- Email: [email protected]
Eosinophil Activation in Patients With Pulmonary Fibrosis
Eosinophil Activation in Patients With Pulmonary Fibrosis* Keisaku Fujimoto, MD; Keishi Kubo, MD; Shinji Yamaguchi, MD; Takayuki Honda, MD; Yukinori Matsuzawa, MD The eosinophil count and concentrations of eosinophil cationic protein (ECP) were measured in bronchoalveolar lavage fluid (BALF) from patients with idiopathic pulmonary fibrosis (IPF), pulmonary fibrosis associated with a collagen vascular disorder (PF-CVD), sarcoidosis, and healthy controls. The patients with IPF and PFCVD showed significant increased eosinophil count and ECP levels in BALF compared with the controls. When the patients with IPF and PF-CVD were subclassified into chronic stable, progressive, and acute progressive subgroups in accordance with the observed progression of pulmonary dysfunction during the preceding 3- to 6-month period, those in the acute progressive subgroup showed significantly elevated recovered eosinophil count and ECP level, as well as recovered lymphocyte count and total protein, albumin, and type III procollagen aminoterminal peptide-related antigens (piiip) in BALF, compared with either of the other two subgroups. Multivariate stepwise logistic regression analysis revealed that, among these variables, only ECP and piiip significantly contributed to discrimination among the
Jt has been shown that a relative large number of eosinophils are recovered in the bronchoalveolar lavage fluid (BALF) in patients with pulmonary fibrosis, 1-4 and the finding of increased number of eosinophils in BALF has been interpreted as a sign of poor prognosis.l-3 The role of these accumulated eosinophils in the pathogenesis of pulmonary fibrosis has recently been investigated. Eosinophils contain various cytotoxic substances and have been suggested as being implicated in tissue injury in various lung diseases. 5 -9 Eosinophil infiltration and the deposition of eosinophil granule proteins have also been shown in fibrous tissue in various fibrotic disorders. 7•9 Eosinophil cationic protein (ECP), a specific component of eosinophil granulocytes, is one of these cytotoxic substances.l 0 In this study, to evaluate the role of accumulated eosinophils in the lungs in the pathogenesis of fibrosis in patients with pulmonary fibrosis, we measured ECP in BALF as a marker of degranulation *From the First Department of Internal Medicine, Shinshu University School of Medicine Matsumoto, Japan. Manuscript received March 9, 1994; revision accepted December 1.
Reprint requests: Dr. Fujimoto, First Dept Internal Medicine, Shinsu University School of Medicine , 3-1-1 Asahi, Matsumoto, japan, 390
48
three subgroups differing in disease activity. These findings suggest that eosinophils are involved in the inflammatory process in pulmonary fibrosis and that the released ECP and other cytotoxic eosinophil products may contribute to the lung injury and development of fibrosis. (CHEST 1995; 108:48-54) BALF=bronchoalveolar lavage fluid; Dco=carbon monoxide diffusing capacity; ECP=eosinophil cationic protein; FEV1%=percent of forced expiratory volume in 1 s; IPF=idiopathic pulmonary fibrosis; LDH=Iactate dehydrogenase; PF-CVD=pulmonary fibrosis associated with a collagen vascular disorder; plllp=type III procollagen aminoterminal peptide-related antigen; TP=total protein; VC=vital capacity
Key words: bronchoalveolar lavage; eosinophil cationic protein; idiopathic pulmonary fibrosis; pulmonary fibrosis associated with a collagen vascular disorder; type III procollagen aminoterminal peptide-related antigen
from activated eosinophils. Furthermore, the associations between the ECP level and progression of pulmonary dysfunction during the 3- to 6-month period preceding the investigation and the levels of type III procollagen aminoterminal peptide-related antigens (piiip) in BALF, as a marker of fibroblast activation,ll·12 were examined. METHODS
The ECP and piiip in BALF were measured in 27 subjects with idiopathic pulmonary fibrosis (IPF) and 19 with pulmonary fibrosis associated with a collagen vascular disorder (PF-CVD), and, for comparison, in 24 patients with sarcoidosis and 13 healthy volunteers. The criteria for IPF or PF-CVD were as follows: (1) no clinical history of exposure to environmental agents known to cause interstitial lung disease, no history suggestive of extrinsic allergic alveoli tis, and no history of chronic lung infection or left ventricular failure; (2) evidence of interstitial infiltrates on chest radiograph and chest CT scan, and pulmonary function test results consistent with a restrictive ventilatory defect and decreased single breath carbon monoxide diffusing capacity (Dco); (3) biopsy specimen evidence of interstitial pneumonitis with varying degrees of interstitial fibrosis without evidence of granulomas. Among the 46 patients with IPF and PF-CVD, 16 underwent open lung biopsy. Of the 46 patients, 27 had the diagnosis of IPF alone and 19 had the diagnosis of PF -CVD. Of the latter patients, five had seropositive rheumatoid arthritis, two had polyarteritis nodosa, two had polymyositis, one had dermatomyositis, two had progressive systemic sclerosis, three had systemic lupus erytheClinical Investigations
Table !-Demographic and Physiologic Characteristics of the Study Population* Group IPF PF-CVD Sarcoidosis Healthy control
N(M, F) 27 19 24 13
(17, 10) (5, 14) (13, ll) (8, 5)
Age, yr 63±2 56±3 47±3 49±4
Smoking (S, EX) (6, (2, (4, (4,
13) 3) 3) O)
Pa02, mm Hg 74.1±2.1' 79.3±3.1 80.4 ± 1.8 86.9± 1.0
%VC 75.9±3.9 1 79.4±5.7' 99.1 ±3.7 110.0±3.8
FEV1%
%Dco
83:-S± 1.5_ 79.0±2.2 83.4±2.2 86.8±2.0
