Journal of Applied Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

J Appl Physiol 100: 1124-1133, 2006. First published December 15, 2005; doi:10.1152/japplphysiol.00562.2005
8750-7587/06 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow All Versions of this Article:
100/4/1124    most recent
00562.2005v1
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (2)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zaldivar, F.
Right arrow Articles by Cooper, D. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zaldivar, F.
Right arrow Articles by Cooper, D. M.

Constitutive pro- and anti-inflammatory cytokine and growth factor response to exercise in leukocytes

Frank Zaldivar,1 Jessica Wang-Rodriguez,2 Dan Nemet,1,3 Christina Schwindt,1 Pietro Galassetti,1 Paul J. Mills,4 Lori D. Wilson,1 and Dan M. Cooper1

1Pediatric Exercise Research Center, Department of Pediatrics, University Children's Hospital, University of California, Irvine; 2Veterans Affairs San Diego Healthcare System, San Diego; Department of Pathology, University of California, San Diego, California; 3University of Tel Aviv, Meir Hospital, Kfar Saba, Israel; and 4Department of Psychiatry, University of California, San Diego, California

Submitted 12 May 2005 ; accepted in final form 6 December 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Leukocytosis following exercise is a well-described phenomenon of stress/inflammatory activation in healthy humans. We hypothesized that, despite this increase in circulating inflammatory cells, exercise would paradoxically induce expression of both pro- and anti-inflammatory cytokines and growth factors within these cells. To test this hypothesis, 11 healthy adult men, 18–30 yr old, performed a 30-min bout of heavy cycling exercise; blood sampling was at baseline, end-exercise, and 60 min into recovery. The percentage of leukocytes positive for intracellular cytokines and growth factors and mean fluorescence intensity was obtained by flow cytometry. Proinflammatory cytokines (IL-1{alpha}, IL-2, IFN-{gamma}, and TNF-{alpha}), a pleiotropic cytokine (IL-6), and anti-inflammatory cytokines and growth factors [IL-4, IL-10, growth hormone (GH), and IGF-I] were examined. Median fluorescence intensity was not affected by exercise; however, we found a number of significant changes (P < 0.05 by mixed linear model and modified t-test) in the numbers of circulating cells positive for particular mediators. The pattern of expression reflected both pro- and anti-inflammatory functions. In T-helper lymphocytes, TNF-{alpha}, but also IL-6, and IL-4 were significantly increased. In monocytes, both IFN-{gamma} and IL-4 increased. B-lymphocytes positive for GH and IGF-I increased significantly. GH-positive granulocytes also significantly increased. Collectively, these observations indicate that exercise primes an array of pro- and anti-inflammatory and growth factor expression within circulating leukocytes, perhaps preparing the organism to effectively respond to a variety of stressors imposed by exercise.

immunology; interleukin-6; growth hormone; intracellular

MANY OF THE HEALTH EFFECTS of exercise are influenced by the balance of stress/inflammatory-related cytokines and growth factors that are known to be altered by physical activity. For example, in elderly humans, higher levels of the circulating cytokine IL-6 are inversely correlated with levels of physical activity, with muscle mass, and with the anabolic mediator IGF-I (3, 19, 54). Brief bouts of heavy exercise also lead to increased numbers of circulating stress/inflammatory cells, such as lymphocytes [T, B, and natural killer (NK) cells], monocytes, and neutrophils, all of which can secrete a variety of cytokines and growth factors (31, 45), and there are new data to suggest that stress activation of circulating leukocytes may play beneficial roles in subsequent poststress challenges, such as tissue injury (55). Leukocytes are increasingly viewed as virtual endocrine, autocrine, or paracrine organs (20), capable of supplying specific tissues with a vast array of mediators that play a role not only in immune responses but also in functions ranging from angiogenesis to bone remodeling (7, 34), all part of the adaptive response to exercise (23, 41). Consequently, the purpose of this study was to examine the impact of brief exercise in healthy young adults on key cytokines and growth factors found within specific leukocyte subpopulations in the circulating blood.

Flow cytometry is used extensively to assess changes in intracellular cytokine production and to identify the specific leukocyte subsets in which these changes are occurring (51). This powerful tool has led to a number of new insights regarding the relationship between intracellular cytokines and leukocyte subtypes. First, it can no longer be assumed that changes in cytokine concentration in the plasma parallel changes observed in those same cytokines within the circulating leukocytes (47). Second, the pattern of intracellular cytokine and mediator response to specific challenges is different within the leukocyte subtypes (e.g., the expression of mediators within monocytes may not parallel the expression of these same mediators in lymphocytes). Thus examining a single population of leukocytes reveals only part of the process.

Most studies involving exercise have examined traditional immune system mediators (24, 27, 35, 44, 46, 48, 49, 52) [e.g., IL-4 and IFN-{gamma} to gauge the impact of exercise on the balance of T-helper-1 (TH1) to T-helper-2 (TH2) type lymphocytes] within a particular leukocyte phenotype (37). Recently, investigators have used flow cytometry to examine lymphocyte expression of heat shock proteins and heme oxygenases in response to exercise (18, 33). Finally, measurement of intracellular IL-6 in monocytes has been used to demonstrate that monocytes were not the source of the increased circulating levels of IL-6 that accompany heavy exercise (47).

There is mounting evidence that the responses to stresses such as exercise likely involve many leukocyte phenotypes and a variety of mediators, even antagonistic mediators. In 1999, Ostrowski et al. (36) noted that strenuous exercise led to increased circulating levels of proinflammatory mediators, but simultaneously, "... cytokine inhibitors and anti-inflammatory cytokines restrict the magnitude and duration of the inflammatory response to exercise." Subsequent investigators have shown that other stressors, such as sepsis (40), also cause a seemingly paradoxical increase in the circulating levels of both pro- and anti-inflammatory cytokines. Studies in adults and children now demonstrate that exercise stimulates growth and stress factors [such as growth hormone (GH) and IL-6] known specifically to antagonize one another (12, 14, 58).

