Selective mobilization of CD14+CD16+ monocytes by exercise

Birgit Steppich1, Farshid Dayyani1, Rudolf Gruber2, Reinhard Lorenz3, Matthias Mack2, and H. W. Löms Ziegler-Heitbrock1

1 Institute for Immunology, 2 Medical Policlinic, Klinikum Innenstadt, and 3 Institute for Prophylaxis and Epidemiology of Cardiovascular Disease, University of Munich, 80336 Munich, Germany


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Strenuous, anaerobic exercise leads to an increase of leukocytes that are mobilized from the marginal pool. We have analyzed in human peripheral blood the effect of exercise on the number of CD14+CD16+ monocytes as determined by two-color immunofluorescence and flow cytometry. We show herein that this type of monocyte responds with a dramatic up to 4.8-fold increase. Mobilization does not occur after 1 min at 100 or 200 W but 1 min at 400 W leads to a twofold increase of the CD14+CD16+ monocytes immediately after exercise. The numbers remain high at 5 min and gradually decrease to reach the initial level at 20 min postexercise. After 20 min of rest, the CD14+CD16+ monocytes can be mobilized again by a second exercise. The CD14+CD16+ monocytes appear to be mobilized from the marginal pool where they preferentially home because of a higher expression of adhesion molecules like CD11d and very late antigen-4. Exercise goes along with an increase of catecholamines, and mobilization of the CD14+CD16+ monocytes can be substantially reduced by treatment of donors with the beta -adrenergic receptor blocker propranolol. Mobilization of CD14+CD16+ monocytes by a catecholamine-dependent mechanism may contribute to the increase of these cells in various clinical conditions.

glucocorticoid; marginal pool; stress


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

WITH THE USE OF CD14 and CD16 antibodies, human monocytes can been divided into two populations. The majority of cells is CD14 strongly positive (CD14++) and CD16 negative, and it represents what previously had been addressed as monocytes. Cells expressing CD14 at low levels together with the CD16 molecule, i.e., the CD14+CD16+ monocytes, usually comprise 10% of all monocytes (21, 29). These cells have distinct properties like a high expression of major histocompatibility complex class II and a lower level of CD11b and CD33 when compared with the CD14++ monocytes. With regard to cytokine production, CD14+CD16+ monocytes are efficient producers of the proinflammatory tumor necrosis factor, but they produce no or only little of the anti-inflammatory interleukin-10 (8). We therefore have addressed these cells as proinflammatory monocytes. This designation fits well with the finding that these cells are strongly elevated in inflammatory diseases like bacterial infections (18), including sepsis (3, 5). Also the CD14+CD16+ monocytes were found to be increased in patients postmajor cardiac surgery, patients that showed features of system inflammation without infection (7).

On the other hand, Gabriel et al. (10) reported that brief periods of exercise will increase low CD14 monocytes by a factor of 2. We have analyzed this observation in detail to elucidate the mechanism of increase. We could demonstrate an average 3.5-fold increase in the number of CD14+CD16+ monocytes by 1 min of excessive exercise. These data show that, under resting conditions, the majority (>75%) of the CD14+CD16+ monocytes are not in peripheral blood, but they reside in the marginal pool.

We hypothesized that the exercise-induced increase is mediated by adrenal hormones. In fact, our blocking studies show that mobilization of the CD14+CD16+ monocytes is mediated in part by catecholamines. The rapid mobilization of these cells into the central pool under conditions of stress may serve to provide a large population of active cells for defense at sites of injury and infection.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Volunteers. Volunteer participants for the study were recruited from laboratory staff and from the students of the University of Munich. The participants were apparently healthy males, aged 32.1 ± 7.6 yr. For studies involving blockade of beta -adrenergic receptors, participants were subject to a medical examination, including electrocardiogram, and written informed consent was obtained from eligible subjects. For treatment with high-dose glucocorticoids, volunteers were subjected to a medical examination, including clinical chemistry for liver and kidney function and blood differential. After written informed consent, glucocorticoids, i.e., 250 mg methylprednisolone (Urbason solubile; Hoechst-Marion-Roussel, Bad Soden, Germany), were administered intravenously over a 15-min period on three consecutive days. The study was cleared by the Ethics Committee of the Medical Faculty, University of Munich. Volunteers did not take any medication in the preceding 4 wk.

