Department of Physiology, College of Medicine, National Cheng-Kung University, Tainan 701, Taiwan, Republic of China
Correspondence: Dr. Chauying J. Jen, Department of Physiology, College of Medicine, National Cheng-Kung University, Tainan 701, Taiwan, Republic of China. E-mail: jen{at}mail.ncku.edu.tw
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R. Although
the blocking effect of MARCO antibody was most pronounced, that of
ICAM-1 antibody was totally reversed by cross-linking CR3. Our results
showed that SE, but not ME, activated BAMs and that the enhanced
nonopsonized phagocytosis was mainly mediated by scavenger receptors
and ICAM-1/CR3.
Key Words: scavenger receptor • CR3 • ICAM-1
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Epidemiological studies have highlighted the importance of physical activity in relation to the incidence of upper respiratory tract infection [2 , 4 ] and the lower prevalence and mortality rates for various site-specific cancers [5 ]. Whether or how exercise affects lung immunity is an important but still unclear issue. In animal studies, treadmill exercise increases the NK-cell cytotoxic activity and decreases the lung retention of tumor cells [6 7 8 ]. However, even after the depletion of NK cells, animals subjected to exercise still show lower retention of tumor cells [8 , 9 ]. Recent studies demonstrate that exercise increases peritoneal macrophage antitumor cytotoxicity [10 , 11 ]. Therefore, it seems likely that bronchoalveolar macrophages (BAMs) may actively participate in the exercise-enhanced lung immunity.
Acute exercise drastically alters physiological conditions in systemic
and pulmonary circulation. It has been shown that the increase of
circulating leukocytes depends on the intensity, duration, and type of
exercise [12
]. However, how the lung-immunity status
changes after a single exercise session is unclear at the present time.
We, therefore, investigated the effects of moderate and severe exercise
(ME and SE, respectively) on the function of murine BAMs, the major
pulmonary immune cells. BAMs are known to function through a variety of
mechanisms, including their abilities to undergo phagocytosis and to
express specific cell-surface receptors [13
]. In this
study, we examined the effects of exercise on BAM number, phagocytic
capacity, as well as the role of various BAM surface receptors,
including scavenger receptors, Fc receptor for immunoglobulin G (IgG;
FcR), complement receptor type 3 (CR3), and intercellular adhesion
molecule-1 (ICAM-1). Although scavenger receptors and FcR are
phagocytosis-related receptors, CR3 and ICAM-1 are usually considered
macrophage-activation markers [14
, 15
].
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Experimental animals and exercise regime
Male BALB/c mice, 7–9 weeks old, were purchased from the
National Cheng Kung University Animal Center (Tainan, Taiwan). The mice
were divided randomly into three groups, namely sedentary control, ME,
and SE. It has been shown that the oxygen consumption of small rodents
running on the treadmill can be predicted by their running speed
[16
]. Based on this study, the following exercise
protocols were used for mice to achieve ME and SE (about 60% and
>80% of maximal oxygen consumption, respectively). The ME group ran
on a treadmill starting at 9 m/min for 3 min followed by a 2 m/min
increment every 3 min until a speed of 17 m/min was reached. Running
was continued for a total duration of 30 min without further increase
in speed. The SE group ran in a similar way until a speed of 17 m/min,
and thereafter continued to run with 1 m/min increments every 3 min
until exhaustion. The SE mice were usually exhausted at 24 m/min with a
total running time of about 36 min. To avoid novel effects, the
sedentary control mice were placed on the treadmill for 10 min without
exercise. The circulating leukocyte count was measured to confirm that
different exercise intensities were indeed effective. Immediately after
SE or ME, the circulating leukocytes increased to >100% or to about
60%, respectively. This parameter returned to its resting value within
24 h.
Isolation of BAMs
Animals were anesthetized by intraperitoneal injection of
0.1–0.15 ml sodium pentobarbital (50 mg/ml). The BAM isolation
procedure was modified from a published method [17
].