50.1±4.9 11 56.2±5.5'' 90.4±4.5 103.1±5.2
*S=smoker; EX=ex-smoker; values are mean± SE. f p<0.01 vs healthy controls. lp<0.01 vs sarcoidosis. matosus, one had mixed connective tissue disease, and three had Sjogren's syndrome. At the time of BAL, only two patients were receiving immunosuppressive or steroid treatment or cytotoxic drug; these two exceptional patients, one with rheumatoid arthritis and one with dermatomyositis, were receiving 10 mg/ d of prednisolone orally. The patients with IPF and PF-CVD were also classified into three subgroups based on the changes in vital capacity (VC) and Dco at 3-month intervals for up to 6 months before the investigation together with the clinical course and chest radiographic and / or CT findings. The chronic stable subgroup consisted of 18 patients who had a clinical course of more than 6 months with less than 15% change of VC and less than 20% change of Dco at 3 months and 6 months. The progressive subgroup consisted of 21 patients with > 15% decrease in VC or >20% decrease in Dco at 6 months but not at 3 months. The acute progressive subgroup consisted of seven patients who showed acute clinical course and increase in interstitial infiltrates of bilateral lungs with > 15% decrease in VC or >20% decrease in Dco at or within 3 months. The correlation of the eosinophil counts in tissue from 16 open lung biopsy specimens (10 in patients with IPF and 6 in patients with PF-CVD) with the recovered eosinophil numbers in BALF and with the ECP level was also examined. The tissue eosinophil count was expressed as the average count for 10 randomly selected high-power (X400) microscopic fields. For comparison, we also evaluated 24 untreated patients with sarcoidosis classified radio-
graphically according to thoracic involvement as having stage 1 (n=10), stage 2!1(n=10), or stage 3 (n =4) disease. In these patients, sarcoidosis had been diagnosed based on the medical history and the findings of physical examination, chest radiography and/ or CT, and histologic examination of the lung biopsy specimen. The demographic and physiologic data for each of four groups are summarized in Table l. The VC and percent of forced expiratory volume in 1 s (FEV 1%) were measured using a water spirometer (Godart Expirograph, Godart-Statham, Bilthoven, Holland). The Dco was measured by the single-breath method (Pulmocorder, model R1551S, Anima, Tokyo, Japan). The VC and Dco are expressed as the percentage of predicted values. 13,l 4 The subjects in all groups were defined as smokers if they currently smoked cigarettes, and as ex-smokers if they had a history of smoking but had not smoked within the preceding 6 months. Compared with that in the control group, the mean percent VC and percent Dco in the IPF and PF-CVD groups and the mean Pa02 in the IPF groups were significantly lower (all, p<0.01). Before bronchoscopy, patients and control subjects were administered atropine subcutaneously, usually combined with morphine .. The upper respiratory tract was anesthetized with 2% lidocaine. A fiberoptic bronchoscope (Olympus BF1 T, Olympus Co, Tokyo, Japan) was wedged in the middle lobe segmental bronchus, and 150 mL of sterile normal saline solution warmed to 37°C was infused in 50-mL boluses. The fluid was aspirated
Table 2-Findings for BALF in Patients With IPF, PF-CVD, Sarcoidosis, and Healthy Control Subjects*
Cell No. Total cells, Xl03 / mL Macrophage, X10S/ mL (%) Lymphocyte, X10S/ mL (%) Neutrophil, X103 / mL (%) Eosinophil, Xl03 / mL (%) CD4/ CD8 ratio Total protein, mg/ dL Albumin, ~tg / mL LDH, U/L ECP, ng/ L piiip, ng/ L
IPF (n=27)
PF-CVD (n=19)
Sarcoidosis (n=24)
Healthy Control (n=13)
260.3±39.1 179.2±22.4 (74.0±3.7') 55.2± 15.9 1 (17.1 ±2.919) 14.9 ± 7.1 !§ (48±1.9§) 9.60±3.27' (3.6±0.81§) 2.1 ±0.:91 30.7±8.6 134.2±50.3 53.5 ± 11.6 106.2±35.81§ 47.1 ±25.o'
328.2±67.2 192.6±43.9 (65.4±5.0') 117.8±50.91 (28.2±50 1) 12.0±4.41§ (4.7 ± 1.7) 5.73±2.39 1 (1.7±06') 4.4±2.511 54.6± 17.9 1 183.1 ±72.01 60.6±14.9 63.5± 18.81 63.1±44.9'
211.0±37.8 114.4±22.8 (66.5 ± 4.8 1) 87.3±23.8' (31.3±4.7 1) 2.3±0.7 (1.0±0.3) 2.95 ± 1.301 (1.2±0.61) 6.5± 1.4 34.8±7.3 138.5±36.1 60.1 ± 21.5 29.3±9.3 13.4±5.1'
147.5±59.3 139.0±56.6 (93.6±0.7) 7.0±2.4 (5.2±0.7) 1.5±0.5 (11±0.2) 0.03±0.01 (0.1 ±0.0) 3.8±0.9 16.1 ± 1.4 52.8±7.8 22.8±5.4 12.3 ± 1.7 0.5 ±0.1
*Values are mean±SE. fp<0.01 vs healthy control group. lp<0.05 vs healthy control group. §p<0.05 vs sarcoidosis group. llp <0.01 vs sarcoidosis group. CHEST / 108 / 1 /JULY,1995
49
Data Analysis
(cella/HPF)
30
•
•
•• j:::
•
:::J
.E
•
20
c :::J
0
u
1
10
'I • 3.1 + 0.27x, r • 0.14, pcO.Ot
c:
1w
40
20
60
80
Eosinophil Count In BALF
100
3 (x!0 cella/ml)
(cella/HPF)
30
•
• ••
j:::
.E
RESULTS
•
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•
20
c
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u
l
10
•
y • 4.3 + 2.5e-2x, r • 0.52, pc0.05
'i 0
w 200
400
600
800 (ng/l)
ECP In BALF
