American Journal of Respiratory and Critical Care Medicine

We partitioned exhaled nitric oxide (NO) into alveolar concentration (Ca) and conducting airway flux (Jnoair,max) in scleroderma (SSc) lung disease and hypothesized that Ca would be elevated. Twenty patients with SSc, 15 with interstitial lung disease (SSc–ILD) alone, and 5 with pulmonary hypertension (SSc–PH) were compared with 20 control subjects. Ca and Jnoair,max were derived from the slope and y intercept, respectively, of the NO output versus expiratory flow rate (V·exh) relationship obtained by measuring exhaled NO (FeNO) at multiple V·exh values of 50–200 ml/second. There were no significant differences in FeNO at any V·exh between the SSc group and control subjects. Jnoair,max was reduced (0.6 ± 0.1 versus 1.2 ± 0.2 nl of NO per second; p = 0.01), whereas Ca was increased (4.7 ± 0.5 versus 1.8 ± 0.2 ppb; p < 0.001) in the SSc group compared with control subjects. No differences were noted between SSc–ILD and SSc–PH. There was a negative correlation between Ca and DlCO among the patients with SSc (R = −0.66, p = 0.002). We conclude that Ca is increased whereas Jnoair,max is decreased in SSc–ILD and SSc–PH. A reduced diffusing capacity of NO from the alveolar space into the blood could explain the observed increase in Ca.

Pulmonary involvement, in the form of interstitial lung disease (ILD) and/or pulmonary hypertension (PH), is currently the leading cause of morbidity and mortality in scleroderma or systemic sclerosis (SSc) (1). The pathogenesis of SSc lung disease is not well understood. Immune activation and vascular endothelial cell injury are thought to be central features of SSc (2). Interstitial and alveolar inflammation is well documented in ILD associated with SSc, which is similar pathologically to idiopathic pulmonary fibrosis (IPF) (3). Inflammatory mechanisms may also play a role in the obliterative vascular wall cell proliferation of PH associated with SSc, which resemble those seen in primary pulmonary hypertension (PPH) (4).

Immune–inflammatory processes are accompanied by cytokine-mediated upregulation of the inducible isoform of nitric oxide synthase (iNOS), leading to the production of large quantities of nitric oxide (NO) (5). Excess NO generation may exert strong proinflammatory and cytotoxic properties (6). NO has been implicated in the pathogenesis of autoimmune diseases (7), including SSc (811), as well as IPF (12). A deficiency in basal NO production by the constitutive endothelial isoform of NOS (eNOS) could promote vasoconstriction and vascular wall thickening and has been implicated in the pathogenesis of PPH (13). Measurement of NO in exhaled air has been used increasingly to assess the potential role of endogenous lung NO in a variety of disease states (14). Several studies have reported an increase in exhaled NO concentrations among patients with ILD associated with SSc (1518). One group reported lower than normal exhaled NO values in patients with SSc with PH (15), whereas another found elevated levels comparable to ILD without PH (16). Some of these studies are hampered by the lack of standardization in expiratory flow rates and exclusion of the nasopharynx, both of which will have a dramatic effect on exhaled NO concentrations (14). Also, measurements at a single expiratory flow rate cannot distinguish an alveolar versus a conducting airway source of NO.

We employed a novel method that is capable of partitioning the alveolar and conducting airway components of lower respiratory tract NO (19, 20) in patients with pulmonary involvement associated with SSc. Our hypothesis was that alveolar concentrations of NO would be elevated, reflecting alveolar and/or interstitial inflammation.

Twenty-two patients with documented SSc (21) and pulmonary involvement (22) were studied. Exclusion criteria included the following: (1) smoking history within the past year; (2) respiratory infection within the past 2 weeks; (3) history of hyper-reactive airways; (4) FEV1/FVC ratio of < 0.7; (5) left heart disease; (6) any intervening acute illness or exacerbation of disease; (7) use of l-arginine, nitrates, or phosphodiesterase inhibitors; and (8) pregnancy. Five patients with PPH (23) were also studied. Twenty healthy, nonsmoking volunteers served as control subjects. Informed written consent was obtained and the study was approved by the Wayne State University (Detroit, MI) Human Investigation Committee.