Our laboratory recently demonstrated that a single bout of heavy exercise in young men substantially alters peripheral blood mononuclear cell (PBMC) gene expression characterized in many cases by a brisk upregulation of genes associated with seemingly antagonistic functions of stress, inflammation, and tissue repair (8). Along these lines, hematopoietic cells have been shown to express growth mediators like GH and IGF-I (22, 59) not typically associated with immune function. Accordingly, we hypothesized that brief bouts of heavy exercise would lead to seemingly paradoxical changes in the pattern of intracellular cytokine and growth factor expression in circulating leukocytes, reflecting both pro- and anti-inflammatory function and growth mediators. Based on our studies and studies in other laboratories, we performed multiparameter flow cytometric studies, which examined nine representative cytokines (15, 17) [four proinflammatory/catabolic (IL-1{alpha}, IL-2, IFN-{gamma}, and TNF-{alpha}), four anti-inflammatory or anabolic (IL-4, IL-10, GH, and IGF-I), and one mixed (IL-6)] in six leukocyte subsets, ranging from neutrophils to monocytes, at three time points (baseline, peak exercise, and recovery).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Subjects.   Healthy young adult men (n = 11), between the ages of 18 and 30 yr, were recruited for this study. Exclusion from participation included history of any chronic medical conditions, use of any medications, or training as a competitive athlete. Our Institutional Review Board approved this study, and written, informed consent was obtained from all participants. The studies were conducted at the University of California Irvine General Clinical Research Center (GCRC).

Height, weight and body mass index.   Standard, calibrated scales and stadiometers were used to determine height, weight, and body mass index (weight/height2).

Measurement of cardiorespiratory fitness and exercise intervention.   Because many metabolic and immune responses to exercise are "dose" dependent (32), i.e., they are influenced by the relative magnitude and duration of the work input, care was taken to scale the exercise to the capability of each subject. We used an approach that has been successful in normalizing the exercise input in subjects with differing exercise capacities (4). We chose 30 min of heavy exercise [defined as work performed above the subject's anaerobic or lactate threshold (LT)] based on our laboratory's previous experience with healthy nonathletes, because exercising above the LT leads to a robust metabolic and cardiorespiratory response (9).

Subjects reported to the GCRC Applied Physiology-Human Performance Laboratory for two visits on separate days. In visit 1, the volunteers performed a ramp-type progressive exercise test on an electronically braked, servo-controlled cycle ergometer to assess maximal exercise tolerance. Gas exchange was measured breath by breath, and the peak oxygen uptake (VO2) was determined as previously described using a Vmax-229 system (SensorMedics) (10). The LT was determined according to the method described by Beaver et al. (5). We included those subjects demonstrating a peak VO2 in the range of 35–45 ml·min–1·kg–1, a level consistent with a moderate level of fitness. On visit 2, separated by at least 48 h, but completed within 1 wk after visit 1, subjects were required to exercise for 30 min at a work rate that corresponded to 50% of the difference between the work rate at the LT and peak work rate. This work rate is equivalent to ~80% of the peak VO2.

Blood sampling and analysis.   During visit 2, blood was sampled from an indwelling catheter placed in the antecubital vein. Separate samples were used for complete blood count, ELISA, or flow cytometry assays. The subjects were instructed to sit quietly for 30 min before baseline draw to ensure that measurable physiological parameters of stress (e.g., heart rate and blood pressure) returned to baseline levels after catheter placement. A baseline blood sample was taken before the start of exercise (Pre sampling time point). Subjects then completed the 30-min exercise bout, and a blood sample was obtained immediately following exercise (End sampling time point). The subject was instructed to sit quietly during the recovery phase and was observed by the GCRC nurses. A final blood sample was drawn after 60 min of exercise (Recovery sampling time point). All serum samples used for ELISA assays were aliquoted and stored at –80°C until use. Samples were only thawed once.

Serum lactate, circulating leukocytes, serum cytokines, and growth factors.   Serum lactate was measured with the YSI lactate analyzer (YSI 1500, Yellow Springs, OH), with a sensitivity of 0.2 mg/dl. Complete blood counts were obtained by standard methods from the clinical hematology laboratory at our institution.

The following serum factors were measured using commercially available kits manufactured by R&D Systems (Minneapolis, MN) and included the following: IL-1{alpha} intra-assay coefficient of variation (CV) 1.1–2.2%, interassay CV 3.4–4.3%, sensitivity ≤1.0 pg/ml; IL-2 intra-assay CV 2.0–4.3%, interassay CV 3.7–5.0%, sensitivity ≤7 pg/ml; IL-4 intra-assay CV 3.4–5.3%, interassay CV 6.1–7.8%, sensitivity ≤10 pg/ml; IFN-{gamma} intra-assay CV 2.6–4.7%, interassay CV 3.7–7.8%, sensitivity ≤8 pg/ml; IL-6 intra-assay CV 3.8–11.1%, interassay CV 9.9–16.5%, sensitivity ≤0.094 pg/ml; IL-10 intra-assay CV 6.6–8.5%, interassay CV 8.1–15.6%, sensitivity ≤0.5 pg/ml; TNF-{alpha} intra-assay CV 5.3–8.8%, interassay CV 10.8–16.7%, sensitivity ≤0.12 pg/ml; GH intra-assay CV 3.3–4.3%, interassay CV 6.3–6.5%, sensitivity 0.03 ng/ml; and IGF-I (total circulating IGF-I was detected by extracting IGF-I from IGF-I binding protein by using a modified acid-ethanol extraction method) intra-assay CV 4.5–7.1%, interassay CV 4.8–8.8%, sensitivity 0.03 ng/ml (GH and IGF-I; Diagnostic System Laboratories, Webster, TX). For IL-{alpha}, IL-4, and IFN-{gamma}, additional dilutions were made to ensure our ability to measure cytokine levels below the company-provided standard curves.

