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Adaptation to eccentric exercise: effect on CD64 and CDllbKD18

expression

FRANCIS X. PIZZA, BRUCE H. DAVIS, STEVEN D. HENRICKSON, JOEL B. MITCHELL, JOHN F. PACE, NANCY BIGELOW, PAUL DILAURO, AND THOMAS NAGLIERI Department of Physical Education, Texas Christian University, Fort Worth, 76129; and Analytical Cytometry Laboratory, Harris Methodist Hospital, Fort Worth, Texas 76104 Pizza, Francis X., Bruce H. Davis, Steven D. Henrickson, Joel B. Mitchell, John F. Pace, Nancy Bigelow, Paul DiLauro, and Thomas Naglieri. Adaptation to eccentric exercise: effect on CD64 and CDllbKD18 expression. J. Appl. Physiol. 80(l): 47-55, 1996.-The primary purpose of the study was to examine circulating neutrophils and monocytes and their plasma membrane expression of CD64, CDllb, and CD18 after two bouts (Bl and B2) of eccentric exercise. Subjects (n = 10) performed 25 forced-lengthened contractions of the forearm flexors on two occasions separated by 3 wk. Blood samples were obtained before exercise and at 1.5,6, 12,24,48, 72, and 96 h of recovery. CD64, CDllb, and CD18 expression was determined via direct immunofluorescence and used as an indicator of neutrophil and monocyte activation. Creatine kinase activity (Bl = 1,390, B2 = 108 U/I), myoglobin (Bl = 163, B2 = 41, ng/dl), and muscle soreness and tenderness were higher (P < 0.01) after Bl compared with B2. Neutrophils at 6,12, and 96 h were higher (P < 0.05) for Bl vs. B2. CDllb expression on neutrophils was 2.7-fold higher at 72 h for Bl vs. B2. CD64 expression on neutrophils at 72 and 96 h was 1.4- and 1.9-fold higher, respectively, for Bl vs. B2. At 72 and 96 h, CD18 and CD64 expression on monocytes was 1.3-fold higher for Bl vs. B2. The observed changes were not significantly correlated with changes in creatine kinase activity or myoglobin. In conclusion, the adaptation to eccentric arm exercise was associated with a reduction in circulating neutrophils and a lower state of neutrophil and monocyte activation. creatine kinase activity; soreness; muscle damage

myoglobin;

delayed-onset

muscle

RECOGNIZED that unaccustomed exercise, particularly eccentric exercise, results in muscle damage and muscle soreness (2, 11, 27). In the days after unaccustomed eccentric exercise, extensive disruption of the ultrastructure of skeletal muscle occurs, as well as the release of myocellular proteins [e.g., creatine kinase (CK) and myoglobin] into the blood and the delayed onset of muscle soreness (DOMS) (2, 11, 13). These indexes of muscle damage are substantially reduced when the same eccentric exercise is performed several weeks later, indicating a rapid adaptation to eccentric exercise (3,7,15,16). Although several mechanisms have been proposed including mechanical overload, Ca2+-mediated proteolysis, and phosphagen depletion, the cause of muscle damage and the mechanism for the adaptation to eccentric exercise is not well understood (2, 11, 27). Several investigators have recently implicated indexes of acute inflammation as a mechanism for Z-line disruption (13), myocellular protein release (4-6, 23), and DOMS (27) after eccentric exercise. IT IS WELL

