Effect of Systemic Inflammation on Inspiratory and Limb ... - ATS Journals

Effect of Systemic Inflammation on Inspiratory and Limb Muscle Strength and Bulk in Cystic Fibrosis Vale´rie Dufresne1, Christiane Knoop1, Alain Van Muylem1, Anne Malfroot2, Michel Lamotte3, Christian Opdekamp3, Gae¨l Deboeck3, Marie Cassart4, Bernard Stallenberg4, Georges Casimir5, Jean Duchateau6, and Marc Estenne1 1 5

Chest Service, 3Department of Physiotherapy, and 4Department of Radiology, Erasme University Hospital; 2Cystic Fibrosis Clinic, UZ-Brussels; Department of Pneumology, HUDERF; and 6Laboratory of Immunology, Brugmann University Hospital, Brussels, Belgium

Rationale: Diaphragm thickness is increased in cystic fibrosis (CF), but it shows a marked variability between patients. The variable response of the diaphragm to loading may reflect the combined and opposite effects of training by the respiratory disease and systemic inflammation. Objectives: To assess the impact of systemic inflammation on diaphragm and limb muscle strength and bulk in adult patients with CF. Methods: In 38 stable patients with CF and 20 matched control subjects, we measured fat-free mass (FFM), inspiratory muscle strength, diaphragm thickness, quadriceps and biceps strength and cross-sectional area, and circulating levels of leukocytes, C-reactive protein, IL-6, IL-8, IL-17, tumor necrosis factor-a, tumor necrosis factor-a soluble receptors, and immunoglobulin G. Measurements and Main Results: Patients had increases in several inflammatory markers that correlated with the severity of lung disease and nutritional depletion. Compared with control subjects, patients with CF had increased diaphragm thickness and inspiratory muscle strength and showed a trend toward a reduction in limb muscle strength and bulk. Multiple regression analyses identified FFM and airway resistance as independent predictors of diaphragm thickness, but systemic inflammation had no (or only a minor) predictive effect on FFM, inspiratory muscle strength, diaphragm thickness, and limb muscle strength and bulk. Conclusions: In patients with CF, the intensity of systemic inflammation does not account significantly for the variance of FFM and diaphragm or limb muscle strength and bulk. Training of the diaphragm in CF occurs despite the presence of systemic inflammation. Keywords: skeletal muscles; airway obstruction; acute phase reaction; lung infection

Skeletal muscle wasting develops in a variety of chronic pulmonary and nonpulmonary disorders, including chronic obstructive pulmonary disease (COPD), congestive heart failure, uremia, cancer, and acquired immunodeficiency syndrome (1, 2). The observation that cachexia develops in such a variety of chronic diseases suggests a common underlying mechanism. Although nutritional deficiency, physical inactivity, and decreased plasma levels of anabolic hormones may contribute to muscle wasting, there is growing evidence that systemic in-

(Received in original form February 7, 2008; accepted in final form April 1, 2009) Supported in part by a grant from the Fonds Erasme and from the Fonds pour la Chirurgie Cardiaque. Correspondence and requests for reprints should be addressed to Marc Estenne, M.D., Chest Service, Erasme University Hospital 808, Route de Lennik B-1070, Brussels, Belgium. E-mail: [email protected] This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org Am J Respir Crit Care Med Vol 180. pp 153–158, 2009 Originally Published in Press as DOI: 10.1164/rccm.200802-232OC on April 2, 2009 Internet address: www.atsjournals.org

AT A GLANCE COMMENTARY Scientific Knowledge on the Subject

Several studies suggest that systemic inflammation may contribute to muscle wasting in respiratory and nonrespiratory chronic diseases. However, studies of chronic obstructive pulmonary disease have not established a causal link between systemic inflammation and skeletal muscle dysfunction. What This Study Adds to the Field

The potential impact of several inflammatory markers on fat-free mass and on the strength and bulk of the diaphragm and two limb muscles in stable patients with cystic fibrosis is very limited. Continued inflammation does not impede the response of the diaphragm to loading.

