Glucocorticoid Resistance (PCS1, PMBC)
Dexamethasone Suppression Test (PMBC, subsample)
Dexamethasone is a synthetic glucocorticoid that demonstrates the same anti-inflammatory effects as cortisol. Accordingly, mitogen-stimulated production of pro-inflammatory cytokines such as interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α can be expected to be suppressed when stimulated cells are cultured in the presence of dexamethasone. Glucocorticoid resistance (GCR)—or insensitivity of the cell to the effects of glucocorticoids, then, is thought to be present when pro-inflammatory cytokine production in the presence of dexamethasone is not reduced relative to production in the absence of dexamethasone.
Funding for measurement of GCR was not included in the original PMBC grants (NHLBI P01 HL65111 and P01 HL65112), but instead was obtained through a supplement awarded from the John D. and Catherine T. MacArthur Foundation (see Supporting Grants). Because the supplemental funding was received after data collection already had begun, GCR data are available for the last 79 participants only.
Blood samples for the assessment of glucocorticoid resistance were collected on Quarantine Day 0, prior to viral challenge.
To measure GCR, 1.8 ml of whole blood was incubated with 200 μl of dexamethasone phosphate dissolved in phosphate buffering solution (PBS) for 30 minutes. The final in-well concentrations of dexamethasone were 0 nM, 1 nM, 50 nM, and 250 nM. 220 μl of lipopolysaccharide (LPS) dissolved in PBS was then added to each sample. The final concentration of LPS was 10 ng/ml.
The samples were then transferred to 24-well plates and incubated for 24 hours at 37°C with 5% CO2 in air. The plates were removed from the incubator and immediately centrifuged for 10 minutes at 2,000 rpm. Plasma samples were then aspirated and stored at –20°C until the end of the study. At that time, samples were thawed and cytokine assays were performed using Biosource multiplex immunoassay kits (Biosource International, Camarillo, CA). The assays were performed in duplicate using a Luminex 100 bead-based immunoassay system. Luminex multiplex technology has been demonstrated to be a valid alternative method for the quantification of cytokines1.
Area under the dexamethasone (dex) curve (AUC) was used as the marker of GCR. Adjusted (adj) cytokine scores were calculated for each dex concentration by subtracting from each the cytokine level of the saline (unstimulated) control. Using IL-6 as an example, AUC was calculated using the following equation:
(IL-6 dex 0 pg/ml adj + IL-6 dex 1 pg/ml adj) + (IL-6 dex 1 pg/ml adj + IL-6 dex 50 pg/ml adj)*49 + (IL-6 dex 50 pg/ml adj + IL-6 dex 250 pg/ml adj)*200)/2.
This is a modified version of a protocol that has been used to examine whether acute bouts of exercise modulate glucocorticoid sensitivity2,3.
1 duPont, N. C., Wang, K., Wadhwa, P. D., Culhane, J. F., & Nelson, E. L. (2005). Validation and comparison of luminex multiplex cytokine analysis kits with ELISA: Determinations of a panel of nine cytokines in clinical sample culture supernatants. Journal of Reproductive Immunology, 66, 175-191.
An indirect index of glucocorticoid resistance (GCR) was created by combining leukocyte data derived from complete blood counts (CBC) and plasma cortisol data. Greater levels of glucocorticoid are associated with higher numbers of circulating neutrophils, lower numbers of circulating lymphocytes, and a lower ratio of neutrophils to lymphocytes—an overall marker of the trafficking of these cells1, 2. Cole and his colleagues3, 4 showed that this association can be used to indirectly assess GCR. The logic of the measure is that there is a strong physiologic correlation between cortisol levels and the number of circulating leukocytes ONLY if leukocyte glucocorticoid receptors are sensitive (i.e., signaling cells to redistribute).
Blood samples for the assessment of glucocorticoid resistance were collected using standard venipuncture techniques on two occasions, with the first blood draw being conducted 2 weeks prior to viral challenge and the second 1 week prior to challenge.
Creation of Index
Leukocyte and cortisol data were averaged across the two sample collections to create reliable measures of each parameter. If a variable is associated with GCR (e.g., stress), one would expect a statistical interaction of cortisol and that variable in predicting the leukocyte counts. For example, in the case of stress, one would expect an interaction where cortisol is related to leukocyte counts in the absence of stress (the usual condition), but not associated with leukocyte counts in the presence of stress (glucocorticoid resistance).
1 Fauci, A. S., Dale, D. C., & Balow, J. E. (1976). Glucocorticosteroid therapy: Mechanisms of action and clinical considerations. Annals of Internal Medicine, 84, 304-315.