0161-7567/96

$5.00

Copyright

o

The cellular phase of acute inflammation consists of predominantly neutrophils and monocytes that adhere to vascular endothelium and enter the damaged and surrounding tissue. When activated, these phagocytes are capable of dissolving muscle and connective tissue via the release of lysosomal enzymes (degranulation) and/or reactive oxygen intermediates (32). The coating, or opsonization, of surfaces with antibodies or complement fragments facilitates the selection and phagocytosis of target cells. Neutrophils and monocytes recognize opsonized surfaces via plasma membrane receptors for complement fragments and immunoglobulins (Ig) (34). Cluster of differentiation CDllb/CDl8 [complement receptor type 3 (CR3), Mac-l], a P2-integrin, is a transmembrane glycoprotein expressed on the plasma membrane of neutrophils, monocytes, and natural killer cells. CDllb/CDl8 functions as a receptor for a fragment of the third component of complement (iC3b) and mediates phagocytosis of iC3b opsonized surfaces and adherence to vascular endothelium (10, 34). Inhibition of CDllb/CDl8 by using monoclonal anti-receptor antibodies has been reported to decrease tissue accumulation of neutrophils and to prevent reperfusion injury in skeletal (8,33,35) and cardiac muscle (26). Infiltration of neutrophils (13) and macrophages (24) into damaged muscle has been reported after eccentric exercise. Based on these observations, it is possible that changes in the expression of CDllb/CDl8 on neutrophils and monocytes occur after eccentric exercise; however, to date, the effect of eccentric exercise on CDllb/CDl8 has not been investigated. Neutrophils and monocytes also recognize opsonized surfaces via plasma membrane receptors for the Fc region of IgG (FcrR) (30,34). FcyRI (CD64), a transmembrane glycoprotein, mediates antibody-dependent cellular cytotoxicity and triggers phagocytosis, superoxide production, and degranulation (1, 25, 30). We recently reported preliminary data in which we observed a significant positive correlation between CK activity and CD64 expression on neutrophils after downhill and level running (23). Based on the observed correlation, the present study was conducted to further characterize changes in CD64 after eccentric exercise. The primary purpose of the study was to examine circulating neutrophils and monocytes and their expression of CD64, CDllb, and CD18 after two bouts of eccentric arm exercise. A secondary purpose was to examine their relationship to CK activity and myoglobin. We hypothesized that if exercise-induced muscle damage is associated with acute inflammation then the adaptation to eccentric exercise [i.e., second bout (Sz)]

1996 the American

Physiological

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be accompanied by an attenuated of acute inflammation.

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response

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METHODS

Subjects. Ten male subjects (20-37 yr of age) participated in this investigation after giving verbal and written informed consent in accordance with federal and institutional guidelines. Subjects had not performed resistive exercises for at least 9 mo before the experimental trials. Subjects were instructed to maintain their normal diet and to abstain from physical activity, alcohol, caffeine, and from taking antiinflammatory or analgesic medicines 48 h before and during the experimental trials. ExperimentaL trials. Subjects performed two bouts of eccentric arm exercise using their nondominant arm. The exercise bouts were separated by 3 wk. The exercise consisted of performing 25 forced-lengthened (i.e., eccentric) contractions of the forearm flexors using a modified arm-curl bench. The arm-curl bench was equipped with a lever arm and a handgrip that were attached to a load cell (Omega International, Stamford, CT). The load cell was interfaced with a digital indicator (Omega International) and a microcomputer. Subjects began with the forearm flexed at -5O”, and the forearm was forcibly extended to 170” in 4 s. Subjects immediately returned their forearm to the starting position without resistance. Peak and average force (in N) were determined for each eccentric contraction. Blood sampling. Blood samples were obtained from an antecubital vein after 10 min of supine rest before exercise (Pre) and at 1.5, 6, 12, 24, 48, 72, and 96 h of recovery. The time of blood collection from the first exercise bout (Bl) was recorded, and corresponding samples for B2 were collected at the same time of day. Two tubes of blood were collected at each sampling period. The whole blood tube (KS EDTA) was stored at room temperature until hematological and flow cytometric analyses were performed. The second tube was allowed to clot before being centrifuged. The serum was stored in a polypropylene tube at -80°C until subsequent analysis. BZood anaZyses. Leukocytes were prepared for phenotyping by using a Coulter Immunoprep leukocyte preparation system (Q-Prep, Coulter Electronics; Hialeah, FL). An aliquot of the cell suspension from the Q-Prep was washed with phosphate-buffered saline and incubated (10 min at room temperature) with saturating amounts of monoclonal antibodies CD64, CDllb, and CD18 (Becton Dickinson, Mountain View, CA and Medarex, Princeton, NJ) within 4 h of collection. The monoclonal antibodies were conjugated with fluorescein isothiocynate (FITC) or phycoerythrin and were specific for the high-affinity receptor for IgG (FcyRI; CD64-FITC) and for the a-chain (CDllb-phycoerythrin) and the P-chain (CDl8FITC) of CR3 (Mac-l). The antibody-labeled cells (20,000 per sample) were analyzed using a Cytoron absolute flow cytometer (Ortho Diagnostics, Raritan, NJ). The expressions of CD64, CDllb, and CD18 on neutrophils and monocytes were calculated by using QuickCal beads (Flow Cytometry Standards, Research Triangle, NC) and linear regression to derive mean equivalent soluble fluorescence units by subtracting the isotypic control stain from the CD antibody-stained intensity (Fig. 1). Data analysis was performed using Winlist analysis program (Verity Software House, Topsham, ME). Complete and differential blood counts were determined with the use of an automated analyzer (Sysmex KIOOO, TOA Electronics, Kobe, Japan). Serum samples were analyzed for myoglobin using a radioimmunoassay procedure (Biomerica, Newport Beach, CA) and CK activity using an enzymatic method (Sigma Chemical, St. Louis, MO).

