The misleading nature of in vitro and ex vivo findings in studying the impact of stress hormones on NK cell cytotoxicity

Highlights

• Exclusion of plasma renders NK cells 10–30 fold more susceptible to stress hormones.

• In vitro (no plasma), physiological levels of CORT, PGE, and Epi suppress NKCC.

• Acute physiological CORT levels are unlikely to affect NKCC in vivo (plasma milieu).

• Prolonged CORT exposure may suppress NKCC in vivo; effects may be inflated ex vivo.

• In vivo profound suppression of NKCC by PGE or Epi would not be detected ex vivo.

Abstract

In vitro and ex vivo studies assessing the impact of stress hormones on immune competence commonly replace the natural milieu of leukocytes with an artificial medium, excluding plasma factors, hormones, and cytokines. Given prevalent inconsistencies between in vitro, ex vivo, and in vivo findings, we studied whether such procedures could yield misleading outcomes regarding the impact of stress hormones on NK cell cytotoxicity (NKCC), using fresh human whole blood samples. We found that in the presence of plasma 10–30-fold higher concentrations of cortisol, epinephrine, and prostaglandin-E2 (PGE2) were required to reach suppression levels evident in the context of artificial medium. Importantly, whereas the NK suppressive effects of PGE2 occurred immediately and remained stable upon prolonged exposure, the suppressive effects of cortisol slowly increased over time. Last, to simulate the exclusion of stress factors in the ex vivo approach, we subjected whole blood to stress hormones (as occurs in vivo), and abruptly removed them. We found that the effects of epinephrine and PGE2 quickly disappeared, while the effects of cortisol persisted. Overall, these findings demonstrate the potential misleading nature of in vitro and ex vivo procedures, and specifically suggest that (i) the common in vitro findings of profound suppression of NKCC by stress hormones are overestimation of their direct effects expected in vivo; and (ii) the common ex vivo approach cannot reflect the direct in vivo suppressive effects of epinephrine and PGE2 on NKCC, while inflating the effects of glucocorticoids. Some of these fallacies may be circumvented by using non-delayed whole blood NKCC assays in humans.

Introduction

The following are authors’ definitions.

In vitro: “in glass” (Latin). Studies in which the experimental manipulations and the assessment of the outcomes are both conducted in test-tubes, petri dishes, and alike. For example, studying the impact of epinephrine on cytotoxicity of purified NK cells – all conducted in testing tubes.

Ex vivo: “out of the living” (Latin). Studies in which the experimental manipulations are conducted in the living organism, followed by in vitro assessments of the measured outcomes. For example, subjecting animals to swim-stress, followed by blood withdrawal and in vitro assessment of NK cytotoxicity.

In vivo: “within the living” (Latin). Studies in which the experimental manipulations are conducted in the living organism, followed by either (i) assessments of outcome indices in the living organism (e.g., imaging of a developing malignant mass or freezing behavior), or (ii) assessment of an index in an approach that undoubtedly preserves and reflects its status in the living organism (e.g., number of lung metastases counted following animal euthanasia, or serum antibody levels).

Stress and stress hormones are known to alter the function of Natural Killer (NK) cells. However, significant inconsistencies are prevalent between in vitro, ex vivo, and in vivo findings regarding the nature and direction of the effects of specific stress hormones or stress paradigms on NK cell cytotoxicity (NKCC) (Ben-Eliyahu et al., 2007). For example, epinephrine was reported to suppress NKCC in vitro, both in human and animal blood (Takamoto et al., 1991, Whalen and Bankhurst, 1990, Hellstrand and Hermodsson, 1989, Meron et al., 2013), through activating NK cell adrenergic receptors and the consequent increase in intracellular cAMP levels (Whalen and Bankhurst, 1990). However, ex vivo human and animal studies reported contradictory results; many have demonstrated that administration of epinephrine, acute stress exposure, or exercise enhances NKCC (Greisen et al., 1999, Pedersen et al., 1988, Schedlowski et al., 1993, Tarr et al., 2012, Tonnesen et al., 1987, Tonnesen et al., 1984), whereas some have reported suppression of NKCC (Shakhar and Ben-Eliyahu, 1998, Ben-Eliyahu et al., 1999, Andersen et al., 1998). Animal studies employing in vivo procedures generally inferred a suppressive effect of epinephrine on NK activity (Shakhar and Ben-Eliyahu, 1998, Ben-Eliyahu et al., 2000, Rosenne et al., 2014, Inbar et al., 2011).