Briefly, murine lungs were filled and flushed five times with 1 ml of
prewarmed phosphate-buffered saline (PBS; 145 mM NaCl, 5 mM KCl, 9.35
mM Na2HPO4, 1.9 mM
KH2PO4, 5.5 mM glucose, pH 7.4). This procedure
was repeated twice for a total vol of about 2 ml of lung lavage per
animal. BAMs were collected by centrifugation at 800 g for 8
min at 4°C and were resuspended in PBS. The percentage of BAM in lung
lavages was measured using Wright’s differential leukocyte staining.
The viability of cells was >98% as revealed by trypan blue exclusion
test.
Nonopsonized and opsonized phagocytosis
Carboxylated fluorescent latex microspheres (2 µm in
diameter) were coated for 1 h with 1% BSA or 0.2 mg/ml mouse IgG
at room temperature, sonicated for 5 min, and thereafter washed twice
in PBS. For the preparation of complement-opsonized beads, the
IgG-coated beads were incubated further for 1 h with normal mouse
serum (fourfold dilution) at room temperature, and then washed twice.
The suspensions were adjusted to a final concentration of 3.2 x
106 beads/10 µl PBS. BAMs were incubated for 1 h
with the bead suspension (cell:bead=1:30) at 37°C in a divalent
cation-containing PBS (0.15 mM CaCl2, 0.1 mM
MgCl2, and 0.03 mM MgSO4). Cells were
subsequently washed with cold PBS to eliminate uningested particles and
were thereafter fixed with 4% paraformaldehyde. The fluorescence
histogram of macrophages was measured by a flow cytometer (FACSort,
Becton Dickinson, San Jose, CA). Results were presented as population
percentage of phagocytic cells or as averaged number of ingested beads
per cell (phagocytosis index). Percentage of phagocytic cells =
number of macrophage that ingest at least one bead/total number of
macrophages x 100. Phagocytosis index = number of ingested
beads/total number of macrophages. Additionally, the same BAMs were
cytospun and stained after phagocytosis. Adhered and phagocytosed beads
were measured by counting the number of cell-associated beads for 200
cells under a 100x oil-immersion objective. Beads were scored as
phagocytosed when they were at the same depth of field as the cell.
Beads were scored as adhered when they were attached to the cell
membrane, either at different depth-of-field compared with the cell or
in profile to be outside the cell membrane [18
].
Immunofluorescence staining and flow cytometry
To examine BAM surface receptors, the cells were incubated for
1 h with various primary antibodies at 4°C. After cold PBS wash,
cells were incubated further for 1 h with fluorescence-labeled
goat antirat IgG or protein G Alexa Fluor 488-conjugate at 4°C. The
BAMs were then washed twice in PBS, fixed with 4% paraformaldehyde,
and thereafter analyzed by the flow cytometer.
Statistics
Data were expressed as mean ± SE, and
n = number of animals in that group. Results were
analyzed by one-way analysis of variance (ANOVA) and considered to be
significantly different when P < 0.05.
Student-Newman-Keuls contrast procedures were performed when
significant main effects were found.
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Table 1. BAM Counts in Sedentary Control and Different Exercise Groups
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Figure 1. BAM phagocytosis of fluorescently labeled latex beads. Flow cytometric
analysis shows BAM phagocytosis of nonopsonized beads. (A) Control, (B)
post-SE 0 h, and (C) post-ME 0 h. Percentage of phagocytic
cells, %P (i.e., number of cells that ingest at least one bead divided
by total cell numbers), and phagocytosis index, PI (total ingested
beads/total number of cells), were calculated and are shown next to the
respective histogram. (D) The morphology of BAMs that did not ingest
any bead (M0) and that ingested one bead (M1), two beads (M2), or
numerous beads (Mx) as viewed under light microscope.
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Figure 2. Effects of SE on BAM phagocytosis. Percentage of phagocytic cells and
phagocytosis index were calculated from flow cytometric results. Data
are presented as mean ± SE (n=6). One-way
ANOVA, *P < 0.05 compared with control and post-SE 4-h
values.
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R-mediated and complement
receptor-mediated phagocytosis. Results in Figure 3
show that the BAM phagocytic capacity was elevated for
opsonin-coated beads. However, the SE effect on phagocytosis of these
opsonized beads was minimal, indicating that SE mainly altered BAM
phagocytosis of nonopsonized beads.