flammation also plays a role. This evidence comes from measurements of circulating cytokines in humans, and from various in vivo and ex vivo experimental models (3–8). For example, in well-functioning elderly subjects with or without COPD, higher circulating levels of tumor necrosis factor (TNF)a and IL-6 are associated with lower quadriceps strength (9). Collectively, these studies suggest that systemic inflammation is able to induce muscle wasting by the combined effects of inequity between muscle protein synthesis and degradation, and impaired muscle regeneration. In cystic fibrosis (CF), systemic inflammation is likely more prolonged and pronounced than in many other chronic diseases. This inflammation results in part from ‘‘spillover’’ of proinflammatory cytokines produced in the chronically infected lungs to the central circulation (5). In a previous study, we assessed quadriceps and diaphragm muscle bulk in 18 adult patients with CF (10). We found that patients with CF had smaller quadriceps mass and greater diaphragm mass than healthy control subjects, but diaphragm muscularity varied widely between patients. By multiple regression analysis, we found that fat-free mass (FFM) and airway resistance explained only approximately 50% of this variability. We hypothesized that the unexplained variance might be accounted for by the combined and opposite effects that systemic inflammation and the chronic increase in load resulting from airflow obstruction may have on the diaphragm; the former is expected to produce muscle wasting, whereas the latter is expected to have a training effect and increase muscle bulk. The primary aim of this study was to test this hypothesis, and the secondary aim was to assess the impact of systemic inflammation on peripheral muscles. We therefore studied 38 stable patients with CF and a wide range of disease severity

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and 24 age-, sex-, and height-matched healthy control subjects. We measured FFM, the strength of inspiratory and limb muscles, the thickness of the diaphragm and the cross-sectional area of the biceps and quadriceps, and circulating levels of several inflammatory markers. We found that systemic inflammation had no independent predictive effect on FFM and on inspiratory or limb muscle strength and bulk. Results of these studies have been reported in part in the form of an abstract (11).

METHODS Subjects We studied 38 adult patients with CF and 24 age-, sex-, and heightmatched healthy control subjects. Patients were clinically stable and were not treated by oral corticosteroids, antiinflammatory agents, or azithromycin, but 28 received inhaled corticosteroids. Our previous study showed that FFM and airway resistance explained 51% of the variance in diaphragm thickness (Tdi) (10). For a third variable (e.g, systemic inflammation) to reduce the residual variance by one fourth (12%) with a power of 0.85 (and a type I error of 0.05), Faul and colleagues (12) indicate that a sample size of 38 patients or more is needed. All subjects gave written informed consent to the investigation, which was approved by the Human Studies Committee of the Erasme University Hospital (Brussels, Belgium).

Pulmonary Function Spirometry and static lung volumes were measured using a computerized spirometer and a body plethysmograph (13). Resting PaO2 and PaCO2 were measured while breathing room air.

Inspiratory Muscle Strength Mouth pressure (PIMAX) was measured in seated posture (14) using a Hyp’Air equipment (Medisoft, Sorinnes, Belgium). Measurements were performed at functional residual capacity in the patients and the control subjects; additional measurements were obtained in the latter at approximately 50 and 25% of inspiratory capacity.

Diaphragm Thickness Tdi was measured by ultrasound using a 6- to 15-MHz linear probe (Aloka, Wallingford, CT) (10). Measurements were made in seated posture at functional residual capacity in the patients and the control subjects; additional measurements were obtained in the latter at approximately 50 and 25% of inspiratory capacity.

(Luminex; R&D Systems, Abingdon, UK), and TNF-a soluble receptors (sTNF-RI and RII) were determined by ELISA (Biosource, Nivelles, Belgium). All data were log transformed.

Data Analysis Data are expressed as means 6 SD unless otherwise specified. In each patient, values of PIMAX and Tdi obtained at FRC were compared with those obtained in a matched control subject at a similar absolute volume. Statistical analyses were made using paired and unpaired t tests, single linear regression analysis, and covariance analysis when appropriate. Multiple stepwise forward regression analyses were performed to investigate the contribution of differences in gender, nutritional status, airflow obstruction, and systemic inflammation to the interpatient variability in FFM and inspiratory and limb muscle strength and bulk. A level of P , 0.05 was considered statistically significant. Additional details on patients and methods are given in the online supplement.