Antibodies used in flow cytometry to detect surface markers and intracellular mediators.   Whole blood was drawn into a vacutainer containing sodium heparin at the Pre, End, and Recovery sampling points. All samples were treated with Golgi stop reagent (Berfeldin A, 10 µg/ml) immediately after phlebotomy and incubated for a total of no more than 4 h before analysis. The samples were not stimulated by mitogens. Surface antigen-specific fluorescent-conjugated monoclonal antibodies (MAbs) CD3 peridinin chlorophyll protein (PerCP)-Cy5.5, CD4 antigen presenting cell (APC), CD8 PerCP-Cy5.5, CD19 APC, and intracellular antibodies to IL-1{alpha} phycoerythrin (PE), IL-2 FITC, IL-4 PE, IL-6 FITC, IL-10 PE, IFN-{gamma} PE, TNF-{alpha} FITC, and all appropriate isotype controls were purchased from Pharmingen/Becton Dickinson (San Diego, CA). Similar fluorochromes were used in separate tubes to generate data. Anti-GH purchased from ICN Biomedicals (Aurora, OH) and anti-IGF-I purchased from R&D Systems (Minneapolis, MN) were custom conjugated to the appropriate fluorochrome by Bethyl Laboratories (Montgomery, TX).

Staining for cell surface antigens and intracellular cytokines.   Briefly, after incubation with Berfeldin A, 100 µl of blood from each sample were added to 12 x 75-mm tubes with specific surface antigen fluorescent-conjugated MAbs, mixed well, and incubated in the dark at room temperature for 15 min. Two milliliters of 1x FACS Lysing Solution (Becton Dickinson, San Jose, CA) were added to lyse red blood cells, mixed gently, and incubated for 10 min at room temperature in the dark. Two milliliters of 1x wash buffer (Becton Dickinson) were added and centrifuged at 500 g for 5 min. Supernatant was removed without disturbing the cell pellet. Five hundred microliters of 1x FACS Permeablizing Solution (Becton Dickinson) were added and mixed well to resuspend the pellet and incubated for 10 min at room temperature in the dark. Three milliliters of 1x wash buffer were added and centrifuged at 500 g for 5 min. Supernatants were removed, and fluorescent-conjugated anti-cytokine/growth factor MAbs were added and incubated for 30 min in the dark. Cells were washed as described above and were resuspended in 500 µl of 5% paraformaldehyde.

Acquisition and analysis.   Samples were acquired by using a FACS Calibur flow cytometer (Becton Dickinson). All surface and intracellular antibodies were optimized for four-color fluorescent antibody conjugates. CaliBRITE beads and FACSComp software were used for setting the photomultiplier tube voltages and the fluorescence compensation, as well as checking instrument sensitivity before use. A forward scatter threshold was used to acquire 100,000 events for each prepared sample. Flow cytometry data analysis was accomplished by using CellQuest software (version 3.2.1). Specific cell populations (neutrophils, monocytes, and lymphocytes) were delineated by various gating strategies. An example of gating strategy is illustrated in Fig. 1. Lymphocyte subpopulations were identified by forward and side scatter and separated by antigenic expressions of CD3+/CD4+ (T-helper lymphocytes), CD3+/CD8+ (suppressor/cytotoxic T lymphocytes), and CD19+ (B lymphocytes). For intracellular cytokine expressions in NK cells, we gated on the CD3– and CD8+ dim population. We confirmed that this gated population included CD3–/CD16+ and CD56+ NK cells by surface expression. Monocytes were defined and gated by CD4+ dim vs. side scatter. We found that this gating strategy included 99% of the CD14+ and CD64+ monocytes. The intracellular mediator data were presented in two ways: 1) in terms of cells positive for a particular mediator (i.e., fluorescence intensity exceeds background) and 2) as the median fluorescence intensity (MFI).


Figure 1
View larger version (33K):
[in this window]
[in a new window]
  		Fig. 1. An example of the gating strategy used to identify cell populations based on surface CD4+, side scatter complexity (SSC), and intracellular mediators from one subject. A: differentiating cell populations based on CD4+ (Y-axis markers) and SSC (X-axis) of cells using flow cytometry. Monoclonal antibodies in CD and cell side scatter are used to identify distinct cell populations. Cell surface events are expressed in the logarithmic scale on the Y-axis from dim expression (≤101) to bright expression (≥101) and scatter on the X-axis. 100,000 events are captured for analysis at Pre (baseline blood sample taken before start of exercise), End (blood sample obtained immediately following exercise), and Recovery (blood sample drawn after 60 min of exercise). Four cell populations are clearly identified: L, T-helper cells (CD4+ bright, low side scatter); M, monocytes (CD4+ dim, moderate side scatter); G, granulocytes (CD4–, high side scatter); and N, non-CD4 positive lymphocytes (CD4–, low side scatter). B: the CD4+ lymphocyte gate was drawn based on CD4+ bright and low side scatter. C: the isotype control labeled with IgG1 phycoerythrin (PE) and IgG2a FITC. Based on the isotype control, the quadrant markers were set for the three samples. D: Pre. E: End. F: Recovery. The percentage of cells positive for growth hormone (GH)/IGF-I in CD4+ cells and the absolute count in the peripheral blood mononuclear cells are listed in the quadrant windows. The absolute count is obtained by multiplying the proportion of CD4+ lymphocytes (%GH or IGF+ x %CD4+) x white blood cell count/µl. APC, antigen presenting cell.

 
Statistical analysis.   We analyzed the effect of exercise on cytokine and growth mediator regulation in total lymphocytes and their subsets, total monocytes, and total granulocytes in the circulation. We applied a mixed linear model, which is a generalization of the standard linear model and is designed for analyzing repeated measurements. Except for modeling the means, a mixed linear model allows for various patterns of variance among correlated observations, such as data collected repeatedly at different time points from the same subject. In our study, time (Pre, End, Recovery) is our primary explanatory variable, and a quadratic form of time (time2) has been added into the model for flexibility in determining the pattern of changes over time. For each variable of interest, an overall test of any mean difference between the three time points was first conducted at significance level 0.05; thus we were able to identify a list of variables that showed significant changes in response to exercise. Then, for those significant variables, pairwise comparisons among the three time points (i.e., Pre-End, Pre-Recovery, End-Recovery) were performed and were considered significant if P = 0.017 (e.g., 0.05/3). Because little is known about the variability in the response of intracellular cytokines to exercise, we had no data on which to accurately calculate statistical power. Nonetheless, since we found significant changes in response to exercise, these data can be used for future studies to better estimate sample size.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Subjects.   Table 1 summarizes anthropometric and exercise test results for the 11 subjects.