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Muscle soreness and tenderness. Muscle soreness and tenderness were assessed before exercise and at 1.5,6,12,24,48, 72, and 96 h from Bl and B2. Muscle soreness was assessed by having an investigator passively move the subject’s arm several times through its range of motion. Subjects were asked to rate their soreness using a 1 (normal) to 10 (very, very sore) scale. Muscle tenderness was assessed by palpating several sites on the upper arm and the forearm. Five sites 4 cm apart were used for the upper arm, beginning 5 cm lateral to the medial epicondyle of the humerus and extending upward to the acromion process. Seven sites 4 cm apart were used for the forearm, beginning 5 cm lateral of the medial epicondyle of the humerus and extending down the brachioradialis to midgirth of the wrist. Sites were marked with a permanent marker to ensure that the same sites were palpated during subsequent measurements. A metal probe 2 cm in diameter attached to a calibrated load cell (Omega International) allowed for the measurement of the force that elicited tenderness at each site. A force of 30 N represented no tenderness, and the mean of two measurements was subtracted from 30 to give a tenderness score for each site. A tenderness score was calculated for the upper arm, forearm, and the total arm by determining the average tenderness score from 5, 7, and 12 sites, respectively. Statistical anaZyses. A repeated-measures analysis of variance was used to analyze the main effects, i.e., conditions (Bl and B2), time (Pre, 1.5, 6, 12, 24, 48, 72, 96 h), and the interaction effect for all dependent measures except muscle soreness and peak and average force (Systat, Evanston, IL). The Huynh-Feldt Epsilon was applied to degrees of freedom to account for violation of the sphericity assumption. If the observed F ratio was statistically significant, a NewmanKeuls post hoc test was used to locate the differences. Muscle soreness was analyzed by a nonparametric Wilcoxon matchedpairs signed-rank test. Average and peak forces for each repetition, averaged over the 25 repetitions, were analyzed with the use of a repeated-measures analysis of variance. Trend analysis was also performed, and polynomial coefficients were calculated for the unequal interval of time (Systat). Ind’ 1~1‘d ua 1 scores for the dependent measures were multiplied by the coefficients, and a weighted score (2 of the transformed scores for Pre, 1.5,6, 12,24,48, 72, and 96 h) for Bl and B2 was calculated for each subject. The relationship of the weighted scores for neutrophils, monocytes, and CD64, CDllb, and CD18 expression to the weighted scores for CK activity and myoglobin was determined by calculating a Pearson product-moment correlation coefficient. Statistical significance was set at P < 0.05. Because of possible diurnal variations in dependent measures at 6 and 12 h of recovery, significant time effects at these time points are not reported. RESULTS

Average and peak forces. Average and peak forces between Bl and B2 for each repetition were similar. Average force and peak force for each repetition averaged over the 25 repetitions for Bl and B2 were not significantly different (average force Bl = 142.7 _t 27.2 N, B2 = 143.7 t 31.3 N; peak force Bl = 191.2 t 37.2 N, B2 = 183 t 42.1 N; means t SD). Muscle soreness and muscle tenderness. Muscle soreness (scale l-10) was higher (P < 0.01) for Bl at 24 h (4.3 ? 2.0>, 48 h (5.6 2 2.0>, 72 h (4.6 t 2.0>, and 96 h (2.7 t 1.6) compared with B2 (24 h: 2.7 _t 0.8; 48 h: 2.1 t 1.1; 72 h: 1.2 t 0.6; 96 h: 1.0 2 0.0; means t SD).

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LFt2-Monocytes[

Gated Region Gated Events Percent Gated Mean MESF

RI 1262 6.31 628

R2 1176 5.88 2791

R3 16169 80.83 1479

R4 954 4.83 1123

R5 1295 6.46 84994

R6 16057 80.23 11387

R4* 943 4.72 1006

R5* 1307 6.52 85107

R6* 15961 79.81 11474

Fig. 1. CD64 expression was quantified by gating on lymphocytes, monocytes, and neutrophils based on either light scatter properties [forward-angle light scatter (FSCl vs. log-side scatter (SSC)] or CD64 expression vs. log-side scatter. Use of Quick-Cal for Winlist and quantum fluorescein isothiocyanate (FITC) beads allows for conversion of fluorescence intensity into FITC mean equivalent soluble fluorescence (MESF) units. Final leukocyte FITC MESF values are derived by subtracting nonspecific binding isotypic control antibody (top panels: Rl, R2, and R3) from CD64 antibody binding. Light scatter gating (middle panels: R4, R5, and R6) and CD64/side scatter gating (bottom panels: R4*, R5”, and R6”) techniques give similar FITC MESF values. PMNs, polymorphonuclear leukocytes.