FIGURE l. Relationship of eosinophil count per high-powerfield (X400) in tissue from open lung biopsy specimens in 10 patients with IPF and 6 patients with PF-CVD to recovered eosinophil count (upper panel) and ECP concentration (lower panel) in BALF. under low suction immediately after each instillation, and then filtered through gauze. One small aliquot of this fluid was used to count the total cell number, and another aliquot was spun in a cytometer (500 rpm for 5 min) and stained by the May-Gri.inwaiiGiemsa method to identify the cell populations. Five hundred nonepithelial cells per slide were identified to establish the differential cell count (Xl,OOO, oil objective lens) with each cell type expressed as a percentage of the 500 cells and as the concentration in total recovered lavage fluid. The remaining BALF was centrifuged at 300xg for 10 min at 4°C, and the supernatant was removed. The pellets from BALF were subjected to analysis ofT-cell subsets of lymphocytes using flow cytometry with monoclonal antibodies to CD4 and CD8 (Becton Dickinson Co). The supernatant was concentrated to 50-fold (using an Amicon, division of W.R. Grace & Co, Danvers, Mass). This concentrated supernatant was provided for the measurements of ECP and PiliP. The ECP and piiip were measured in duplicate using 1251-ECP radioimmunoassay (RIA) kits (Pharmacia Diagnostics, Uppsala, Sweden) and 125 I-piiip RIA kit (Behringwerke AG, Marburg, Germany). The total protein (TP), albumin, and lactate dehydrogenase (LDH) concentrations in BALF were also measured. This study was performed according to the Declaration of Helsinki, and informed written consent was obtained first from all volunteers and patients.
50
The values shown in the text, tables, and figures are expressed as mean± SE. The data distribution of the variables in each group was first assessed using Bartlett's test. When the data for the variables showed normal distribution, the comparison was performed using one-way analysis of variance, and then multiple comparisons were performed by the Tukey-Kramer method. When the data for the variables did not show normal distribution, the variables were compared using Kruskal-Wallis test, and then multiple comparisons among groups were performed by the nonparametric Tukey-Kramer method. The patients with IPF and PF-CVD were classified into the three subgroups in accordance with disease activity described above. In the identification of the parameters contributing to the disease activity or the parameters distinguishing among the three subgroups, multivariate stepwise logistic regression was performed using a procedure (STEPDISC) with statistical software programs (SAS). The correlation between variables was examined by calculating Pearson's product correlation coefficient. A p value of less than 0.05 was considered significant for all statistical tests.
Bronchoalveolar Lavage Cellularity, CD4 / CD8 Ratio, and Chemical Substances in Patients With IPF, PF-CVD, and Sarcoidosis and Healthy Controls The total number of recovered cells per milliliter of BALF in all patient groups was higher than that in the control group (Table 2). In the differential cell count, the lymphocyte count was significantly increased in all patient groups compared with that in the healthy controls, and the highest mean value was seen in the PF -CVD group. The neutrophil count in the IPF and PF -CVD groups was significantly increased compared with that in the sarcoidosis and control group (all, p<0.05). Eosinophils were rarely detected in BALF from controls and did not exceed 0.3% in any control subject. In contrast, the eosinophil count was> 1% of the total cells recovered in 17 (63%) of the 27 patients with IPF, 6 (32%) of the 19 patients with PF-CVD, and 5 (21%) of the 24 patients with sarcoidosis; mean percent eosinophils was 3.6% (range, 0 to 13.0%), 1.7% (0 to 9.5%), and 1.2% (0 to 12.8%), respectively. Both the percent eosinophils and absolute eosinophil count in the patients with IPF were significantly higher than those in the controls (p<0.01), and a significant increase in the percent eosinophils in the IPF group compared with that in the sarcoidosis group was also observed (p<0.05). The percent and number of eosinophils in BALF were significantly correlated with the eosinophil count per high-power field in tissue obtained at open lung biopsy in the 16 patients with IPF and PF-CVD (r=0.64, p<0.01) (Fig 1). The CD4/ CD8 ratio in the patients with IPF was significantly lower than that in the patients with sarcoidosis who showed slightly elevated ratio compared with the healthy controls. All patient groups showed elevated concentrations Clinical Investigations
(ng/L)
800
II.
600
=
400
..J c( ID
~
u
.. + 0
0
0
w 200
0 0 0
IPF
0
PF-CVD Sarcoldoala Control
FIGURE 2. The ECP concentrations in BALF in individual patients with idiopathic pulmonary fibrosis (IPF, n=27), pulmonary fibrosis associated with a collagen vascular disorder (PF-CVD, n=l9), and sarcoidosis (n=24), and in healthy control subjects (n= 13). The horizontal bar represents the mean value in each group. Asterisk=p<0.05; two asterisks=p
of TP, albumin, and LDH in BALF. The PF-CVD group showed significantly increased TP (p<0.01) and albumin (p<0.05) concentrations compared with the healthy controls. The mean BALF ECP concentration was 106.2±35.8 ng/ L (range, <10 to 767.8 . ng/ L) in the patients with IPF and 63.5 ±18.8 ng/ L (range, <10 to 620.0 ng/ L) in those with PF-CVD (Fig 2) . The ECP concentration in both groups was higher than that in the controls (12.3 ± 1.7 ng/ L;
range, <10 to 31.0 ng/ L) and patients with sarcoidosis (29.3±9.3 ng/ L; range, <10 to 210.4 ng/ L), and the IPF group showed significant difference from the values in controls (p<0.01) and patients with sarcoidosis (p<0.05). The patients with sarcoidosis showed a relative increase in ECP concentration compared with that in the controls, but the difference was not significant. The median plllp concentration in BALF was 47.1 ± 25.0 ng/ L (range, <0.5 to 463.0 ng/ L) in the patients with IPF, 63.1 ±44.9 ng/ L (range, <0.5 to 826.4 ng / L) in those with PF-CVD , and 13.4±5.1 ng/ L (range, <0.5 to 85.5 ng/ L) in those with sarcoidosis; the piiip level was significantly higher than that in the controls (0.5 ± 0.1 ng/ L; range, <0.5 to 1.68 ng/ L).