RESULTS Tables 1 and E1 in the online supplement summarize the characteristics of the patients with CF and the control subjects. They were well matched for age, height, and sex. Weight and BMI were significantly reduced in the patients with CF; FFM and FM showed a similar trend, but differences with the control subjects did not reach statistical significance. On average, the degree of airflow obstruction was moderate, but there was a wide range of disease severity (FEV1 ranged between 15 and 113% of predicted). The level of daily physical activity and peak oxygen uptake were also reduced in the patients with CF. Table 2 shows that patients with CF had increased circulating levels of leukocytes; neutrophils; CRP; IL-6, -8, and -17; and sTNF-RI and RII. The degree of systemic inflammation varied considerably between patients; for example, CRP ranged between , 0.07 and 4.0 mg/dl, IL-6 between 0.1 and 13.9 pg/ml, and TNF-a between 0.1 and 4.82 pg/ml. Although inhaled corticosteroids may reduce the level of systemic inflammation in COPD (16), inflammatory markers in this study did not differ significantly between treated versus untreated patients. Values of leukocytes, neutrophils, CRP, IL-6, and IL-8 were increased in male and female patients; in contrast, values of IL-17, TNF-a, and sTNF-RI and RII were increased only in female (Table E4). TABLE 1. DETAILS OF STUDY POPULATION

Biceps and Quadriceps Strength The maximum isometric strength of the biceps and quadriceps of the dominant arm and leg was measured using a hand-held dynamometer (Compufet; Biometrics, The Netherlands).

Biceps and Quadriceps Cross-Sectional Area The cross-sectional area of the biceps and quadriceps of the dominant arm and leg was measured using computerized tomography (10).

Nutritional Status Nutritional status was assessed by computing body mass index (BMI) and by measuring FFM and fat mass (FM) by electrical bioimpedance (Xitron Technologies Corporation, San Diego, CA).

Physical Activity We measured the level of leisure and occupational physical activity using the Modifiable Activity Questionnaire (15) and peak oxygen uptake by cycle ergometry.

Markers of Systemic Inflammation High-sensitivity C-reactive protein (CRP) and immunoglobulin G levels were measured according to standard techniques. Serum levels of IL-6, -8, and -17 and TNF-a were measured by multiplex assays

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Age, years Sex, males/females Height, m Weight, kg BMI, kg/m2 FFM, kg FM, kg FFMI, kg/m2 TLC, % pred FRC, % pred FEV1, % pred FEV1/VC, % RV, % pred sRaw, % pred PaO2, mm Hg PaCO2, mm Hg Peak VO2, % pred MAQ, hours/week

Patients with CF (n 5 38)

Control Subjects (n 5 24)

P Value

29.1 6 6.6 20/18 1.68 6 0.08 56.5 6 11.2 19.8 6 2.8 40.0 6 11.0 16.4 6 6.0 13.9 6 2.9 105 6 14 142 6 29 49 6 25 60 6 14 215 6 65 374 6 298 81 6 11 40 6 8 75 6 15 8.5 6 11.8

30.2 6 6.1 12/12 1.69 6 0.08 62.5 6 11.2 21.6 6 2.6 44.6 6 11.2 17.9 6 5.9 15.3 6 2.9 102 6 8 109 6 16 100 6 9 83 6 5 105 6 21 108 6 57 — — — 21.4 6 19.1

NS NS NS ,0.05 0.01 NS NS NS NS ,0.001 ,0.001 ,0.001 ,0.001 ,0.001 — — — ,0.005

Definition of abbreviations: BMI 5 body mass index; CF 5 cystic fibrosis; FFM 5 fat-free mass; FFMI 5 fat-free mass index; FM 5 fat mass; MAQ 5 modifiable activity questionnaire (13); peak VO2 5 peak oxygen uptake (obtained in 31 patients); RV 5 residual volume; sRaw 5 specific airway resistance.