View this table:
[in this window]
[in a new window]
  		Table 1. Anthropometric characteristics

 
Hct and serum lactate.   Serum lactate was measured to determine the relative metabolic effect of the external work performed by each subject. The exercise bout caused a 10-fold increase (P < 0.05) in serum lactate levels (Pre: 12 ± 0.70 mg/dl vs. End: 121 ± 8 mg/dl, P < 0.001) and approached baseline at Recovery (23 ± 2 mg/dl, P < 0.001). Mean values for Hct were significantly different at Pre vs. End and End vs. Recovery (Pre: 43 ± 0.8 and 47 ± 0.9, P < 0.007; Recovery: 41 ± 0.8, P < 0.01).

Serum cytokines and growth factors.   We found significant exercise effects on circulating levels of serum cytokines and growth factors (Table 2).


View this table:
[in this window]
[in a new window]
  		Table 2. Effect of exercise on circulating cytokines and growth factors

 
Leukocyte response to exercise.   Table 3 summarizes the impact of exercise on total leukocytes, lymphocytes, monocytes, and granulocytes. Table 4 shows the effect of exercise on intracellular levels of the nine inflammatory cytokines and growth factors within the major groups of leukocytes (i.e., lymphocytes, monocytes, and granulocytes). Table 5 shows the effect of exercise on intracellular levels of cytokines and growth factors within lymphocyte subpopulations. For a variety of reasons, including the cost of performing all of the analyses, we were not able to measure all mediators, as indicated in Table 5 by ND.


View this table:
[in this window]
[in a new window]
  		Table 3. Impact of exercise on circulating leukocytes

 

View this table:
[in this window]
[in a new window]
  		Table 4. Impact of exercise on intracellular cytokines and mediators in leukocytes

 

View this table:
[in this window]
[in a new window]
  		Table 5. Impact of exercise on intracellular cytokines and mediators in lymphocytes

 
Figures 2–5 show the impact of exercise on the proportional changes as a percent change relative to baseline of the key leukocyte cell types and of the specific intracellular cytokines and growth factors. For comparison, the exercise effect on the leukocyte cell type is shown by the thick gray lines, and the changes in individual cytokines and growth factors by the thinner dotted or dashed lines in each of these figures. Finally, we calculated the ratio of TH2 (T-helper lymphocytes positive for IL-4) to TH1 (T-helper lymphocytes positive for IFN-{gamma}). There was no significant effect of exercise on this ratio (Pre, 1.1 ± 0.2; End, 1.8 ± 0.6; Recovery, 1.9 ± 0.5).


Figure 2
View larger version (18K):
[in this window]
[in a new window]
  		Fig. 2. Effects of exercise on intracellular mediator levels in T-helper lymphocytes (solid thick line). A: T-helper-1 proinflammatory: IL-6 (shaded circle), IL-1{alpha} (solid diamond), TNF-{alpha} (solid square), and IFN-{gamma} (shaded square). B: T-helper-2 anti-inflammatory: IL-4 (solid inverted triangle), GH (open triangle), IGF-I (open inverted triangle), and IL-10 (open diamond). Values are means ± SE.

 

Figure 5
View larger version (13K):
[in this window]
[in a new window]
  		Fig. 5. Effects of exercise on intracellular mediator expression in granulocytes (solid thick line). GH (open triangle) is shown.

 
In contrast to the percent positive cells, we found no significant differences in MFI between any intracellular mediators at Pre, End, and Recovery time points (Tables 6 and 7).


View this table:
[in this window]
[in a new window]
  		Table 6. Impact of exercise on intracellular cytokines and mediators in lymphocytes

 

View this table:
[in this window]
[in a new window]
  		Table 7. Impact of exercise on intracellular cytokines and mediators in leukocytes

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
By the end of 30 min of heavy exercise in this sample of young adults, the number of stress/inflammatory cells, including T and B lymphocytes, NK cells, monocytes, and neutrophils, had, as expected, increased in the circulating blood. But rather than reflecting a predominantly proinflammatory pattern, the increases in the numbers of cells positive for intracellular cytokines and growth factors within these cells revealed a mixed response in which significant increases in intracellular proinflammatory cytokines (IL-1{alpha}, IL-2, TNF-{alpha}, and IFN-{gamma}) in at least one of the leukocyte subpopulations were balanced by increases in intracellular anti-inflammatory and anabolic factors (like IL-4, IL-10, GH, and IGF-I). IL-6, a cytokine known to possess both pro- [catabolic (21, 25)] and anti-inflammatory (39) functions and produced directly by exercising skeletal muscle, was significantly changed in monocytes and in all of the lymphocyte subpopulations studied. Thus our data show that the response of leukocytes and their intracellular cytokines and growth factors to exercise in healthy individuals support Ostrowski's hypothesis that the proinflammatory responses to exercise are attenuated by concomitant anti-inflammatory responses.

In the present study, we did not stimulate leukocytes in vitro with immunogenic agents such as lipopolysaccharide. The latter technique is common (38) and is predicated on the notion that immunological function of leukocytes is best observed by measuring their response to a specific antigen or other immunological stimulus. But the goal of this study was to determine the extent to which exercise itself acted as a stimulus to intracellular cytokine levels in leukocytes, and we, therefore, elected to use flow cytometry to detect intracellular cytokines before, immediately after, and during recovery in unstimulated cells. Other researchers are using this approach as well; for example, Vredevoe et al. (56) recently examined leukocyte expression of various cytokines in patients with heart failure. Like us, those investigators used unstimulated leukocytes and found that the constitutive expression of IL-6 was substantially increased in the patients.

A weakness of the study is the lack of a nonexercised control group. However, we have examined changes in peripheral leukocyte counts over a 30-min rest period for two subjects and observed virtually no changes. There are spontaneous changes in leukocyte counts, but these occur in a circadian pattern over hours and are much smaller in magnitude than the acute changes found with exercise (1). Although our data do not reveal the mechanism for the changes in circulating leukocyte cell types and their intracellular cytokines, some inferences about these mechanisms can be made. As shown in Fig. 5, the proportional increase of granulocytes within the circulation following exercise precisely paralleled the increases in GH-positive granulocytes. A similar parallel proportion was observed for T-helper lymphocytes (Fig. 2A) and their intracellular IL-1{alpha}; cytotoxic T-cells (Fig. 3A) and their intracellular IL-6; NK cells (Fig. 3B) and their intracellular IL-6; and monocytes and their intracellular IFN-{gamma} (Fig. 4A). In these cases, our data suggest that exercise mobilized specific cells from pools where these cells were already expressing certain cytokines and growth factors, and the changes in the circulating pool of the leukocyte cell type and their intracellular mediators reflected this proportional mobilization.


