Journal of Applied Physiology
Vol. 83, No. 3, pp. 845-850, September 1997
EXERCISE AND MUSCLE

Modulation of NK cell cytolytic activity by macrophages in chronically exercise-stressed mice

Sally E. Blank¹, T. Bucky Jones¹, Eric G. Lee¹, C. Jayne Brahler¹, Randle M. Gallucci², Marne L. Fox¹, and Gary G. Meadows²

Departments of ¹ Kinesiology and ² Pharmaceutical Sciences, Washington State University, Pullman, Washington 99164-1410

ABSTRACT

INTRODUCTION

MATERIALS AND METHODS

RESULTS

DISCUSSION

ACKNOWLEDGEMENTS

FOOTNOTES

REFERENCES

ABSTRACT

Blank, Sally E., T. Bucky Jones, Eric G. Lee, C. Jayne Brahler, Randle M. Gallucci, Marne L. Fox, and Gary G. Meadows. Modulation of NK cell cytolytic activity by macrophages in chronically exercise-stressed mice. J. Appl. Physiol. 83(3): 845-850, 1997.This study was designed to investigate the effects of moderate-intensity endurance training on basal natural killer (NK) cell cytolytic activity in murine splenocytes that were enriched for 1) NK1.1⁺ cells or 2) macrophages and NK1.1⁺ cells. Mice were assigned to sedentary (Sed), treadmill control (TM), or treadmill-trained (Trn) groups. Splenocyte number, the percentages of NK1.1⁺, large granular lymphocytes (NK1.1⁺, LGL-1⁺), and other subpopulations did not change in Trn mice. Approximately 70% of cells enriched for NK1.1⁺ expressed this surface antigen. Lytic units (LU) expressed per LGL-1⁺ cell were significantly lower in Trn [83.9 ± 3.2 (SE)] compared with Sed (109.5 ± 7.5) and TM (101.3 ± 6.4) groups. When macrophages remained in the in vitro assay, LU per LGL-1⁺ cell did not differ across groups. The results indicate that highly enriched NK1.1⁺ cells from Trn mice had lower NK cell activity compared with Sed mice. No differences in NK cell activity were observed when cells were enriched for NK1.1⁺ cells and macrophages. These findings support the hypothesis that macrophage modulation of NK cells may be one mechanism contributing to augmented basal NK cell activity in endurance-trained individuals.

endurance training; juxtacrine and paracrine regulation

INTRODUCTION

ENDURANCE TRAINING IS OFTEN associated with enhanced basal natural killer (NK) cell cytolytic activity in human subjects (19, 21, 23). In rodents, splenic NK cell cytolytic activity is also generally enhanced after endurance training, although the magnitude of adaptation may be strain dependent (10, 12). However, when splenic NK cells are enriched in vitro by nylon wool nonadherence (NWNA), NK cell cytolytic activity is lower in cells from endurance-trained mice compared with sedentary mice (2). Macrophages and B cells are adherent to nylon wool, but not all macrophages are removed from the cellular suspension by NWNA. Thus the previous experiments did not decisively demonstrate that removal of macrophages from the in vitro cytolytic assay was responsible for the lower NK cell activity in endurance-trained groups. Within recent years, development of cellular enrichment techniques such as monoclonal antibody (MAb) conjugation to magnetic beads improved purification yields and created methods for investigating discreet mechanisms between different cells in in vitro assays. In the present study, splenocytes were highly enriched for either NK1.1⁺ cells or for NK1.1⁺ cells plus macrophages by negative depletion with MAbs conjugated to magnetic beads. The purpose of the study was to examine the in vitro cytolytic activity of highly enriched NK1.1⁺ cells and to further pursue the role that macrophages may play in the regulation of NK cells in endurance-trained subjects.

MATERIALS AND METHODS

Three separate experiments were conducted by using 6-wk-old female C57/BL6 mice obtained from Jackson (Bar Harbor, ME) or Charles River (Wilmington, MA) Laboratories. In experiment 1, 120 mice were used and splenocytes were enriched for only NK1.1⁺ cells. In experiment 2, splenocytes were enriched for NK1.1⁺ cells plus macrophages by using the same experimental design (n = 120 mice). The purpose of experiment 3 was to replicate the results obtained in experiment 2 (n = 80 mice).