Forms of exercise. For the first type of exercise employed in our study, participants were asked to run at the highest possible speed from the basement to the roof in a stairway consisting of 129 stairs with a total height of 21.3 m. The average time required for the task was 36 s (range 35-37 s). The calculated work ranged from 426 to 506 W depending on the body weight of the participant. Blood was drawn before and immediately after the exercise.

For a graded and defined workload, we used a home trainer bike (Ergo-bike; Daum-Electronic, Veitsbronn, Germany) equipped with an eddy current brake for selection of different levels of exercise intensity. Duration of exercise ranged from 45 s to 2 min at 100, 200, or 400 W. For continuous sampling, participants received a permanent venous access (Venflow 2; Ohmeda, Helsingborg, Sweden) that was kept open by slow infusion of a balanced salt solution. Blood was drawn before and at 1, 5, 10, and 20 min after the exercise.

For the study of blockade of beta -adrenergic receptors, volunteers after the first exercise at 400 W for 45 s took 5 × 40 mg of propranolol (Dociton 40; Zeneca, Plankstadt, Germany) over a period of 24 h. The last dose of propranolol was taken 1 h before the second exercise at 400 W for 45 s. For these studies, blood was drawn before and 5 min after the exercise.

In the glucocorticoid depletion study, volunteers exercised to their maximum capability (75-90 s at 400 W for these individuals) on day 0, they received glucocorticoid infusion on days 1, 2, and 3, and they again exercised for the same period of time at 400 W on day 4.

Leukocyte analysis. Two-milliliter EDTA-blood samples were taken. The total leukocyte count was determined with a cell dyne counter (Abbott, Wiesbaden, Germany). For determination of monocyte populations, 100-µl blood samples were washed one time with PBS and then admixed with the CD14 antibody My-4 conjugated to FITC (no. 6603511; Coulter Electronics, Krefeld, Germany) and with the CD16 antibody B73.1 conjugated to phycoerythrin (no. 347617; Becton Dickinson, Heidelberg, Germany) at saturating concentrations. After incubation for 20 min on ice, the erythrocytes were lysed, and cells were analyzed using a fluorescence-activated cell-sorter (FACS, Becton Dickinson). Compensation was adjusted using the two control stains. With scatter gates around leukocytes, we acquired 5,000 CD14+CD16+ monocytes plus CD14++ monocytes according to the green/yellow analysis. Percentages of the CD14+CD16+ monocytes and the CD14++ monocytes were calculated from the two-color plot. Absolute number of monocytes per microliter of blood was calculated based on the cell dyne number and the FACS scatter gate for all leukocytes.

For three-color immunofluorescence, whole blood samples were stained with two different combinations. For detection of CD11d, anti-CD11d (antibody 217L, kindly provided by W. Michael Gallatin and Darcey Clark, ICOS, Bothell, WA), or the respective isotype, control was employed for a first incubation on ice for 20 min. After cells were washed, goat anti-mouse-Ig-FITC (no. M35001; Medac, Hamburg, Germany) was added for 20 min. Next, mouse IgG (no. M-9269; Sigma, Munich, Germany) was added at 50 µg/ml, followed by addition of anti-CD14 antibody My-4 conjugated to phycoerythrin (no. 6603262; Coulter) plus anti-CD16 antibody conjugated to the energy-transfer complex PC-5 (no. 1851; Coulter/Immunotech). After a final 15-min incubation, erythrocytes were lysed.