Total muscle tenderness followed a similar pattern as muscle soreness, where differences (P = 0.02) between the exercise bouts were observed at 24 h (Bl = 5.1 t 4.0, B2 = 2.4 + 2.61, 48 h (Bl = 8.0 + 5.3, B2 = 2.4 I? 2.91, 72 h (Bl = 6.7 t: 6.3, B2 = 0.9 t 1.61, and 96 h (Bl = 4.3 t 5.0, B2 = 0.7 t 1.3; means + SD). Muscle tenderness for Bl was principally experienced throughout the upper arm with minimal tenderness in the forearm (data not reported). CK activity and myoglobin. Differences (P < 0.01) between Bl and B2 in myoglobin and CK activity became apparent at 12 and 24 h, respectively (Fig. 2). For Bl, CK activity at 72 h was higher compared with 48 and 96 h (Fig. 2A), whereas myoglobin peaked at 72 h of recovery from Bl, which was not different compared with results at 48 and 96 h (Fig. 2B). No significant correlations between changes in CK activity

and myoglobin with changes in circulating neutrophils, monocytes, and their expression of CD64, CDllb, and CD18 were observed. Neutrophils and monocytes. The number of circulating neutrophils was higher (P = 0.05) for Bl at 6, 12, and 96 h compared with B2 (Fig. 3). No significant main effects or interactions were observed for the number of circulating monocytes; however, they tended to be higher for Bl (P = 0.056) (Table 1). CDllb,

CD18, and CD64 expression

on neutrophils.

CDllb expression on neutrophils was 2.7-fold higher (P = 0.05) for Bl at 72 h compared with B2 (Fig. 4A). For Bl, CDllb expression was elevated above Pre levels at 24, 72, and 96 h, whereas for B2, Cllb expression was higher at 24 and 96 h compared with Pre levels. No significant interaction or main effects were observed for CD18 expression on neutrophils (Fig.

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Fig. 2. Creatine kinase activity (A) and myoglobin means 2 SE. Bl, bout 1; B2, bout 2. #Significance 0.05) between Bl and B2 at specified time point.

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(B); (P < 350 -

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4B). CD64 expression on neutrophils was 1.4- and 1.9-fold higher (P = 0.04) for Bl at 72 and 96 h, respectively, compared with B2 (Fig. 4C). For Bl, CD64 expression on neutrophils was increased above Pre values at 48,72, and 96 h. CD11 b, CDl8, and CD64 expression on monocytes. No significant differences between the bouts for CDllb expression on monocytes were observed; however, CDllb was elevated (P < 0.001) above Pre levels at 24 h after Bl and B2 (Fig. 5). At 72 and 96 h, CD18 and CD64 expression on monocytes was 1.3-fold higher (P < 0.05) for Bl compared with B2 and was elevated for Bl at these time points compared with Pre. DISCUSSION

The major finding in the present study was that an unaccustomed bout of eccentric arm exercise (Bl) relative to the same bout performed 3 wk later (B2) was associated with higher circulating neutrophils and increased plasma membrane expression of CD64, CDllb, and CD18 on neutrophils and monocytes. The

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(h)

observed changes, however, were not associated with changes in CK activity or myoglobin. The methodology in the present study was based on the assumption that Bl would result in ultrastructural disruption, whereas the degree of ultrastructural disruption would be substantially less after B2 (15, 16). Changes in dependent measures after Bl were hypothesized to represent the combined effect of eccentric exercise and muscle damage, whereas changes after B2 were presumed to represent the effect of eccentric exercise. Therefore, differences between the exercise bouts were hypothesized to be predominantly the result of exercise-induced muscle damage. Although no direct evidence of muscle damage can be provided to support our hypothesis, the significant attenuation in DOMS, muscle tenderness, CK activity (Fig. 2A), and myoglobin (Fig. 2B) after B2 provides indirect evidence that differences in muscle damage existed between the eccentric exercise bouts (11, 27). The magnitude of the difference in CK activity between Bl and B2 (Fig. 2A), however, cannot be inferred to