All patients underwent pulmonary function tests (PFTs) within 6 months of study, with no significant clinical changes occurring since the time of testing. PFTs were performed according to American Thoracic Society guidelines (2426) on a SensorMedics (Yorba Linda, CA) Vmax 22 system. Predicted values were taken from Crapo and coworkers (27) and Miller and coworkers (28). ILD was considered present if a chest radiograph or high-resolution computed tomogram (HRCT) of the chest demonstrated compatible changes in reticular or air space opacities (29). PH was defined by a right ventricular systolic pressure of > 35 mm Hg on echocardiogram and/or a mean pulmonary artery pressure of > 25 mm Hg at right heart catheterization (30). In the absence of either study, PH was considered absent if there were no suggestive clinical signs. Immunosuppressive treatment was defined as current use or therapy for 3 months or longer during the preceding year.

The method recommended for online measurement of the fractional exhaled NO concentration (FeNO) in adults was employed (14), using a chemiluminescent NO analyzer (model 280 NOA; Sievers, Boulder, CO) (19). After a full inspiration from room air, subjects exhaled against positive pressures of 11–16 cm H2O to generate flow rates (V·exh) of 50, 100, 150, and 200 ml/second (FeNO,50–200). Ambient NO levels were always less than 20 parts per billion (ppb), which have been shown not to affect FeNO readings (14). For each V·exh, the NO output (V·no = V·exh × FeNO) was calculated. FeNO is inversely related to V·exh, whereas V·no varies directly as a function of V·exh. At flow rates of > 50 ml/second, the latter relationship is linear (31) and can be expressed as V·no = Ca × V·exh + Jnoair,max (19, 20). The positive slope of this relationship (Ca) represents the steady state alveolar concentration of NO in parts per billion and the y intercept (Jnoair,max) approximates the maximum total molar flux of NO (in nanoliters per second) from the airway wall into the lumen that would occur at infinite V·exh.

Data are presented as means ± standard error. One-way analysis of variance was used to evaluate differences between three or more groups with the Bonferroni correction for multiple comparisons. Two-sample t tests using Satterthwaite's correction for unequal variances were used to compare differences between two groups with the Bonferroni–Holm correction for multiple comparisons. Pearson correlation coefficients were used to assess the association between exhaled NO measures and PFT parameters with adjustment for multiple comparisons.

Subject Characteristics

Two patients with SSc were excluded because of inability to achieve an acceptable FeNO plateau at the highest flow rate. The demographic and clinical features of the remaining 20 patients with SSc, 5 patients with PPH, and 20 control subjects are presented in Table 1

TABLE 1. Demographic and clinical features of study subjects

SSc Group (n = 20)

SSc–ILD Group
 (n = 15)
SSc–PH Group
 (n = 5)
PPH Group
 (n = 5)
Control Group
 (n = 20)
Age, yr 50 (2) 63 (3) 52 (6)42 (2)
Sex, female/male9/64/15/012/8
Skin involvement, limited/diffuse8/73/2
RVSP range, mm HgNone with PH61–8562–97
FVC, % predicted 72 (3)91 (10) 94 (10)
FEV1/FVC ratio 0.84 (0.02) 0.82 (0.04) 0.74 (0.02)
TLC, % predicted 81 (3) 89 (5) 100 (4)
DlCO, % predicted 56 (3) 26 (3) 69 (5)
Vasoactive medications,
Immunosuppressive therapy, n

Definition of abbreviations: CCBs = calcium channel blockers; PPH = primary pulmonary hypertension; RSVP = right ventricular systolic pressure estimated by Doppler echocardiography; SSc = scleroderma; SSC–ILD sclerodoma with interstitial lung disease alone; SSc–PH = sclerodoma with pulmonary hypertension.

