Download Leukocyte IGF-1 Receptor Expression during Muscle Recovery

Leukocyte IGF-1 Receptor Expression during
Muscle Recovery
MAREN S. FRAGALA, ADAM R. JAJTNER, JEREMY R. TOWNSEND, ADAM M. GONZALEZ, ADAM J. WELLS,
LEONARDO P. OLIVEIRA, JAY R. HOFFMAN, JEFFREY R. STOUT, and DAVID H. FUKUDA
Department of Educational and Human Sciences, Institute of Exercise Physiology and Wellness, Sport and Exercise Science,
University of Central Florida, Orlando, FL
ABSTRACT
BASIC SCIENCES
FRAGALA, M. S., A. R. JAJTNER, J. R. TOWNSEND, A. M. GONZALEZ, A. J. WELLS, L. P. OLIVEIRA, J. R. HOFFMAN, J. R.
STOUT, and D. H. FUKUDA. Leukocyte IGF-1 Receptor Expression during Muscle Recovery. Med. Sci. Sports Exerc., Vol. 47, No. 1,
pp. 92–99, 2015. Introduction: The insulin-like growth factor 1 (IGF-1) system plays a central role in anabolic cellular processes.
Recently, a regulatory role of IGF-1 in the immune response for muscle repair has been suggested, but how it modulates the inflammatory process is largely unknown. We evaluated changes in leukocyte expression of IGF-1 receptors (IGF-1R) during recovery from
resistance exercise to determine whether changes in the potential for IGF-1 interactions with leukocytes may mediate the role of IGF-1 in
muscle repair. Methods: Twenty resistance-trained men (18–35 yr) performed resistance exercise followed by cold water immersion
(CWI) or control treatment (CON) on three consecutive days. Blood was sampled at baseline (PRE), immediately (IP), 30 min (30P),
24 h (24H), and 48 h after (48H) exercise. Circulating IGF-1 was assayed, and IGF-1 receptor expression (CD221) on gated circulating
leukocytes (monocytes, granulocytes, and lymphocytes) was measured by flow cytometry. Time and treatment effects were analyzed
with ANCOVA. Results: Circulating IGF-1 significantly increased from PRE to IP as a result of resistance exercise, but no differences
between CON and CWI were observed. Mean fluorescence intensity of CD221 on monocytes and granulocytes and percent of CD221+
granulocytes significantly increased at 30P (P G 0.000) and returned to preexercise levels by 24H. No treatment effects on monocytes or
granulocytes were observed. On lymphocytes, mean fluorescence intensity of CD221+ significantly increased from PRE to 30P in CWI.
Conclusions: Changes in IGF-1 and its receptor on monocytes and granulocytes seem to be part of the mechanism that facilitates
recovery from resistance exercise during earlier stages of muscle recovery. In addition, CWI seems to alter IGF-mediated responses on
slower-acting lymphocytes, suggesting that its effects may be seen in later stages of muscle repair. Key Words: MUSCLE REPAIR,
IMMUNE FUNCTION, COLD WATER IMMERSION, CRYOTHERAPY, RESISTANCE EXERCISE, INFLAMMATION
T
previous research has demonstrated a regulatory role of
IGF-1 in the immune response during muscle repair (11,
23,44) where both IGF-1 messenger RNA and protein expression are markedly up-regulated in injured muscles (23)
and the up-regulation of IGF-1 in skeletal muscle seems to
be protective against muscle damage (11) and beneficial for
regeneration (44).
Among the several cell types that contain IGF-1 receptors
(IGF-1R) to elicit cellular growth are immune cells (38). IGF1R are prevalent on the surface of human peripheral blood
mononuclear cells identified by cell differentiation marker
221 (CD221). Their presence signifies IGF-1’s communication with the immune system in muscle repair processes (38).
Earlier work has shown that the cell surface receptors for
IGF-1 are required for regulating cellular immune responses
including proliferation and differentiation (22). In addition,
IGF-1R on immune cells are known to play a role in inflammation through regulating macrophage infiltration and
accumulation (23). The macrophages that infiltrate damaged
skeletal muscles are known to express high concentrations of
IGF-1 and play a role in muscle regeneration (23).
The mechanical stress of physical exercise causes physical disruption to the muscle’s myofilaments and connective
tissue, which initiates an inflammatory cascade (10,40). Consequently, treatments to facilitate recovery have traditionally
he insulin-like growth factor 1 (IGF-1) system contains a family of proteins essential to mediating anabolic and metabolic cellular processes (29). Among
its ubiquitous functions, IGF-1 is an anabolic growth factor
important for initiating the sequence of events involved in
muscle repair and remodeling (11,44). The binding of IGF-1
to its cellular receptor (IGF-1R) triggers signaling via the
Akt/mTOR pathway for muscle hypertrophy to proceed (16).
IGF-1 exerts its hypertrophic function by both increasing
protein synthesis and decreasing protein degradation (1).
Whereas IGF-1 is known to play a central role in the
mechanisms underlying muscle hypertrophy, its role in immune communication is largely unknown. Interestingly,
Address for correspondence: Maren S. Fragala, Ph.D., Institute of Exercise Physiology and Wellness, Sport and Exercise Science, University of
Central Florida, 4000 Central Florida Blvd., Orlando, FL 32816; E-mail:
[email protected].
Submitted for publication March 2014.
Accepted for publication May 2014.