Bronchoalveolar Lavage Cellularity, CD4 / CD8 Ratio, and Chemical Substances in the Three Subgroups of Patients With IPF and PF-CVD In the analysis according to the progression of pulmonary dysfunction in the patients with IPF and PF-CVD (Table 3), those in the acute progressive subgroup showed significantly increased counts of total cells (p<0.05), lymphocytes (p<0.01), and eosinophils (p<0.05) , and significantly decreased CD4/ CD8 ratio (p<0.05) compared with both the chronic stable and progressive subgroups. However, there were no significant differences between the chronic stable and progressive subgroups in any cell population or the CD4/ CD8 ratio. The acute progressive subgroup showed the high-
Table 3-Comparison of BALF Findings in the Three Subgroups of Patients With IPF and PF-CVD Classified
According to the Progression of Pulmonary Dysfunction*
Cell No. Total cells, X103 / mL Macrophage, X103 / mL (%) Lymphocyte, X103 / mL (%) Neutrophil, X103 / mL (%) Eosinophil, X103 / mL (%) CD4/ CD8 ratio Total protein, mg/ dl Albumin, !Lg/mL LDH, U/ L ECP, ng/L piiip, ng/ L
Chronic Stable (n=18)
Progressive (n=21 )
Acute Progressive (n =7)
284.6±65.2 214.3±46.6 (78.7±3.0) 51.9± 19.6 (15.9±2.8) 12.9±8.6 (3.0± 1.0) 5.6±2.2 (2.3 ±0 7) 2.6± 0.7 21.7±2.3 84.4± 11.3 36.9±8.6 24.1 ±5.6 1.7±0.5
214.9± 29.6 155.8±28.9 (72.1±4.5) 41.2±12.0 (189±4.0) 12.6±6.3 (6.2 ±2.5) 4.6± 1.3 (2.5± 0.7) 4.3 ±2.2 24.7±4. 1 82.4 ±9.3 58.4± 14.4 94.2±32.01 3.7 ± l.l
521.7±116.811 200.5± 17.9 (44.9± 5.7§11) 275.3 ± 121.3§11 (44.6±7.5§11) 18.6±8.4 (4.7 ±2.4) 23.7 ± 10.911 (4.9±2.0) 0.7±0.211 131.0 ± 42.5§11 603.2±243.9§11 101.0±31.7 237.6 ± 93.5H 337.7 ± 105.2§11
*Chronic Stable=VC~l5 % and % Dco~ 20% at 3 and 6 months; progressive=VC>I5% or %Dco >20% at 6 months but not at 3 months; acute progressive=VC> 15% or %Dco>20% at orwithin 3 months. Values are mean± SE. lp<0.05 vs chronic s table. lp<0.05 vs progressive. §p
51
(ng/L)
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Progr .. alve
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80
Eosinophil Count in BALF
80
3
100
(x10 cello/ml)
FIGURE 3. Relationship between the concentration of ECP and absolute recovered eosinophil count in individual patients with IPF and PF-CVD classified into the three subgroups of chronic stable (triangles), :::::15% VC and :::::20% Dco for 6 months; progressive (open circles),> 15% VC or >20% Dco for 6 months; and acute progressive (closed circles),> 15% VC or >20% Dco for <3 months.
est level of ECP as well as piiip, TP, albumin, and LDH concentration in BALF, corresponding to the disease activity. The acute progressive subgroup showed significantly elevated TP, albumin, ECP, and piiip in comparison with the chronic stable subgroup (all, p<0.01) and significantly increased TP (p<0.01), albumin (p<0.01) , ECP (p<0.05), and piiip (p<0.01) in comparison with the progressive subgroup. The latter subgroup also showed significantly increased ECP level compared with the chronic stable subgroup (p<0.05). Multivariate stepwise logistic regression revealed that , of all variables, including recovered cell analysis variables, only ECP and piiip significantly contributed to discrimination among the three subgroups (p=O.Oll and p<0.001, respectively), and piiip made the greatest contribution to discrimination according to the disease activity class. In patients with IPF and PF -CVD, there was a significant correlation of the BALF ECP level with the recovered eosinophil number (r=0.62; p<0.001) (Fig 3), the eosinophil count in tissue per high-powerfield (r=0.52, p<0.05) (Fig 1), the CD4/ CD8 ratio (r=-0.40, p<0.01), and the LDH (r=0.59, p<0.01) and piiip (r=0.45, p<0.01) (Fig 4) levels in BALF. The plllp values in patients with IPF and PF -CVD were significantly correlated with total recovered cells (r=0.54; p
Our finding of increased eosinophil number in 52
BALF in patients with pulmonary fibrosis, especially IPF , is in agreement with previous studies.l- 4 The recovered eosinophil count was significantly correlated with the eosinophil infiltration in lung tissue in the 16 open lung biopsy specimens. The role of these accumulated eosinophils in the lungs in patients with pulmonary fibrosis , however, has not been clearly elucidated. It was recently suggested that eosinophilia in BALF may be a marker of progressive lung disease in patients with IPF and PF-CVD 1-3 and can be used to identify patients with poor response to therapy. 1•2 Peterson et al 2 have reported that BAL eosinophilia, but not increased number of polymorphonuclear leukocytes in BAL, predicted a greater than 15% reduction in VC over a 6-month period in patients with IPF and PF -CVD. In this study, the activity of disease of IPF and PF-CVD was defined in accordance with the changes in VC and Dco at 3-month intervals over a 6-month period in patients with no immunosuppressive or steroid therapy. In the patients who showed acute progression of pulmonary dysfunction, the recovered eosinophil number and lymphocyte count in BALF were significantly higher than those in the chronic stable and progressive subgroups. However, there was no difference between the chronic stable and progressive subgroups in either the recovered eosinophil or lymphocyte count, and the eosinophil count did not contribute to discrimination between the two subgroups. The eosinophil represents a potent arsenal of highly cytotoxic products that may be involved in the development of tissue damage.5- 9 Eosinophil cationic protein , a specific constituent of the eosinophil granulocyte, is one of these cytotoxic components.l 0