For detection of very late antigen-4 (VLA-4) and CD62L, the reagents anti-VLA-4 (no. 0764; Coulter-Immunotech, Hamburg, Germany) and anti-CD62L (no. 347440; Becton Dickinson) or the respective isotype control was employed for a first incubation on ice for 20 min and then cells were washed. Goat anti-mouse-Ig-Tricolor (no. 35006; Medac) was added for 20 min. Next, mouse IgG (no. M-9269; Sigma) was added at 50 µg/ml, followed by addition of anti-CD14 My-4-FITC and anti CD16-phycoerythrin. After a final 15-min incubation, erythrocytes were lysed.

In FACS analysis for both types of staining, gates were set around the two monocyte populations in the two-color plot for CD14 and CD16, and the signals for the third marker were analyzed in single-parameter histograms. Here the specific median fluorescence intensity was calculated by subtracting the median for the isotype control from the median for the specific antibody. With the type of data processing used herein, an increase of the fluorescence intensity by 77 channels reflects a doubling of antigen expression.

Lactate. Blood was drawn in a sodium-heparin vacutainer (Becton Dickinson) and centrifuged immediately at 4°C, and plasma was stored without delay at -20°C for not more than 4 wk. Lactate concentration was determined with a commercially available test kit based on an enzymatic reaction on an autoanalyzer (TDx; Abbott).

Catecholamines. Plasma catecholamines were determined in blood samples collected in vials containing EGTA (1.8 mg/ml final) and reduced glutathione (2.4 mg/ml final). Samples were chilled on ice immediately, centrifuged in the cold, and stored at -80°C until analysis. An established method (26) with some modifications was employed. This involved addition of an internal standard (dihydroxybenzylamine) and adsorption to aluminum oxide followed by reverse-phase HPLC and electrochemical detection.

Statistics. For statistical analysis of paired data, the Wilcoxon test was employed. For analysis of time course data, the Bonferroni-Holm test for multiple data points was used.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Mobilization of CD14+CD16+ monocytes by an upstairs run. Staining of whole blood samples with CD14 and CD16 antibodies allows for detection of two monocyte populations. One population is strongly positive for CD14 and lacks CD16 (Fig. 1A). These cells are the classical monocytes. A second cell population coexpresses the CD14 antigen at a low density together with the CD16 antigen. These double positive cells usually account for 10% of all monocytes and for 50.3 ± 19.5 cells/µl blood in apparently healthy donors (n = 10).


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Fig. 1.   Monocyte subpopulations before and after excessive exercise. Whole blood samples of one donor were stained with anti-CD14 and anti-CD16 antibodies, and fluorescence-activated cell-sorter (FACS) analysis was performed with scatter gates set around monocytes. A: before; B: after running upstairs (36 s, 21.3 m in height, 129 stairs). The CD14+CD16+ monocytes increased from 34 to 124 cells/µl in this example.

After an excessive exercise, i.e., an upstairs run of 21.3 m in height (129 stairs) in about 36 s, the CD14+CD16+ monocytes show a dramatic increase (Fig. 1B). The average increase in seven donors is 3.5-fold, with an average of 50.5 ± 19.7 CD14+CD16+ monocytes/µl before and 175.9 ± 71.2 monocytes/µl after the run (Fig. 2). The strongest increase observed was 4.8-fold.


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Fig. 2.   Effect of excessive exercise on CD14+CD16+ monocytes. Given is the number of CD14+CD16+ monocytes/µl of blood before and after an upstairs run (36 s, 21 m in height, 129 stairs). The average numbers were 50.5 ± 19.7 before and 175.9 ± 71.2 after the exercise (P < 0.01). The average increase was 3.5-fold.

These data show that, under resting conditions, only a small fraction of all CD14+CD16+ monocytes is detectable in peripheral blood. A three times larger fraction is recruited to appear in blood after a short period of excessive exercise.

