for example , vitorino et al.13 found no effect of shark liver oil supplementation , exercise or their combination on the innate immune system.13 this response depends on variables of physical exercise , such as intensity , duration and frequency , along with the exercise type .
in addition , few studies have evaluated the immunomodulatory effect of exercise training on peritoneal macrophage function , as activated macrophages are important sources of proinflammatory factors .
several studies , however , have shown that regular moderate exercise stimulates many aspects of the immune response.11,14,15 dos santos et al.16 showed that the increase in macrophage function induced by exercise is supported by enhanced glutamine consumption and metabolism ( animals trained 1  h / day for six weeks at moderate intensity ) . during exercise bouts , animals swam with an extra load equivalent to 5.5% of their total body weight attached to their tail to enhance aerobic training .
physical exercise can improve the macrophagemediated immune response to pathogen attack ( including antigen presentation ) in animals with metabolic syndrome.17 the abovementioned studies , however , measured macrophage function 1 and 72  h after exercise , respectively , and neutrophil phagocytosis was not accessed .
these exercise traininginduced changes in the immune response can be influenced by multiple factors , including mode , duration and intensity of exercise ; they also vary according to the time since the last exercise session.18   considering that the observed differences between study protocols are determinants for immune responses to exercise , the phagocytic response to prolonged exercise is not completely understood , especially for cases in which an adaptation period and different intensities are added .
given that exercise relies on the interplay between both intensity and duration , an experimental design that incorporates both of these variables would increase our understanding of this immunological response .
we hypothesized that after adaptation to exercise training , a decrease in the stressor effect of exhausting exercise would occur .
therefore , the purpose of this investigation was to analyze the effects of exhausting longduration physical exercise of different durations and intensities on the number and phagocytic capacity of macrophages and neutrophils in rats .
albinus , rodentia , mammalia ) with a mean weight of 200  g were used .
the animals had free access to water and chow and were kept in collective cages ( three rats per cage ) at a constant temperature of 23 (  2 ) c and a cycle of 12  h of light and 12  h of dark ( lights on from 06:00 h to 18:00 h ) .
this research was approved by the federal university of so carlos committee of experimental animals .
the animals were divided into two groups : untreated rats ( macrophage study ) and oyster glycogentreated rats ( neutrophil study ) . in each group , five subgroups were formed ( 10 rats per group ) : unexercised controls , an unadapted lowintensity exercise group , an unadapted moderateintensity exercise group , a preadapted lowintensity exercise group and a preadapted moderateintensity group .
the animals in the exercised groups performed swimming until exhaustion , and the animals in the preadaptation groups were submitted to four adaptation sessions lasting 5 , 15 , 30 or 45  min before the exhaustion session.19 the rats in the neutrophil study were treated with oyster glycogen approximately 4  h before exercise .
each animal received an intraperitoneal injection of 10  ml of type ii oyster glycogen ( 2% ) in pbs to induce the migration of circulating neutrophils to the peritoneal cavity.20   the physical exercise model chosen was swimming in a tank with a water temperature of 30  2 c during the afternoon .
the unadapted groups were submitted to physical exercise at low or moderate intensity only once ( exagl and exagm , respectively ) .
the animals in the preadapted groups were submitted to four days of adaptation with low and moderateintensity exercise ( exadl and exadm , respectively ) .
conversely , the groups submitted to moderateintensity exercise had a load of 5% of their body weight placed on their backs , which corresponded to an intensity below the point of inflection of the lactate threshold curve.21,22 the collection of cells was performed immediately after the exercise .
differential leukocyte counts were accomplished with an optical microscope using a blood smear colored with maygrnwald and giemsa ( sigma , st .
pbs ( 10  ml , ph  7.2 ) was injected into the animal 's peritoneum , and the area was massaged .
after the sacrifice , the peritoneum was then longitudinally sectioned , and the injected liquid was collected with a pasteur pipette .
the liquid was placed in a glass tube and centrifuged for 1  min at 2000  rpm until a pellet was formed .
the supernatant was discarded , and the pellet was suspended again in 10  ml of pbs ( 10 dilution , tube 1 ) .