0195-9131/15/4701-0092/0
MEDICINE & SCIENCE IN SPORTS & EXERCISEÒ
Copyright Ó 2014 by the American College of Sports Medicine
DOI: 10.1249/MSS.0000000000000392
92
Copyright © 2014 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
METHODS
Participants. Twenty resistance-trained men volunteered
to participate in this study. Recruitment criteria required that
participants be age 18–35 yr, have at least 1 yr of resistance
training experience (including the barbell back squat), refrain
from using nutritional supplements (i.e., creatine, amino acids,
Beta-hydroxy-beta-methylbutyrate [HMB], herbal supplements)
or medication (i.e., prescribed or over-the-counter medications
such as cold medications or nonsteroidal anti-inflammatory
drugs [NSAID]) during the study, and agree to not engage in
any additional recovery strategies (i.e., saunas, stretching,
foam rollers, massage, and supplementation) outside the study
procedures. Before enrollment in the study, all volunteers gave
a written informed consent after being informed of all study
procedures, risk, and benefits. The study protocol was approved by the university’s institutional review board for the
protection of human participants. Volunteers were randomized into one of two treatment groups: control (CON) (n = 10,
23.8 T 3.0 yr, 178 T 6 cm, 85.7 T 5.4 kg) or CWI (n = 10,
22.5 T 3.0 yr, 171 T 7 cm, 77.1 T 7.7 kg). Participants had an
average of 5.7 T 3.4 yr (CWI) and 7.6 T 4.2 yr (CON) of
resistance training experience using multiple-set periodized
programs including heavy-load phases. Participants had squat
strength (one-repetition maximum (1RM)) of 148.0 T 30.9 kg
(CON) and 148.6 T 31.9 kg (CWI), which corresponded to
relative squat strength to body mass of 1.73 T 0.35 (CON)
and 1.94 T 0.43 (CWI).
LEUKOCYTE IGF-1 RECEPTOR
Protocol. Using a randomized controlled design, all
participants performed a high-volume resistance exercise
protocol on day 1, which consisted of four sets of up to 10
repetitions of the barbell back squat at 80% of their previously determined 1RM and barbell dead lift and split squat
at 70% of their previously determined 1RM, with 90-s rest
intervals allowed between each set and exercise. On days 2
(24 h after) and 3 (48 h after), participants again performed
resistance exercise consisting of four sets of up to 10 repetitions of the barbell back squat exercise only at 80% of
1RM with 90-s rest intervals. Immediately after exercise on
days 1 and 2, assigned treatment was administered as described in the following sections. All testing was performed
in the morning after a 10-h overnight fast (except for water).
Each participant was scheduled for testing at the same time
on days 1, 2, and 3 (between 07:00 and 10:00 a.m.).
CWI. Participants randomized to the CWI treatment group
were immersed in a cold water ice bath in a metal tub
(58.4 121.9 cm) for 10 min after exercise on days 1 and 2.
The water temperature was maintained at 10-C to 12-C (31),
and water depth was set to 22.9 cm, which fully immersed
the lower body while seated and reached the approximate
height of the umbilicus. After the bath, participants remained
in the laboratory for 20 additional minutes until collection
of data at 30 min after exercise on day 1.
Control. Participants randomized to the control group
completed the same resistance exercise protocol at the same
relative intensity as those assigned to the CWI treatment
group. However, after exercise on days 1 and 2, those
assigned to the control group received no treatments after
exercise and remained in the laboratory until collection of
data at 30 min after exercise on day 1.
Biological sampling. Blood samples were collected
from a superficial forearm vein at the following time points:
before exercise (PRE), immediately after exercise (IP), 30 min
after exercise (30P), 24 h after exercise (24H), and 48 h after
exercise (48H). During the first exercise session on day 1, the
blood samples collected at PRE, IP, and 30P were obtained
using a 20-gauge Teflon cannula with a three-way stopcock
and male luer lock adapter. A 15-min equilibration period
after cannula insertion was allowed with the participant lying
supine before collection of the PRE blood sample. The participant returned to the supine position for the IP and 30P
blood draws to reduce the influence of fluid volume shifts.
The cannula was kept patent between blood draws using an
isotonic saline solution. The cannula was removed after the
30P blood sample. Participants returned to the laboratory for
blood sample collection on days 2 (24H) and 3 (48H). Blood
samples at 24H and 48H were collected before exercise using
a 20-gauge disposable needle equipped with a VacutainerÒ
tube holder (Becton Dickinson, Franklin Lakes, NJ), with
the participant in a supine position for at least 15 min before
collection. All blood samples were collected into prechilled
plasma collection VacutainerÒ tubes containing K2EDTA. A
volume of 350 KL of K2EDTA-treated fresh whole blood
was reserved for isolation of peripheral blood mononucleated
Medicine & Science in Sports & Exercised
Copyright © 2014 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
93
BASIC SCIENCES
focused on attenuating inflammation. One common treatment used by athletes to alleviate muscle soreness is cryotherapy via cold water immersion (CWI). CWI is believed
to modulate tissue damage by attenuating the inflammatory
response (33). Its anti-inflammatory actions are primarily
attributed to reflexive vasoconstriction, which reduces local
blood flow (14). In addition, CWI has been shown to attenuate pro-inflammatory cytokine signaling (31) and leukocytosis (30). Although previous studies investigating the
role of IGF-1–related signaling in immune responses have
yielded important mechanistic insights, it is not presently
known how these mechanisms apply to exercise-induced
muscle recovery or how recovery treatments, such as CWI,
may influence these processes. Thus, the purpose of the present investigation was to evaluate changes in leukocyte expression of IGF-1R during recovery from resistance exercise
with and without CWI treatment to determine whether
changes in the potential for IGF-1 interactions with specific
immune cells may mediate the role of IGF-1 in muscle repair.