Dufresne, Knoop, Van Muylem, et al.: Inflammation and Skeletal Muscles in CF TABLE 2. LEUKOCYTOSIS AND SERUM INFLAMMATORY MARKERS IN STUDY POPULATION

Leukocytes, per mm3 PMN, per mm3 ESR, mm/hour CRP, mg/dl IL-6, pg/ml IL-8, pg/ml IL-17, pg/ml TNF-a, pg/ml sTNF-aRI, ng/ml sTNF-aRII, ng/ml

Patients with CF

Control Subjects

P Value

9,600 6 3,600* 6,400 6 3,200 21 6 20 0.8 (0.2–2.6) 2.3 (0.7–8.0) 1.7 (0.2–13.6) 0.2 (0.02–2.0) 2.4 (1.1–5.0) 1.9 (1.4–2.6) 5.7 (4.0–8.1)

6,200 6 2,400 3,600 6 2,200 665 0.1 (0.06–0.2) 0.04 (0.004–0.3) 0.08 (0.005–1.1) 0.05 (0.06–0.5) 1.7 (0.5–5.3) 1.5 (1.2–1.8) 4.4 (3.2–5.9)

,0.001 ,0.001 0.001 ,0.001 ,0.001 ,0.01 0.019 0.1 ,0.001 0.005

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higher values of Tdi (P , 0.001). Figure 1 also shows that the correlation between diaphragm thickness and FFM was less tight in patients with CF than in control subjects due to a greater between-subject variability in Tdi. Single regression analyses were performed in the patients between inflammatory markers and muscle strength and bulk. Some negative correlations were found for limb muscle (e.g., between biceps strength or quadriceps cross-sectional area and erythrocyte sedimentation rate or IL-17), but r values were low

Definition of abbreviations: CF 5 cystic fibrosis; CRP 5 C-reactive protein; ESR 5 erythrocyte sedimentation rate; PMN 5 polyneutrophils; sTNF-RI and sTNF-RII 5 TNF-a soluble receptors; TNF-a 5 tumor necrosis factor-a. * Data are presented as mean 6 SD for number of leukocytes and polyneutrophils and for erythrocyte sedimentation rate; for the other variables, data are presented as geometric mean (geometric interval).

Body mass index and FEV1 were positively correlated with each other (r 5 0.6; P 5 0.002); on the other hand, FEV1 was negatively correlated with logIL-17 (r 5 20.43; P , 0.05) and logCRP (r 5 20.48; P 5 0.024), and BMI was negatively correlated with logIL17 (r 5 20.5; P 5 0.02) and logIL-8 (r 5 20.32; P , 0.05). Levels of physical activity were correlated with quadriceps cross-sectional area (r 5 0.49; P , 0.01) but not with quadriceps strength (r 5 0.27; P 5 NS). Table 3 summarizes values of inspiratory muscle strength, diaphragm thickness, and limb muscle strength and bulk in the two groups. Biceps and quadriceps strength and cross-sectional area tended to be reduced in the patients with CF, but differences with the control subjects did not reach significance. Figures 1A and 1B illustrate that biceps and quadriceps crosssectional area was tightly correlated with FFM in the two groups and that for any given FFM patient with CF and control subjects had similar muscle bulk. PIMAX and Tdi were greater in patients with CF than in control subjects. The difference in Tdi was observed when values were compared at a similar absolute lung volume and at FRC irrespective of the patient’s gender. Differences were more significant for comparisons made at FRC and when Tdi was normalized by FFM (Table E5). Otherwise stated, for any given FFM, patients with CF had thicker diaphragms than control subjects (Figure 1). By covariance analysis, the regression line in the patients with CF was displaced toward TABLE 3. STRENGTH AND BULK OF RESPIRATORY AND LIMB MUSCLES IN STUDY POPULATION Patients with CF cm2