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Fig. 3. Circulating neutrophils (X 10%); mean + SE. #Significance (P < 0.05) bet ween Bl and B2 at specified time point; ‘Gsignificance compared with preexercise.

indicate a similar magnitude of change in the ultrastructure, since CK does not correlate with histological evidence of skeletal muscle damage (13,31). Neutrophils. In the course of acute inflammation, neutrophils are mobilized to the site of injury or infection where they adhere to endothelium and enter the injured or infected tissues (32). The greater mobilization of neutrophils for Bl (Fig. 3), therefore, indicates a greater acute inflammatory response relative to B2. The neutrophilia after endurance exercise has been attributed to an increased blood flow and/or epinephrine resulting in the demargination of neutrophils from small blood vessels, particularly in the lungs (14,21). It is unlikely that differences in blood flow and epinephTable 1. Circulating

monocytes

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rine levels could account for the difference in circulating neutrophils between Bl and B2, since the amount of work performed was similar and differences in neutrophils became apparent after blood flow and epinephrine typically return to preexercise levels. It is well established that chemoattractants (e.g., complement fragments and leukotrienes) and activated endothelium are potent stimulators of neutrophil traffic (10). Canno n et al. (4) reported a positive correlation between C3a des Arg and neutrophils and suggested that complement activation contributes to the mobilization of neutrophils after damaging exercise. Despite differences in methodology, the results in the present investigation are consistent with the findings of previous investigators who have reported that an unaccustomed bout of eccentric exercise relative to concentric exercise is associated with a sustained mobilization of neutrophils (23, 28). We have recently reported that downhill, relative to level, running (70% maximal O2 consumption, 60 min) resulted in a higher number of circulating neutrophils at 1.5 and 12 h of recovery (23). In the present investigation, however, neutrophils were not significantly different between Bl and B2 at 1.5 h of recovery. The difference between the studies in neutrophils at 1.5 h of recovery may be explained by inherent differences between concentric and eccentric contractions (i.e., tension per active motor unit and possibly the neuroendocrine response) and the mode and duration of the exercise. At 12 h, the difference in neutrophils between Bl and B2 was similar in magnitude (12%) to the difference between level running and downhill running (18%) in our previous report (23). CDllb/CDl6. CDllb/CDl8 is a member of a family of leukocyte adhesion molecules (CDll/CDl8), which also includes leukocyte function-associated antigen-l (LFA-1; CDlla/CD18) and p150,95 (CDllcED18) (10, 34). CDllb/CDl8, consisting of a 160-kDa a-chain (CDllb) noncovalently linked to a 95-kDa p-chain (CD18), mediates adherence and phagocytosis of surfaces opsonized with iC3b and adherence to endothelial cells (10, 34). The importance of CDlUCD18 in host defense is underscored in individuals with a genetic defect in CD18. Leukocytes from these individuals are defective in chemotaxis, aggregation, adherence, and phagocytosis and, as a result, these subjects suffer from recurrent, often life-threatening bacterial infection (10, 34). The clinical consequence of the molecular defect in CD18 illustrates the biological importance of CDll/ CD18 in mediating normal inflammatory function. The number of CDllb/CD18 receptors on unstimulated phagocytes is relatively low; however, during the

(XIOg/l) Recovery,

Bl B2 Values

Pre

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0.58 + 0.04 0.50 + 0.05

0.57 + 0.05 0.50 + 0.05

are means

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2 SE; 12 = 10 measurements.

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0.60 + 0.05 0.55 2 0.04

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0.57 + 0.04 0.53 + 0.04

Pre, preexercise;

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67oooO -

47oMxl-

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Fig. 4. CDllb (A), CD18 (B), and CD64 (C) expression on neutrophils; means 2 SE. #Significance (P < 0.05) between Bl and B2 at specified time point; ‘i:significance compared with preexercise.

& g

iiO(K)

4WO-

22%

2ooo-

1C

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lsw

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I PKE

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I

I

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initial stage of phagocytosis, CDllb is translocated to the plasma membrane from an intracellular pool (specific granules) (12, 19, 29). Degranulation from the specific granules has been reported to increase the plasma membrane expression of CDllb by 3- to lo-fold (19, 29). Alth ough CD18 is also contained within the specific granules, the mechanism for why CDllb and CD18 are not expressed simultaneously on the plasma membrane after degranulation is not well understood (29). The higher expression of CDllb and CD18 on neutrophils and monocytes, respectively, for Bl relative to B2, suggests greater degranulation from specific granules (Figs. 4 and 5). Fielding et al. (13) and Round et al. (24) have reported infiltration of neutrophils and macrophages, respectively, into damaged muscle after an unaccustomed bout of eccentric exercise. Fielding et al.