The resistance exercise protocol was expected to result in
at least two- to threefold increases in circulating creatine
kinase at 24 h after exercise, in line with previous acute resistance exercise protocols (21,43). Similarly, perceived
muscle soreness was expected to significantly increase over
preexercise ratings at 24 h after exercise, similar to previous heavy acute resistance exercise protocols in resistancetrained volunteers (21,36).
BASIC SCIENCES
cells (PBMC). Remaining blood was centrifuged at 3000g
for 15 min. Resulting plasma was aliquot into separate 1.6-mL
microcentrifuge tubes and frozen at j80-C for subsequent analysis.
Biochemical analysis. Plasma IGF-1 was assayed by
commercially available QuantikineÒ enzyme-linked immunosorbent assay (Catalog No: DG100; R&D Systems,
Minneapolis, MN) according to manufacturer specifications.
Before the assay, 20 KL of plasma samples were pretreated
with the acidic dissociation solution to release IGF-1 from
binding proteins and reconstituted with a protein buffer.
Reconstituted samples of 50 KL were added to each well for
the assay. All samples were run in duplicate and thawed
only once before an analysis. The sensitivity of the assay
was 0.026 ngImLj1, and intraassay precision was 4.6%.
Cell staining. Peripheral whole blood treated with
K2EDTA was used to identify leukocytes and quantify the
IGF-1 receptor expression by direct immunofluorescence
and flow cytometry. Erythrocytes were lysed from 350 KL
of K2EDTA-treated whole blood with BD Pharm Lyse solution (BD Biosciences, Franklin Lakes, NJ) within 30 min
of collection. Remaining leukocytes were then resuspended
in 100 KL BD Pharmingen stain buffer (BD Biosciences)
containing phosphate buffered saline (PBS), fetal bovine serum, metabolic inhibitor, and sodium azide (NaN3) to block
Fc receptors. Direct staining methods were used to label CD221
with PE-conjugated anti-CD221 (catalog #555999, Clone 1H7;
BD Pharmingen). Staining was performed by adding 10 KL of
directly conjugated PE-anti-CD221 to the cell suspension and
incubating in the dark for 30 min at 20-C. Cells were resuspended
in 1.0 mL of stain buffer for flow cytometry analysis.
Flow cytometry. Flow cytometry analysis of stained
cells was performed on a BD C6 Accuri flow cytometer (BD
Biosciences, San Jose, CA) equipped with BD Accuri analysis
software (BD Biosciences). Cell scatter and fluorescence
were collected using two lasers providing excitation at 488
and 640 nm. Leukocyte populations (monocytes, granulocytes,
and lymphocytes) were gated (Fig. 1) using forward scatter
versus side scatter, as previously described (13). Gating
was set to exclude dead cell debris. A minimum of 10,000
cells per gate were obtained with each sample. Analysis of
CD221+ cell subpopulations was completed by quadrant
analyses. Mean fluorescence intensity (MFI) of CD221+ on
cell populations was recorded, representing the mean density
of IGF-1R per cell. To control for fluorescence spillover,
correction subtraction values were applied when necessary
as per manufacturer instructions (BD Biosciences). Data are
reported as the percent of CD221+ positive cells and MFI
of CD221 on positively stained cells.
Statistical analysis. Effects of exercise and treatment
were analyzed using two-way (group–time) repeatedmeasures ANCOVA. All analyses used the PRE measure as
the covariate. In the event of a significant F score, post hoc
one-way repeated-measures ANOVA and Tukey comparisons were run. Before analysis, all data were assessed for
homogeneity of variance and sphericity. If the assumption of
94
Official Journal of the American College of Sports Medicine
sphericity was violated, a Greenhouse–Geisser correction
was applied. An alpha level of P e 0.05 was considered
statistically significant for all comparisons. P e 0.10 was
interpreted as a meaningful trend in the analysis to reduce
the chance of making a type 2 error and missing an important change because of the heterogeneity of immune response in humans. Thus, post hoc analyses were run when
main effects were P e 0.10. All data are reported as mean T
SE unless otherwise noted.
RESULTS
Participants assigned to the CWI and CON groups were
similar in physical characteristics including years of resistance training experience (7.1 T 3.8 yr) and squat 1RM
(145.6 T 30.3 kg). Subject characteristics and performance
data have been previously described in a related study (15).
Briefly, the exercise protocol resulted in significant increases
in circulating creatine kinase (154.7 T 139.8 UILj1 at PRE to
468.9 T 358.7 UILj1 at 24H in CON and 103.4 T 62.2 UILj1
at PRE to 510.9 T 385.2 UILj1 at 24H in CWI), with no
significant difference seen between the groups.
Circulating IGF-1 (Fig. 2) increased from PRE (72.0 T
14.8 ngImLj1) to IP (75.5 T 13.9 ngImLj1) (P = 0.045) and
returned to preexercise levels at 30P (72.8 T 13.3 ngImLj1).
At 24H (74.4 T 12.5 ngImLj1) and 48H (75.2 T 15.1 ngImLj1),
circulating IGF-1 tended to increase above PRE levels, albeit
not significantly (P = 0.129 and 0.083, respectively). No
differences between treatment groups were observed for circulating IGF-1.
Granulocytes. MFI of CD221 (relative expression of
IGF-1R) on granulocytes increased at 30P (5568.9 T 940.6)
from PRE (4724.6 T 713.3) (P e 0.000) and returned to PRE
levels at 24H (4938.8 T 932.4) (Fig. 3a). No differences
FIGURE 1—Gating of peripheral blood mononuclear cell populations
(granulocytes, monocytes, and lymphocytes) by forward (FSC-A) and
side scatter (SSC-A) characteristics.
http://www.acsm-msse.org
Copyright © 2014 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
FIGURE 2—Circulating IGF-1 before and after resistance exercise.