Figure 3
View larger version (15K):
[in this window]
[in a new window]
  		Fig. 3. Effects of exercise on intracellular mediator levels in CD8+ cytotoxic lymphocytes (A), natural killer (NK) cells (B), and CD19+ B-lymphocytes (C). Values are means ± SE.

 

Figure 4
View larger version (15K):
[in this window]
[in a new window]
  		Fig. 4. Effects of exercise on intracellular mediator expression in monocytes (solid thick line). A: proinflammatory: IL-1{alpha} (solid diamond), IL-2 (solid triangle ), TNF-{alpha} (solid square), IFN-{gamma} (shaded square), and IL-6 (shaded circle). B: anti-inflammatory: IL-4 (solid inverted triangle). Values are means ± SE.

 
Our results also indicate possibly different patterns of mobilization for other leukocyte types and their intracellular cytokines. For example, in a unique case, NK cells and NK cells expressing IL-6 increased substantially following exercise (by ~80–100%), whereas NK cells expressing IGF-I actually fell significantly and remained below baseline by 60 min into recovery (Fig. 3B). In B-cells (Fig. 3C), the proportion of cells expressing GH and IGF-I far exceeded the increases in circulating B-cells alone; similarly, in T-helper cells (Fig. 2), the proportion of cells expressing IL-4, IL-6, IFN-{gamma}, and TNF-{alpha} exceeded the increase in T-helper cells. In these cases, leukocytes may have been mobilized disproportionately from pools where the expression of cytokines and growth factors differed.

Alternatively, exercise itself (perhaps through the effect of circulating factors like GH or IL-6) influences the intracellular expression of cytokines. In contrast to the number and proportion of cells positive for a given mediator, we found no effect of brief exercise on MFI (Tables 6 and 7). This would tend to support the idea that the predominant influence of exercise is to mobilize distinct populations of cells from marginal pools where they are already expressing mediators at a particular level, rather than altering the cytokine production of the cells themselves. The mechanism of this remarkable regulation of the mobilization of leukocytes and their intracellular cytokines has yet to be elucidated.

Our data suggest that the leukocyte-associated proinflammatory and anti-inflammatory response to exercise is robust, multifactorial, and brief, all features consistent with the global stress/immune activation associated with the "danger" concept of initial immune regulation, leading, ultimately, to either tolerance or activation (28). We found, for example, that TH2 cells increased significantly with exercise, but the ratio of TH1 to TH2 cells did not significantly change, suggesting that, in healthy individuals, a balance of TH1 and TH2 activity is maintained, despite the overall perturbation in stress/inflammatory cells and their intracellular cytokines. An imbalance between TH1 and TH2 cells has been linked to a variety of diseases, including allergy and asthma (30). An intriguing, but as yet unanswered, question is whether or not individuals who are susceptible to the common entity of exercise-induced asthma (11) maintain TH1/TH2 balance in response to brief exercise as did our nonasthmatic subjects (note: none of our subjects experienced wheezing following the exercise test).

GH and IGF-I have been detected in stress and immune cells (22, 59), but no previous studies have examined the impact of brief exercise on the numbers of GH or IGF-I-positive cells in the circulation. Granulocytes positive for GH increased significantly with exercise, as did GH-positive B-cells. IGF-I expressing B-cells also increased, but, as noted above, IGF-I-positive NK cells decreased following exercise, despite an overall increase in the numbers of circulating NK cells themselves. The importance of GH and IGF-I in the maintenance of muscle mass is well documented (53); more recent work also suggests that these growth factors can aid in angiogenesis, tendon strength, tissue healing, and repair, all functions that are important in the adaptation to exercise (13, 16, 29). In addition, GH serves as a cytokine by aiding in lymphoid proliferation and preventing stress-induced apoptosis (26).

White cells are known to migrate to exercising tissue during the recovery phase (42). A recent study by Viswanathan and Dhabhar (55) elegantly demonstrated that migration of leukocytes to the site of a surgical wound is significantly increased in those animals that had been psychologically stressed. Whether or not intracellular GH or IGF-I act in an autocrine or paracrine manner to promote tissue adaptation to exercise has not been studied. Of related interest is the role of circulating angiogenic precursor cells that are now known to be stimulated by exercise (43) and capable of supplying key growth factors to support new capillary formation in a localized manner (2).

Our data demonstrate that exercise-induced changes in the intracellular leukocyte cytokines do not necessarily parallel the changes in circulating levels of those same cytokines and growth factors. The largest increases in circulating cytokines and growth factors occurred for GH and IL-6 (Table 2), which are known to be produced in response to exercise by the pituitary and skeletal muscle, respectively. No significant increase was found for circulating IGF-I, IL-2, or IFN-{gamma}, despite relatively large and significant changes in intracellular expression of these mediators in some cell types. We did find significant increases in circulating levels of the proinflammatory cytokine TNF-{alpha} and the anti-inflammatory cytokine IL-10, and a significant decrease in IL-4. These observations also support Ostrowski's notion that the initial stress/inflammatory response to exercise involves both pro- and anti-inflammatory activity. Of the cytokines we examined, IL-6 is the mediator most consistently reported to be elevated within the circulation after exercise. In the case of the other mediators that we measured, circulating levels seem to be more sensitive to a variety of factors, including exercise intensity, type, and duration; changes in plasma volume (note: we did find, as expected, an increase in Hct following exercise in our subjects); and, perhaps, the sensitivity of the assay used for detection (6). The extent to which mediators originating from circulating leukocytes contribute to circulating levels of these mediators is simply not known.