Glucocorticoids in physiological concentrations were repeatedly shown to markedly suppress human and animal NKCC in vitro (Cox et al., 1983, Gatti et al., 1986, Shakhar et al., 2007). However, several ex vivo studies in humans and animals have suggested that no such suppression occurs in vivo (Bodner et al., 1998, Melamed et al., 2005), and a recent in vivo animal study has supported this suggestion, indicating specific conditions under which corticosterone may exerts some effects (Rosenne et al., 2014).

Unlike catecholamines and glucocorticoids, the release of prostaglandins (PGs) is not controlled centrally. Rather, PGs are released locally by a variety of cells (Liedtke, 1988, Ueda et al., 1994), including malignant cells (Wojtowicz-Praga, 1997), and as a response to tissue damage (Buvanendran et al., 2006). Under some conditions (e.g., surgery) local release can markedly increase systemic PGs levels (Baxevanis et al., 1994, Vitoratos et al., 1996). In vitro studies showed that prostaglandin-E2 (PGE2) can markedly suppresses NK activity (Gatti et al., 1986, Baxevanis et al., 1993, Skibinski et al., 1992), and in vivo studies reported deleterious impacts of PGs on resistance to cancer metastases (Melamed et al., 2005), which is allegedly mediated through in vivo suppression of NK cells (Yakar et al., 2003). However, in a recent study in rats, we have provided evidence indicating that a direct in vivo suppressive effect of PGE2 on NKCC cannot be evident in an ex vivo assessment of NKCC (Meron et al., 2013).

We hypothesize that most inconsistencies regarding the impact of stress hormones on NKCC originate from methodological obstacles and specific procedures that yield misleading outcomes. These procedures include: (1) exclusion or distortion of the natural milieu when conducting ex vivo or in vitro testing, such as replacement of plasma with a hormone- and cytokine-free artificial medium, or testing cytotoxicity in purified NK cells; (2) overlooking the kinetics of the effects of a hormone in its presence and following its exclusion; (3) disregarding the in vivo effects of a stress hormone on NK cell trafficking, which may manifest itself as a change in function; and (4) disregarding the existence of different NK cell subpopulations with different cytotoxicity capacity, in conjunction with stress-induced redistribution of NK cells that is subpopulation-specific (specifically in ex vivo studies). Some alleged inconsistencies result from differences in stress paradigms or hormone levels/concentrations, which we do not consider as inconsistent findings, but rather as reflecting the complexity of the effects of stress.

Although the impact of stress on immune competence should ideally be studied in vivo, in humans this approach is commonly impractical. Thus, it is important to elucidate the degree to which the standard in vitro and ex vivo approaches, used in human studies of NKCC, would reflect in vivo outcomes. It would be instrumental to point at specific distortions caused by these approaches, if exist. To start addressing these issues in humans, we herein simulated several critical procedural aspects of the standard in vitro and ex vivo approaches, employing fresh whole human blood. Admittedly, such an in vitro study may seem limited and paradoxical in examining potential limitations of in vitro and ex vivo approaches. Thus, we restricted the scope of the study to the assessment of aspects that can be simulated or examined by this methodology, aiming at identifying inherent impediments in standard in vitro and ex vivo approaches. Specifically, we address the potential effects of (i) studying human NKCC in the absence of plasma factors, and (ii) disregarding the kinetics of the impact of stress hormones and of their removal from the assay medium. We address these issues employing three different putative immune modulating stress hormones acting through different cellular or intracellular mechanisms, namely epinephrine, PGE2, and cortisol.