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Figure 3. Effects of SE on BAM phagocytosis of nonopsonized and opsonized beads.
Data are presented as mean ± SE (n=4).
One-way ANOVA, *P < 0.05 compared with nonopsonized
values in the same group, and #P < 0.05 relative to
the corresponding control.
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Table 2. Effects of Severe Exercise on the Expression of BAM Surface Receptors
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Figure 4. Effects of scavenger receptor antibodies and inhibitors on BAM
phagocytosis. After a 20-min preincubation with 20 µg/ml ED31
(anti-MARCO), 5 µg/ml 2F8 (anti-type I/II), or 30 µg/ml poly-I or
heparin (negative control), the BAM phagocytosis of nonopsonized beads
was determined. Percent difference from vehicle was calculated as the
reduction of phagocytosis index compared with saline-treated BAMs. Data
are presented as mean ± SE (n=4). One-way
ANOVA, *P < 0.05 compared with vehicle value in the
same group, and #P < 0.05 relative to the
corresponding control.
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R on BAMs. As shown in Table 2
, SE
increased the BAM surface expression of CR3 (labeled with an antibody
against its 
chain CD11b) and ICAM-1 but not FcR. Moreover, the
increase in the level of CR3 was diminished gradually in hours
(unpublished results). It is interesting to know whether CR3 and ICAM-1
modulate the SE-enhanced BAM phagocytosis. Our results showed that the
inhibitory effect of anti-ICAM-1 antibody on BAM phagocytosis was also
more pronounced in SE compared with control (Fig. 5A
). Conversely, the anti-CR3 antibody used in this study did not
have any neutralizing effect.
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Figure 5. The role of ICAM-1 and CR3 in BAM phagocytosis of nonopsonized
beads. (A) Effects of neutralizing antibody against ICAM-1 on BAM
phagocytosis. After 20 min incubation with antibody (0.1–10 µg/ml)
at 4°C, BAMs were incubated further with fluorescently labeled,
albumin-coated latex beads, and the phagocytosis index was measured by
flow cytometry. Normal mouse IgG was used as control antibody. The
percent difference from vehicle was calculated based on the reduction
of phagocytosis index by antibody treatment. (B) Interaction between
ICAM-1 and CR3. In the presence or absence of 3 µg/ml neutralizing
antibody against ICAM-1, BAM-surface CD11b molecules were first
cross-linked for 20 min with anti-CD11b antibody at 4°C. BAMs were
incubated subsequently for 20 min with a secondary antibody
F(ab')2, and their phagocytosis index values were
determined. Phagocytosis index values were compared with those obtained
in the absence of any antibodies. Data are presented as mean ±
SE (n=4). One-way ANOVA, *P <
0.05 compared with the value of combined treatment with anti-ICAM-1
antibody and CD11b cross-linking in the same group.
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A single bout of strenuous running caused a transient murine BAM phagocytosis elevation as well as a transient decrease in BAM number. Studies using human subjects or animals have shown that exercise affects a wide range of immune parameters [1 , 2 ]. Most notably, acute exercise induces a rapid interchange of leukocytes between peripheral lymphoid tissues and the circulation, as indicated by a biphasic leukocytosis [12 ]. However, we show that there was no obvious infiltration of circulating leukocytes into the lung after SE. It is likely that the decrease in BAM number (collected in the first 2 ml of lavages) immediately after SE may result from the stronger adhesion between BAM and epithelium and/or a deeper movement of BAM into the lower airway. We have collected BAMs from additional lung flushed, up to 6 ml of lavages, and found no difference in total BAM number between control and exercised animals (unpublished results). Moreover, these BAMs from SE group also showed elevated phagocytic capacity. Therefore, all BAMs collected were apparently activated during SE and were more phagocytic.
Although the detail cause of exercise-induced lung immunity alteration cannot be investigated thoroughly in a single study, our results clearly show that SE, but not ME, enhances BAM phagocytosis. Various stress hormones and cytokines are known to be elevated in response to different exercise protocols [2 ]. One would assume that once the threshold of a certain hormone or cytokine is reached, BAMs become stimulated. However, the exercise effects are most likely to be more complicated than this assumption. Not only do these hormones and cytokines interact with one another, but also the exercise effects on certain physiological parameters can be in different directions as dictated by the exercise intensities. Our previous study showed the different effects between these two exercise intensities on platelet function in humans, where ME was found to inhibit platelets, and SE activated them [22 ].
Of all immune cells, NK cells, neutrophils, and macrophages (of the innate system) appear to be most responsive to acute exercise, in terms of cell number and function [3 , 11 ]. Several studies have demonstrated that exercise increases the antitumor cytotoxicity of alveolar and peritoneal macrophages [6 , 10 , 11 ]. Resident macrophages are the first line of defense against microbial invaders and malignancies by virtue of their phagocytic, cytotoxic, and intracellular killing capacity. Therefore, the growth reduction of site-specific tumors, especially in the lungs [6 , 23 ], by exercise could be partially attributed to the elevated phagocytosis of macrophages. In the lung, SE may be beneficial to cytolysis of tumor cell but not to the viral infection [24 ]. The latter effect of SE may be attributed to the suppression of macrophage-antigen presentation [25 ]. Although SE might not affect different pathogenic infections equally, the SE-enhanced phagocytosis of nonopsonized particles should lead to a faster clearance of foreign pathogens in general and decrease the possibility of acute lung injury.
Our results indicated that the SE effect on BAMs was most pronounced with regard to nonopsonized phagocytosis and that this effect was likely mediated by elevated scavenger receptors (especially MARCO) and CR3/ICAM-1 on their surface. Whether the exercise-enhanced phagocytic activity is generally mediated by the modulation of surface receptors in immune cells is an interesting question. The scavenger receptor class A has been shown to mediate the ingestion of nonopsonized latex beads, environmental particles, and certain bacteria [19 , 26 , 27 ]. These scavenger receptors may thus play a major role in safeguarding against large amounts of airborne particles, which are likely to be encountered during severe exercise. To our knowledge, the possible exercise effects on macrophage scavenger receptors have not been shown before. Results from this study have confirmed that the scavenger receptor-A is constitutively expressed in murine BAMs [28 ]. In addition, we demonstrated that SR-A type I/II were the major subtypes (Table 2) . It is interesting that although MARCO was relatively sparse on control BAM surface, this scavenger receptor subtype was largely upregulated after SE and appeared to play an important role in the SE-enhanced BAM phagocytosis. Conversely, the interaction of BAM activation markers, CR3 and ICAM-1, may also play a role in the SE effect because both receptors were upregulated as well. Previous studies have shown that severe exercise causes elevated expression of CR3 in circulating granulocytes [29 30 31 ] and also of ICAM-1 in monocytes [32 ]. In contrast, moderate exercise leads to decreased expression of ß1 and ß2 integrins in leukocytes [33 ].
In this study, we showed that BAM phagocytosis was suppressed by blocking ICAM-1 and that this suppression was reversed by cross-linking CD11b (Fig. 5) . This finding suggests that ICAM-1 may affect phagocytosis via its counter-receptor CR3 by outside-in signaling in the same cell type. In general, these two receptors are expressed in different cell types, for instance CR3 on leukocytes and ICAM-1 on endothelial/epithelial cells, for the mediation of leukocyte adhesion and extravasation. The stimulatory effects of ICAM-1 on BAM phagocytosis [34 ] and cytokine production [35 ] are probably mediated by the surface ß2 integrin family including CR3. When CR3 but not other ß2 integrins is cross-linked with specific antibodies to either one of its subunits (CD11b or CD18), it can trigger intracellular tyrosine phosphorylation [36 ]. Besides, CR3 can serve as signaling partners for other receptors or cytokines to induce a synergistic activation of macrophage [21 , 37 ].
In summary, acute SE, but not ME, increases murine BAM phagocytic capacity. This exercise effect is likely mediated by the upregulation of BAM surface receptors. However, we cannot rule out the possible participation of other factors, such as cytokine levels and pulmonary surfactant amount/composition, which also change during physical exercise.
Received June 12, 2000; revised August 30, 2000; accepted August 31, 2000.
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