Effect of graded workload on mobilization of CD14+CD16+ monocytes. Next, we asked what extent of exercise is required for the increase of the CD14+CD16+ monocytes to occur. For this, participants exercised on a home trainer bike for 1 min at 100 W followed by 20 min of rest. They then exercised for 1 min at 200 W, and after another 20 min of rest they exercised for 1 min at 400 W. Both the exercise at 100 and at 200 W did not result in a significant increase in the number of CD14+CD16+ monocytes (Fig. 3). An exercise for 1 min at 400 W, however, resulted in a 2.1-fold increase of these cells. This increase was evident immediately after the exercise, persisted for 5 min, and declined to the initial level after 20 min (Fig. 3). The increase of monocytes in response to exercise was quite specific to the CD14+CD16+ monocytes since the classical CD14++ monocytes showed only a moderate 1.3-fold increase (Fig. 3, bottom).


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Fig. 3.   Effect of graded workload on the two monocyte populations. Donors received a permanent peripheral venous access and exercised on a home trainer bike for 1 min each (bars) at 100, 200, and 400 W. Blood was drawn at various time points, and CD14+CD16+ (first 3 panels) and CD14++ (last panel) monocyte numbers were determined by FACS analysis (average of 3 donors ± SD, * P < 0.05). At 400 W, the average increase for the CD14+CD16+ monocytes was 2.1-fold, and for the CD14++ monocytes it was 1.3-fold.

We then asked whether the exercise leading to the increase of the CD14+CD16+ monocytes was really exhaustive such that it exceeded the individual's aerobic capacity. The average lactate levels were 1.06 ± 0.35 mM before and 5.39 ± 2.99 mM after 1 min at 400 W (P < 0.05), indicating that the exercise was anaerobic.

Effect of repeated exercise on mobilization of CD14+CD16+ monocytes. We next analyzed the effect of extending the time of exercise on the increase of CD14+CD16+ monocyte numbers. Donors exercised on the bike for 1 min at 400 W, and after 20 min of rest they exercised for 2 min at 400 W. As shown in Fig. 4, the average 2.1-fold increase after 1 min at 400 W is exceeded by a 2.8-fold increase of the CD14+CD16+ monocytes after 2 min of exercise. In addition to demonstrating an increased mobilization when increasing the duration of the exercise, these data also show that the cells that are mobilized after the first exercise disappear with rest and then can be mobilized once again.


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Fig. 4.   Effect of increasing duration of exercise on mobilization of CD14+CD16+ monocytes. Donors received a permanent peripheral venous access and exercised for 1 min at 400 W; 20 min later they exercised for 2 min at 400 W. Blood was drawn at various time points, and CD14+CD16+ monocytes were determined by FACS analysis (average of 3 donors, * P < 0.05).

Expression of adhesion molecules by monocyte populations. Adhesion molecules may be involved in localization of cells to a compartment like the marginal pool. We therefore analyzed adhesion molecules on the CD14+CD16+ monocytes compared with the CD14++ cells. Expression of CD11d was clearly higher on the CD14+CD16+ monocytes as was VLA-4 (2.7- and 3.4-fold, respectively). At the same time, expression of CD62L was virtually absent from these cells (Fig. 5). Additional integrin molecules that were found to be upregulated in the CD14+CD16+ monocytes were CD11a and CD11c (Table 1). By contrast, as noted earlier (28), CD11b was substantially lower, and CD18, the common beta -chain for CD11a-d, showed a similar level of expression for the CD14+CD16+ monocytes and the CD14++ cells. Also, in addition to CD62L, the CD15 and CD15s markers were found to be almost absent in the CD14+CD16+ monocytes (Table 1).


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Fig. 5.   Expression of adhesion molecules on the two monocyte populations. Whole blood samples were stained in 3-color immunfluorescence with VLA-4, CD11d, and CD62L or with isotype control followed by fluorescein-labeled secondary antibody. This was followed by blockade with an excess of mouse IgG and then by staining with CD14-phycoerythrin and CD16-Tricolor; 5,000 CD14+CD16+ monocytes plus CD14++ monocytes were analyzed per sample. Staining for adhesion molecules is shown for CD14++ (A) and CD14+CD16+ (B) cells. The specific median fluorescence intensity given in the top right of the histogram was calculated by subtracting the median for the isotype staining from the median for the specific staining. Results are representative of 6-14 donors.