a 1  ml sample was extracted from tube 1 and deposited in another tube with 9  ml of pbs ( 10 dilution , tube 2 ) .
after this step , 100  ml of the sample in tube 2 was collected and placed in a plastic tube .
louis , mo , usa ) was added to the plastic tube , and the contents were homogenized with a pipette ( 2 dilution )
. the neubauer chamber was filled , and the numbers of peritoneal macrophages and neutrophils ( 4  h before counting , the animals received an intraperitoneal injection of 10  ml of a solution of type ii oyster glycogen ) were determined using a microscope .
the results are reported as number of cellsn  10 [ number found at the sum of the four quadrants , 4 ( number of quadrants )  50 ( dilution  =   10  10  2 )  =   number  10 ] .
macrophages or neutrophils were incubated with 10  ml of pbs containing zymosan ( zymosan : 50  l , sigma , st .
after incubation , a 100  l aliquot was extracted and deposited in a plastic assay tube to which 100  l of tryplan blue was added . the neubauer chamber was filled , and 100 cells ( macrophages ) were identified .
the number of cells among the 100 identified macrophages that had incorporated zymosan was determined ; the macrophages were only considered valid if at least three zymozan particles had been incorporated .
therefore , the counting defined the percentage of peritoneal macrophages that had incorporated at least three particles of zymosan , according to the methodological descriptions provided by curi et al.20   the normality of distribution of all parameters was checked with the kolmogorovsmirnov test .
we compared the exercise groups with the control group , the exercise groups given the same duration of exercise to each other and the exercise groups with different protocols . when a significant value was observed , tukey 's posthoc test was used .
albinus , rodentia , mammalia ) with a mean weight of 200  g were used .
the animals had free access to water and chow and were kept in collective cages ( three rats per cage ) at a constant temperature of 23 (  2 ) c and a cycle of 12  h of light and 12  h of dark ( lights on from 06:00 h to 18:00 h ) .
this research was approved by the federal university of so carlos committee of experimental animals .
the animals were divided into two groups : untreated rats ( macrophage study ) and oyster glycogentreated rats ( neutrophil study ) . in each group , five subgroups were formed ( 10 rats per group ) : unexercised controls , an unadapted lowintensity exercise group , an unadapted moderateintensity exercise group , a preadapted lowintensity exercise group and a preadapted moderateintensity group .
the animals in the exercised groups performed swimming until exhaustion , and the animals in the preadaptation groups were submitted to four adaptation sessions lasting 5 , 15 , 30 or 45  min before the exhaustion session.19 the rats in the neutrophil study were treated with oyster glycogen approximately 4  h before exercise .
each animal received an intraperitoneal injection of 10  ml of type ii oyster glycogen ( 2% ) in pbs to induce the migration of circulating neutrophils to the peritoneal cavity.20
the physical exercise model chosen was swimming in a tank with a water temperature of 30  2 c during the afternoon .
the unadapted groups were submitted to physical exercise at low or moderate intensity only once ( exagl and exagm , respectively ) .
the animals in the preadapted groups were submitted to four days of adaptation with low and moderateintensity exercise ( exadl and exadm , respectively ) .
conversely , the groups submitted to moderateintensity exercise had a load of 5% of their body weight placed on their backs , which corresponded to an intensity below the point of inflection of the lactate threshold curve.21,22 the collection of cells was performed immediately after the exercise .
differential leukocyte counts were accomplished with an optical microscope using a blood smear colored with maygrnwald and giemsa ( sigma , st .
pbs ( 10  ml , ph  7.2 ) was injected into the animal 's peritoneum , and the area was massaged . after the sacrifice ,
the peritoneum was then longitudinally sectioned , and the injected liquid was collected with a pasteur pipette .
the liquid was placed in a glass tube and centrifuged for 1  min at 2000  rpm until a pellet was formed .
the supernatant was discarded , and the pellet was suspended again in 10  ml of pbs ( 10 dilution , tube 1 ) .
a 1  ml sample was extracted from tube 1 and deposited in another tube with 9  ml of pbs ( 10 dilution , tube 2 ) .
after this step , 100  ml of the sample in tube 2 was collected and placed in a plastic tube .