BicCSA, BicCSA/FFM, cm2/kg BicPT, N QuadCSA, cm2 QuadCSA/FFM, cm2/kg QuadPT, N Tdi,* mm Tdi/FFM, mm/kg PIMAX,* cm H2O

8.4 0.21 194 51.8 1.30 356 1.5 0.039 100

6 6 6 6 6 6 6 6 6

3.3 0.04 72 15.2 0.14 127 0.4 0.011 29

Control Subjects 9.4 0.21 226 59.3 1.35 402 1.4 0.031 83

6 6 6 6 6 6 6 6 6

3.4 0.03 83 13.1 0.20 128 0.3 0.007 28

P Value NS NS NS 0.05 NS NS 0.04 ,0.001 0.002

Definition of abbreviations: BicCSA 5 cross-sectional area of biceps muscle; BicPT 5 peak torque of biceps muscle; CF 5 cystic fibrosis; FFM 5 fat-free mass; PIMAX 5 maximum static inspiratory mouth pressure; QuadCSA 5 cross-sectional area of quadriceps muscle; QuadPT 5 peak torque of quadriceps muscle; Tdi 5 diaphragm muscle thickness. * Tdi and PIMAX were measured at a similar absolute lung volume in the patients with CF and the control subjects.

Figure 1. Plots of (A) biceps cross-sectional area, (B) quadriceps crosssectional area, and (C ) diaphragm thickness versus fat-free mass in 38 patients with cystic fibrosis (open circles) and 24 control subjects (solid circles). The regression lines obtained in the two groups are superimposed to each other in A and B, but in C the regression line obtained in the patients is displaced toward higher values of diaphragm muscle thickness (P , 0.001 by covariance analysis).

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(,0.5). For inspiratory muscle strength and diaphragm thickness, no correlation with inflammatory markers was found. Results of multiple regression analyses performed in the patients are given in Table 4. FFM was primarily predicted by gender and 2sRaw, which accounted for 59 and 10% of the total variance, respectively; diaphragm thickness was best predicted by sRaw and FFM, which accounted for 25 and 22% of the total variance, respectively. Inflammatory markers were not selected as independent predictors of FFM and diaphragm thickness. The regression equations for biceps and quadriceps strength and bulk identified FFM as a primary independent variable, accounting for 69 to 84% of the total variance. Other factors, such as inflammatory markers or sRaw, accounted for very small percentages (1–7%) of the total variance.

DISCUSSION This study shows that, in adult patients with mild to moderate CF (1) the intensity of systemic inflammation correlates positively with the degree of airflow obstruction and the alteration in nutritional status; (2) the inspiratory muscles are stronger and the diaphragm is thicker in patients with CF than in control subjects, which is consistent with a training effect and suggests that continued systemic inflammation does not prevent the response of the muscle to loading; and (3) the level of systemic inflammation is not an independent predictor of FFM, Tdi, inspiratory muscle strength, and limb muscle strength and bulk.

CRITIQUE OF METHODS Patient Selection

This study compared patients with CF with healthy adults. Caution is required before extrapolating these results to other chronic respiratory diseases, such as non-CF bronchiectasis, because CF is a genetic disorder that might affect many organ systems, including the skeletal muscles (17, 18; see also the DISCUSSION in the online supplement). On average, the patients studied here had less advanced disease than the patients of our previous study (10), as evidenced by a higher FEV1 (49 vs. 39% predicted) and a greater BMI (19.8 vs. 17.8 kg/m2). This is explained by the selection of patients with a large range of disease severity, including a few without significant airflow obstruction. Assessment of Tdi

Because diaphragm length is sensitive to lung volume (19), the diaphragm is expected to be thicker in hyperinflated patients than in control subjects when measurements are made at FRC.