(h)

(13) also reported a significant positive correlation between neutrophil infiltration and Z-line disruption. Whether the greater induction of CDllb and CD18 for Bl resulted in a greater accumulation of neutrophils and macrophages in damaged muscle at 72 and 96 h of recovery cannot be determined from our data. Several investigators, however, have demonstrated that monoclonal antibodies directed at CDllb and/or CD18 reduce the infiltration of phagocytes and the extent of muscle damage in reperfusion-injury models (8,26,33,35). CD64. Neutrophils, monocytes, and natural killer cells express several receptors for the Fc region of IgG molecules (FcyR), FcyRI (CD64), FcyRII (CD32), and FcyRIII (CD16) (30). CD64 exhibits high-affinity binding to human IgGl and IgG3, and its expression is regulated by interferon-y (IFN-y), an inflammatory

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Fig. 5. CDllb (A), CD18 (B), and CD64 (C) expression on monocytes; means + SE. #Significance (P < 0.05) between Bl and B2 at specified time point; *significance compared with preexercise.

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I

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cytokine secreted from activated lymphocytes (1, 30). Neutrophils possess fewer than 1,000 CD64 sites per cell, and after incubation with IFN-y the number of sites per cell has been reported to increase 5- to ZO-fold (17,22). A 2- to lo-fold induction of CD64 on monocytes has also been reported after IFN-y incubation (17). The IFN-y-induced expression of CD64 is slow and dependent on both RNA and protein synthesis with induction occurring within 6 h and maximal expression occurring 24-48 h after IFN-y treatment (9,22). CD64 is capable of generating a transmembrane phagocytic signal that initiates superoxide generation, antibody-dependent cellular cytotoxicity, and phagocytosis (1,25,30). Akerley et al. (1) has recently reported a strong relationship between oxidative burst and CD64 expression. The 1.5- to 2-fold increase in CD64 expression for Bl and B2 indicates IFN-y-induced activation of neutrophils and monocytes (Figs. 4 and 5). The 1.3-fold greater induction of CD64 on neutrophils and monocytes at 72 and 96 h of Bl relative to B2 suggests that

the adaptation to eccentric exercise was associated with a lower state of neutrophil and monocyte activation. Whether the greater neutrophil and monocyte activation after Bl contributed to muscle damage and/or the removal of cellular debris is unkown. FcyRmediated phagocytosis and FcyR-triggered generation of reactive oxygen intermediates, however, has been suggested to contribute to organ damage in diseases such as vasculitis, rheumatoid arthritis, and systemic lupus erythematosus (l&30). Correlational analyses. Previous investigators have reported significant positive correlations between the number of circulating neutrophils (4, 23), superoxide release from isolated neutrophils (6), complement fragment C3a des Arg (4), and muscle interleukin-1P (13) with plasma CK activity after eccentric exercise. Cannon et al. (4) have suggested that neutrophil activation contributes to the increased myocellular membrane permeability and, thus, to the release of CK into the blood after eccentric exercise. The lack of a significant

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correlation between CK activity and neutrophil number in the present investigation is in contrast to our previous investigation (23) and that of Cannon et al. (4). In addit ion, the lack of a significant correlation between CD64 expression on neutrophils with CK activity is not consistent with preliminary data reported from our laboratory (23). Conflicting results from our laboratory might be related to mode of eccentric exercise (downhill running vs. eccentric arm exercise), and hence the magnitude and the associated variability in CK activity. Conchsions. The greater induction of CD64, CDllb, and CD18 on neutrophils and monocytes at specific time points of Bl relative to B2 indicates that the adaptation to eccentric arm exercise is associated with a lower state of neutrophil and monocyte activation. The lack of significant correlations between changes in circulating neutrophils and monocytes and their expression of CD64, CDllb, and CD18 with CK activity and myoglobin suggests that neutrophils and monocytes do not contribute to CK or myoglobin efflux after eccentric arm exercise. Further investigation is needed to determine the biological significance of the reported changes in CD64, CDllb, and CD18. We gratefully acknowledge the Analytical Cytometry Laboratory at Harris Methodist Hospital for their technical assistance. A special thanks to Dr. David Cross for his assistance with the statistical analyses. This research project was supported by the Research Fund at Texas Christian University. Address for reprint requests: F. X. Pizza, Texas Christian Univ., Dept. Physical Education, Box 32901, Fort Worth, TX 76129. Received

12 December

1994; accepted

in final

form

1 August

1995.