then , 100  ml of tryplan blue ( sigma , st . louis , mo , usa ) was added to the plastic tube , and the contents were homogenized with a pipette ( 2 dilution ) .
the neubauer chamber was filled , and the numbers of peritoneal macrophages and neutrophils ( 4  h before counting , the animals received an intraperitoneal injection of 10  ml of a solution of type ii oyster glycogen ) were determined using a microscope .
the results are reported as number of cellsn  10 [ number found at the sum of the four quadrants , 4 ( number of quadrants )  50 ( dilution  =   10  10  2 )  =   number  10 ] .
macrophages or neutrophils were incubated with 10  ml of pbs containing zymosan ( zymosan : 50  l , sigma , st .
after incubation , a 100  l aliquot was extracted and deposited in a plastic assay tube to which 100  l of tryplan blue was added .
the number of cells among the 100 identified macrophages that had incorporated zymosan was determined ; the macrophages were only considered valid if at least three zymozan particles had been incorporated .
therefore , the counting defined the percentage of peritoneal macrophages that had incorporated at least three particles of zymosan , according to the methodological descriptions provided by curi et al.20
we compared the exercise groups with the control group , the exercise groups given the same duration of exercise to each other and the exercise groups with different protocols .
in the macrophage study , the mean time of swimming until exhaustion was 425.0  3.0  min . in the study of neutrophils , the swim time was 35.0  4.0  min .
when compared to the control groups , significant leukocytosis was observed in the unadapted acutely exercised and preadapted groups following exercise to exhaustion at both low and moderate intensities for both protocols ( p  0.05 , see figure 1 ) . for the neutrophil groups ,
a general increase in the percentage of monocytes and neutrophils in the unadapted groups at low and moderate intensities was observed in the differential leukogram . in addition , a decrease in the percentage of neutrophils in the preadapted groups at low and moderateintensity exercise was observed .
macrophage study : there was a significant decrease in the total number of peritoneal macrophages in the unadapted lowintensity group as compared to the control group .
conversely , in the preadapted lowintensity group , the total number of peritoneal macrophages increased , with no changes in the moderateintensity groups .
a significant increase was observed in macrophage phagocytosis in all exercised rats in the macrophage study ( figure 2 ) .
neutrophil study : the total number of peritoneal neutrophils significantly decreased in the unadapted and preadapted lowintensity groups and increased in the unadapted and preadapted moderateintensity groups . a significant decrease was observed in neutrophil phagocytosis in the unadapted moderateintensity group ; however , an increase in the preadapted moderateintensity group was observed , with no changes in the lowintensity groups ( figure 2 ) .
when compared to the control groups , significant leukocytosis was observed in the unadapted acutely exercised and preadapted groups following exercise to exhaustion at both low and moderate intensities for both protocols ( p  0.05 , see figure 1 ) . for the neutrophil groups , a general increase in the percentage of monocytes and neutrophils in the unadapted groups at low and moderate intensities was observed in the differential leukogram .
in addition , a decrease in the percentage of neutrophils in the preadapted groups at low and moderateintensity exercise was observed .
macrophage study : there was a significant decrease in the total number of peritoneal macrophages in the unadapted lowintensity group as compared to the control group .
conversely , in the preadapted lowintensity group , the total number of peritoneal macrophages increased , with no changes in the moderateintensity groups .
a significant increase was observed in macrophage phagocytosis in all exercised rats in the macrophage study ( figure 2 ) .
neutrophil study : the total number of peritoneal neutrophils significantly decreased in the unadapted and preadapted lowintensity groups and increased in the unadapted and preadapted moderateintensity groups . a significant decrease was observed in neutrophil phagocytosis in the unadapted moderateintensity group ; however , an increase in the preadapted moderateintensity group was observed , with no changes in the lowintensity groups ( figure 2 ) .
leukocytosis associated with exhausting longduration exercise is well described in the literature and was first reported in 1902.23 the increase in immune cell count may be caused by a combination of effects of glucocorticoids and catecholamines or merely a redistribution phenomenon.24,25 in this study , leukocytosis was observed in both studies regarding macrophages and neutrophils at low and moderate exercise intensities ( figures 1a and b ) .