Our data show a small change in Hct with the relatively heavy exercise bout. Some investigators have used these changes as a way of estimating changes in plasma volume and as evidence of hemoconcentration (57). In humans, as in other mammals, heavy exercise is accompanied by an infusion of highly concentrated red blood cells from the spleen, and the use of Hct to "correct" for concentrations of other mediators in the blood has been recently questioned (50). The observed changes in Hct probably have little if any substantial impact on our data. For example, in the case of circulating cytokines, IL-4 significantly decreased, the opposite of the effect of hemoconcentration. Moreover, the mean change in Hct was only 8.7%, whereas the percent increases in cytokines were 40% for IL-1{alpha}, 228% for IL-6, 64% for IL-10, and 580% for GH. Similarly, the percent changes for intracellular cytokines ranged roughly from 25 to 600%.

In summary, the current data show that the constitutive response of intracellular mediators within stress/inflammatory cells, traditionally thought to be part of a proinflammatory response, actually represent a balanced mixture of pro- and anti-inflammatory mediators, consistent with the paradoxical nature of the immediate immune response to brief exercise. These results corroborate our recent findings that a single bout of heavy exercise (in a separate group of young adults using the same protocol as outlined here) substantially altered expression of over 300 genes in PBMCs, characterized by a brisk activation and deactivation of genes associated with stress, inflammation, and tissue repair (8). Even with a focus on only nine cytokines and growth factors, we found that brief exercise led to substantial changes in the numbers and intracellular cytokine expression of leukocytes in the circulating blood. Cells positive for the predominantly proinflammatory cytokines IL-1{alpha}, IL-2, IFN-{gamma}, and TNF-{alpha} were increased, but so were cells positive for anti-inflammatory and growth mediators like IL-4, IL-10, GH, and IGF-I. It thus appears that brief, heavy exercise leads to a sizeable mobilization of leukocytes expressing a variety of mediators, some of which appear to be antagonistic.

Collectively, these observations indicate that exercise increases the numbers of circulating cells positive for both pro/anti-inflammatory and growth factors, perhaps contributing to the organism's ability to effectively respond to a variety of postexercise challenges, such as tissue injury and adaptation.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by National Heart, Lung, and Blood Institute Grant RO1-HL-080947 and the University of California Irvine Satellite General Clinical Research Center Grant MO1 RR-00827.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. Cooper, Center for the Study of Health Effects of Exercise in Children, Dept. of Pediatrics, Bldg. 25, 2nd Floor, 101 The City Drive, Orange, CA 92868 (e-mail: dcooper{at}uci.edu)