Data are covaried by a PRE value of 72.02 ngImLj1. *Indicates P G 0.05
for time point from PRE (CWI, solid line; CON, dashed line).
DISCUSSION
The expression of IGF-1R on target cells provides important insights into the physiological role of IGF-1. This
investigation sought to examine its relative expression on
leukocyte populations in response to resistance exercise and
CWI therapy. Whereas the endocrine–immune interactions
of other hormones and corresponding receptors have been
previously reported (12,13), the potential of IGF-1 immune
interactions in humans, to our knowledge, has not been reported
and may be the key to explaining the endocrine–immune
FIGURE 3—A, MFI of CD221+ (IGF-1R) on circulating granulocytes before and after resistance exercise. Data are covaried by a PRE value of 4724.61. B,
Percent CD221+ (IGF-1R) granulocytes before and after resistance exercise. Data are covaried by a PRE value of 31.58%. *Indicates P G 0.05 from PRE
(CWI, solid line; CON, dashed line).
LEUKOCYTE IGF-1 RECEPTOR
Medicine & Science in Sports & Exercised
Copyright © 2014 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
95
BASIC SCIENCES
between treatment groups were observed for MFI of CD221
on granulocytes. When collapsed across groups, percent of
granulocytes expressing IGF-1R (Fig. 3b) significantly decreased from PRE (31.6% T 10.7%) to IP (27.2% T 10.0%)
(P = 0.004) and increased at 30P (41.7% T 12.8%) from PRE
(P G 0.000) and IP (P = 0.019). At 24H (33.0% T 10.4%)
and 48H (32.7% T 11.4%), percent of granulocytes expressing
IGF-1R decreased from 30P (P = 0.006, P G 0.000) and
returned to preexercise levels. No treatment–time interaction
(P = 0.284) was observed for percent of granulocytes expressing IGF-1R, although at 48H postexercise CWI (30.9% T
12.0%), it seemed to reduce percent of granulocytes expressing IGF-1 compared with CON (34.6% T 11.0%) at 48H.
Monocytes. MFI of CD221 (relative expression of
IGF-1R) on monocytes showed a significant time effect
(P = 0.002) where MFI on monocytes significantly increased at 30P (5846.6 T 1346.2) from PRE (4514.1 T
760.1) (P G 0.000) and IP (4753.6 T 1039.7) (P = 0.001)
levels (Fig. 4a). By 24H (4983.1 T 1223.8) (P = 0.015) and
48H (4847.2 T 950.3) (P = 0.001), MFI had decreased from
30P and returned to preexercise levels. No treatment–time
interaction was observed in MFI of CD221 on monocytes.
Percent of monocytes expressing IGF-1R (Fig. 4b) showed a
trend toward a time effect (P = 0.094) where percent of monocytes expressing IGF-1 decreased from PRE (6.1% T 1.8%)
at IP (5.7% T 1.4%) (P = 0.015) and at 30P (4.8% T 1.3%)
(P G 0.000) and returned to preexercise levels at 24H
(6.0% T 2.3%) and 48H (5.7% T 2.0%). No treatment–time
interaction was observed in percent of monocytes expressing IGF-1R.
Lymphocytes. MFI of CD221 (relative expression of
IGF-1R) on lymphocytes showed a significant time effect
(P = 0.012) and trended toward a treatment–time interaction (P = 0.071) (Fig. 5a). Both groups tended to increase
MFI from PRE (1621.4 T 165.3) to IP (1706.8 T 206.9) (P =
0.087). At 30P, MFI increased only in the CWI group
(1868.9 T 333.7) (P = 0.004) and remained above PRE at
24H (1751.5 T 52.6) (P = 0.041). In the CON group, MFI
did not significantly increase above PRE at 30P (1694.4 T
174.4). Percent of lymphocytes expressing IGF-1R (Fig. 5b)
increased from PRE (42.6% T 10.7%) to IP (48.7% T 10.9%)
(P = 0.003) and decreased at 30P (35.8% T 11.1%) (P G
0.000) in both groups. Percent of lymphocytes expressing
IGF-1R (43.4% T 11.0%) returned to preexercise levels by
24H. No significant treatment–time interaction was observed
in percent of lymphocytes expressing IGF-1R.
BASIC SCIENCES
FIGURE 4—A, MFI of CD221+ (IGF-1R) on circulating monocytes before and after resistance exercise. Data are covaried by a PRE monocyte MFI
value of 4514.42. B, Percent of CD221+ (IGF-1R) monocytes before and after resistance exercise. Data are covaried by a PRE value of 6.06%.
*Indicates P G 0.05 from PRE (CWI, solid line; CON, dashed line).
interactions in muscle recovery. Because IGF-1 has been
previously shown to be protective against muscle damage
(11) and beneficial for regeneration (44), we hypothesized
that the stress of the resistance exercise would result in altered cellular expression of IGF-1R that may be attenuated
by CWI treatment after exercise. The main finding of this
investigation was that resistance exercise seems to increase
IGF-1R expression on monocytes and granulocytes, supporting
the evidence for IGF-1’s role in communicating with immune cells in muscle tissue repair. In addition, CWI therapy
after resistance exercise did not seem to alter IGF-1R expression on monocytes and granulocytes. However, CWI
therapy after resistance exercise seemed to alter IGFmediated responses on lymphocytes, the immune cells that
are important for the latter stages of tissue repair and immunosuppression observed after injury (35).