ra0.45, p<0.01
(log(ng/L)) • Chronic Stobie 0 Progreaalve
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e
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FIGURE 4. Relationship of the concentration of ECP to piiip in BALF in individual patients with IPF and PF-CVD classified into the three subgroups as defined in the previous figure of chronic stable (triangles), progressive (open circles), and acute progressive (closed circles). Clinical Investigations
In this study, the significantly increased BALF ECP level in patients with IPF and PF -CVD was correlated with eosinophilia in BALF. Hallgren et aF 5 reported that increased ECP level in BALF was correlated with the eosinophil number in BALF in patients with IPF, and a significant correlation was found between BALF ECP and BALF myeloperoxide (MPO) or percent Dco. Although our study did not demonstrate any correlation of BALF ECP with percent VC (r=O.l3, NS), FEY 1% (r=O.OO, NS), or percent Dco (r=O.l9, NS) in patients with IPF and PF -CVD at the time that BALF was obtained, the ECP level showed a close association with the progression of pulmonary dysfunction . The patients who showed acute deterioration of pulmonary dysfunction showed the highest BALF ECP level, which was significantly increased compared with those in the chronic stable and progressive subgroups. The ECP level in the progressive subgroup was also significantly higher than that in the chronic stable subgroup. These findings are supported by the findings reported by Pohl et al, 16 who observed that the changes in ECP level paralleled the clinical course and that successful treatment resulted in a significant decrease of BALF ECP concentration, while clinical deterioration or treatment failure was associated with an increase of ECP level. We also evaluated the concentration of total protein, albumin, LDH, and piiip in BALF . Pilip, produced mainly by fibroblasts,l7,1B has been postulated to be a potential marker of activated fibroblasts or an expanded fibroblast mass associated with pulmonary fibrosis 11 ,1 2 early in the disease process. 19 It has been demonstrated that the levels of piiip in BALF increase in respect to the disease activity.l 2·20 A significantly larger amount of piiip was recovered in the patients with IPF, PF -CVD, and sarcoidosis compared with the healthy control subjects. In the patients with IPF and PF-CVD, the level of piiip increased in relation to the disease activity; significantly increased piiip, TP , and albumin levels, compared with the chronic stable and progressive subgroups, were observed in the acute progressive subgroup. Correlations of the ECP levels with LDH and with piiip and of the piiip levels with TP and albumin in BALF were also observed. Multivariate stepwise logistic regression analysis revealed that, of all the parameters, only BALF ECP and piiip levels significantly contributed to discrimination among the three subgroups. These findings suggest that the ECP and piiip levels in BALF are important indices of disease activity in patients with IPF and PF -CVD . The increased ECP levels in BALF reflect degranulation from activated eosinophils in the lung. These findings indicate that eosinophils are activated in the lung in patients with IPF and PF -CVD and that cy-
totoxic granule proteins, as reflected by ECP, are released from the activated eosinophils. Eosinophil infiltration and extracellular eosinophil granule deposition have been observed in fibrous tissue in various fibrotic disorders, including pulmonary fibrosis.6,7,9,15 These substances may cause tissue injury and participate in the development of pulmonary fibrosis. Eosinophil extracts may stimulate fibroblast proliferation21 and eosinophil-derived neurotoxin, an eosinophil granule protein, can also stimulate fibroblast proliferation.22 ECP has no direct action on fibroblast proliferation , but can alter proteoglycan metabolism , which may contribute to fibrosis in concert with other growth factors. 23 However, human lung fibroblast-derived granulocyte macrophage colony-stimulating factor mediates the in vitro survival of the human eosinophil. 24 There are apparent interactions between fibroblasts and eosinophils. Our study demonstrated the close association between elevated ECP levels in BALF and progression of pulmonary dysfunction, decreased CD4/ CD8 ratio, and elevated BALF LDH and piiip levels in IPF and PF -CVD , suggesting that eosinophils are involved in the inflammatory process in pulmonary fibrosis and that the released ECP and other cytotoxic eosinophil products may contribute to the lung injury and development of fibrosis. REFERENCES