To control for this confounding effect, a comparison of Tdi between groups was made at a similar absolute lung volume corresponding to the patients’ FRC. However, for this comparison to be valid, the increase in FRC resulting from CF and the increase in volume achieved in the control subjects to match the patients’ FRC should be similarly accommodated by the rib cage and the diaphragm–abdomen pathways and hence produce similar changes in diaphragm length and configuration. Whether this assumption is true or not is unknown, but Bellemare and colleagues (20) recently reported that male and female patients with CF do not accommodate pulmonary hyperinflation in the same way. Although both pathways shared the increase in FRC in male patients, it was primarily accommodated by rib cage expansion with preservation of diaphragm length in female patients. Based on these observations, it is difficult to know how Tdi should be compared between patients with CF and control subjects. For this reason, we provide values of Tdi obtained at a similar absolute lung volume and at FRC for each gender separately (Table E5). Table E5 shows that Tdi was greater in patients with CF than in control subjects, irrespective of gender and of the volume at which the comparison was made, although differences were more significant when Tdi was normalized by FFM and when comparisons were made at FRC. Inflammatory Markers

Because the aim of this study was to assess the potential effects of systemic inflammation on respiratory and limb muscles, it was appropriate to measure the levels of inflammatory markers in the serum rather than in the bronchoalveolar lavage fluid or the sputum. We measured circulating levels of CRP, IL-6, IL-8, and TNF-a, which are well recognized markers of systemic inflammation in CF (21). Furthermore, measuring levels of IL-6 and TNF-a was pertinent in the context of this study because these cytokines have the potential to induce skeletal muscle proteolysis via the ubiquitin–proteasome pathway (1). We also assessed IL-17; this is a newly described proinflammatory cytokine, which, like IL-8, is critical for pulmonary neutrophil recruitment and host defense against gram-negative bacteria and is increased in the sputum during acute exacerbations of CF (22).

PREVIOUS STUDY In this study, we assessed overall inspiratory muscle, rather than diaphragm strength. We found that patients had increased PIMAX values, which contrasts with our previous finding that twitch transdiaphragmatic pressure is reduced in CF (10). The most likely explanation for this apparent discrepancy is that comparison of PIMAX between groups was made at a similar absolute lung volume, whereas twitch transdiaphragmatic pres-

TABLE 4. RESULTS OF MULTIPLE REGRESSION ANALYSES IN PATIENTS WITH CYSTIC FIBROSIS

FFM Tdi PIMAX BicCSA BicPT QuadCSA QuadPT

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Best Equations

R 2*

P Value

39.18 1 (15.24 3 gender) – (0.9 3 Raw) – (0.09 3 ESR) 0.47 1 (0.01 3 sRaw) 1 (0.02 3 FFM) 44.18 1 (1.66 3 FFM) – (0.45 3 sRaw) 1 (7.2 3 logIL-8) 22.52 1 (0.26 3 FFM) – (0.52 3 logIL-8) 1 (0.076 3 Raw) 251.1 1 (5.75 3 FFM) 1 (0.54 3 sRaw) – (13.9 3 logCRP) 50.12 1 (1.21 3 FFM) – (14.33 3 logIgG) – (0.06 3 sRaw) 2141.7 1 (9.89 3 FFM) 1 (280.1 3 logsTNF-RI) 1 (0.96 3 sRaw)

0.59/0.10/0.02 0.25/0.22 0.38/0.07/0.05 0.81/0.02/0.01 0.78/0.02/0.01 0.84/0.01/0.01 0.69/0.07/0.02

,0.001 ,0.001 ,0.001 ,0.001 ,0.001 ,0.001 ,0.001

Definition of abbreviations: BicCSA 5 cross-sectional area of biceps muscle; BicPT 5 peak torque of biceps muscle; ESR 5 erythrocyte sedimentation rate; FFM 5 fat-free mass; PIMAX 5 mouth pressure; QuadCSA 5 cross-sectional area of quadriceps muscle; QuadPT 5 peak torque of quadriceps muscle; sTNF-RI 5 tumor necrosis factor-a–soluble receptor; Tdi 5 diaphragm muscle thickness. * R2 values are given for each independent variable in the order they appear in the different equations; the sum of these values yields the R2 of the equation.