Circulating IGF-1 somewhat increased in response to the
acute resistance exercise protocol on day 1. Similar to our
findings, previous research has also reported that resistance
exercise results in marginal increases in circulating IGF-1
levels in men (28). Interestingly, some research has also shown
that the effects of resistance exercise on IGF-1 seem to be in
the partitioning of IGF-1 among its binding proteins (28). This
work has suggested that the bioavailability of IGF-1 in response to resistance exercise may be more attributable to
changes in its circulating binding proteins than the free or
total IGF-1 in circulation (28). Regardless of the mechanism
for increased circulating IGF-1 in response to exercise, the
observed increase in circulating IGF-1 at IP suggests that it
may have a role in subsequent anabolic muscle regeneration
processes. IGF-1 is known to act as a stimulator of both the
skeletal muscle regeneration and hypertrophy (26) via the
activation of satellite cells (27). However, in order for IGF-1
to stimulate myofiber hypertrophy, mechanical loading
seems necessary (5). The inflammatory process triggered by
muscle-damaging exercise may help explain this connection.
Muscle-damaging exercise triggers the release of chemoattractive
factors to recruit leukocytes (neutrophils and macrophages),
FIGURE 5—A, MFI of CD221+ (IGF-1R) on circulating lymphocytes before and after resistance exercise. Data are covaried by a PRE value of 1621.36. B,
Percent of CD221+ (IGF-1R) lymphocytes before and after resistance exercise. Data are covaried by a PRE value of 42.63%. *Indicates P G 0.05 from PRE.
#Indicates P G 0.05 from PRE in the CWI group (CWI, solid line; CON, dashed line).
96
Official Journal of the American College of Sports Medicine
http://www.acsm-msse.org
Copyright © 2014 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
LEUKOCYTE IGF-1 RECEPTOR
Monocytes. MFI of CD221 (relative expression of IGF1R) on monocytes increased at 30P and returned to preexercise levels by 24H. Increased expression of the IGF-1R
on monocytes increases the availability for IGF-1’s interaction with its receptor to mediate a response. Thus, our data
would support that at 30P, there is an increased potential for
IGF-1–mediated responses in circulating monocytes. Monocytes are immune cells in circulation fated to become macrophages once they enter the damaged tissue (34). Once they
infiltrate the damaged tissue, macrophages play an important
role in phagocytosis (25). Previous research has shown that
IGF-1 can enhance phagocytosis and the oxidative burst of
monocytes (4). Thus, it may be that the purpose of increased
expression of IGF-1R on monocytes at 30P is to ‘‘prime’’
these future macrophages in their phagocytic roles once they
enter the tissue.
Interestingly, at the same time that the relative expression
of IGF-1R was increasing, the percent of monocytes expressing the IGF-1R decreased at IP and 30P. While the percent of CD221+ positive monocytes (approximately 5%) is
relatively low compared with that of granulocytes (approximately 35%) and lymphocytes (approximately 40%) in the
present study, it seems that the decrease in the percent of
CD221+ (expressing the IGF-1R) monocytes may be due
to the interaction of IGF-1 with its receptor on monocytes.
If IGF-1 is interacting with its receptor, the percentage of
monocytes expressing IGF-1R would potentially decline.
This may explain the decline in CD221+ monocytes. In addition, the decrease in CD221+ monocytes from circulation
may be due to their movement into the tissue in response to
the exercise to elicit their local effects. When muscle damage
occurs, injured skeletal muscle recruits monocytes for phagocytosis and for stimulation of myogenesis and muscle fiber
growth (2). Previous research has shown that intense exercise
lasting for only 1 min can significantly increase the monocytes
in circulation (37) partly because of the exercise-induced
elevations in catecholamines (13,37). However, monocyte/
macrophage infiltration does not seem to be evident until
90 min to 24 h after tissue damage (13). In addition, although
CWI treatment was hypothesized to affect circulatory responses and thus immune cells in circulation, no differences in
IGF-1R expression on monocytes were observed in those receiving CWI treatment after exercise compared with those receiving no treatment. Thus, CWI effects for recovery likely act
on pathways other than IGF-1–mediated monocyte responses.
Because this is the first study, to our knowledge, to evaluate
IGF-1R on monocytes in response to acute exercise, further
studies are needed to elaborate on our results to determine
the specific monocytes that are responsible for this effect.
Lymphocytes. MFI of CD221 on lymphocytes increased at IP. At the same time, the percent of lymphocytes
expressing IGF-1 increased at IP. Increased relative expression of IGF-1R on lymphocytes and percent of lymphocytes
expressing IGF-1R at IP would increase the opportunity for
IGF-1–mediated responses in these immune cells. During
the muscle repair processes, lymphocytes are typically not
Medicine & Science in Sports & Exercised
Copyright © 2014 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
97
BASIC SCIENCES
which both remove the damaged tissue and activate satellite cells to repair the tissue (10,40). Most leukocyte subpopulations express IGF-1R (9), and previous research has
shown that IGF-1 mediates muscle repair and regeneration
by attenuating chronic inflammatory response (26). Thus,
taken together, in addition to its role in muscle hypertrophy,
IGF-1 may be an important mediator of the inflammatory
cascade to repair muscle tissue in response to exercise.
However, although CWI therapy was expected to attenuate
the inflammatory response after resistance exercise, it did
not seem to alter the circulating IGF-1 response in the present
study. Thus, in the present study, CWI treatment did not seem
to alter the acute endocrine response to resistance exercise.