1

RESULTS

In each experiment, all mice gained body weight with no apparent group effect (Table 1). Average daily food intake was similar among the groups (~15-17 cal/day) and was within the range of values reported by our laboratory for Sed mice (17). Spleen weight normalized to body weight was not significantly different among the groups (~3.4 mg/g for Sed mice). There was large variability in splenic cellularity that was not related to treatment effects (Table 1). This observation was consistent with previous experiments (2).

Table  1.   Descriptive data n Final Body Wt, g Splenocyte No./mg Spleen (×107) Experiment 1  NK1.1+-enriched   Sed 10 20.8 ± 0.5  4.6 ± 0.4    TM 10 21.5 ± 0.3 5.0 ± 0.3    Trn 10 20.3 ± 0.3  4.6 ± 0.4  Experiment 2  NK1.1+- and macrophageenriched   Sed 10 22.4 ± 0.2  3.2 ± 0.3    TM 10 22.5 ± 0.2  3.0 ± 0.3    Trn 9 21.6 ± 0.2  2.2 ± 0.2* Experiment 3  NK1.1+- and macrophageenriched   TM 10 21.9 ± 0.2  2.0 ± 0.3    Trn 10 22.2 ± 0.2  2.3 ± 0.2 Values are means ± SE from pooled samples for sedentary (Sed), treadmill control (TM), and treadmill-trained (Trn) groups; n, no. of animals. * Significantly different from other groups within experiment, P < 0.05.  Significantly different from Trn group within experiment, P < 0.05.

Approximately 4% of the nonenriched cells were NK1.1⁺ (Table 2). The enrichment yield for NK1.1⁺ cells in experiment 1 was 1.1-1.5% of the splenocytes placed over the magnetic column (Table 2), and this range was in agreement with reported values (7). Cell yields increased to 1.7-2.4% in experiments 2 and 3 when splenocytes were enriched for macrophages and NK1.1⁺ cells (Table 2). Differences in cellular recovery were inversely related to the percentage of macrophages retained in the in vitro assay. Cellular yield was not influenced by group effects.

Table  2.   Enrichment yield Group Experiment 1  Experiment 2  Experiment 3  Sed 1.49 ± 0.21  1.71 ± 0.16  TM 1.19 ± 0.15  1.73 ± 0.12  2.31 ± 0.50  Trn 1.11 ± 0.18  1.66 ± 0.28  2.40 ± 0.82 Values are means (%cells eluted from magnetic column) ± SE for Sed, TM, and Trn groups.

The lymphocyte profile for pooled nonenriched splenocytes was generally unaffected by chronic exercise (Tables 3 and 4). These data are consistent with results from single spleen analyses (3). In each experiment, samples were omitted from analyses if the contaminating cellular populations were >5% of the total yield. An exception was made in experiment 2, in which the average percentage of CD4⁺ cells equaled 10% in the Trn group. Statistical analyses indicated that samples containing >5% CD4⁺ cells did not influence the group average for NK cell cytolytic activity, and therefore these data were included. The percentages of other contaminating lymphocyte subpopulations were not statistically different among groups (Table 4).

Table  3.   Lymphocyte subpopulation percentages in splenocytes (experiment 1) Enrichment n CD4+ CD8+ B220+ F4/80+ NK1.1+ LGL-1+ Nonenriched   Sed 10 22.6 ± 0.5  14.6 ± 0.7  51.3 ± 1.9  ND 3.8 ± 0.2  1.5 ± 0.1    TM 10 21.7 ± 0.7  13.9 ± 0.6  52.9 ± 2.0  ND 4.0 ± 0.2  1.5 ± 0.1    Trn 10 23.6 ± 1.0  15.7 ± 1.0  48.9 ± 2.2  ND 4.0 ± 0.2  1.6 ± 0.1  NK1.1+   Sed 10 4.2 ± 2.4  0.3 ± 0.1  0.7 ± 0.2  4.1 ± 2.0  69.5 ± 2.1  28.2 ± 1.3    TM 9 1.1 ± 0.3  0.2 ± 0.0  0.5 ± 0.2  2.8 ± 0.5  66.5 ± 2.3  26.2 ± 1.3    Trn 10 4.0 ± 1.9  0.2 ± 0.1  0.5 ± 0.1  4.6 ± 1.4  67.0 ± 2.6  25.3 ± 1.9 Values are means ± SE from pooled samples for Sed, TM, and Trn groups. n, No. of animals; LGL, large granular lymphocyte; ND, not determined.