Data are presented as means with SEM in parentheses unless otherwise indicated.

. All patients with SSc had at least some degree of interstitial changes detectable by chest radiograph and/or HRCT consistent with ILD. Fifteen had no evidence of PH (SSc–ILD group) and the other five had clinically significant PH (SSc–PH). Four of these patients had minimal radiographic evidence of ILD and TLC > 80% predicted. Immunosuppressive therapy in the patients with SSc consisted of daily oral or monthly intravenous cyclophosphamide (n = 4), low-dose (10–15 mg) weekly methotrexate (n = 4), low-dose (5–10 mg/d) prednisone (n = 6), and hydroxychloroquine (n = 1).

Exhaled NO

Visual inspection of the regression line and data points of the FeNO-versus-V·exh plots indicated no gross departure from the assumption of linearity for the vast majority of subjects. Mean R2 values of this relationship were 0.93 ± 0.02, 0.85 ± 0.03, and 0.96 ± 0.01 for the SSc, control, and PPH groups, respectively. Plots of the mean values for the SSc and control groups are demonstrated in Figure 1

. Table 2

TABLE 2. Summary of exhaled nitric oxide measurements

 (n = 20)

 (n = 15)

 (n = 5)

 (n = 5)

Control Subjects
 (n = 20)
FeNO,50, ppb17.4 (2)16.9 (1.5)18.8 (7.2)20.2 (6.5) 26 (3)
FeNO,100, ppb10.8 (1)10.3 (0.8)12.4 (3.5)11.3 (3.2) 14 (1.7)
FeNO,150, ppb8.7 (0.8)8.2 (0.7)10.2 (2.5)8.0 (2.1) 10.1 (1.2)
FeNO,200, ppb7.9 (0.6)7.4 (0.6)9.3 (1.6)6.9 (1.6) 7.8 (0.9)
y Intercept,
 NO (nl/s)0.6 (0.1)*0.6 (0.1)0.6 (0.4)0.9 (0.3)1.2 (0.2)
Slope, ppb
4.7 (0.5)
4.3 (0.6)
6.0 (0.7)
2.3 (0.1)
 1.8 (0.2)

*p = 0.01 compared with control subjects.

p < 0.001 compared with control subjects.

p < 0.01 compared with PPH group.

Definition of abbreviations: Subject group abbreviations as in Table 1; FeNO,50–200 = exhaled NO concentration at expiratory flow rates of 50–200 ml/second.

Values represent means with SEM in parentheses.

summarizes the exhaled NO measurements. FeNO at the three slower flow rates (FeNO,50–150) were somewhat lower in the SSc group compared with control subjects, but not significantly different. The resultant y intercepts, however, of the V·no-versus-V·exh plots were significantly reduced in patients with SSc, indicating decreased conducting airway flux of NO into the airway lumen. In contrast, the slopes of the patients with SSc were markedly increased, compared with the control group, indicating a higher Ca. There was no relationship between the y intercept and any lung function parameter in the SSc group. Slope was negatively correlated with DlCO (R = −0.66, p = 0.002; Figure 2) , but not with TLC (R = −0.1, p = 0.78). No significant differences were observed in any exhaled NO parameter between patients with SSc with ILD alone versus those with PH. There were also no differences between those who received immunosuppression and those who had not or between those with diffuse versus limited skin involvement. The Ca and Jnoair,max of the PPH group were similar to those of the control subjects. We compared the five SSc–PH patients with the PPH group. No differences in any of the FeNO values or y intercept were noted. Ca was significantly higher whereas DlCO was considerably lower in the SSc–PH group (Table 1).

The main findings of this study are that in patients with scleroderma lung disease (1) maximum conducting airway flux of NO is decreased; (2) alveolar NO concentrations are increased; (3) Ca is inversely correlated with DlCO; and (4) exhaled NO measurements do not differ between patients with PH compared with those with ILD alone.