monocytes are precursors of macrophages in circulation , and they can be found in free or marginated forms.26 in response to acute exercise , monocyte numbers increase as a result of catecholaminemediated demargination.1,27 exhausting exercise reduces the number of peritoneal macrophages in rats , and the magnitude of this effect is correlated with serum corticosterone concentration .
 optimal  physiologic concentration at which glucocorticoids can stimulate macrophages , which is consistent with the idea that low glucocorticoid levels can enhance immunity instead of suppressing it.2,28,29 in this study , we observed a tendency for monocytosis in the exercise groups ( figure 1a ) .
we also observed a decrease in the total count of peritoneal macrophages in the unadapted animals submitted to lowintensity exercise and an increase in the animals preadapted to the same exercise regimen ( figure 2a ) .
one potential explanation for this finding could be that muscular training may induce physiological stress during acute exercise , resulting in an increase in the total number of peritoneal macrophages ( figure 2a ) .
physical exercise increases the plasmatic concentration of many substances , including glucocorticoids , catecholamines , prolactin ( prl ) , thyroid hormones and endorphins.30,31 all of these substances can enhance the phagocytic capacity of macrophages.1 according to sanders,32 most studies addressing the direct effects of catecholamines and the sympathetic nervous system on immunity have examined their effects on adaptive immunity ; however , little is known about adrenergic effects on innate immunity .
it has been observed that moderate treadmill training upregulates stimulantinduced production of nitric oxide ( no ) and cytokines essential for antimicrobial defense in cells of the monocyte / macrophage lineage , suggesting a reduced risk of infection.11   another possible mechanism occurs via mitogenactivated protein kinase ( mapk ) , which mediates the production of proinflammatory cytokines , whereas mapk phosphatase ( mkp)1 plays an essential role in intracellular homeostasis by negatively regulating macrophage mapk activation .
exercise training upregulates basal macrophage mkp1 expression , accelerates lpsevoked mkp1 upregulation , and impacts lpsevoked immune responses in mice.14 unfortunately , as a limitation of the present study , hormones and intracellular pathways were not studied ; however , there was an increase in the phagocytic capacity of macrophages in all of the exercised groups ( macrophage study ) ( figure 2a ) .
similarly , another study reported that phagocytic capacity and anion superoxide production by peritoneal macrophages were significantly increased in exercisetrained mice following 12  weeks of moderate exercise training ( 15120  min of treadmill running at a speed of 13  m / min for 5  days / week).15 comparisons between our results and those of other studies , however , are difficult to interpret because physiological responses depend on variables of physical exercise , such as intensity , duration and frequency as well as exercise type .
in addition , in other studies , the time at which the immune response is analyzed following exercise is widely variable .
catecholamines have been shown to modulate neutrophil cell levels.25 an increase in catecholamines depends on the exercise intensity , duration and adaptation .
moreover , catecholamines promote acute exercise effects on neutrophils , whereas cortisol may be responsible for the maintenance of neutrocytosis after longduration exercise.33 in this study , we observed neutrocytosis in unadapted animals submitted to an exercise session at low and moderate intensities ( neutrophil study ) and neutropenia in animals that were preadapted to the same exercise regimens ( figure 1b ) . regarding the total number of peritoneal neutrophils , a decrease occurred in the unadapted and preadapted groups submitted to lowintensity exercise ( neutrophil study ) , and
an increase was noted in the groups that exercised at moderate intensity ( figure 2b ) .
it is possible that the magnitude of the physiological stress caused a redistribution of neutrophils during exercise and adaptation , as was observed in our preadapted groups submitted to both exercise intensities ( neutrophil study ) ( figure 2b ) .
an increase in the total number of peritoneal neutrophils in the groups that completed moderateintensity exercise ( unadapted and preadapted ) was observed , and the migration of neutrophils to the peritoneal cavity increased proportionally with the intensity of exercise , consistent with previous findings.33   neutrophil phagocytic capacity is stimulated mainly by glucocorticoids and catecholamines , as neutrophils have receptors for these substances.1 adrenaline is a potent modulator of the immune system , and its level increases with exercise intensity .
