth King for assistance with immunologic assays, Joel Sorosky, David Bender, and Michael Goodheart for assistance with patient recruitment, and Anna Hoffman for assistance in study administration.

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