The measurement of exhaled NO as a biomarker for inflammatory lung diseases has been used extensively. Several factors need to be taken into consideration for the accurate measurement and interpretation of exhaled NO. Exclusion of the nasopharynx and the recording of a plateau reading at a constant V·exh are crucial technical factors (14). At any given V·exh, FeNO reflects both an alveolar and conducting airway source. Measuring FeNO at multiple V·exh, and then plotting V·no versus V·exh, can partition these components according to the method described by Tsoukias and coworkers (19, 20). Elevated FeNO in asthma has been clearly demonstrated to originate from the conducting airways (31) and has been correlated with airway hyperresponsiveness (32). Only one previous report has studied alveolar NO concentrations in patients with interstitial lung disease. Lehtimaki and coworkers (32) reported a mean Ca of 4.1 ppb in 17 patients with IPF and hypersensitivity pneumonitis, compared with 1.1 ppb in control subjects; values comparable to our data in the SSc and control groups, respectively. Similar to our study, these authors found an inverse correlation between Ca and DlCO, suggesting that high levels of alveolar NO may play a role in the pathogenesis of ILD. Five previous studies have evaluated FeNO in SSc lung disease (Table 3)

TABLE 3. Results of previous studies of exhaled nitric oxide in scleroderma


Type of

Control of

Exclusion of

FeNO Findings

Kharitonov and
 coworkers (15)All*/23 (6 PH)NoNoInc in non-PH; Dec in PHPeak value recorded
Fajac and
 coworkers (16)All/14 (4 PH)NoNoInc in allTidal breathing
Paredi and
 coworkers (17)ILD only/17YesYesInc with BAL alveolitis onlyV·exh of 83–100 ml/second
Rolla and
 coworkers (18)All/47 (16 PH)NoYesInc in non-PH; normal in PH; PH < non-PH; PH with no ILD < PH with ILDV·exh of 5–15 L/minute;
 inverse correlation
 between RVSP and FeNO
Moodley and
 Lalloo (33)ILD/noneNoYesNormal in ILD; Inc in no ILD groupV·exh of 10–15 L/minute;
 no ILD group had alveolitis by BAL

*All includes ILD, PH, both, or none (no lung involvement).

Definition of abbreviations: BAL = bronchoalveolar lavage; Dec = decreased; FeNO = fractional exhaled NO concentration; ILD = interstitial lung disease; Inc = increased; PH = pulmonary hypertension; RVSP = right ventricular systolic pressure; V·exh = expiratory flow rate.

(1518, 33). Most, but not all (33), have detected modest elevations in patients with ILD. Unfortunately, these studies used variable methodology, making their interpretation difficult. Partitioning of FeNO into its alveolar and conducting airway sources has not been previously reported in SSc lung disease.

We were unable to detect a difference in FeNO at any V·exh, but did demonstrate that the y intercept of the V·no-versus-V·exh relationship in the SSc group was half of that observed in control subjects, indicating a reduced Jnoair,max. This was an unexpected and surprising finding given the results of previous studies. The determinants of Jnoair,max include the airway surface area, the transfer coefficient of NO from airway tissue to lumen, and the airway wall NO concentration. The latter is related to the wall production rate of NO relative to its uptake by bronchial blood flow (20, 31). Small airway disease has been described in a large proportion of patients with SSc (34, 35). Increased thickness of the bronchial/bronchiolar wall from subtle airway disease would be expected to reduce the transfer coefficient and hence Jnoair,max. Chronic airway disease could also alter the expression or function of NOS in bronchial epithelial cells. Alternatively, increased amounts of superoxide could be present in airway lining fluid that would promote the catabolism of NO (36). Decreased airway surface area as a result of smaller lung volumes in these patients could also lower Jnoair,max, although we found no correlation between the latter and TLC.