this hormone is capable of reducing the phagocytic capacity of peritoneal neutrophils in vitro.34 in addition , noradrenaline seems to stimulate neutrophil functionality .
a study with rats subjected to sympathetic inhibition demonstrated a reduction in chemotaxis and phagocytosis as compared to control rats.6,7 in the context of our investigation , a reduction was observed in peritoneal neutrophil phagocytic capacity in the group submitted to a moderateintensity exercise session , whereas an increase was observed in the preadapted group subjected to the same level of exercise ( neutrophil study ) ( figure 2b ) .
the increase associated with the preadapted group submitted to moderateintensity exercise may have resulted from a decreased physiological stress response because of the adaptation to physical exercise ( figure 2b ) .
the major limitation of our data is that hormone levels were not measured . despite this limitation ,
the current data demonstrate important novel findings regarding exercise intensity and adaptation prior to exhaustive endurance exercise .
in general , exhausting longduration physical exercise was found to reduce the total number of peritoneal macrophages following lowintensity exercise ; however , this variable was increased in a separate group of animals submitted to four adaptation sessions .
conversely , the total number of peritoneal neutrophils decreased in the groups submitted to lowintensity exercise and increased in the groups performing moderateintensity exercise .
in addition , the phagocytic capacity decreased in the sedentary group following acute moderateintensity exercise , whereas an increase was observed in the preadapted groups .
it is possible that the adaptive response to exercise training resulted in a decrease in the stressor effect of exhausting exercise . in sedentary rats , neutrophils and macrophages
, adaptation sessions may be important for individuals who are beginning an exercisetraining program , to thus avoid excessive physiological stress on the immune system .
we thank fapesp , pibic / cnpq , fapic / unimep , fap / unimep and capes / prosup for financial support and all of the individuals that participated direct or indirectly in this research .
 objective : to analyze the effects of exhausting longduration physical exercise ( swimming ) sessions of different durations and intensities on the number and phagocytic capacity of macrophages and neutrophils in sedentary rats.introduction:exercise intensity , duration and frequency are important factors in determining immune response to physical effort . 
 thus , the effects of exhausting longduration exercise are unclear.methods:wistar rats were divided into two groups : an untreated group ( macrophage study ) and oyster glycogentreated rats ( neutrophil study ) . in each group , 
 the animals were subdivided into five groups ( 10 rats per group ) : unexercised controls , an unadapted lowintensity exercise group , an unadapted moderateintensity exercise group , a preadapted lowintensity exercise group and a preadapted moderateintensity exercise group . 
 all exercises were performed to exhaustion , and preadaptation consisted of 5 , 15 , 30 and 45  min sessions.results:macrophage study : the number of peritoneal macrophages significantly decreased ( 9.22  1.78  106 ) after unadapted exercise but increased ( 21.50  0.63  106 ) after preadapted lowintensity exercise , with no changes in the moderateintensity exercise group . 
 phagocytic capacity , however , increased by more than 80% in all exercise groups ( low / moderate , unadapted / preadapted ) . 
 neutrophil study : the number of peritoneal neutrophils significantly decreased after unadapted ( 29.20  3.34  106 ) and preadapted ( 50.00  3.53  106 ) lowintensity exercise but increased after unadapted ( 127.60  5.14  106 ) and preadapted ( 221.80  14.85  106 ) moderate exercise . 
 neutrophil phagocytic capacity decreased by 63% after unadapted moderate exercise but increased by 90% after corresponding preadapted sessions , with no changes in the lowintensity exercise groups.conclusion:neutrophils and macrophages of sedentary rats respond differently to exerciseinduced stress . 
 adaptation sessions reduce exerciseinduced stress on the immune system . 
INTRODUCTION
MATERIALS AND METHODS
Animals
Experimental Groups
Physical Exercise
Leukometry and Differential Leukogram
Total Number of Peritoneal Macrophages and Neutrophils
Phagocytic Capacity of Peritoneal Macrophages and Neutrophils
Statistical Analysis
RESULTS
4.1. Leukometry and Differential Leukogram
Total Number and Phagocytic Capacity of Peritoneal Macrophages and Neutrophils
DISCUSSION
ACKNOWLEDGEMENTS