Table  4.   Splenic subpopulations percentages (experiments 2 and 3) Enrichment n CD4+ CD8+ B220+ F4/80+ NK1.1+ LGL-1+ Experiment 2  Nonenriched   Sed 10 24.5 ± 0.5  14.7 ± 0.5  46.5 ± 1.2  16.1 ± 0.5  3.4 ± 0.2  1.4 ± 0.1    TM 10 24.3 ± 0.6  14.5 ± 0.5  46.0 ± 1.7  16.5 ± 0.4  3.1 ± 0.2  1.8 ± 0.2    Trn 9 25.1 ± 0.8  18.1 ± 0.9* 43.3 ± 1.8  19.7 ± 0.5* 4.3 ± 0.5  1.7 ± 0.1  NK1.1+ and macrophages   Sed 9 3.2 ± 0.9  0.3 ± 0.1  0.0 ± 0.0  15.1 ± 1.1  68.9 ± 1.5  30.5 ± 1.0    TM 10 4.9 ± 1.0  0.3 ± 0.1  0.0 ± 0.0  17.8 ± 1.4  69.6 ± 0.7  29.7 ± 1.1    Trn 7 10.1 ± 2.5* 0.5 ± 0.1* 0.0 ± 0.0  21.5 ± 3.5  69.6 ± 3.3  26.4 ± 1.5  Experiment 3  Nonenriched   TM 10 22.1 ± 1.1  17.0 ± 2.1  34.8 ± 1.8  15.6 ± 2.0  4.7 ± 0.6  2.0 ± 0.3    Trn 10 20.3 ± 1.9  13.4 ± 1.2  28.2 ± 3.5  22.1 ± 2.5  5.1 ± 0.4  2.0 ± 0.3  NK1.1+ and macrophages   TM 10 1.2 ± 0.3  0.6 ± 0.1  0.2 ± 0.0  33.4 ± 3.7  26.8 ± 0.4  10.3 ± 1.4    Trn 10 0.7 ± 0.2  0.6 ± 0.1  0.3 ± 0.1  34.2 ± 3.1  21.5 ± 3.3  8.0 ± 0.7 Values are means ± SE from pooled samples for Sed, TM, and Trn groups; n, no. of animals. Percentages of F4/80+ were determined from gated lymphocyte population in experiment 2 and from gated leukocyte population in experiment 3.* Significantly different from all other groups within experiment, P < 0.05.

In experiments 1 and 2, 70% of the gated lymphocytes were NK1.1⁺, whereas in experiment 3, ~25% of the gated leukocytes were NK1.1⁺ cells. Macrophages represented ~18 and 33% of the gated cells in experiments 2 and 3, respectively (Table 4). The differences in percentages of NK1.1⁺ cells and macrophages among experiments coincided with gated lymphocyte and leukocyte populations used in flow cytometric analyses.

In enriched NK1.1⁺ cells, NK cell cytolytic activity expressed as LU per LGL-1⁺ cell was significantly lower in Trn compared with Sed and TM groups (Fig. 2). Furthermore, lytic activity did not differ between Sed and TM groups. In contrast, when macrophages remained in the assay, NK cell cytolytic activity did not differ among Trn, Sed, and TM groups (Figs. 3 and 4). In experiment 3, the ratio of LGL-1⁺ cells to macrophages varied among samples and contributed to the large within-group variability for NK cell cytolytic activity. Normalized to the LGL-1⁺-to-macrophage ratio, LU variability was reduced within groups (Fig. 4), and NK cell cytolytic activity was not different between Tr and TM groups.