The increased alveolar concentrations of NO detected in the SSc group suggest either increased NO production by alveolar epithelial and/or inflammatory cells (V·lNO), reduced diffusing capacity of NO from the alveolar space into the pulmonary capillary blood (DlNO), or decreased catabolism of NO (20). The latter possibility appears unlikely given the reduced Jnoair,max. An important limitation of this study is the absence of a direct measurement of DlNO (37). Other studies such as bronchoalveolar lavage fluid (BALF) and lung tissue analysis for NO metabolites, nitrotyrosine (a marker of protein nitration by peroxynitrite), and NOS expression would also help delineate the basis for our observation of increased Ca in SSc-related lung disease.

Although the negative correlation between Ca and DlCO may relate to increasing severity of disease associated with higher NO levels, low DlCO may also account for high Ca. In normal individuals and in patients with chronic obstructive lung disease, the DlNO/DlCO ratio is consistently ∼ 4.3 (38, 39). If we assume that this relationship is applicable to our population, DlNO can be calculated on the basis of the measured DlCO. Subsequently, V·lNO can be determined as the product of Ca and DlNO (37). We performed this calculation (using 100% of the predicted DlCO for the control group) as a post hoc analysis and found no differences in V·lNO among the groups studied (Table 4)

TABLE 4. Calculation of production and diffusion of alveolar nitric oxide from measured dlco and ca






DlCO, ml/min per mm Hg11.7 (1)13.7 (1)5.5 (0.6) 17 (1)27.2 (1.1)*
DlNO, ml/min per mm Hg 50 (5) 59 (4)23.5 (3) 73 (5) 117 (5)
Ca, mm Hg (btps)3.5 (0.4)3.1 (0.5)4.4 (0.5)1.7 (0.1)1.3 (0.2)
V·lNO, μl/min
0.15 (0.02)
0.17 (0.02)
0.11 (0.02)
0.12 (0.02)
0.15 (0.01)

*Control group DlCO assumed to be 100% of predicted.

Definition of abbreviations: Group abbreviations as in Table 1; Ca = alveolar concentration of NO; DlNO = diffusing capacity of NO from the alveoli into blood, calculated from the DlCO (see text for details) ; V·lNO = alveolar production rate of NO.

Data presented are means with SEM in parentheses.

. Several assumptions, however, are required to exclude an increased V·lNO as a contributing factor to the observed elevation in Ca in the SSc group. Because NO combines with hemoglobin much faster than CO, DlNO is relatively independent of pulmonary capillary blood volume (Vpc) and the specific blood transfer conductance (θ), depending mostly on the membrane component (Dm) of diffusing capacity (38, 39). If the reduced DlCO in our patients were due to a disproportionate loss in Vpc relative to Dm, then the calculated DlNO would be underestimated. A reduction in Vpc is the primary mechanism for decreased DlCO in PPH (40) and likely also predominates in SSc-related PH where minimal ILD exists. The relative contributions of Vpc and Dm to the observed reduction of DlCO in ILD vary, but Dm often predominates (41). Even in the presence of an actual measurement of DlNO, the contribution of V·lNO to Ca maybe difficult to ascertain completely, particularly in diseased lung. Morphometric studies of lung biopsy samples in ILD have shown that the reduced Dm is related more to reduced alveolar surface area than to membrane thickening (41). This would be expected to reduce both NO production by the alveolar compartment as well as diffusion into the capillary blood, thereby offsetting the effect on Ca (20). Also, functional inhomogeneities in diffusion, lung volume, and distribution of ventilation can lead to considerable error (both over- and underestimation) of true diffusing capacity (42). Thus, although a reduction in DlNO is a likely explanation for the observed increase in Ca in the SSc group, increased V·lNO is not excluded.