Granulocytes. MFI of CD221 (relative expression of
IGF-1R) on granulocytes increased at 30P and returned to
preexercise levels by 24H. At the same time, the percent of
granulocytes expressing IGF-1R similarly increased at 30P
and returned to preexercise levels by 24H. Both the observed increased expression of the IGF-1 receptor and increased number of cells expressing the IGF-1 receptor after
exercise increase the potential for IGF-1–mediated responses
in granulocytes after exercise. Although the mechanisms for
the interaction of IGF-1 with granulocytes require further
investigation previous research has shown that IGF-1 may be
important in promoting granulocyte activity in several ways.
IGF-1 has been shown to increase the longevity of granulocytes by inhibiting their spontaneous apoptosis (18). In addition, IGF-1 has been reported to enhance other granulocyte
functions such as phagocytosis, degranulation, and oxidative
burst (6). Moreover, previous work has shown that IGF-1
enhances interleukin 8 (IL-8) cytokine secretion by peripheral blood mononuclear cells (18). Because granulocytes
produce large amounts of IL-8 (8), it may be that the role of
increased expression of IGF-1R is to help mediate the secretion of IL-8, an important cytokine for neutrophil accumulation in muscle repair (3). Thus, although speculative,
our observed increase in IGF-1R on granulocytes after resistance exercise may be important for promoting granulocyte longevity and activity mediated by IGF-1.
CWI treatment did not seem to affect the MFI of CD221
on granulocytes or the percent of granulocytes expressing
IGF-1R compared with control after exercise. We had expected to see an attenuation in IGF-1R expression in the cold
water treatment group because granulocytes (mainly composed of neutrophils) are still being recruited to the damaged
tissue if the damage is sustained at 24H and 48H (24) and
cold may modulate damage by reducing the inflammatory
response intensity and preserving morphology (33). Nevertheless, the observed reduction in the percent of granulocytes
expressing IGF-1R and percent of CD221+ granulocytes at
24 and 48 h from 30P in both groups could mean that subsequent inflammatory actions of IGF-1 occurred within the
first 24 h during recovery. Considering that this is the first
study to attempt to explore the time sequence, future studies
incorporating more time points during the first day of the
recovery process may provide further insights.
BASIC SCIENCES
recruited into the muscle until several days after exercise
(10,40). However, IGF-1 has been shown to influence the
development and function of T and B lymphocytes in earlier
stages of lymphocyte involvement (17,20). Considering that
IGF-1 has been shown to enhance lymphopoiesis, cell proliferation, and cell survival (9,19), IGF-1–mediated responses
on lymphocytes may be occurring before the lymphocytes
infiltrate the damaged tissue. In support of the delayed response in tissue repair, previous research has shown that
IGF-1 is involved in the initiation of T lymphocyte activation (7). In addition, previous studies have shown that IGF-1
both inhibits apoptosis of activated T lymphocytes (42) and
acts as a chemotactic factor for T lymphocytes (39). Thus, it
is likely that the early increases in IGF-1R on lymphocytes
observed in the present study are indicative of acute IGF-1–
mediated interactions with lymphocytes that are ‘‘informing’’
the cells early of their subsequent responsibilities in the regeneration cascade.
Interestingly, CWI treatment immediately after acute resistance exercise seemed to increase the MFI of CD221 on
lymphocytes at 30 min and 24H whereas those receiving no
treatment had levels return to preexercise levels by 30P. The
physiological significance of this difference is not known
but likely indicates differences in IGF-1–mediated actions in
lymphocytes after CWI treatment. Because CWI is known
to reduce local blood flow (14), attenuate pro-inflammatory
cytokine signaling (32,41), and decrease leukocytosis (30),
CWI treatment may have altered the population of lymphocytes in circulation after exercise. However, both activated
and inactivated lymphocytes have been shown to contain
functional IGF-1R (39). Although the subsequent effects on
muscle regeneration remain to be elucidated, differences in
IGF-1R expression may mediate lymphocyte action through
chemotaxis and lymphocyte growth (39). In addition, because lymphocytes are not required for the initiation of repair but are essential for a normal outcome of tissue repair
(35), the cold water treatment may alter the repair outcome.
Although it is unclear whether the alteration is ultimately
beneficial or detrimental particularly because our study only
provides limited snapshots in time during the recovery process, enhanced lymphocyte action may potentially facilitate
repair outcomes.
Although this investigation is the first, to our knowledge,
to report on the IGF-1R expression on immune cells to an
acute resistance exercise stress, it is important to interpret
the results within the specific context of this study and to consider the potential limitations. First, this investigation sought
to explore the potential mechanisms of IGF-1’s interaction
with immune cells. Although a novel and relevant question,
it is important to keep in mind that several other hormones
and cytokines contribute to the entire scope of how immune
cells respond to tissue damage. In addition, it is important to
acknowledge that IGF-1 functions as a system containing
various binding proteins, which determine its bioavailability. Thus, results are intended to contribute evidence to the
larger paradigm of endocrine–immune interactions. Second,
as the first study of its kind, we attempted to somewhat
globally identify immune cell populations as granulocytes,
monocytes, and lymphocytes. However, it is important to
consider that within each leukocyte subpopulation, there are
various cells with specialized functions. Although our data
may drive hypotheses for future work, further investigation
is needed to more closely examine targeted immune cell
populations. Finally, interpreting immune data in human volunteers is complex because of the heterogeneity of data. Thus, we
opted to interpret the present data using covariate analysis with a
more lenient P value of 0.10 to reduce the likelihood of missing
a potentially relevant change. Because optimal statistic tools to
evaluate heterogeneous data are a topic of debate, we opted to
report all P values to allow different interpretations of the data.