                              
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Table 1.   Expression of adhesion molecules by monocyte subpopulations

We next asked whether the CD14+CD16+ monocytes mobilized from the marginal pool show an even higher expression of adhesion molecules. To study this question, we used cells from donors that had performed the upstairs run, since under these conditions >75% of the CD14+CD16+ monocytes represent mobilized cells.

In these studies, we found similar levels of expression for adhesion molecules before and after exercise (Fig. 6). The data suggest that the CD14+CD16+ monocytes preferentially localize to the marginal pool because of their higher expression of adhesion molecules compared with the classical CD14++ cells. The expression of these molecules is, however, similar on the CD14+CD16+ monocytes in both compartments.


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Fig. 6.   Comparison of adhesion molecules for CD14+CD16+ monocytes before (A) and after (B) excessive exercise. Blood was taken from donors before and after running upstairs (36 s, 21.3 m, 129 stairs), and adhesion molecule expression was determined as described in legend to Fig. 6. After exercise, the CD14+CD16+ cells were increased by a factor of 3.3. Therefore, the majority, i.e., >75% of the CD14+CD16+ has been newly recruited cells, and the adhesion molecule expression largely represents that of the mobilized CD14+CD16+ cells. The specific median fluorescence intensity given in the top right of the histogram was calculated by subtracting the median for the isotype staining from the median for the specific staining. Results are representative of 3-5 donors.

Role of catecholamines in mobilization of CD14+CD16+ monocytes. Excessive exercise is associated with the release of stress hormones like catecholamines. We have determined the levels of both epinephrine and norepinephrine in blood of donors before and after 1 min of exercise at 400 W. As can be seen in Table 2, both hormones were increased severalfold immediately after exercise.

                              
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Table 2.   Effect of exercise on blood catecholamine levels

We have therefore asked whether these hormones might be involved in the mobilization of the CD14+CD16+ monocytes. For this, volunteer donors exercised for 45 s at 400 W before and after five doses of 40 mg of the nonspecific beta -adrenergic receptor blocker propranolol over a period of 24 h. Of eight donors studied, two showed no effect of beta -blockade on these cells (Fig. 7) although a reduction of the increase of the heart rate after exercise was achieved in all donors, including these two (data not shown). In six donors, beta -propranolol clearly blocked the increase in the CD14+CD16+ monocytes after exercise, with a near-complete ablation in some, i.e., the degree of increase is close to one (Fig. 7). Hence, it appears that catecholamines are involved to some extent in the mobilization of the CD14+CD16+ monocytes in most donors.


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Fig. 7.   Effect of blockade of beta -adrenergic receptors on mobilization of CD14+CD16+ monocytes. Blood was taken from donors before and after a 45-s exercise at 400 W. After the initial exercise, donors took 5 × 40 mg of propranolol over a period of 24 h. One hour after the last dose, donors exercised once again for 45 s at 400 W. Data are expressed as %change compared with the preexercise level of CD14+CD16+ monocytes. Left, before; right, after propranolol. Mobilization after beta -propranolol was significantly reduced (P < 0.05).

Effect of glucocorticoids on mobilization of CD14+CD16+ monocytes. Previous studies have shown that high-dose therapy with glucocorticoids will selectively deplete the CD14+CD16+ monocytes (6). Depletion of these cells in the central pool could be due to a shift into the marginal pool. We have therefore treated healthy volunteers with methylprednisolone at 250 mg/day for three consecutive days. Twenty-four hours before the first and 24 h after the last dose, the volunteers were subjected to a maximum exercise on the home trainer bike at 400 W. The first exercise led to a 3.5-fold increase from 33 to 109 cells/µl, i.e., 348% (Fig. 8). The 3-day treatment with glucocorticoids reduced the number of CD14+CD16+ monocytes to 8 cells/µl. Exercise on day 4 also led to a substantial enhancement of cell numbers by a factor of 3.5, but the maximum level achieved was only 98% of the starting level (Fig. 8).