REFERENCES 1. Akerley, W. L. III, P. M. Guyre, and B. H. Davis. Neutrophil activation through high-affinity Fey receptor using a monomeric antibody with unique properties. Blood 77: 607-6151991. 2. Armstrong, R. B. Initial events in exercise-induced muscular injury. Med. Sci. Sports Exert. 22: 429-435, 1990. 3. Bymes, W. C., P. M. Clarkson, J. S. White, S. S. Hsieh, P. N. Frykman, and R. J. Maughan. Delayed onset muscle soreness following repeated bouts of downhill running. J. AppZ. Physiol. 59: 710-715,1985. 4. Cannon, J. G., M. A. Fiatarone, R. A. Fielding, and W. J. Evans. Aging and stress-induced changes in complement activation and neutrophil mobilization. J. Appl. Physiol. 76: 26162620,1994. 5. Cannon, J. G., S. N. Meydani, R. A. Fielding, M. A. Fiatarone, M. Meydani, M. Farhangmehr, S. F. Orencole, J. B. Blumberg, and W. J. Evans. Acute phase response in exercise. II. Association between vitamin E, cytokines, and muscle proteolysis. Am. J. Physiol. 260 (Regulatory Integrative Comp. Physiol. 29): R1235-R1240,1991. 6. Cannon, J. G., S. F. Orencole, R. A. Fielding, M. Meydani, S. N. Meydani, M. A. Fiatarone, J. B. Blumberg, and W. J. Evans. Acute phase response in exercise: interaction of age and vitamin E on neutrophils and muscle enzyme release. Am. J. Physiol. 259 (Regulator-y Integrative Comp. PhysioZ. 28): Rl214R1219,1990. 7. Clarkson, P. M., K. Nosaka, and B. Braun. Muscle function after exercise-induced muscle damage and rapid adaptation. Med. Sci. Sports Exert. 24: 512-520, 1992. 8. Carden, D. L., J. K. Smith, and R. J. Korthuis. Neutrophilmediated microvascular dysfunction in postischemic canine skeletal muscle. Circ. Res. 66: 1436-1444, 1990.