1 Rudd RM, Haslam PL, Turner-Warwick M. Cryptogenic fibrosing alveolitis: relationships of pulmonary physiology and bronchoalveolar lavage to response to treatment and prognosis. Am Rev Respir Dis 1981; 124:1-8 2 Peterson MW, Monick M, Hunninghake GW. Prognostic role of eosinophils in pulmonary fibrosis. Chest 1987; 92:51-6 3 Watters LC, Schwarz Ml, Cherniack RM, et a!. Idiopathic pulmonary fibrosis: pretreatment bronchoalveolar lavage cellular constituents and their relationship with lung histopathology and clinical response to therapy. Am Rev Respir Dis 1987; 135:696-704 4 Schwartz DA, Helmers RA, Dayton CS, eta!. Determinants of bronchoalveolar lavage cellularity in idiopathic pulmonary fibrosis. J Appl Physiol1991 ; 71:1688-93 5 Ayars GH, Altman LC, Gleich G, et a!. Eosinophil and eosinophil granule mediated pneumocyte injury. J Allergy Clin Immunol1985; 76:595-604 6 Hallgren R, Samuelsson T, Venge P, eta!. Eosinophil activation in the lung is related to lung damage in adult respiratory distress syndrome. Am Rev Respir Dis 1987; 135:639-42 7 Tai P-C, Ackerman SJ, Spry CJF, eta!. Deposits of eosinophil granule proteins in cardiac tissues of patients with eosinophilic endomyocardial disease. Lancet 1987; 1:643-47 8 Fujimoto K, Yoshikawa S, Martin S, et a!. Oxygen radical scavengers protect against eosinophil-induced injury in isolated perfused rat lungs. J Appl Physiol 1992; 73:687-94 9 Noguchi H , Kephart GM, Colby TV, eta!. Tissue eosinophilia and eosinophil degranulation in syndrom es associated with fibrosis. Am J Pathol1992; 140:521-28 10 Venge P, Dahl R, Fredens K, eta!. Eosinophil cationic proteins in health and disease. In: Yoshida T, Torisu M, eds. Immunobiology of the eosinophil. New York: Elsevier Science Publish-
CHEST / 108 / 1/ JULY,1995
53
ing, 1983; 163-79 11 Bjermer L , Engstrom- Laurent A, Lundgren R, et al. Hyaluronate and type III procollagen peptide in bronchoalveolar lavage fluid as markers of disease activity in farmer 's lung. BMJ 1987; 295:803-06 12 Bjermer L, Lundgren R, Hallgren R. Hyaluronan and type III procollagen peptide concentrations in bronchoalveolar lavage fluid in idiopathic pulmonary fibrosis. Thorax 1989; 44:126-31 13 Baldwin EO, Cournand A, Richards DW. Pulmonary insufficiency: physiological classification, clinical methods of analysis, standard values in normal subjects. Medicine 1948; 27:243-78 14 Burrows BB, Kasik JE, Niden AH, et al. Clinical usefulness of the single-breath pulmonary diffusion capacity test. Am Rev Respir Dis 1961; 84:789-806 15 Hallgren R, Bjermer L, Lundgren R, et al. The eosinophil component of the alveolitis in idiopathic pulmonary fibrosis: signs of eosinophil activation in the lung are related to impaired lung function. Am Rev Respir Dis 1989; 139:373-77 16 Pohl WR, Schenk E, Umek H, et al. Diagnostic value of secretory products of eosinophils and neutrophils in bronchoalveolar lavage in patients with idiopathic lung fibrosis. Wien Klin Wochenschr 1993; 105:387-92 17 Rennard SI, Ferrans VJ, Bradley KH, et al. Lung connective
54
18 19
20
21 22
23
24
tissue. In: Witchi H, ed. Mechanisms in respiratory toxicology (vol 2). Boca Raton, Fla: CRC Reviews, 1982; 115-53 Laurent GJ. Lung collagen: more than scaffolding. Thorax 1986; 41:418-28 Kirk JME, Heard BE, Kerr I, et al. Quantitation of types I and Ill collagen in biopsy lung samples from patients with cryptogenic fibrosing alveolitis. Collagen Rei Res 1984; 4:169-82 Cantin AM, Boileau R, Begin R. Increased procollagen Ill aminoterminal peptide-related antigens and fibroblast growth signals in the lungs of patients with idiopathic pulmonary fibrosis. Am Rev Respir Dis 1988; 137:572-78 Pincus SH, Ramesh KS, Wyler DJ. Eosinophils stimulate fibroblast DNA synthesis. Blood 1987; 70:572-74 Noguchi H, Hoerl BJ, Gleich GJ. Eosinophil-derived neurotoxin (EDN) stimulates fibroblast proliferation [abstract]. F ASEB J 1990; 4:A1943 Hernnas J, Sarnstrand B, Lindroth P, et al. Eosinophil cationic protein alters proteoglycan metabolism in human lung fibrosis cultures. Eur J Cell Bioi 1992; 59:352-63 Vancheri C, Gauldie J, Bienenstock J, et al. Human lung fibroblast-derived granulocyte-macrophage colony stimulating factor (GM-CSF) mediates eosinophil survival in vitro. Am J Respir Cell Mol Bioi 1989; 1:289-95
Clinical Investigations
Jt has been shown that a relative large number of eosinophils are recovered in the bronchoalveolar lavage fluid (BALF) in patients with pulmonary fibrosis, 1-4 and the finding of increased number of eosinophils in BALF has been interpreted as a sign of poor prognosis.l-3 The role of these accumulated eosinophils in the pathogenesis of pulmonary fibrosis has recently been investigated. Eosinophils contain various cytotoxic substances and have been suggested as being implicated in tissue injury in various lung diseases. 5 -9 Eosinophil infiltration and the deposition of eosinophil granule proteins have also been shown in fibrous tissue in various fibrotic disorders. 7•9 Eosinophil cationic protein (ECP), a specific component of eosinophil granulocytes, is one of these cytotoxic substances.l 0 In this study, to evaluate the role of accumulated eosinophils in the lungs in the pathogenesis of fibrosis in patients with pulmonary fibrosis, we measured ECP in BALF as a marker of degranulation *From the First Department of Internal Medicine, Shinshu University School of Medicine Matsumoto, Japan. Manuscript received March 9, 1994; revision accepted December 1.