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Fig. 8.   Effect of high-dose glucocorticoid treatment on mobilization of the CD14+CD16+ monocytes. Donors exercised for 75-90 s (filled bars) at 400 W before (A) and after 3 days (B) of glucocorticoid treatment at 250 mg daily, and the number of CD14+CD16+ monocytes was followed over time. Results are expressed as percentage of the initial value on day 0. The difference between the initial value (average of 33 cells/µl) and the depleted value on day 4 (average of 8 cells/µl) was significant, with P < 0.05 (average of 3 donors ± SD, * P < 0.05 compared with the value immediately before exercise).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The existence of distinct monocyte populations in human blood is now well established (27). In the present report, we have analyzed the mobilization of monocytes by anaerobic exercise, and we demonstrate a dramatic increase (average 3.5-fold) of the CD14+CD16+ blood monocyte subpopulation within seconds of an upstairs run. This type of exercise, although constant with respect to altitude to be covered, is not very well controlled with respect to work load. Also, it is not very well suited for continuous monitoring of parameters. We have therefore used graded levels of exercise on a home trainer bike. Here we could show that a 200-W exercise for 1 min is not sufficient for a significant mobilization of the CD14+CD16+ monocytes. Rather, 400 W for 1 min are required. After 20 min of rest, the CD14+CD16+ monocytes can be mobilized again by a second round of exercise at 400 W for 2 min. This indicates that the CD14+CD16+ monocytes are mobilized from a dynamic compartment.

Similar observations have been made for neutrophils and for lymphocytes (17). Among the latter, the natural killer (NK) cells show the most pronounced increase (9, 12). These cells are considered to be mobilized from the marginal pool, a pool that has been shown in experimental animals to contain granulocytes, NK cells, and monocytes (14). We suggest that the CD14+CD16+ monocytes recruited after excessive exercise stem from this marginal pool.

The assignment of the cells to the marginal pool can, however, not be demonstrated directly in this system. Here distribution analysis with tagged cells might give direct evidence. Direct demonstration of the marginal pool can, however, only be achieved in an animal model, where the central pool can be defined by bleeding and the marginal pool by extensive perfusion of the vasculature (14).

The pig has recently been demonstrated as a useful animal model, since here a homologue of the CD14+CD16+ monocytes, i.e., CD163+ cells, has been identified (23). Hence, the pig model may be employed for the study of the CD14+CD16+ homologue in the marginal pool.

Our findings show that mobilization from the marginal pool mainly applies to the CD14+CD16+ monocytes while the CD14++ cells are hardly affected. What then is the basis of this selectivity?

One possibility is the higher expression of adhesion molecules on the CD14+CD16+ monocytes. These cells do show clearly higher levels of CD11d and VLA-4. VLA-4 is an integrin that mediates rolling on and adhesion to vascular cell adhesion molecule (VCAM)-1 on endothelial cells (1, 16). CD11d is a recently identified beta 2-integrin that can interact with endothelial intercellular adhesion molecule-3 (22, 25) but also with VCAM-1 (11). The question is whether such interactions can occur with resting endothelial cells. It is evident, however, that rolling of leukocytes does occur on endothelium in vivo in the absence of inflammation (15, 19). Our concept that the CD14+CD16+ monocytes preferentially localize to the marginal pool is also supported by the finding that these cells fail to express CD62L. Palecanda et al. (20), on the basis of cross-linking studies, have suggested that leukocytes, when rolling on endothelial cells, will shed the L-selectin, leading to the presence of soluble serum L-selectin in the absence of inflammation. Other selectins still may be involved in the interaction of the CD14+CD16+ monocytes with endothelium, since these cells do have a higher expression of P-selectin-glycoprotein-ligand-1 that may allow for interaction with P-selectin on endothelium.

The finding that the monocytes with a higher expression of adhesion molecules are found mainly in the marginal pool is very similar to findings in the rat. Here the frequency of cells with high leukocyte-function antigen-1 expression was low in the central pool and very high in the marginal pool (14). When adhesion molecules, in fact, mediate localization to the marginal pool, the question is how cell attachment to endothelial cells can change so rapidly. Here changes in affinity of the adhesion molecules on the leukocytes are a likely explanation (4, 13).