DISCUSSION

This study is part of ongoing research designed to elucidate the mechanisms by which chronic exercise stress may modulate NK cell cytolytic activity. In previous experiments using the same exercise model, nonenriched murine splenocyte NK cell cytolytic activity was not significantly increased in treadmill-trained mice (3), whereas chronic treadmill exercise was associated with a 40% reduction in NK cell cytolytic activity in NWNA splenocytes (2). NWNA enriches NK1.1⁺ cells twofold by selective depletion of the majority of B cells and macrophages (2). We originally hypothesized that the in vitro enrichment of NWNA splenocytes eliminated paracrine regulation of NK cells by macrophages (hypothesis 1). The lower NK cell cytolytic activity in NWNA splenocytes from chronically exercised mice indicated that endurance training may alter macrophage paracrine regulation of NK cells in vivo. Alternatively, it is possible that the inherent cytolytic activity of NK cells is changed by the effects of chronic exercise stress and that partial enrichment of NK1.1⁺ cells amplified this functional adaptation (hypothesis 2).

To test these hypotheses, splenocytes were highly enriched for NK1.1⁺ cells by using the method of Gallucci et al. (7). The enriched cell population consisted of >70% NK1.1⁺ cells and was nearly depleted of CD4⁺, CD8⁺, and B220⁺ cells. The contaminating population of CD4⁺ cells (1-4%) was not significantly different among the groups and was not associated with higher NK cell cytolytic activity in those samples having higher percentages of this subpopulation. Subsequent experiments in our laboratory have established that the average contaminating macrophage population in enriched cells is ~5% (S. E. Blank, E. G. Lee, R. M. Gallucci, and G. G. Meadows, unpublished observations). The difficulty in determining the exact percentage of the macrophage population is because of unavailability of an MAb specific to an epitope exclusively expressed on murine monocytes/macrophages. The F4/80 antigen is a seven-transmembrane segment glycoprotein expressed on mouse macrophages, Langerhans cells, and dendritic cells (8, 14). Macrophages that are F4/80⁺ are located in several body compartments including the spleen red pulp, peritoneal cavity, liver, and brain (8). Specific functions associated with this cell surface molecule are not known, but F4/80 expression may be related to monocyte/macrophage activation state and adhesion events (8). Surface expression of the F4/80 antigen is generally downregulated on monocytes and on activated macrophages, whereas "resting" resident macrophages have high expression of this molecule.

The residual null cells (CD4, CD8, NK1.1, B220, and F4/80) constitute up to 30% of the cellular population (7). It is possible that enrichment of the null cell population may have a modulating role in in vitro NK cell cytolytic activity. Yet, little is known about the functional characteristics of null cells and their role in regulation of NK cell cytolytic activity (11).

In splenocytes that were highly enriched NK1.1⁺ cells, chronic exercise stress was associated with a significant reduction (20%) in NK cell cytolytic activity. These results are consistent with previous experiments using NWNA splenocytes and support the hypothesis that chronic exercise stress may modulate intrinsic NK cytolytic activity as indicated by lower LU per LGL-1⁺ cell. It is appropriate to express NK cell cytolytic activity in this manner because it normalizes target lysis to the predominantly cytolytically active subpopulation of NK1.1⁺ cells (15, 16). Others have attempted to estimate cytolytic activity per individual NK cell from chronically (25) and acutely (20, 26) exercised subjects. In these studies, in vitro NK cell cytolytic activity was adjusted per NK cell from nonenriched cell populations, and lytic activity was higher in cells from endurance-trained subjects compared with controls. Hoffman-Goetz and colleagues (10) reported that strain differences in mice also influence the training-associated changes in NK cell cytolytic activity. There are smaller training-associated increases in NK cell cytolytic activity in mouse strains having inherently low cytolytic activity or in those having large interanimal variability, such as in outbred strains.