Reprint requests: Dr. Fujimoto, First Dept Internal Medicine, Shinsu University School of Medicine , 3-1-1 Asahi, Matsumoto, japan, 390
48
three subgroups differing in disease activity. These findings suggest that eosinophils are involved in the inflammatory process in pulmonary fibrosis and that the released ECP and other cytotoxic eosinophil products may contribute to the lung injury and development of fibrosis. (CHEST 1995; 108:48-54) BALF=bronchoalveolar lavage fluid; Dco=carbon monoxide diffusing capacity; ECP=eosinophil cationic protein; FEV1%=percent of forced expiratory volume in 1 s; IPF=idiopathic pulmonary fibrosis; LDH=Iactate dehydrogenase; PF-CVD=pulmonary fibrosis associated with a collagen vascular disorder; plllp=type III procollagen aminoterminal peptide-related antigen; TP=total protein; VC=vital capacity
Key words: bronchoalveolar lavage; eosinophil cationic protein; idiopathic pulmonary fibrosis; pulmonary fibrosis associated with a collagen vascular disorder; type III procollagen aminoterminal peptide-related antigen
from activated eosinophils. Furthermore, the associations between the ECP level and progression of pulmonary dysfunction during the 3- to 6-month period preceding the investigation and the levels of type III procollagen aminoterminal peptide-related antigens (piiip) in BALF, as a marker of fibroblast activation,ll·12 were examined. METHODS
The ECP and piiip in BALF were measured in 27 subjects with idiopathic pulmonary fibrosis (IPF) and 19 with pulmonary fibrosis associated with a collagen vascular disorder (PF-CVD), and, for comparison, in 24 patients with sarcoidosis and 13 healthy volunteers. The criteria for IPF or PF-CVD were as follows: (1) no clinical history of exposure to environmental agents known to cause interstitial lung disease, no history suggestive of extrinsic allergic alveoli tis, and no history of chronic lung infection or left ventricular failure; (2) evidence of interstitial infiltrates on chest radiograph and chest CT scan, and pulmonary function test results consistent with a restrictive ventilatory defect and decreased single breath carbon monoxide diffusing capacity (Dco); (3) biopsy specimen evidence of interstitial pneumonitis with varying degrees of interstitial fibrosis without evidence of granulomas. Among the 46 patients with IPF and PF-CVD, 16 underwent open lung biopsy. Of the 46 patients, 27 had the diagnosis of IPF alone and 19 had the diagnosis of PF -CVD. Of the latter patients, five had seropositive rheumatoid arthritis, two had polyarteritis nodosa, two had polymyositis, one had dermatomyositis, two had progressive systemic sclerosis, three had systemic lupus erytheClinical Investigations
Table !-Demographic and Physiologic Characteristics of the Study Population* Group IPF PF-CVD Sarcoidosis Healthy control
N(M, F) 27 19 24 13
(17, 10) (5, 14) (13, ll) (8, 5)
Age, yr 63±2 56±3 47±3 49±4
Smoking (S, EX) (6, (2, (4, (4,
13) 3) 3) O)
Pa02, mm Hg 74.1±2.1' 79.3±3.1 80.4 ± 1.8 86.9± 1.0
%VC 75.9±3.9 1 79.4±5.7' 99.1 ±3.7 110.0±3.8
FEV1%
%Dco
83:-S± 1.5_ 79.0±2.2 83.4±2.2 86.8±2.0
50.1±4.9 11 56.2±5.5'' 90.4±4.5 103.1±5.2
*S=smoker; EX=ex-smoker; values are mean± SE. f p<0.01 vs healthy controls. lp<0.01 vs sarcoidosis. matosus, one had mixed connective tissue disease, and three had Sjogren's syndrome. At the time of BAL, only two patients were receiving immunosuppressive or steroid treatment or cytotoxic drug; these two exceptional patients, one with rheumatoid arthritis and one with dermatomyositis, were receiving 10 mg/ d of prednisolone orally. The patients with IPF and PF-CVD were also classified into three subgroups based on the changes in vital capacity (VC) and Dco at 3-month intervals for up to 6 months before the investigation together with the clinical course and chest radiographic and / or CT findings. The chronic stable subgroup consisted of 18 patients who had a clinical course of more than 6 months with less than 15% change of VC and less than 20% change of Dco at 3 months and 6 months. The progressive subgroup consisted of 21 patients with > 15% decrease in VC or >20% decrease in Dco at 6 months but not at 3 months. The acute progressive subgroup consisted of seven patients who showed acute clinical course and increase in interstitial infiltrates of bilateral lungs with > 15% decrease in VC or >20% decrease in Dco at or within 3 months. The correlation of the eosinophil counts in tissue from 16 open lung biopsy specimens (10 in patients with IPF and 6 in patients with PF-CVD) with the recovered eosinophil numbers in BALF and with the ECP level was also examined. The tissue eosinophil count was expressed as the average count for 10 randomly selected high-power (X400) microscopic fields. For comparison, we also evaluated 24 untreated patients with sarcoidosis classified radio-