Such an affinity decrease may be brought about by catecholamines. We have shown herein that catecholamines rapidly increase after excessive exercise. Furthermore, blockade of beta -adrenergic receptors reduced mobilization of the CD14+CD16+ monocytes or even prevented it in some donors. One might argue that beta -blockade will reduce the intensity of work since catecholamines cannot stimulate energy supply appropriately. In our experiments, the donors exercised, however, at a submaximal level with a fixed work load before and after the beta -blockade. Hence, the reduction of CD14+CD16+ monocytes appears to be a direct effect of beta -blockade at the level of the leukocyte. A role for catecholamines has been clearly demonstrated also for the mobilization of NK cells after epinephrine infusion (24). Also, mobilization of NK cells after psychological stress was successfully blocked by propranolol (2). When looking at the effect of beta -propranolol on catecholamine levels, we noted in preliminary experiments with three donors an increase of the basal level of norepinephrine from 64 ± 36 to 109 ± 48 pg/ml, whereas exercise led to a similar increase before (364 ± 257 pg/ml) and after the beta -blockade (261 ± 192 pg/ml).

We failed to demonstrate a blockade of CD14+CD16+ monocyte mobilization in two of eight donors. Albeit less pronounced compared with the six donors with a propranolol suppression of CD14+CD16+ monocyte mobilization, the CD16+ NK cells in these subjects were also decreased by propranolol treatment in these two donors (data not shown). Hence, it is clear that the beta -blocker did work in these donors. Therefore, it is possible that in these donors mobilization is independent of catecholamines.

The depletion of the CD14+CD16+ monocytes by glucocorticoids as reported earlier (6) has been confirmed in this study. This depletion could be due to a shift of the cells into the marginal pool. If this is true, then exercise-induced mobilization should result in similarly high levels of CD14+CD16+ monocytes both before and after glucocorticoid treatment. We found, however, that the average mobilization after glucocorticoid treatment only reached the basal level of CD14+CD16+ monocyte numbers. This then indicates that glucocorticoids do not shift these cells into the marginal pool. Rather they deplete the cells in both the central and the marginal pool. Still one can argue that glucocorticoids will shift the CD14+CD16+ monocytes into the marginal pool in a fashion that renders them resistant to mobilization, i.e., the cells might be sticking tightly to the endothelium. To analyze this possibility, in situ analysis in an animal model is required.

Taken together, the data herein show that CD14+CD16+ monocytes are selectively mobilized from the marginal pool in a rapid catecholamine-dependent fashion.

The CD14+CD16+ monocytes have been shown to increase in clinical conditions like sepsis, human immunodeficiency infection, and postsurgical systemic inflammatory syndrome and in patients with atherogenic lipid profile (27). This increase was interpreted to reflect a net increase of the cells in blood, but based on the current findings such increases in number could also be due to a shift from the marginal pool into the central pool.


    ACKNOWLEDGEMENTS

We acknowledge the generous provision of anti-CD11d antibodies by ICOS (Bothell, WA). We also thank M. Blumenstein (Munich, Germany) for helpful discussion and H. Gabriel (Jema, Germany) for critically reading the manuscript.


    FOOTNOTES

This work was supported by grants from the Ernst and Berta Grimmke-Foundation (Düsseldorf, Germany), from Deutsche Forschungs-gemeinschaft (SFB 217, A6), from the Graduierten-Kolleg "Infektion und Immunität" at the University of Munich, and from Hoechst-Marion-Roussel (Bad Soden, Germany).

The data presented herein are part of the thesis of B. Steppich.

Address for reprint requests and other correspondence: H. W. L. Ziegler-Heitbrock, GSF-National Research Center, Institute for Inhalation Biology, Cooperation Group Aerosols in Medicine, Robert-Koch-Allee 6, 82131 Gauting, Germany (E-mail: ziegler{at}gmx.de).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Received 22 June 1999; accepted in final form 20 March 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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