To our knowledge, the present experiments are the first to use isolated NK cells through in vitro enrichment and to examine the influence of chronic exercise on the in vitro cytolytic activity of highly enriched NK cells. Two enrichment techniques have been used in our experiments, NWNA and magnetic bead separation. The results were consistent between the methods, both yielding lower NK cytolytic activity in enriched splenocytes from Trn mice compared with controls (Sed and TM). Furthermore, B cells and macrophages were either partially removed or nearly depleted from the in vitro assays, indicating a probable modulatory role for one or both of these cell types on in vitro NK cell cytolytic activity.

B cells can secrete interleukin-12 (IL-12) (6); in the in vitro assay where B cells are present, NK cell cytolytic activity could be regulated by these cells. However, it is unknown whether B cells contribute to training-associated increases in NK cell cytolytic activity. Macrophages are known to modify NK cell cytolytic activity through secretion of products from the 5-lipoxygenase pathway (4) and via cytokines such as IL-1, IL-2, IL-12, IL-15, tumor necrosis factor-, and interferon (INF)- (5, 6, 11, 13). Unique regulation of NK cell cytolytic activity can also occur through cell-to-cell contact with macrophages (4, 9).

There is evidence that macrophage/monocyte-mediated paracrine regulation of NK cell cytolytic activity occurs in response to acute exercise stress. Pedersen et al. (22) reported transient suppression of in vitro peripheral blood NK cell cytolytic activity after strenuous exercise by human subjects. Reduced cytolytic activity was attributed to monocyte-secreted prostaglandins, and NK cell cytolytic activity was restored to preexercise values by indomethacin treatment. In the present study, NK cell cytolytic activity in splenocytes enriched for macrophages and NK1.1⁺ cells was unaffected by the effects of endurance training, whereas in highly enriched NK1.1⁺ cells, NK cell cytolytic activity was significantly lower in cells from Trn mice. In total, these data support the hypothesis that the effects of endurance training may augment the macrophage paracrine modulation of NK cell cytolytic activity against YAC-1 targets.

To address the question of whether macrophage activation and subsequent paracrine regulation of NK cell activity can occur within a 4-h in vitro assay, cytosolic second messengers can be induced within 5 min of INF- binding to its surface receptor on the macrophage (1). Within 5 min to 4 h after receptor binding, increased gene transcription, stabilization of mRNA, and synthesis of certain proteins, such as tumor necrosis factor- can occur in macrophages. During this time frame, the macrophage activation and secretion of proteins can be enhanced or suppressed by numerous factors. Various secretagogues of macrophages can be detected within 4-24 h after initiation of the cascade (1). In the presence of IL-12, a macrophage-derived cytokine, NK cell cytolytic activity is activated within a few hours (5). Direct evidence of macrophage augmentation of NK cell cytolytic activity during a standard 4-h ⁵¹Cr-release assay was observed by Nelson et al. (18) and attributed to I-A⁺ macrophage secretion of IFN-. Thus it is plausible that paracrine modulation of NK cell function by macrophages could occur within the duration of a 4-h assay.

In conclusion, the results of this study provide support for the hypothesis that the intrinsic cytolytic activity of NK cells is modified by the effects of chronic exercise stress. In light of previous experiments demonstrating equal or greater NK cell cytolytic activity in nonenriched splenocytes from Trn mice compared with controls, we hypothesize that the effects of chronic endurance exercise may alter paracrine regulation of NK cells by macrophages. It is possible that production of several secretagogues from macrophages and NK cells is altered in the adaptation to chronic exercise stress, and their secretion results in both autocrine and paracrine modulation of NK cell cytolytic function. Whether chronic exercise stress alters production of macrophage- and NK cell-derived cytokines, their soluble receptors, and the associated receptors on cell surface membranes has yet to be adequately investigated.

ACKNOWLEDGEMENTS

We thank Tarun Kshetrapal for excellent laboratory and clerical assistance.

FOOTNOTES

Address for reprint requests: S. E. Blank, Dept. of Kinesiology, PO Box 641410, Washington State Univ., Pullman, WA 99164-1410 (E-mail: Blank{at}mail.wsu.edu).

Received 8 January 1997; accepted in final form 6 May 1997.

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