Stress and skin leukocyte trafficking as a dual-stage process

Abstract

Stress responses are known to modulate leukocyte trafficking. In the skin, stress was reported both to enhance and reduce skin immunity, and the chronicity of stress exposure was suggested as a key determining factor. We here propose a dual-stage hypothesis, suggesting that stress, of any duration, reduces skin immunity during its course, while its cessation is potentially followed by a period of enhanced skin immunity. To start testing this hypothesis, rats were subcutaneously implanted with sterile surgical sponges for four-hours, during or after exposure to one of several acute stress paradigms, or to a chronic stress paradigm. Our findings, in both males and females, indicate that numbers of sponge-infiltrating leukocytes, and their specific subsets, were reduced during acute or chronic stress, and increased after stress cessation. Studying potential mediating mechanisms of the reduction in leukocyte numbers during acute stress, we found that neither adrenalectomy nor the administration of beta-adrenergic or glucocorticoid antagonists prevented this reduction. Additionally, administration of corticosterone or epinephrine to adrenalectomized rats did not impact skin leukocyte numbers, whereas, in the blood, these treatments did affect numbers of leukocytes and their specific subsets, as was also reported previously. Overall, our findings support the proposed dual-stage hypothesis, which can be evolutionally rationalized and accounts for most of the apparent inconsistencies in the literature regarding stress and skin immunity. Other aspects of the hypothesis should be tested, also using additional methodologies, and its predictions may bear clinical significance in treatment of skin disorders related to hyper- or hypo-immune function.

Figure 1. Illustration of cell populations in flow cytometric analysis

Cell populations in blood and sponge samples are marked by ovals. Lymphocytes and granulocytes were identified based on their forward and side scatter properties. T cells were identified as CD5 positive, NK cells as CD161^bright lymphocytes, and NKT cells as positive for both markers. Within sponge-infiltrating leukocytes, neutrophils were identified as CD161^dim. Polystyrene microbeads were added to each sample and used to assess the absolute numbers of cells per sample studied (see Methods). At least 30,000 events from each sample were analyzed (the illustration shows a smaller number for clarity of presentation). “Debris from sponge and cells” does not contain CD161+ or CD5+ events, and does not form a coherent population when displayed on a CD5 by CD161 axes.

Figure 2. Experimental design and timeline for Experiment 1

Figure 3. Experimental design and timeline for Experiment 2

Figure 4. Effects of a four-hour wet-cage stress exposure – during its course, immediately after, or four-hours after its cessation, on numbers of total sponge-infiltrating leukocytes and their specific subsets in male rats

During its course, wet-cage stress significantly reduced numbers of total sponge-infiltrating leukocytes (A), and specific subsets (B). These numbers returned to baseline immediately following stress, and significantly increased four-hours following its cessation. Data are presented as mean + SEM. * indicates a significant decrease from the control group and # indicates a significant increase.

Figure 5. Effects of an acute four-hour stress paradigm and a chronic stress paradigm, during their course, or two-hours after their cessation, on total sponge-infiltrating leukocyte numbers and on serum corticosterone levels

Stress exposure, during its course, significantly reduced numbers of total sponge-infiltrating leukocytes, in both the four-hour wet-cage paradigm (by ~50%, A), and the chronic alternating paradigm (by ~20%, C). At the endpoint of stress exposure corticosterone serum levels were significantly increased in both paradigms (by four-fold in the acute paradigm, B; by twofold in the chronic one, D). In sponges implanted two-hours following stress cessation, leukocyte numbers were significantly increased to baseline levels in the chronic paradigm (B) and exceeded these levels in the acute (A) stress paradigm. Six hours after stress cessation, corticosterone levels were reduced beyond baseline levels in the acutely stressed rats (C), and retured to baseline levels in the chronically stressed rats (D). Data are presented as mean + SEM. * indicates a significant decrease from the control group and # indicates a significant increase.

Figure 6. Experimental design and timeline for Experiment 5

Daily four-hour stress sessions along the first 24 days alternated between the “wet-cage” and the “restraint” stress paradigms (see Methods).

Figure 7. Comparing the effects of four-hour wet-cage and restraint stress paradigms, during their course, on sponge-infiltrating leukocyte numbers, in male rats

Both wet-cage stress and restraint stress, during their course, significantly reduced numbers of total sponge-infiltrating leukocytes, compared to controls. Data are presented as mean + SEM. * indicates a significant difference from the control group.

Figure 8. Comparing the effects of four-hour wet-cage and surgical stress paradigms, during their course, on sponge-infiltrating leukocyte numbers, in female rats

Both wet-cage stress and surgical stress, during their course, significantly reduced numbers of total sponge-infiltrating leukocytes, compared to controls. Data are presented as mean + SEM. * indicates a significant difference from the control group.

Figure 9. Effects of a beta-adrenergic or a glucocorticoid blockade (employing nadolol or mifepristone) on the impact of a four-hour wet-cage paradigm on numbers of sponge-infiltrating leukocytes

Wet-cage stress significantly reduced numbers of sponge-infiltrating leukocytes during its course. Nadolol, mifepristone, or their combination, did not block this effect, nor changed baseline levels, compared to vehicle controls. Data are presented as mean + SEM. * indicates a significant difference from the control group.

Figure 10. Effects of a four-hour wet-cage paradigm, or of administration of epinephrine or corticosterone in adrenalectomized rats, on numbers of sponge-infiltrating leukocytes

Wet-cage stress, during its course, significantly reduced numbers of sponge-infiltrating leukocytes both in sham-operated and in adrenalectomized (ADX) rats. In contrast, administration of epinephrine or corticosterone to adrenalectomized rats did not affect these numbers. Data are presented as mean + SEM. * indicates a significant difference from the control group.

Figure 11. Effects of a four-hour wet-cage paradigm, or of administration of epinephrine or corticosterone in adrenalectomized rats, on numbers of circulating leukocyte subsets

Adrenalectomy (ADX) prevented the reduction in numbers of circulating lymphocytes caused during stress, and specific lymphocyte subset numbers (NK, NKT and T cells) were even increased in adrenalectomized stressed rats. Consistently, administration of epinephrine or corticosterone to adrenalectomized rats reduced numbers of circulating lymphocytes. Data are presented as mean + SEM. * indicates a significant decrease from the relevant control group and # indicates a significant increase.