In conclusion, changes in IGF-1 and it receptor expression on leukocyte populations seem to be part of the mechanism that helps facilitate recovery from resistance exercise.
Resistance exercise seems to increase IGF-1R expression
on monocytes and granulocytes, suggesting that IGF-1
likely mediates their role in the earlier stages of muscle recovery. Thus, in addition to its anabolic role, IGF-1 seems to
communicate with the immune system via cellular receptors
to facilitate recovery. In addition, CWI treatment after resistance exercise seems to increase the potential for IGFmediated responses on lymphocytes. Whether this increase
is ultimately favorable or detrimental to muscle recovery
outcomes remains unclear. However, the observed increase
in potential for IGF-mediated responses on lymphocytes
may contribute to the mechanisms underlying the previously
reported benefits of CWI treatment for recovery. Because
this study aimed to explore for the first time the in vivo
potential for IGF-1 interaction with leukocytes during recovery from resistance exercise, further work is needed to
elucidate the outcomes of these effects.
This research was supported by the Young Investigator Grant
awarded by the National Strength and Conditioning Association.
The authors have no conflicts of interest to report.
The results of the present study do not constitute endorsement by
the American College of Sports Medicine.
REFERENCES
1. Allen RE, Boxhorn LK. Regulation of skeletal muscle satellite
cell proliferation and differentiation by transforming growth factorbeta, insulin-like growth factor I, and fibroblast growth factor. J Cell
Physiol. 1989;138(2):311–5.
2. Arnold L, Henry A, Poron F, et al. Inflammatory monocytes
recruited after skeletal muscle injury switch into antiinflammatory
98
Official Journal of the American College of Sports Medicine
macrophages to support myogenesis. J Exp Med. 2007;204(5):
1057–69.
3. Baggiolini M, Clark-Lewis I. Interleukin-8, a chemotactic and inflammatory cytokine. FEBS Lett. 1992;307(1):97–101.
4. Balteskard L, Unneberg K, Halvorsen D, Hansen JB, Revhaug A.
Effects of insulin-like growth factor 1 on neutrophil and monocyte
http://www.acsm-msse.org
Copyright © 2014 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
5.
6.
7.
8.
9.
10.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
LEUKOCYTE IGF-1 RECEPTOR
25. McLennan IS. Degenerating and regenerating skeletal muscles
contain several subpopulations of macrophages with distinct spatial and temporal distributions. J Anat. 1996;188(Pt 1):17–28.
26. Mourkioti F, Rosenthal N. IGF-1, inflammation and stem cells:
interactions during muscle regeneration. Trends Immunol. 2005;26(10):
535–42.
27. Musarò A, McCullagh K, Paul A, et al. Localized Igf-1 transgene
expression sustains hypertrophy and regeneration in senescent
skeletal muscle. Nat Genet. 2001;27(2):195–200.
28. Nindl BC, Kraemer WJ, Marx JO, et al. Overnight responses of
the circulating IGF-I system after acute, heavy-resistance exercise.
J Appl Physiol (1985). 2001;90(4):1319–26.
29. Nindl BC, Pierce JR. Insulin-like growth factor I as a biomarker
of health, fitness, and training status. Med Sci Sports Exerc.
2010;42(1):39–49.
30. Peake J, Peiffer JJ, Abbiss CR, et al. Body temperature and its
effect on leukocyte mobilization, cytokines and markers of neutrophil activation during and after exercise. Eur J Appl Physiol.
2008;102(4):391–401.
31. Pournot H, Bieuzen F, Duffield R, Lepretre PM, Cozzolino C,
Hausswirth C. Short term effects of various water immersions on
recovery from exhaustive intermittent exercise. Eur J Appl Physiol.
2011;111(7):1287–95.
32. Pournot H, Bieuzen F, Louis J, et al. Time-course of changes in
inflammatory response after whole-body cryotherapy multi exposures following severe exercise. PLoS One. 2011;6(7):e22748.
33. Puntel GO, Carvalho NR, Amaral GP, et al. Therapeutic cold: an
effective kind to modulate the oxidative damage resulting of a
skeletal muscle contusion. Free Radic Res. 2011;45(2):125–38.
34. Saclier M, Cuvellier S, Magnan M, Mounier R, Chazaud B.
Monocyte/macrophage interactions with myogenic precursor cells
during skeletal muscle regeneration. FEBS J. 2013;280(17):4118–30.
35. Schäffer M, Barbul A. Lymphocyte function in wound healing and
following injury. Br J Surg. 1998;85(4):444–60.
36. Spiering BA, Kraemer WJ, Vingren JL, et al. Responses of criterion
variables to different supplemental doses of L-carnitine L-tartrate.
J Strength Cond Res. 2007;21(1):259–64.
37. Steppich B, Dayyani F, Gruber R, Lorenz R, Mack M, ZieglerHeitbrock HW. Selective mobilization of CD14(+)CD16(+)
monocytes by exercise. Am J Physiol Cell Physiol. 2000;279(3):
C578–86.
38. Stuart CA, Meehan RT, Neale LS, Cintron NM, Furlanetto RW.
Insulin-like growth factor-I binds selectively to human peripheral
blood monocytes and B-lymphocytes. J Clin Endocrinol Metab.
1991;72(5):1117–22.
39. Tapson VF, Boni-Schnetzler M, Pilch PF, Center DM, Berman JS.
Structural and functional characterization of the human T lymphocyte receptor for insulin-like growth factor I in vitro. J Clin
Invest. 1988;82(3):950–7.
