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

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.

Stress impairs the efficacy of immune stimulation by CpG-C: Potential neuroendocrine mediating mechanisms and significance to tumor metastasis and the perioperative period

“…This effect of CORT was observed beginning at low physiological levels (10 −9 M) that characterized baseline (no stress) conditions. These in vitro findings correspond with the in vivo findings that blocking stress hormones in the absence of external stressor markedly potentiate the efficacy of CpG-C, although it is hard to extrapolate in vitro to in vivo outcomes [48]. …”

Stress-Induced In Vivo Recruitment of Human Cytotoxic Natural Killer Cells Favors Subsets with Distinct Receptor Profiles and Associates with Increased Epinephrine Levels

“…Previous studies variably associated acute stress with enhanced [ 6 ], or decreased NK cell activity [ 24 , 25 ], which could relate to different assay conditions (e.g. not including human plasma in the culture media, cryopreservation [ 26 ]), some of which were, however, inevitable in order to test all samples from a donor simultaneously with the same target cell conditions. Reduced function has moreover been attributed to glucocorticoid-induced epigenetic changes, which we might have missed by testing NK function within three hours post-stressor [ 27 ].…”

Maintaining unperturbed cerebral blood flow is key in the study of brain metastasis and its interactions with stress and inflammatory responses

“…3c), which indicate immunocytes trafficking or augmented leukocyte apoptosis (Bronte and Pittet, 2013). While the direct effects of this reduction in splenocytes number has not been addressed in the current study, it is acknowledged that such immune perturbations may have profound effects on tumor and metastasis progression, and on therapeutic interventions (Candeias and Gaipl, 2016; Dhabhar et al, 2012; Gotlieb et al, 2015; Melamed et al, 2010). Moreover, while the addition of laparotomy affected animals undergoing aECAi in a pattern similar to the effects evident in non-injected animals, in ICAi operated animals, laparotomy had diverse, often an exaggerated response, compared to control and aECAi operated animals.…”