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. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Abo T, Kawate T, Itoh K, and Kumagai K. Studies on the bioperiodicity of the immune response. I. Circadian rhythms of human T, B, and K cell traffic in the peripheral blood. J Immunol 126: 1360–1363, 1981.[ISI][Medline]
  2. Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, Witzenbichler B, Schatteman G, and Isner JM. Isolation of putative progenitor endothelial cells for angiogenesis. Science 275: 964–967, 1997.[Abstract/Free Full Text]
  3. Barbieri M, Ferrucci L, Ragno E, Corsi A, Bandinelli S, Bonafe M, Olivieri F, Giovagnetti S, Franceschi C, Guralnik JM, and Paolisso G. Chronic inflammation and the effect of IGF-I on muscle strength and power in older persons. Am J Physiol Endocrinol Metab 284: E481–E487, 2003.[Abstract/Free Full Text]
  4. Barstow TJ, Cooper DM, Sobel E, Landaw E, and Epstein S. Influence of increased metabolic rate on 13C-bicarbonate washout kinetics. Am J Physiol Regul Integr Comp Physiol 259: R163–R171, 1990.[Abstract/Free Full Text]
  5. Beaver WL, Lamarra N, and Wasserman K. Breath-by-breath measurement of true alveolar gas exchange. J Appl Physiol 51: 1662–1675, 1981.[Abstract/Free Full Text]
  6. Cannon JG, Nerad JL, Poutsiaka DD, and Dinarello CA. Measuring circulating cytokines. J Appl Physiol 75: 1897–1902, 1993.[Abstract/Free Full Text]
  7. Carmeliet P. Angiogenesis in health and disease. Nat Med 9: 653–660, 2003.[CrossRef][ISI][Medline]
  8. Connolly PH, Caiozzo VJ, Zaldivar F, Nemet D, Larson J, Hung SP, Heck JD, Hatfield GW, and Cooper DM. Effects of exercise on gene expression in human peripheral blood mononuclear cells. J Appl Physiol 97: 1461–1469, 2004.[Abstract/Free Full Text]
  9. Cooper DM, Barstow TJ, Bergner A, and Lee WNP. Blood glucose turnover during high- and low-intensity exercise. Am J Physiol Endocrinol Metab 257: E405–E412, 1989.[Abstract/Free Full Text]
  10. Cooper DM, Weiler-Ravell D, Whipp BJ, and Wasserman K. Aerobic parameters of exercise as a function of body size during growth in children. J Appl Physiol 56: 628–634, 1984.[Abstract/Free Full Text]
  11. Cummiskey J. Exercise-induced asthma: an overview. Am J Med Sci 322: 200–203, 2001.[CrossRef][ISI][Medline]
  12. De Benedetti F, Meazza C, and Martini A. Role of interleukin-6 in growth failure: an animal model. Horm Res 58, Suppl 1: 24–27, 2002.
  13. Delafontaine P, Song YH, and Li Y. Expression, regulation, and function of IGF-1, IGF-1R, and IGF-1 binding proteins in blood vessels. Arterioscler Thromb Vasc Biol 24: 435–444, 2004.[Abstract/Free Full Text]
  14. Denson LA, Held MA, Menon RK, Frank SJ, Parlow AF, and Arnold DL. Interleukin-6 inhibits hepatic growth hormone signaling via upregulation of Cis and Socs-3. Am J Physiol Gastrointest Liver Physiol 284: G646–G654, 2003.[Abstract/Free Full Text]
  15. Dinarello CA. Therapeutic strategies to reduce IL-1 activity in treating local and systemic inflammation. Curr Opin Pharmacol 4: 378–385, 2004.[CrossRef][ISI][Medline]
  16. Edmondson SR, Thumiger SP, Werther GA, and Wraight CJ. Epidermal homeostasis: the role of the growth hormone and insulin-like growth factor systems. Endocr Rev 24: 737–764, 2003.[Abstract/Free Full Text]
  17. Elenkov IJ and Chrousos GP. Stress hormones, proinflammatory and antiinflammatory cytokines, and autoimmunity. Ann NY Acad Sci 966: 290–303, 2002.[Abstract/Free Full Text]
  18. Fehrenbach E, Niess AM, Passek F, Sorichter S, Schwirtz A, Berg A, Dickhuth HH, and Northoff H. Influence of different types of exercise on the expression of haem oxygenase-1 in leukocytes. J Sports Sci 21: 383–389, 2003.[ISI][Medline]
  19. Ferrucci L, Penninx BW, Volpato S, Harris TB, Bandeen-Roche K, Balfour J, Leveille SG, Fried LP, and Md JM. Change in muscle strength explains accelerated decline of physical function in older women with high interleukin-6 serum levels. J Am Geriatr Soc 50: 1947–1954, 2002.[CrossRef][ISI][Medline]
  20. Goldsby RA, Kindt TJ, Osborne BA, and Kuby J. Cytokines. In: Immunology. New York: Freeman, 2003, p. 276–298.
  21. Haddad F, Zaldivar F, Cooper DM, and Adams GR. IL-6-induced skeletal muscle atrophy. J Appl Physiol 98: 911–917, 2005.[Abstract/Free Full Text]
  22. Hattori N, Ikekubo K, Ishihara T, Moridera K, Hino M, and Kurahachi H. Spontaneous growth hormone (GH) secretion by unstimulated human lymphocytes and the effects of GH-releasing hormone and somatostatin. J Clin Endocrinol Metab 79: 1678–1680, 1994.[Abstract]
  23. Hughes-Fulford M. Signal transduction and mechanical stress. Sci STKE 2004: RE12, 2004.
  24. Ibfelt T, Petersen EW, Bruunsgaard H, Sandmand M, and Pedersen BK. Exercise-induced change in type 1 cytokine-producing CD8+ T cells is related to a decrease in memory T cells. J Appl Physiol 93: 645–648, 2002.[Abstract/Free Full Text]
  25. Janssen SP, Gayan-Ramirez G, Van den BA, Herijgers P, Maes K, Verbeken E, and Decramer M. Interleukin-6 causes myocardial failure and skeletal muscle atrophy in rats. Circulation 111: 996–1005, 2005.[Abstract/Free Full Text]
  26. Jeay S, Sonenshein GE, Postel-Vinay MC, Kelly PA, and Baixeras E. Growth hormone can act as a cytokine controlling survival and proliferation of immune cells: new insights into signaling pathways. Mol Cell Endocrinol 188: 1–7, 2002.[CrossRef][ISI][Medline]
  27. Lancaster GI, Khan Q, Drysdale PT, Wallace F, Jeukendrup AE, Drayson MT, and Gleeson M. Effect of prolonged exercise and carbohydrate ingestion on type 1 and type 2 T lymphocyte distribution and intracellular cytokine production in humans. J Appl Physiol 98: 565–571, 2005.[Abstract/Free Full Text]
  28. Matzinger P. The danger model: a renewed sense of self. Science 296: 301–305, 2002.[Abstract/Free Full Text]
  29. Molloy T, Wang Y, and Murrell G. The roles of growth factors in tendon and ligament healing. Rev Environ Health 33: 381–394, 2003.
  30. Ngoc LP, Gold DR, Tzianabos AO, Weiss ST, and Celedon JC. Cytokines, allergy, and asthma. Curr Opin Allergy Clin Immunol 5: 161–166, 2005.[ISI][Medline]
  31. Nielsen HB. Lymphocyte responses to maximal exercise: a physiological perspective. Sports Med 33: 853–867, 2003.[CrossRef][ISI][Medline]
  32. Nieman DC, Miller AR, Henson DA, Warren BJ, Gusewitch G, Johnson RL, Davis JM, Butterworth DE, and Nehlsen-Cannarella SL. Effects of high- vs. moderate-intensity exercise on natural killer cell activity. Med Sci Sports Exerc 25: 1126–1134, 1993.[ISI][Medline]
  33. Niess AM, Fehrenbach E, Schlotz E, Sommer M, Angres C, Tschositsch K, Battenfeld N, Golly IC, Biesalski HK, Northoff H, and Dickhuth HH. Effects of RRR-alpha-tocopherol on leukocyte expression of HSP72 in response to exhaustive treadmill exercise. Int J Sports Med 23: 445–452, 2002.[CrossRef][ISI][Medline]
