Norephinephrine modulates dendritic cell activity by altering chemokine.
Previous work from our laboratory and others has reported a dramatic increase in the circulating concentration of natural killer (NK) cells during long-duration (>1-hr), high-intensity (>70% V[O.sub.2peak]) aerobic exercise [1-4]. NK cells are large, granular lymphocytes of the innate immune system that function by eliminating virally infected and cancerous cells and by inhibiting the colonization of several types of pathogenic organisms . This elevation in circulating NK cell levels is the result of a redistribution from peripheral lymphoid organs [6, 7]. Although it is suspected that this mobilization of NK cells is mediated by catecholamines including norepinephrine (NE), the exact mechanism has yet to be fully elucidated .
NK cells interact with activated dendritic cells (DC) in draining lymph nodes and spleen [9-11]. DC are dedicated antigen presenting cells that mature in peripheral tissues following exposure to microbial antigen such as lipopolysaccharide (LPS) . DC then migrate to the peripheral lymphoid organs where they recruit and reciprocally activate other leukocytes including NK cells . Murine experiments have also demonstrated that NK cells are recruited into subcutaneous B16 melanoma tumors by DC and that this process is mediated by the release of the chemokines; Macrophage Inflammatory Protein-1 a (MIP-1[alpha]), MIP-1[beta], and Regulated upon Activation, Normal T-cell Expressed, and Secreted (RANTES) . All three chemokines bind the CCR5 receptor on granulocytes, T-cells, and NK cells and induce chemotaxis to site of microbial invasion and inflammation.
Physiological stress such as high-intensity aerobic exercise induces a "fight or flight" response during which time the sympathetic nervous system (SNS) prepares the body for the rigors of physical activity [13-17]. NE is released from the pre-synaptic fibers of the SNS and stimulates numerous physiological responses including improved cardiac function, elevated blood glucose levels resulting from liver glycogenolysis, and vasodilation of skeletal muscle vasculature [15-17]. Both lymph nodes and spleen possess extensive sympathetic innervations [18, 19]. The leukocytosis observed during high-intensity exercise is thought to be part of the "fight or flight" response, increasing the circulating concentrations of white blood cells (including NK cells), thus providing additional protection against possible infections resulting from increased ventilation, food and water consumption, or injury [20, 21].
Past research has established that NE alters DC production of several cytokines in both murine cells, in vivo, and human cells in vitro [22-24]. It is possible therefore, that Ne may inhibit DC release of MIP-1[alpha], MIP-1[beta], and RANTES within the peripheral lymphoid organs, contributing to the stimuli that result in a migration of NK cells from these tissues into the peripheral circulation. The purpose of this study was to assess the effect of NE on the chemokine release of LPS-activated DC in vitro.
Human DC were purchased from MatTEK Corp. (Ashland, MA). Upon receipt, they were allowed to acclimate for 24-h, (37[degrees]C, 5% C[O.sub.2], humidified environment). All incubations were completed in triplicate in 24-well plates. The DC were seeded at a density of 1.65 x [10.sup.5] cells per well in final volume of 1 mL in complete media (DCMM, MatTEK Corp., Ashland, MA). All reagents were purchased from Sigma-Aldrich (Saint Louis, Mo) unless otherwise stated. LPS Escherichia coli serotype 055:B5 (specific activity of 1.2 x [10.sup.6] units/[micro]g) was used at a concentration of 5 [micro]g/mL). DL-NE hydrochloride was used at a concentration of [10.sup.-6] M. Following a 24-h incubation, (37[degrees]C, 5% C[O.sub.2], humidified environment), the plates were centrifuged and the supernatants aspirated and stored at -80[degrees]C until assessment of MIP-1[alpha], MIP-1[beta], and RANTES concentrations by ELISA. Due to limited resources, testing multiple concentrations of stimulants and inhibitors was not possible. A search of the relevant literature revealed a study by Goyarts et al., which utilized a similar protocol. We used the concentrations that Goyarts et al. found to produced optimal responses in DC .
Enzyme-Linked Immuno-Sorbent Assay (ELISA)
The kits and reagents were purchased from R&D Systems (Minneapolis, MN). Controls (both commercially available and laboratory generated) were included with each batch of samples to determine the intra and inter-assay coefficients of variation, all of which were below 10%.
Statistical analysis was completed by using SPSS v15.0 (SPSS, Chicago, IL). All measurements of MIP-1[alpha], MIP-1[beta], and RANTES concentrations were analyzed by using a one-way ANOVA. The independent variable was the incubation condition, i.e. medium only, LPS only, or LPS + NE. Significance was set at P < 0.05. When significance was found, a Student's t-test with Bonferroni correction for multiple comparisons was used to determine the location of significance. Bivariate Pearson correlations was also completed to assess the degree of interaction among the various dependent variables. All values are presented as means [+ or -] SE.
LPS stimulation significantly increased the production of MIP-1[alpha] (385%, P < 0.001, Figure 1), MIP-1[beta] (158%, P = 0.002, Figure 2) and RANTES (2757%, P < 0.001, Figure 3), when compared to the control condition. NE significantly inhibited the production of MIP-1[beta] (74%, P = 0.001) and RANTES (-93%, P < 0.001), but not MIP-1[alpha] (-17%, P = 0.227) relative to LPS stimulation alone.
[FIGURE 1 OMITTED]
Our hypothesis was partially supported by our results. NE significantly decreased the LPS-stimulated production of MIP-1[beta] and RANTES, but not MIP-1[alpha] in DC. To our knowledge, this is the first study to demonstrate that this aspect of DC function can be modified by a neurotransmitter of the sympathetic nervous system.