40. Tidball JG. Inflammatory cell response to acute muscle injury.
Med Sci Sports Exerc. 1995;27(7):1022–32.
41. Townsend JR, Fragala MS, Jajtner AR, et al. A-hydroxy-Amethylbutyrate (HMB)-free acid attenuates circulating TNF-> and
TNFR1 expression postresistance exercise. J Appl Physiol (1985).
2013;115(8):1173–82.
42. Tu W, Cheung PT, Lau YL. Insulin-like growth factor 1 promotes
cord blood T cell maturation and inhibits its spontaneous and
phytohemagglutinin-induced apoptosis through different mechanisms. J Immunol. 2000;165(3):1331–6.
43. Wolf MR, Fragala MS, Volek JS, et al. Sex differences in creatine
kinase after acute heavy resistance exercise on circulating granulocyte estradiol receptors. Eur J Appl Physiol. 2012;112(9):3335–40.
44. Ye F, Mathur S, Liu M, et al. Overexpression of insulin-like
growth factor-1 attenuates skeletal muscle damage and accelerates
muscle regeneration and functional recovery after disuse. Exp
Physiol. 2013;98(5):1038–52.
Medicine & Science in Sports & Exercised
Copyright © 2014 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
99
BASIC SCIENCES
11.
functions in normal and septic states. JPEN J Parenter Enteral
Nutr. 1998;22(3):127–35.
Bamman MM, Shipp JR, Jiang J, et al. Mechanical load increases
muscle IGF-I and androgen receptor mRNA concentrations in
humans. Am J Physiol Endocrinol Metab. 2001;280(3):E383–90.
Bjerknes R, Aarskog D. Priming of human polymorphonuclear neutrophilic leukocytes by insulin-like growth factor I: increased phagocytic capacity, complement receptor expression,
degranulation, and oxidative burst. J Clin Endocrinol Metab. 1995;
80(6):1948–55.
Brocardo MG, Schillaci R, Galeano A, et al. Early effects of
insulin-like growth factor-1 in activated human T lymphocytes.
J Leukoc Biol. 2001;70(2):297–305.
Cassatella MA. Neutrophil-derived proteins: selling cytokines by
the pound. Adv Immunol. 1999;73:369–509.
Clark R. The somatogenic hormones and insulin-like growth factor-1:
stimulators of lymphopoiesis and immune function. Endocr Rev.
1997;18(2):157–79.
Clarkson PM, Sayers SP. Etiology of exercise-induced muscle
damage. Can J Appl Physiol. 1999;24(3):234–48.
Dumont N, Frenette J. Macrophages protect against muscle atrophy and promote muscle recovery in vivo and in vitro: a mechanism partly dependent on the insulin-like growth factor-1 signaling
molecule. Am J Pathol. 2010;176(5):2228–35.
Fragala MS, Kraemer WJ, Denegar CR, Maresh CM, Mastro AM,
Volek JS. Neuroendocrine-immune interactions and responses to
exercise. Sports Med. 2011;41(8):621–39.
Fragala MS, Kraemer WJ, Mastro AM, et al. Leukocyte A2adrenergic receptor expression in response to resistance exercise.
Med Sci Sports Exerc. 2011;43(8):1422–32.
Gregson W, Black MA, Jones H, et al. Influence of cold water
immersion on limb and cutaneous blood flow at rest. Am J Sports
Med. 2011;39(6):1316–23.
Jajtner AR, Hoffman JR, Gonzalez AM, Worts P, Fragala MS,
Stout JR. Comparison of electrical stimulation versus cold water
immersion on treatment of muscle soreness following resistance
exercise. J Sports Rehabil. 2014 Mar 12. [Epub ahead of print].
Kalista S, Schakman O, Gilson H, et al. The type 1 insulin-like
growth factor receptor (IGF-IR) pathway is mandatory for the
follistatin-induced skeletal muscle hypertrophy. Endocrinology.
2012;153(1):241–53.
Kimata H, Fujimoto M. Growth hormone and insulin-like growth
factor I induce immunoglobulin (Ig)E and IgG4 production by
human B cells. J Exp Med. 1994;180(2):727–32.
Kooijman R, Coppens A, Hooghe-Peters E. Igf-I inhibits spontaneous
apoptosis in human granulocytes. Endocrinology. 2002;143(4):1206–12.
Kooijman R, Hooghe-Peters EL, Hooghe R. Prolactin, growth
hormone, and insulin-like growth factor-I in the immune system.
Adv Immunol. 1996;63:377–454.
Kooijman RK, Scholtens LE, Rijkers GT, Zegers BJ. Differential
expression of type I insulin-like growth factor receptors in different
stages of human T cells. Eur J Immunol. 1995;25(4):931–5.
Kraemer WJ, Flanagan SD, Comstock BA, et al. Effects of a whole
body compression garment on markers of recovery after a heavy
resistance workout in men and women. J Strength Cond Res.
2010;24(3):804–14.
Long E, Huynh HT, Zhao X. Involvement of insulin-like growth
factor-1 and its binding proteins in proliferation and differentiation
of murine bone marrow-derived macrophage precursors. Endocrine. 1998;9(2):185–92.
Lu H, Huang D, Ransohoff RM, Zhou L. Acute skeletal muscle
injury: CCL2 expression by both monocytes and injured muscle is
required for repair. FASEB J. 2011;25(10):3344–55.
Marsolais D, Cote CH, Frenette J. Neutrophils and macrophages
accumulate sequentially following Achilles tendon injury. J Orthop
Res. 2001;19(6):1203–9.