High Ca may play a role in the pathogenesis of SSc lung disease, inducing cellular toxicity mediated directly by NO and via the formation of reactive nitrogen intermediates, such as peroxynitrite (6). Previous studies suggest increased NO production in SSc. Serum concentrations of nitrite and nitrate (stable end oxidation by-products of NO) are increased in SSc (810) and there is abnormal expression of iNOS and nitrotyrosine in microvascular endothelial cells, infiltrating mononuclear cells, and fibroblasts of affected skin, which correlates with the histologic severity of fibrosis (10, 43). Enhanced spontaneous and interleukin 1β-stimulated release of nitrite from SSc peripheral blood mononuclear cells in vitro has also been demonstrated (11). Large quantities of nitrotyrosine and iNOS have been detected in inflammatory cells and alveolar epithelium of patients with early to intermediate stages of IPF (12). Immune–inflammatory mechanisms may also contribute to the vascular remodeling of SSc–PH (4), and excess NO may participate in vascular tissue injury. In this regard, oxidants, such as peroxynitrite, may antagonize the vasorelaxant effect of NO, thereby contributing to vasoconstriction (44). Thus, excess NO within the alveolar space could initiate and/or perpetuate the inflammatory, fibrotic, and vascular manifestations of SSc lung disease.

In contrast to the findings of other authors (15, 18), we did not detect any differences in exhaled NO between patients with SSc with ILD alone compared with those with concomitant PH. Although all of our SSc–PH patients had at least minimal changes of ILD by HRCT, four of them (with TLC > 80% predicted) would have been considered to be free of ILD using the definition of Rolla and coworkers (18). The one patient in this group with significant ILD as well as PH had the lowest Jnoair,max (0.03 nl of NO per second), whereas the Ca (5.6 ppb) was close to the mean value for both the SSc–ILD and SSc–PH groups. On the basis of our data, it does not appear that exhaled NO differs between patients with SSc–ILD and patients with SSc–PH, although the latter group was small. For comparison with SSc–PH, we studied five patients with PPH. Ca was nearly three-fold higher in the SSc–PH group compared with the PPH group, despite a similar degree of pulmonary hypertension. DlCO, however, was significantly lower in SSc–PH, possibly accounting for the difference in Ca. Both Ca and Jnoair,max in the PPH group were similar to those of control subjects. Decreased expression of vascular endothelial NOS has been demonstrated in PPH (45). Exhaled NO analysis is likely a poor index of the intravascular compartment because most of the NO produced by vascular endothelial cells would be expected to immediately bind to hemoglobin, rather than diffuse into the alveolar compartment (46).

Our patients with SSc were not characterized regarding the presence or extent of alveolitis. The cellular profile in BALF has been proposed as a useful measure for this purpose (47). It is possible that most of our patients did not have active alveolitis and this could have accounted for the lack of elevated FeNO described in other reports (1518). However, this would not explain our findings of an actual decrease in Jnoair,max or the elevated Ca. Four of our patients with SSc–ILD did undergo BAL for clinical purposes immediately after exhaled NO analysis. Three had an abnormal BALF cellular profile consistent with alveolitis (17, 47), with granulocyte percentages of 16–23%. None of these patients had a Jnoair,max above the mean value for the control group and all had Ca values well above the mean found in normal subjects, consistent with data obtained from the SSc group as a whole. The absence of BALF analysis, therefore, does not impact significantly on the implications of our findings.

In summary, we have demonstrated increased alveolar concentrations of NO in SSc–ILD patients with or without concomitant PH. Although this can be potentially explained by a reduction in the diffusion of NO into capillary blood, an actual increase in NO production within the alveolar space remains possible. This elevated Ca, whether resulting from decreased diffusion or increased production, may participate in the pathogenesis of the disease. In contrast, the maximal conducting airway flux of NO was reduced, which may be related to some intrinsic airway abnormality.

Supported by the Department of Internal Medicine, Wayne State University (Detroit, MI).

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Correspondence and requests for reprints should be addressed to Reda E. Girgis, M.B., B.Ch., Division of Pulmonary and Critical Care Medicine, Johns Hopkins University, 600 N. Wolfe St., Blalock 910, Baltimore, MD 21287. E-mail:


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