Dufresne, Knoop, Van Muylem, et al.: Inflammation and Skeletal Muscles in CF

sure was compared at FRC. Another difference with our earlier study is that we measured diaphragm thickness rather than mass. Results of the two studies, however, were remarkably consistent in showing that CF is associated with an increase in diaphragm bulk (Table 3 and Figure 1C) and does not similarly affect respiratory and limb muscles (Figure E1). In addition, multiple regression analyses in both studies identified airway resistance as the principal independent predictor of diaphragm bulk, which strongly supports the view that thickening of the muscle occurs in response to loading.

CURRENT STUDY As in earlier studies (23–28), this group of adult patients with CF who were clinically stable showed increases in several inflammatory markers that correlated with the severity of lung disease and the reduction in BMI or FFM (i.e., the lower the FEV1 and the BMI, the higher the degree of systemic inflammation). On the other hand, multiple regression analyses showed no independent predictive effect of inflammatory markers on the strength and bulk of inspiratory and limb muscles (Table 4). This negative finding is particularly relevant in the context of this study, which was designed with several strengths and novelties to reveal the potential effects of systemic inflammation. First, we studied a group of patients who showed wide ranges of disease severity and inflammation and were free of treatment with oral corticosteroids, antiinflammatory agents, or azithromycin at the time of studies. Second, in addition to the impact of inflammation on FFM, we assessed the thickness of the diaphragm and the cross-sectional area of two limb muscles, which had not been done in earlier studies. Finally, we used multiple stepwise regression analyses to look for independent associations between inflammation and skeletal muscle function. The results of the correlation analyses reported in Table 4 do not necessarily imply that systemic inflammation has no detrimental effect on skeletal muscles in CF. First, measuring levels of circulating cytokines may not be the best way to assess the intensity of inflammation because soluble receptors and natural inhibitors may modulate their bioactivity. In addition, inflammatory cytokines may exert part of their negative effects by increasing the generation of free radical species (29), which were not measured here. More importantly, measurements of cytokines were obtained at a single time point and hence cannot be considered as reflecting accurately the level of inflammation to which the patient (and his skeletal muscles) was exposed during the course of his longstanding disease. Ideally, a marker reflecting the level of inflammation over time, as does glycosylated hemoglobin for serum glucose, would be better suited, but such a marker does not exist. With this in mind, we included in the regression analyses two markers of sustained infection/ inflammation: the levels of immunoglobulin G measured during the 2-yr period preceding the study and the number of days of intravenous antibiotic treatment received during the same time period (Table E1); these markers, however, did not perform better than circulating cytokines in predicting muscle function or bulk. A last issue that may confound the demonstration of a negative effect of inflammation on skeletal muscles is the complex interplay between systemic and intramuscular factors, as discussed in previous reviews on patients with COPD (30, 31). The former may include the duration and severity of the respiratory disease, the degree of hypoxemia and nutritional imbalance, and the circulating levels of cytokines, free radicals and anabolic hormones; the latter may include the degree of inactivity versus muscle loading, muscle acidosis, and local concentrations of cytokines and free radicals (32). This complex interplay will eventually

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determine if inflammation leads to muscle anabolism or catabolism. An illustration of this phenomenon is the observation that, depending on the context, local expression of TNF-a in skeletal muscles of patients with COPD may be beneficial or detrimental to muscle regeneration (33, 34). The limitations discussed above may apply to studies performed in patients with COPD and explain why these studies failed to establish unequivocally a causal link between systemic inflammation and skeletal muscle dysfunction (31, 35, 36). By showing that this link cannot be demonstrated in a disease like CF, which produces much more pronounced and longstanding inflammation than COPD, the current studies emphasize the need for a better understanding of the influence of inflammation on muscle injury, regeneration, and healing in chronic pulmonary disorders (31, 34). In summary, the current studies show that in patients with mild to moderate CF, the intensity of systemic inflammation does not account significantly for the variance in FFM, Tdi, or limb muscle strength and bulk. The presence of continued systemic inflammation does not seem impede the response of the diaphragm to loading. Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Acknowledgment: The authors thank Mrs. L. Schandene and Mr. J. Tresnie for technical assistance.

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