  34. O'Gradaigh D and Compston JE. T-cell involvement in osteoclast biology: implications for rheumatoid bone erosion. Rheumatology (Oxford) 43: 122–130, 2004.
  35. Ogawa K, Oka J, Yamakawa J, and Higuchi M. Habitual exercise did not affect the balance of type 1 and type 2 cytokines in elderly people. Mech Ageing Dev 124: 951–956, 2003.[CrossRef][ISI][Medline]
  36. Ostrowski K, Rohde T, Asp S, Schjerling P, and Pedersen BK. Pro- and anti-inflammatory cytokine balance in strenuous exercise in humans. J Physiol 515: 287–291, 1999.[Abstract/Free Full Text]
  37. Pala P, Hussell T, and Openshaw PJM. Flow cytometric measurement of intracellular cytokines. J Immunol Methods 243: 107–124, 2000.[CrossRef][ISI][Medline]
  38. Perfetto SP, Currier J, and Birx DL. T-lymphocyte activation and cell signaling. In: Manual of Clinical Laboratory Immunology, edited by Rose NR, Hamilton RG, and Detrick B. Washington, DC: ASM, 2002, p. 224–237.
  39. Petersen AM and Pedersen BK. The anti-inflammatory effect of exercise. J Appl Physiol 98: 1154–1162, 2005.[Abstract/Free Full Text]
  40. Pinsky MR. Sepsis: a pro- and anti-inflammatory disequilibrium syndrome. Contrib Nephrol 132: 354–366, 2001.
  41. Prior BM, Yang HT, and Terjung RL. What makes vessels grow with exercise training? J Appl Physiol 97: 1119–1128, 2004.[Abstract/Free Full Text]
  42. Raastad T, Risoy BA, Benestad HB, Fjeld JG, and Hallen J. Temporal relation between leukocyte accumulation in muscles and halted recovery 10–20 h after strength exercise. J Appl Physiol 95: 2503–2509, 2003.[Abstract/Free Full Text]
  43. Rehman J, Li J, Parvathaneni L, Karlsson G, Panchal VR, Temm CJ, Mahenthiran J, and March KL. Exercise acutely increases circulating endothelial progenitor cells and monocyte-/macrophage-derived angiogenic cells. J Am Coll Cardiol 43: 2314–2318, 2004.[Abstract/Free Full Text]
  44. Rhind SG, Castellani JW, Brenner IK, Shephard RJ, Zamecnik J, Montain SJ, Young AJ, and Shek PN. Intracellular monocyte and serum cytokine expression is modulated by exhausting exercise and cold exposure. Am J Physiol Regul Integr Comp Physiol 281: R66–R75, 2001.[Abstract/Free Full Text]
  45. Shephard RJ. Adhesion molecules, catecholamines and leucocyte redistribution during and following exercise. Sports Med 33: 261–284, 2003.[CrossRef][ISI][Medline]
  46. Starkie RL, Angus DJ, Rolland J, Hargreaves M, and Febbraio MA. Effect of prolonged, submaximal exercise and carbohydrate ingestion on monocyte intracellular cytokine production in humans. J Physiol 528: 647–655, 2000.[Abstract/Free Full Text]
  47. Starkie RL, Rolland J, Angus DJ, Anderson MJ, and Febbraio MA. Circulating monocytes are not the source of elevations in plasma IL-6 and TNF-alpha levels after prolonged running. Am J Physiol Cell Physiol 280: C769–C774, 2001.[Abstract/Free Full Text]
  48. Starkie RL, Rolland J, and Febbraio MA. Effect of adrenergic blockade on lymphocyte cytokine production at rest and during exercise. Am J Physiol Cell Physiol 281: C1233–C1240, 2001.[Abstract/Free Full Text]
  49. Steensberg A, Toft AD, Bruunsgaard H, Sandmand M, Halkjaer-Kristensen J, and Pedersen BK. Strenuous exercise decreases the percentage of type 1 T cells in the circulation. J Appl Physiol 91: 1708–1712, 2001.[Abstract/Free Full Text]
  50. Stewart IB and McKenzie DC. The human spleen during physiological stress. Rev Environ Health 32: 361–369, 2002.
  51. Sunil KA. Analysis of intracellular cytokines using flowcytometery. Methods Cell Sci 24: 37–40, 2002.[CrossRef][Medline]
  52. Vassilakopoulos T, Karatza MH, Katsaounou P, Kollintza A, Zakynthinos S, and Roussos C. Antioxidants attenuate the plasma cytokine response to exercise in humans. J Appl Physiol 94: 1025–1032, 2003.[Abstract/Free Full Text]
  53. Veldhuis JD, Anderson SM, Shah N, Bray M, Vick T, Gentili A, Mulligan T, Johnson ML, Weltman A, Evans WS, and Iranmanesh A. Neurophysiological regulation and target-tissue impact of the pulsatile mode of growth hormone secretion in the human. Growth Horm IGF Res 11, Suppl A: S25–S37, 2001.
  54. Visser M, Pahor M, Taaffe DR, Goodpaster BH, Simonsick EM, Newman AB, Nevitt M, and Harris TB. Relationship of interleukin-6 and tumor necrosis factor-alpha with muscle mass and muscle strength in elderly men and women: the Health ABC Study. J Gerontol A Biol Sci Med Sci 57: M326–M332, 2002.[Abstract/Free Full Text]
  55. Viswanathan K and Dhabhar FS. Stress-induced enhancement of leukocyte trafficking into sites of surgery or immune activation. Proc Natl Acad Sci USA 102: 5808–5813, 2005.[Abstract/Free Full Text]
  56. Vredevoe DL, Widawski M, Fonarow GC, Hamilton M, Martinez-Maza O, and Gage JR. Interleukin-6 (IL-6) expression and natural killer (NK) cell dysfunction and energy in heart failure. Am J Cardiol 93: 1007–1011, 2004.[CrossRef][ISI][Medline]
  57. Wilkerson JE, Gutin B, and Horvath SM. Exercise-induced changes in blood, red cell, and plasma volumes in man. Med Sci Sports Exerc 9: 155–158, 1977.
  58. Wu X, Herndon DN, and Wolf SE. Growth hormone down-regulation of interleukin-1 beta and interleukin-6 induced acute phase protein gene expression is associated with increased gene expression of suppressor of cytokine signal-3. Shock 19: 314–320, 2003.[CrossRef][ISI][Medline]
  59. Yang Y, Guo L, Ma L, and Liu X. Expression of growth hormone and insulin-like growth factor in the immune system of children. Horm Metab Res 31: 380–384, 1999.[ISI][Medline]



This article has been cited by other articles:


Home page
 AMERICAN JOURNAL OF LIFESTYLE MEDICINEHome page
M. G. Flynn, B. K. McFarlin, and M. M. Markofski
State of the Art Reviews: The Anti-Inflammatory Actions of Exercise Training
American Journal of Lifestyle Medicine, May 1, 2007; 1(3): 220 - 235.
[Abstract] [PDF]

Home page
 Ann. N. Y. Acad. Sci.Home page
R. HAYUN, S. SHPUNGIN, H. MALOVANI, M. ALBECK, E. OKUN, U. NIR, and B. SREDNI
Novel Involvement of the Immunomodulator AS101 in IL-10 Signaling, via the Tyrosine Kinase Fer
Ann. N.Y. Acad. Sci., January 1, 2007; 1095(1): 240 - 250.
[Abstract] [Full Text] [PDF]

Home page
 CJASNHome page
M. H. Rosner and J. Kirven
Exercise-Associated Hyponatremia
Clin. J. Am. Soc. Nephrol., January 1, 2007; 2(1): 151 - 161.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow All Versions of this Article:
100/4/1124    most recent
00562.2005v1
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (2)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zaldivar, F.
Right arrow Articles by Cooper, D. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation