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The Journal of Immunology, 2001, 167: 5084-5091.
Copyright © 2001 by The American Association of Immunologists

C57BL/6 and BALB/c Bronchoalveolar Macrophages Respond Differently to Exercise1

Shu-Hui Su, Hsiun-ing Chen and Chauying J. Jen2

Department of Physiology, College of Medicine, National Cheng-Kung University, Tainan, Taiwan, Republic of China


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Macrophages from prototypical Th1 strains (e.g., C57BL/6) and Th2 strains (e.g., BALB/c) are classified as M-1 and M-2 phenotypes. We investigated the different phagocytic responses between M-1 and M-2 bronchoalveolar macrophages (BAMs) under resting and two various exercise conditions. At rest, M-1 BAMs showed higher phagocytic capacity of unopsonized particles, higher expression of MARCO (macrophage receptor with collagenous structure), and higher generation of NO than M-2 BAMs. Severe exercise, but not moderate exercise, significantly enhanced both phagocytosis of unopsonized particles and expression of MARCO in M-2 BAMs. In contrast, M-1 BAMs were unaffected by either exercise protocol. The phagocytosis of unopsonized particles was largely mediated by MARCO, especially in M-1 BAMs. Secreted products from cultured M-2 BAMs isolated after severe exercise, but not those from M-1 BAMs, enhanced BAM phagocytosis. The cultured M-1 BAMs secreted phagocytosis inhibitors, and this effect could be blocked by NO antagonists. Moreover, the extent of phagocytosis suppression induced by M-1 BAM-secreted products correlated with their production of nitrite/nitrate. Exogenous NO donors as well as NO derivatives, nitrite and nitrate, suppressed the BAM phagocytosis. We propose that while the severe exercise-enhanced phagocytosis in M-2 BAMs was largely mediated by MARCO up-regulation and secretion of stimulators, the lack of exercise effect in M-1 BAMs could be partially due to the constitutive secretion of NO-related suppressors. In conclusion, genetically different mice use different strategies in regulating BAM activity under resting conditions and in response to various exercise paradigms.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Physical exercise exerts profound effects on the immune responses. Although regular exercise seems to be a panacea for health, its effects on immune competence have been reported in both positive and negative ways (1). Numerous reports emphasize the quantitative and functional variation of either circulating blood cells (including NK cells, lymphocytes, neutrophils, and platelets) or plasma mediators (including neuroendocrine hormones, cytokines, and Igs) (2, 3, 4). These observations show that the exercise-induced influence depends on the type/intensity/duration of exercise, the species of animals, and the inherent difference and body conditions of individuals. Epidemiological studies have highlighted the importance of the physical activity in relation to the incidence of upper respiratory tract infections (2, 5), and lower prevalence and mortality rates for various site-specific cancers (6). In animal studies, treadmill exercise increases the NK cell cytotoxic activity and decreases the lung retention of tumor cells (7, 8). However, even after the depletion of NK cells, animals subjected to exercise still show lower retention of tumor cells (8). Therefore, it seems likely that bronchoalveolar macrophages (BAMs)3 may actively participate in the exercise-enhanced lung immunity.

BAMs reside in a unique respiratory environment in which they are exposed to high ambient oxygen concentrations, to pulmonary surfactant that is rich in both lipids and opsonins, and to large amounts of inhaled particles (9). These cells represent a primary line of defense against the adverse effects of inhalation of bacteria and foreign particles, and play an important role in regulation of the pulmonary immune responses. BAMs are one of the alternatively activated macrophages under healthy conditions (10). Moreover, the alternatively activated macrophages preferentially express the receptors of innate immunity with broad specificity for foreign Ags, including scavenger receptors, and show enhanced capacity for phagocytosis than the classically activated macrophages. Besides the residing microenvironment of macrophages, their genetic characteristics also determine their functional properties. M-1 and M-2 phenotypes of macrophages from prototypical Th1 and Th2 strains of inbred mice (e.g., C57BL/6 and BALB/c mice, respectively) show different immunological and pathological responses (11, 12).

The most noted characteristics of C57BL/6 and BALB/c mice are their distinct responses after infection with intracellular pathogens and feeding with high-cholesterol diet (12, 13, 14). C57BL/6 mice are naturally resistant to intracellular pathogen infection (such as Leishmania major and Mycobacterium tuberculosis), but susceptible to diet-induced atherosclerosis. As a comparison, BALB/c mice show the opposite reactions. It is plausible to assume that the macrophages from C57BL/6 and BALB/c mice are different in their functional behavior. When infected by M. tuberculosis, T lymphocytes and macrophages of C57BL/6 mice preferentially produce IL-12 and Th1 cytokines (e.g., IFN-{gamma}) that are necessary for inducing macrophage cytotoxicity (15). However, BALB/c mice tend to produce Th2 cytokines (e.g., IL-4 and IL-10) that inhibit the generation of Th1 cytokines. Macrophages determine the immunological outcomes by modulating Th responses (16) and their own activities (17). Compared with M-2 macrophages, M-1 macrophages in culture are capable of producing larger amounts of NO when exposed to either IFN-{gamma} or LPS (11). NO acts in the immune system to kill foreign bodies, such as bacteria and tumor cells (18), and to modulate the generation of cellular molecules, such as cytokines and reactive oxygen intermediates (19). Moreover, M-1/M-2 phenotypes are independent of T or B lymphocytes, because macrophages from C57BL/6 and BALB/c NUDE or SCID mice also exhibit these phenotypes (11). BAM phagocytosis plays an important role in maintaining the optimized functional condition of the lung either at rest or during challenged conditions. Whether the phagocytic capacities of M-1 and M-2 BAMs are different or not, and how they are regulated under various exercise paradigms have not been investigated before.

It is interesting to know not only how exercise affects the various components of immune system, but also under which condition exercise will be clinically beneficial or deleterious. Several studies have demonstrated that exercise increases the phagocytic capacity and antitumor cytotoxicity of peritoneal macrophages (20). We have reported that BAMs from BALB/c mice are activated by severe exercise (SE), but not by moderate exercise (ME) (21). This SE-enhanced phagocytosis in M-2 BAMs is mediated by macrophage receptor with collagenous structure (MARCO) and the interaction between complement receptor type 3 and ICAM-1. MARCO, a particular type of scavenger receptor, mediates the binding of unopsonized particles to BAMs (21, 22), and it can be induced by bacterial infection (23). In the present study, we have examined in detail how exercise differentially affects BAM functions in C57BL/6 and BALB/c mice. Our results indicated that phagocytic and secreted activities of M-1 and M-2 BAMs were quite different at rest and were regulated distinctively under various exercise conditions.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

Carboxylate-modified fluorescent latex beads (2 µm in diameter), BSA, purified mouse IgG, bovine hemoglobin, sodium nitroprusside (SNP), and L-nitro-arginine methyl ester (L-NAME) were purchased from Sigma (St. Louis, MO). S-nitro-N-acetylpenicillamine was purchased from RBI (Natick, MA). Rat anti-mouse MARCO (ED31) and SR-A (scavenger receptor-A) type I/II (2F8) mAbs, and FITC conjugate of F(ab')2 goat anti-rat IgG were obtained from Serotec (Oxford, U.K.). Protein G-Alexa Fluor 488 conjugate and protein A-Alexa Fluor 546 conjugate were purchased from Molecular Probes (Eugene, OR). All other chemical reagents used in this study were purchased from Merck (Darmstadt, Germany).

Experimental animals and exercise regime

This study was conducted in conformity with the policies and procedures detailed in the Guide for Animal Care and Use of Laboratory Animals. Male 9- to 12-wk-old BALB/c (H-2d) or C57BL/6 (H-2b) mice were purchased from the National Cheng Kung University Animal Center (Tainan, Taiwan). The mice were randomly divided into three groups, namely sedentary control, ME, and SE. The exercise intensity of mice is closely related to the running speed of animal (24). As described in our previous report (21), the following exercise protocols were employed for mice to achieve ME and SE. The ME group of BALB/c mice 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. In the ME group of C57BL/6 mice, 1-m/min increment and final speed of 14 m/min were used instead. The SE groups ran in similar ways at the beginning, but continued to run with 1-m/min increment every 3 min until exhaustion. The SE group of BALB/c mice was usually exhausted at 24 m/min with a total running time of ~36 min, while the SE group of C57BL/6 mice was usually exhausted at 20 m/min with a total running time of ~33 min. Since the body weight and maximal exercise capacity of C57BL/6 mice were less than that of BALB/c mice in the same age, the lower running speed of C57BL/6 could also reach to the similar intensity of exercise. In these two strains of mice, the SE- or ME-induced increase of circulating leukocytes was ~100 or 50%, respectively. To avoid novel effects, the sedentary control mice were placed on the treadmill for 10 min without exercise.

Isolation of BAMs

Animals were anesthetized by i.p. injection of 0.1–0.15 ml sodium pentobarbital (50 mg/ml). The murine lungs were filled and flushed with 1 ml prewarmed PBS (145 mM NaCl, 5 mM KCl, 9.35 mM Na2HPO4, 1.9 mM KH2PO4, 5.5 mM glucose, pH 7.4). To have enough BAMs for our experiments, this procedure was repeated to obtain a total volume of ~6 ml bronchoalveolar lavage per animal. BAMs were collected by centrifugation at 800 x g for 8 min at 4°C, and were resuspended in RPMI for culture or in PBS for phagocytosis assay. The percentage of BAM in bronchoalveolar lavage was measured using Wright’s differential leukocyte staining. BAMs were the major cell type (95%) collected in all murine bronchoalveolar lavage. The viability of cells was greater than 98%, as revealed by trypan blue exclusion test. There was no difference in BAM count from the 6-ml lavage either between C57BL/6 and BALB/c mice or between control and exercise groups; ~8 x 105 cells were collected from each mouse.

BAM culture and conditioned medium pretreatment

The collected BAMs from first 2-ml lavage (two 1-ml collection) were resuspended in serum-free RPMI 1640 to exclude the possible interfering effects from TGF-{beta}1 (11). We only collected the BAMs from the first 2-ml lavage for cell culture, because the activating effect of SE was most significant on them in BALB/c mice (21). Cells were plated in triplicate at the density of 2 x 105/ml in 0.2-ml flat-bottom microtiter wells. The supernatant of culture medium (conditioned medium, CM) was collected after 1-, 2-, or 4-h incubation of M-1 or M-2 BAMs at 37°C in an atmosphere of 5% CO2. Before carrying out a phagocytosis experiment, BAMs from 6-ml lavage of another control BALB/c mouse were incubated for 30 min with freshly collected CM at 37°C. The remaining CMs were stored at -40°C until use to determine their concentrations of nitrite/nitrate.

Phagocytosis of unopsonized and opsonized latex beads by BAMs

The unopsonized and opsonized beads were prepared, as described in detail previously (21). BAMs were incubated for 1 h with the bead suspension (cell:bead ratio = 1:30) in divalent cation-containing PBS (0.15 mM CaCl2, 0.1 mM MgCl2, and 0.03 mM MgSO4) at 37°C. Cells were subsequently washed with cold PBS to eliminate free particles, and thereafter fixed with 4% paraformaldehyde. The fluorescence histogram of macrophages was measured by a flow cytometer (FACSort; BD Biosciences, San Jose, CA). In our hands, BAMs with fluorescence intensity below 50 U did not ingest any bead, and each ingested bead contributed to ~100 fluorescence U. Results were presented as population percentage of phagocytic cells or as averaged number of ingested beads per cell (phagocytosis index).

That is, 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.

Immunofluorescence staining, flow cytometry, and confocal microscopy for examining the expression of BAM surface receptors

To examine the expression of surface receptors, BAMs were incubated for 1 h with various primary Abs (20 µg/ml) at 4°C. After cold PBS wash, cells were further incubated for 1 h with fluorescence-labeled secondary Ab at 4°C. The BAMs were then washed twice in PBS, fixed with 4% paraformaldehyde, and thereafter analyzed by flow cytometry. An aliquot of stained BAMs was cytospun on a slide, and the locations of fluorescence-labeled receptors were observed by confocal microscopy (Leica-TCS-SP2, Heidelberg, Germany). The dual fluorescence of phagocytic BAMs, including fluorescence of latex beads (in green) and that of surface MARCO (in red), was also examined using a series of confocal images.

Measurement of nitrite and nitrate concentration

The amounts of NO produced by BAMs were determined indirectly by measuring its stable oxidation products, nitrite and nitrate. The amount of NO accumulated in the BAM culture for up to 4 h, or in the supernatant from BAM phagocytosis for 1 h, was measured by using a colorimetric assay kit (Cayman, Ann Arbor, MI). Results were calculated based on a standard curve (r2 > 0.99) that was constructed from sodium nitrate standards. While RPMI 1640 acted as the negative control for samples collected from BAM culture CM, PBS served as the control for samples collected from the supernatant of BAM phagocytosis. It was noted that RPMI 1640, but not PBS, contained significant amounts of nitrite and nitrate (~5 µM in total).

Data presentation

Data were shown as mean ± SEM, and n = number of animals in that group. Results were analyzed by one-way ANOVA and considered to be significantly different when p < 0.05. Student-Newman-Keuls contrast procedures were performed when significant main effects were found. Inverse correlation statistics (Pearson’s correlation coefficient and p value) were obtained from a simple regression analysis.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of various intensity of exercise on the phagocytosis of unopsonized and opsonized particles by M-1 and M-2 BAMs

At rest, the phagocytic capacity of unopsonized particles by M-2 BAMs from BALB/c mice was much less than that of M-1 BAMs from C57BL/6 mice (Table IGo). Immediately after SE, but not ME, the phagocytic capacity of unopsonized particles by M-2 BAMs was significantly enhanced up to the resting level of M-1 BAMs. In contrast, the phagocytic activities of unopsonized particles by M-1 BAMs were unaffected by either exercise regime. Although the BAM phagocytic capacities of opsonized beads were higher than that of unopsonized beads in either type of BAMs, SE or ME exerted little effect on the phagocytosis of opsonized beads.


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  		Table I. Phagocytic capacity of M-1 and M-2 BAMs under resting or postexercise conditions1

 
Effects of SE on the expression and function of scavenger receptors in M-1 and M-2 BAMs

The expression of scavenger receptors MARCO and SR-A I/II at rest or immediately after SE was determined by labeling with the mAb ED31 and 2F8, respectively. The MARCO expression of M-1 BAMs at rest was higher than that of M-2 BAMs, and it was unchanged by SE (Fig. 1GoA). It was noticed that the fluorescence intensities associated with SR-A I/II in M-1 BAMs were quite low. A recent report indicates that SR-A I/II in M-1 macrophages might be incompletely stained by mAb 2F8 (25). The direct comparison of its staining intensity with other markers could be misleading, and hence not conducted in this study. In comparison, both MARCO and SR-A I/II were up-regulated in M-2 BAMs under the post-SE condition. However, a dual labeling using the same fluorescently labeled secondary Ab for both primary mAbs did not raise the fluorescence intensity to levels above that of the SR-A I/II labeling alone. This is probably due to the colocalization of both scavenger receptor types. Fig. 1GoB shows a confocal image of dual labeled M-2 BAMs, indicating that a major portion of MARCO labeling (red) overlapped with SR-A I/II labeling (green). Moreover, BAMs with ingested beads showed reduced MARCO labeling when compared with bead-free BAMs (Fig. 1GoC).



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  		FIGURE 1. The expression of MARCO and SR-A I/II on M-1 or M-2 BAMs under either control or post-SE conditions. A, MARCO and SR-A I/II of BAMs were labeled by incubation with 20 µg/ml mAbs, ED31 and 2F8, respectively, and subsequently labeled by FITC-conjugated secondary Ab, with normal mouse IgG serving as a negative control. Data were analyzed by one-way ANOVA (n = 3). *, p < 0.05 (post-SE 0 h vs control in the same strain); #, p < 0.05 (M-2 vs M-1). B, Confocal image showing SR-A I/II (green) and MARCO (red) distribution on the surface of control M-2 BAMs. Orange spots indicate the colocalizations of these two receptors. Bar equals 10 µm. C, MARCO expression (red) and the ingestion of FITC-labeled beads (green) in control M-2 BAMs. The arrow indicates an ingested bead. Bar equals 5 µm.

 
Furthermore, we investigated the functional roles of MARCO and SR-A I/II in BAM phagocytosis by blocking them with specific Abs or inhibitors. Fig. 2Go shows that MARCO, but not SR-A I/II, plays an important role in the phagocytosis of unopsonized particles by BAMs, especially in M-1 BAMs. In controls, the MARCO contributed ~75% and 25% of the phagocytosis of unopsonized particles in M-1 and M-2 BAMs, respectively. Immediately after SE, ED31 (an Ab against MARCO) completely abolished exercise-enhanced phagocytosis in M-2 BAMs. In comparison, the SR-A I/II-mediated phagocytosis was less than 20% in BAMs from all groups, as evidenced by the experiments using the Ab 2F8. Consistent with the dual labeling results (Fig. 1Go), the inhibitory effects of ED31 and 2F8 were not additive (Fig. 2Go). This is most likely due to the interactive epitope binding between ED31 and 2F8. MARCO and SR-A I/II contain a similar collagenous structure that binds to polyanionic molecules (26). The effects of polyinosinic acid, a polyanionic inhibitor of scavenger receptors, were similar to that of Abs. The negative controls, normal mouse IgG and heparin, were totally ineffective on BAM phagocytosis (data not shown).



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  		FIGURE 2. The role of MARCO and SR-A I/II in BAM phagocytosis. The phagocytosis of unopsonized latex beads by M-1 (upper panel) or M-2 BAMs (lower panel) was examined after a 20-min preincubation with 20 µg/ml ED31 (against MARCO), 2F8 (against SR-A I/II), or 30 µg/ml polyinosinic acid. The percent difference from vehicle was calculated as the reduction of phagocytosis index normalized against the vehicle-treated BAMs. Data were analyzed by one-way ANOVA (n = 4). *, p < 0.05 (post-SE 0 h vs control); #, p < 0.05 (M-2 vs M-1); {dagger}, p < 0.05 (compared with vehicle in the same group).

 
Effects of soluble factors on BAM phagocytosis

As BAMs are surrounded by the pulmonary surfactant in vivo, soluble factors in the surfactant may mediate the functional change of BAMs (9). We therefore examined the effects of the supernatant from bronchoalveolar lavage on the phagocytosis in autologous control BAMs. Only the first ml lavage supernatant, but not additionally flushed supernatants, from the post-SE BALB/c mice induced a significant increase in BAM phagocytosis (16 ± 6%, n = 10). Lavage supernatants from C57BL/6 mice under either resting or post-SE conditions were ineffective.

In an attempt to examine whether BAMs from exercised animals would release bioactive substances to modulate phagocytosis, CM from cultured BAMs were analyzed for their bioactivity. In this part of study, we used M-2 BAMs isolated from control BALB/c mice to assay CM activity. While CM from cultured M-1 BAMs generally showed a suppressive effect on BAM phagocytosis, CM from cultured M-2 BAMs generally showed stimulatory activity instead (Fig. 3Go). The inhibitory bioactivity was long lasting if the CM were derived from cultured M-1 BAMs isolated from control mice. In contrast, the stimulatory bioactivity was long lasting if the CM were derived from cultured M-2 BAMs isolated from post-SE mice.



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  		FIGURE 3. Effects of CM on BAM phagocytosis. The phagocytosis of unopsonized latex beads was examined using control M-2 BAMs preincubated with freshly collected CM derived from either M-1 or M-2 BAMs (2 x 105 cells/ml) for 30 min at 37°C. The percent difference from vehicle was calculated as the reduction of phagocytosis index normalized against RPMI-pretreated specimens. Data were analyzed by one-way ANOVA (n = 4). *, p < 0.05 (post-SE 0 h vs control).

 
NO mediated the suppressive effects in the CM from cultured M-1 BAMs

As mentioned earlier, exercise did not affect BAM phagocytosis in C57BL/6 mice. This could be due to either the release of inhibitory factors by M-1 BAMs or the lack of release of stimulatory factors. The most pronounced inhibitory effect of control M-1 BAM-secreted products was observed in the 2-h culture condition (Fig. 3Go). This culture condition was chosen to identify the possible candidates of phagocytosis suppressors in the present study. Several studies report that endogenous NO and NO donors are able to inhibit leukocyte adhesion and phagocytosis (27, 28). We therefore tested the possibility that the suppression of phagocytosis by the CM from cultured M-1 BAMs was mediated by NO. During the 2-h culture, this medium was either supplied with additional L-arginine (500 µM), or treated with L-NAME (100 µM). In some experiments, the CM was preincubated with hemoglobin (0.2 mg/ml) or methylene blue (5 µM) to block NO or cGMP effects. Results are shown in Fig. 4Go. Since the suppressive effect was reversed by L-NAME (a NO synthase inhibitor) or hemoglobin (a NO scavenger), and enhanced by the addition of L-arginine (the NO precursor), these results suggested that the suppressive activity of M-1 BAM-derived CM was modulated by NO. Methylene blue, a guanylyl cyclase inhibitor, was unable to alter this suppressive activity. Furthermore, the inhibition of phagocytosis remained effective in CM stored for >1 h at 4°C (data not shown), indicating that the inhibitory effect could not be solely mediated by the short-lived NO.



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  		FIGURE 4. Suppression of phagocytosis by CM derived from control M-1 BAMs. Phagocytic activity of control M-2 BAMs was assayed after being incubated at 37°C for 30 min with various CM. CM were collected from control M-1 BAMs cultured under the following conditions: 1) medium alone, 2) in the presence of 100 µM L-NAME, or 3) in the presence of 500 µM L-arginine. Hemoglobin (Hb, 0.2 mg/ml) or methylene blue (MB, 5 µM) was added to the CM in certain experiments. Results were normalized against phagocytosis index of RPMI-pretreated BAMs (n = 4). Data were analyzed by one-way ANOVA. {dagger}, p < 0.05 (compared with RPMI control); §, p < 0.05 (compared with CM alone). RPMI with various additives did not affect BAM phagocytosis.

 
To test whether the differential exercise effects between M-1 and M-2 BAMs were due to their secretion of different levels of NO metabolites, we then measured the amount of nitrite and nitrate in some CM samples. Results in Fig. 5Go indicated that cultured M-1 BAMs, but not cultured M-2 BAMs, released NO metabolites to the CM. Moreover, we also noticed that the nitrite/nitrate levels in CM were similar from cultured BAMs isolated under either resting or post-SE conditions. The presence of either L-NAME or L-arginine in BAM culture modulated the release of NO metabolites to the CM. To examine whether BAMs would secrete NO during the phagocytosis assay or not, we also measured the NO production from either BAM phenotype in 1-h incubation with or without latex beads. Compared with the same cells under cultured conditions, M-1 BAMs in suspension generated less, but significant amounts of NO products during the phagocytosis assay (~2.5 µM nitrite/nitrate from 106 cells/ml). In contrast, M-2 BAMs produced undetectable amount of nitrite/nitrate during the phagocytosis assay in the presence or absence of CM. Finally, we compiled results from various experiments mentioned previously and plotted the amount of nitrite and nitrate in M-1 BAM CMs in each experiment against its inhibitory activity (Fig. 6Go). The amounts of nitrite/nitrate in all CMs collected from cultured M-1 BAMs isolated under resting conditions correlated closely with the suppression of phagocytosis (correlation coefficient = -0.78, p < 0.0001). All these results support our speculation that NO was involved in the CM-induced phagocytosis inhibition.



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  		FIGURE 5. The levels of nitrite and nitrate in CM derived from cultured M-1 or M-2 BAMs. CM were collected from 2-h cultures of either BAM phenotype isolated under control or post-SE conditions. In certain experiments, CM were collected from cultures containing L-NAME (100 µM) or L-arginine (500 µM). Data were analyzed by one-way ANOVA (n = 4). #, p < 0.05 (M-2 vs M-1); {dagger}, p < 0.05 (compared with RPMI control).

 


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  		FIGURE 6. The correlation between M-1 BAM-derived nitrite/nitrate level and its suppressive effect on phagocytosis. BAM-derived nitrite/nitrate levels were calculated by subtracting the basal nitrite/nitrate level in the RPMI. Data were analyzed by Pearson’s method (n = 43, correlation coefficient = -0.78, p < 0.0001).

 
Exogenous NO and its metabolites suppressed the BAM phagocytic activity

To exclude the possibility of different sensitivities between M-1 and M-2 BAMs to NO-related suppressors, we examined the effects of NO donors on phagocytosis in both M-1 and M-2 BAMs. The dose-dependent suppression of SNP on phagocytosis was similar between M-1 and M-2 BAMs (Fig. 7GoA). When S-nitroso-N-acetylpenicillamine (another NO donor) was used, similar results were obtained (data not shown). The BAM phagocytosis was also tested in the presence of SNP (100 µM), sodium nitrite, or nitrate (30 µM each) with or without the addition of either hemoglobin or methylene blue (Fig. 7GoB). All three reagents, SNP, nitrite, and nitrate, showed inhibitory effects on BAM phagocytosis, and their suppressive effects could be partially reversed by hemoglobin, but not by methylene blue.



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  		FIGURE 7. Suppression of BAM phagocytosis by exogenous NO and its derivatives. A, M-1 or M-2 BAMs were preincubated with freshly prepared SNP (10 µM-1 mM) at 37°C for 30 min before the phagocytosis assay. SNP showed similar dose-dependent suppression of unopsonized phagocytosis between M-1 and M-2 BAMs. B, The effects of hemoglobin (Hb, 0.2 mg/ml) or methylene blue (MB, 5 µM) on the suppression of BAM phagocytosis by SNP (100 µM), sodium nitrite (30 µM), or sodium nitrate (30 µM). Data were analyzed by one-way ANOVA (n = 3). {dagger}, p < 0.05 (compared with the corresponding vehicle); §, p < 0.05 (compared with the absence of Hb or MB).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we have demonstrated the differences of phagocytic activity, receptor expression, and NO generation between unprimed M-1 and M-2 BAMs. Compared with M-2 BAMs, M-1 BAMs showed higher basal phagocytic activity of unopsonized particles, higher MARCO expression, and higher NO generation. The high MARCO expression on resting M-1 BAMs contributed to their high phagocytosis of unopsonized particles, but the NO-related suppressive factors from these cells might have prevented them from showing the SE-induced augmentation of phagocytosis. As a comparison, M-2 BAMs exhibited an enhanced phagocytosis with broad specificity after a single SE session, largely due to the up-regulation of scavenger receptors (especially MARCO) and the secretion of stimulating factors. The genetic/phenotypic differences between BAMs from C57BL/6 and BALB/c strains play an important role in their responses to exercise.

BAM phagocytosis in the respiratory system is the determining step for the removal of inhaled foreign particles. It is interesting to note that the BAM phagocytic activity may be modulated in opposite ways via expressing the surface receptor MARCO or secreting NO derivatives. Our results indicated a positive correlation between the phagocytic capacity of unopsonized particles and the amount of surface MARCO in either BAM phenotype (Figs. 1Go and 2Go). Besides, the M-1 BAM phagocytosis of unopsonized beads was predominantly MARCO mediated, i.e., nearly 80% of M-1 BAM phagocytosis could be blocked by anti-MARCO Ab. Moreover, MARCO appeared to be internalized when bead ingestion happened, as indicated in our confocal image. Existing evidence supports a role for scavenger receptor class A, including MARCO and SR-A I/II, in phagocytic recognition of certain microorganisms, environmental particles, and apoptotic thymocytes in the absence of opsonins (22, 26, 29). However, MARCO, but not SR-A I/II, plays an essential role in BAM binding of unopsonized environmental particles (30). Besides, after bacterial infection, MARCO expression is up-regulated in various types of macrophages, regardless whether they previously express MARCO or not (31). Therefore, the intrinsically high phagocytic activity along with high MARCO expression in M-1 BAMs could be partially responsible for the resistance of tuberculosis in C57BL/6 mice.

Results from the current study indicated that the secreted products from cultured M-1 BAMs, but not from cultured M-2 BAMs, were inhibitory to phagocytosis and that these phagocytosis suppressors were NO related ( Figs. 3–6GoGoGoGo). M-1 BAMs isolated under either resting or post-SE conditions produced considerable amounts of NO and its derivatives when cultured in serum-free medium. Furthermore, we showed that not only NO, but also its stable metabolites, nitrite and nitrate, were effective inhibitors for BAM phagocytosis. To our knowledge, this is the first report indicating that nitrite and nitrate affected BAM phagocytosis. In biologic systems or at air-aqueous interfaces, NO generation is marked by the formation of NO2- (nitrite) and NO3- (nitrate) (32). In addition to forming nitrite/nitrate, NO may be stabilized by forming covalent binding with thiols (33). Moreover, the S-nitrosothiols can act as NO•, NO+, and NO- donors under physiological conditions (34). Most NO-related biological effects, including cytotoxicity and vasorelaxation, cannot be substituted by using nitrite/nitrate (18, 32). Recently, NO derivative-mediated tyrosine nitration of cellular proteins, e.g., membrane-bound receptors, has been shown to affect cellular behavior via altering tyrosine phosphorylation (35). Besides the NO derivatives, other macrophage products whose synthesis, secretion, or actions are regulated by NO may also mediate the suppressive effect on BAM phagocytosis. For example, there is cross-talk between NO and products of the cyclooxygenase pathways, such as PGE2 (36). PGE2 has been shown to serve as an effector molecule in NO-mediated effects, such as macrophage phagocytosis and growth factor secretion (37). However, most related studies focus on the role of the inducible NO production in activated macrophages (38), instead of constitutive NO production in unprimed or nonactivated macrophages. The NO generated from unprimed BAMs has been reported to suppress the responses of lymphocytes to general encounters in air or to modulate cellular metabolism (39, 40, 41). Based on our findings, NO and its long-lived derivatives from unprimed M-1 BAMs were capable of suppressing BAM phagocytosis.

Immunomodulating effects of exercise mostly involve the innate immune system, such as the tissue macrophages, circulating phagocytes, and the acute phase plasma proteins/cytokines (4, 20, 42). In the current study, neither ME nor SE induced infiltration of circulating blood cells into the lung, suggesting that the effects from our exercise paradigms were nonpathological. SE, but not ME, enhanced M-2 BAM phagocytosis of unopsonized particles and surface expression of scavenger receptors, especially MARCO. The increased expression of scavenger receptors in M-2 BAMs may thus offer a protection from large amounts of air-borne particles, which are likely to be encountered during SE. However, similar changes were absent in M-1 BAMs after either intensity of exercise, perhaps due to sufficient MARCO expression even under resting conditions. Presently, few mediators that regulate the expression of scavenger receptors have been identified. Although MARCO up-regulation in macrophages is known to occur under inflammation, e.g., bacteria infection in vivo and LPS stimulation in vitro, the proinflammatory cytokines IL-1, IL-6, TNF-{alpha}, and IFN-{gamma} show little effect on MARCO expression in vitro (43). Perhaps the MARCO expression in M-1 BAMs can only be up-regulated under pathological stimulations, e.g., bacteria infection, but not under physiological stimulations, such as SE. SR-A I/II expression in macrophages is enhanced by M-CSF (44) and is down-regulated by IFN-{gamma} or TNF-{alpha} (45). Previous studies have demonstrated that some exercise-augmented hormones, such as corticosterone, prolactin, and thyroid hormone, enhance phagocytic activity in peritoneal macrophages (46, 47). In addition, endogenous norepinephrine regulates TNF-{alpha} production from murine peritoneal macrophages in vitro (48). Therefore, the SE effects in M-2 BAMs observed in the current study might be mediated by a variety of neuroendocrine hormones or cytokines.

As mentioned earlier, the secreted products derived from cultured M-2 BAMs, especially those from post-SE BAMs, showed enhanced effect on BAM phagocytosis (Fig. 3Go). In contrast, the secreted products from cultured post-SE M-1 BAMs did not stimulate BAM phagocytosis. There are several possible explanations for these differential SE effects on different murine strains. First, significant amounts of NO-related suppressors were produced in M-1 BAMs. The NO-mediated inhibitory effects might offset the SE-induced up-regulation of phagocytosis-stimulating factors. The inhibitory effect of CM from M-1 BAM culture was only seen in 1-h incubation in post-SE group, but lasting for >4 h in the control (Fig. 3Go). Perhaps post-SE M-1 BAMs secreted some stimulating factors or NO scavengers to counterbalance NO effect after 2-h incubation in culture. Second, it is possible that the constitutive NO production in M-1 BAMs modulates the effects of SE, a relatively weak immune stimulation, and prevented the further elevation of MARCO expression and phagocytic capacity. Although the surface expression of MARCO in M-1 BAMs was already high compared with resting M-2 BAMs, up-regulation of MARCO expression in these cells actually happened under certain circumstances, such as during bacterial sepsis or after LPS injection (43). Besides, the phagocytic capacity also increased significantly when M-1 macrophages were exposed to inflammatory stimulants (49). Third, different murine strains may release different amounts/types of SE-induced hormones or cytokines. Or, the susceptibility of different BAMs to these systemic modulators may be variable. Finally, the sensitivity of M-1 and M-2 BAMs to BAM-secreted bioactive substances, either activators or suppressors, may be different. Leukocytes from Th1 and Th2 murine strains show different sensitivity to PGE2 (50). However, we do not favor the last explanation. The effects of BAM-secreted products on the phagocytosis of unopsonized particles were identical when assayed by using either M-1 or M-2 resting BAMs (data not shown).

In summary, inbred mice with distinct genetic/phenotypic characteristics offer attractive models to examine the mechanism of immunoregulation in the respiratory system. M-1 and M-2 BAMs are intrinsically different, and they use distinct strategies to regulate their own function. M-1 BAMs have relatively abundant scavenger receptors on their surface and show high phagocytic activity even in resting conditions. However, they constitutively secrete NO and its metabolites, which may serve as a protection to prevent excessive phagocytosis during SE. In contrast, M-2 BAMs have relatively few scavenger receptors and show low basal phagocytic activity. They secrete little NO-related substances and respond to SE by elevating their scavenger receptor expression and phagocytic capability, reaching the level of M-1 BAMs.


   Acknowledgments
 
We are indebted to the helpful discussions from Drs. H. Y. Lei and K. L. Chang, and to the critical reading of the manuscript from Dr. R. Moldzio.


   Footnotes
 
1 This study was funded by grants from National Science Council and National Health Research Institute, Taiwan, Republic of China (Grants NSC 89-2320-B-006-125, NSC 89-2320-B-006-134, and NHRI-GT-EX90-8834SL). Back

2 Address correspondence and reprint requests to Dr. Chauying J. Jen, Department of Physiology, College of Medicine, National Cheng-Kung University, Tainan 701, Taiwan, Republic of China. E-mail address: jen{at}mail.ncku.edu.tw Back

3 Abbreviations used in this paper: BAM, bronchoalveolar macrophage; CM, conditioned medium; L-NAME, L-nitro-arginine methyl ester; MARCO, macrophage receptor with collagenous structure; ME, moderate exercise; SE, severe exercise; SNP, sodium nitroprusside; SR-A, scavenger receptor-A. Back

Received for publication April 23, 2001. Accepted for publication August 28, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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