MIP-1[alpha], MIP-1[beta], and RANTES were specifically chosen because of their ability to directly stimulate the migration of NK cells into subcutaneous B16 melanoma tumors in mice. Liu et al. found that DC injected directly into these tumors and stimulated with CpG (unmethylated DNA found in bacteria) produced large quantities of MIP-1[alpha], MIP-1[beta], and RANTES . Within 48-hrs, NK cell infiltration of the tumors had significantly increased. This response was greatly attenuated when the experiment was repeated with CCR5-/- mice that do not express the common receptor for MIP-1[alpha], MIP-1[beta], and RANTES.
Early research in this area revealed a relationship between periods of elevated psychological stress and inhibition of DC immunoregulatory functions [25-27]. Subsequent mechanistic studies determined that NE can inhibit several aspects of DC function. Our results are in agreement with three previous studies using both mouse and human DC [22-24]. Maestroni et al. (2002) using mouse Langerhans cells (DC found in the skin) stimulated with both LPS and keyhole limpet hemocyanin (KLH) in vivo determined that Ne inhibited the release IL-12, a cytokine that plays a major role in stimulating NK cell function . Conversely, the release of IL-10, an anti-inflammatory cytokine that inhibits the migration of DC to the lymph nodes, was increased . In a separate study, Maestroni et al. (2003) found that LPS-stimulated, bone marrow derived mouse DC incubated with Salbutamol, an agonist of the [[beta].sub.2] receptor for NE, inhibited the DC release of the inflammatory chemokines CCL19 and CCL21 in vitro . Finally, Goyarts et al. found that the release of several inflammatory cytokines (IL-23, TNF-[alpha], and IL-6) and the chemotactic protein IL-12p40 were significantly inhibited in LPS-stimulated human DC following incubation with NE in vitro . The hypothesized mechanism of the SNS-directed, transient inhibition of DC function during long-duration (>1-hr), high-intensity (>70% V[O.sub.2peak]) aerobic exercise resulting in an increased circulating concentration of NK cells is diagramed in Figure 4. Under "normal" conditions, i.e. at rest and in the absence of foreign antigen, NK cells move between the tissues and peripheral circulation, surveying for signals of microbial invasion or damage. DC present in the lymph nodes and other peripheral lymphoid organs release a baseline concentration of several cytokines and chemokines including MIP-1[alpha], MIP-1[beta], and RANTES. When foreign antigen is sequestered in the lymph nodes by DC, their production of these three chemokines is greatly enhanced. These mediators induce circulating NK cells to increase their expression of several adhesion molecules that allow entry into the lymph nodes where they reciprocally activate the DC . Conversely, physiological stress leads to an initiation of the "fight-or-flight" response. NE is released from post-synaptic fibers of the SNS, inhibiting the DC release of MIP-1[alpha], MIP-1[beta], and RANTES. Decreased expression of adhesion molecules induces NK cells to migrate from the peripheral lymphoid organs and enter the blood, thus providing additional, immediate protection against possible pathogenic infiltration during exercise.
[FIGURE 2 OMITTED]
[FIGURE 3 OMITTED]
Although there was a trend towards an inhibition of MIP-1[alpha] release, (-17%) this effect did not reach significance (P = 0.227). Although it is possible that NE does not affect MIP-1[alpha] in the same manner as it does MIP-1[beta] and RANTES, this is unlikely considering that they share a redundant receptor and signaling pathway. The most logical explanation for this was our small sample size. We were only able to run the experiment in triplicate and the variability between samples for this particular ELISA was higher than for the other two chemokines measured. It is also possible that the threshold concentration of NE required to inhibit MIP-1[beta] and RANTES ([10.sup.-6] M) is lower than that required to inhibit MIP-1[alpha].
Clearly, the scope of this study was limited to this particular aspect of the hypothesized mechanism. The complete pathway is likely to be more complex, including several redundant signaling cascades mediated by multiple neurotransmitters, chemokines, and cytokines acting on both DC and NK cells, and perhaps other leukocytes. For example, Schedlowski et al. observed that infusion of NE and epinephrine (Epi) increased NK cell activity in humans for 30-min (NE) and 60-min (Epi) post-infusion . There is also evidence that high intensity exercise inhibits the NK cell-surface expression of several adhesion molecules (CD54, CD18, and CD53), potentially allowing them to migrate from peripheral lymphoid organs into the circulation . These observations support the contention that the increased circulating concentration of NK cells during exercise is a protective adaptation designed to rapidly introduce large numbers of NK cells and other leukocytes to sites of infection that may occur during a "fight-or-flight" response. Future research should seek to further elucidate the complete mechanism.
[FIGURE 4 OMITTED]
In conclusion, LPS-stimulated DC incubated for 24-hrs with NE produced significantly less MIP-1[beta] and RANTES, but not MIP-1[alpha] in vitro. These data support the contention that the exercise-induced increase in circulating NK cells is a protective adaptation mediated by the sympathetic nervous system.
The authors would like to acknowledge the contribution of Timothy Raabe PhD for his invaluable assistance during this project.
Address for correspondence: Hutchison AT, PhD, University of Texas Health Science Center at Houston, Department of Internal Medicine, Division of Infectious Diseases, Houston, Texas, USA, 77030. Phone (713) 500-7225; Email Alexander.T.Hutchison@uth.tmc.edu
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ALEXANDER HUTCHISON , STEPHANIE GARCIA , CASSANDRA HUERTA 
 University of Texas Health Science Center at Houston, Department of Internal Medicine, Division of Infectious Diseases,  Department of Exercise & Sport Science/St. Mary's University, San Antonio, USA
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|Title Annotation:||Systems Physiology--Immune Function|
|Author:||Hutchison, Alexander; Garcia, Stephanie; Huerta, Cassandra|
|Publication:||Journal of Exercise Physiology Online|
|Date:||Feb 1, 2010|
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