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9. Cassetella, M. A., R. Cappelli, V. D. Bianca, M. Grzeskowiak, S. Dusi, and G. Berton. Interferon-gamma activates human neutrophil oxygen metabolism and exocytosis. ImmunoZogy 63: 499-506,1988. 10. Cronstein, B. N., and G. Weissmann. The adhesion molecules of inflammation. Arthritis Rheum. 36: 147-157,1993. 11. Ebbeling, C. B., and P. M. Clarkson. Exercise-induced muscle damage and adaptation. Sports Med. 7: 207-234,1989. 12. English, D., and K Graves. Simultaneous mobilization of Mac-l (CDllb/CD18) and formyl peptide chemoattractant receptors in human neutrophils. Blood 80: 776-787, 1992. 13. Fielding, R. A., T. J. Manfredi, W. Ding, M. A. Fiatarone, W. J. Evans, and J. G. Cannon. Acute phase response in exercise. III. Neutrophil and IL-2p accumulation in skeletal muscle. Am. J. Physiol. 265 (ReguZatory Integrative Comp. Physiol. 34): R166-R172, 1993. 14. Foster, N. K., J. B. Martyn, R. E. Rangno, J. C. Hogg, and R. L. Pardy. Leukocytosis of exercise: role of cardiac output and catecholamines. J. AppZ. Physiol. 61: 2218-2223,1986. 15. Friden, J. Changes in human skeletal muscle induced by long-term eccentric exercise. CeZZ Tissue Res. 236: 365-372, 1984. 16. Friden, J., J. Seger, M. Sjiistriim, and B. Ekblom. Adaptative response in human skeletal muscle subjected to prolonged eccentric training. Int. J. Sports Med. 4: 177-183, 1983. 17. Guyre, P. M., P. M. Morganelli, and R. Miller. Recombinant immune interferon increases immunoglobulin Fc receptors on cultured human mononuclear phagocytes. J. CZin. Invest. 72: 393-397,1983. 18. Haynes, B. F. Vasculitis: pathogenic mechanisms of vessel damage. In: Inflammation: Basic Principles and CZinicaZ Correlates, edited by J. I. Gallin, I. M. Goldstein, and R. Snyderman. New York: Raven, 1992, p. 921-941. 19. O’Shea, J. J., E. J. Brown, B. E. Seligmann, J. A. Metcalf, M. M. Frank, J. I. Gallin. Evidence for distinct intracellular pools of receptors for C3b and C3bi in human neutrophils. J. ImmunoZ. 134: 2580-2587,1985. 20. Monboisse, J. C., R. Garnotel, A. Randoux, J. Dufer, and J. P. Borel. Adhesion of human neutrophils to and activation by type-1 collagen a p2 integrin. J. Leukocyte BioZ. 50: 373-380, 1991. 21. Muir, A. L., M. Cruz, B. A. Martin, H. Thommasen, A. Belzberg, and J. C. Hogg. Leukocyte kinetics in the human lung: role of exercise and catecholamines. J. AppZ. Physiol. 57: 711-719,1984. 22. Perussia, B., M. Kobayashi, M. E. Rossi, I. Anegon, and G. Trinchieri. Immune interferon enhances functional properties of human granulocytes: role of Fc receptors and effect of lymphotoxin, tumor necrosis factor, and granulocyte-macrophage colonystimulating factor. J. Immunol. 138: 765-774, 1987. 23. Pizza, F. X., J. B. Mitchell, B. H. Davis, R. D. Starling, R. W. Holtz, and N. Bigelow. Exercise-induced muscle damage: effect on circulating leukocyte and lymphocyte subsets. Med. Sci. Sports Exert. 27: 363-370, 1995. 24. Round, J. M., D. A. Jones, and G. Cambridge. Cellular infiltrates in human skeletal muscle: exercise induced damage as a model for inflammatory muscle disease. J. Neural. Sci. 82: l-11,1987. 25. Shen, L., P. M. Guyre, and M. W. Fanger. Polymorphonuclear leukocyte function triggered through the high affinity Fc receptor for monomeric IgGl. J. ImmunoZ. 139: 534-538,1987. 26. Simpson, P. J., R. F. Todd III, J. C. Fantone, J. K. Mickelson, J. D. Griffin, and B. R. Lucchesi. Reduction of experimental canine myocardial reperfusion injury by a monoclonal antibody (anti-Mol, anti-CDllb) that inhibits leukocyte adhesion. J. CZin. Invest. 81: 624-629, 1988. 27. Smith, L. L. Acute inflammation: the underlying mechanism in delayed onset muscle soreness? Med. Sci. Sports Exert. 23: 542-551,199l. 28. Smith, L. L., M. McCammon, S. Smith, M. Chamness, R. G. Israel, and K. F. O’Brien. White blood cell response to uphill walking and downhill jogging at similar metabolic loads. Eur. J. AppZ. Physiol. Occup. Physiol. 58: 833-837, 1989. 29. Todd, R. F. III, M. A. Arnaout, R. E. Rosin, C. A. Crowley, W. A. Peters, and B. M. Babior. Subcellular localization of the

INFLAMMATORY

RESPONSE

large subunit of Mol (Molol; formerly gp llO), a surface glycoprotein associated with neutrophil adhesion. J. CZin. Invest. 74: 1280-1290,1984. 30. Unkeless, J. C., P. Boros, and M. Fein. Structure, signaling and function of FcyR. In: Inflammation: Basic Principles and CZinicaZ Correlates, edited by J. I. Gallin, I. M. Goldstein, and R. Snyderman. New York: Raven, 1992, p. 921-941. 3 1. Van Der Meulen, J. H., H. Kuipers, and J. Drukker. Relationship between exercise-induced muscle damage and enzyme release in rats. J. AppZ. Physiol. 71: 999-1004, 1991. 32. Weiss, S. J. Tissue destruction by neutrophils. N. EngZ. J. Med. 320: 365-376,1989.

TO MUSCLE

DAMAGE

55

33. Weselcouch, E. O., R. I. Grove, C. D. Demusz, and A. J. Baird. Effect of in vivo inhibition of neutrophil adherence on skeletal muscle function during ischemia in ferrets. Am. J. Physiol. 261 (Heart Circ. PhysioZ. 30): H1178-H1183, 1991. 34. Wright, S. D. Receptors for complement and the biology of phagocytosis. In: Inflammation: Basic Principles and Clinical Correlates, edited by J. I. Gallin, I. M. Goldstein, and R. Snyderman. New York: Raven, 1992, p. 477-495. 35. Yokota, J. J., P. Minei, G.A. Fantini, and G. T. Shires. Role of leukocytes in reperfusion injury of skeletal muscle after partial ischemia. Am. J. PhysioZ. 257 (Heart Circ. Physiol. 30): Hl068H1075,1989.