graphically according to thoracic involvement as having stage 1 (n=10), stage 2!1(n=10), or stage 3 (n =4) disease. In these patients, sarcoidosis had been diagnosed based on the medical history and the findings of physical examination, chest radiography and/ or CT, and histologic examination of the lung biopsy specimen. The demographic and physiologic data for each of four groups are summarized in Table l. The VC and percent of forced expiratory volume in 1 s (FEV 1%) were measured using a water spirometer (Godart Expirograph, Godart-Statham, Bilthoven, Holland). The Dco was measured by the single-breath method (Pulmocorder, model R1551S, Anima, Tokyo, Japan). The VC and Dco are expressed as the percentage of predicted values. 13,l 4 The subjects in all groups were defined as smokers if they currently smoked cigarettes, and as ex-smokers if they had a history of smoking but had not smoked within the preceding 6 months. Compared with that in the control group, the mean percent VC and percent Dco in the IPF and PF-CVD groups and the mean Pa02 in the IPF groups were significantly lower (all, p<0.01). Before bronchoscopy, patients and control subjects were administered atropine subcutaneously, usually combined with morphine .. The upper respiratory tract was anesthetized with 2% lidocaine. A fiberoptic bronchoscope (Olympus BF1 T, Olympus Co, Tokyo, Japan) was wedged in the middle lobe segmental bronchus, and 150 mL of sterile normal saline solution warmed to 37°C was infused in 50-mL boluses. The fluid was aspirated
Table 2-Findings for BALF in Patients With IPF, PF-CVD, Sarcoidosis, and Healthy Control Subjects*
Cell No. Total cells, Xl03 / mL Macrophage, X10S/ mL (%) Lymphocyte, X10S/ mL (%) Neutrophil, X103 / mL (%) Eosinophil, Xl03 / mL (%) CD4/ CD8 ratio Total protein, mg/ dL Albumin, ~tg / mL LDH, U/L ECP, ng/ L piiip, ng/ L
IPF (n=27)
PF-CVD (n=19)
Sarcoidosis (n=24)
Healthy Control (n=13)
260.3±39.1 179.2±22.4 (74.0±3.7') 55.2± 15.9 1 (17.1 ±2.919) 14.9 ± 7.1 !§ (48±1.9§) 9.60±3.27' (3.6±0.81§) 2.1 ±0.:91 30.7±8.6 134.2±50.3 53.5 ± 11.6 106.2±35.81§ 47.1 ±25.o'
328.2±67.2 192.6±43.9 (65.4±5.0') 117.8±50.91 (28.2±50 1) 12.0±4.41§ (4.7 ± 1.7) 5.73±2.39 1 (1.7±06') 4.4±2.511 54.6± 17.9 1 183.1 ±72.01 60.6±14.9 63.5± 18.81 63.1±44.9'
211.0±37.8 114.4±22.8 (66.5 ± 4.8 1) 87.3±23.8' (31.3±4.7 1) 2.3±0.7 (1.0±0.3) 2.95 ± 1.301 (1.2±0.61) 6.5± 1.4 34.8±7.3 138.5±36.1 60.1 ± 21.5 29.3±9.3 13.4±5.1'
147.5±59.3 139.0±56.6 (93.6±0.7) 7.0±2.4 (5.2±0.7) 1.5±0.5 (11±0.2) 0.03±0.01 (0.1 ±0.0) 3.8±0.9 16.1 ± 1.4 52.8±7.8 22.8±5.4 12.3 ± 1.7 0.5 ±0.1
*Values are mean±SE. fp<0.01 vs healthy control group. lp<0.05 vs healthy control group. §p<0.05 vs sarcoidosis group. llp <0.01 vs sarcoidosis group. CHEST / 108 / 1 /JULY,1995
49
Data Analysis
(cella/HPF)
30
•
•
•• j:::
•
:::J
.E
•
20
c :::J
0
u
1
10
'I • 3.1 + 0.27x, r • 0.14, pcO.Ot
c:
1w
40
20
60
80
Eosinophil Count In BALF
100
3 (x!0 cella/ml)
(cella/HPF)
30
•
• ••
j:::
.E
RESULTS
•
:::J
•
20
c
&
u
l
10
•
y • 4.3 + 2.5e-2x, r • 0.52, pc0.05
'i 0
w 200
400
600
800 (ng/l)
ECP In BALF
FIGURE l. Relationship of eosinophil count per high-powerfield (X400) in tissue from open lung biopsy specimens in 10 patients with IPF and 6 patients with PF-CVD to recovered eosinophil count (upper panel) and ECP concentration (lower panel) in BALF. under low suction immediately after each instillation, and then filtered through gauze. One small aliquot of this fluid was used to count the total cell number, and another aliquot was spun in a cytometer (500 rpm for 5 min) and stained by the May-Gri.inwaiiGiemsa method to identify the cell populations. Five hundred nonepithelial cells per slide were identified to establish the differential cell count (Xl,OOO, oil objective lens) with each cell type expressed as a percentage of the 500 cells and as the concentration in total recovered lavage fluid. The remaining BALF was centrifuged at 300xg for 10 min at 4°C, and the supernatant was removed. The pellets from BALF were subjected to analysis ofT-cell subsets of lymphocytes using flow cytometry with monoclonal antibodies to CD4 and CD8 (Becton Dickinson Co). The supernatant was concentrated to 50-fold (using an Amicon, division of W.R. Grace & Co, Danvers, Mass). This concentrated supernatant was provided for the measurements of ECP and PiliP. The ECP and piiip were measured in duplicate using 1251-ECP radioimmunoassay (RIA) kits (Pharmacia Diagnostics, Uppsala, Sweden) and 125 I-piiip RIA kit (Behringwerke AG, Marburg, Germany). The total protein (TP), albumin, and lactate dehydrogenase (LDH) concentrations in BALF were also measured. This study was performed according to the Declaration of Helsinki, and informed written consent was obtained first from all volunteers and patients.
50
The values shown in the text, tables, and figures are expressed as mean± SE. The data distribution of the variables in each group was first assessed using Bartlett's test. When the data for the variables showed normal distribution, the comparison was performed using one-way analysis of variance, and then multiple comparisons were performed by the Tukey-Kramer method. When the data for the variables did not show normal distribution, the variables were compared using Kruskal-Wallis test, and then multiple comparisons among groups were performed by the nonparametric Tukey-Kramer method. The patients with IPF and PF-CVD were classified into the three subgroups in accordance with disease activity described above. In the identification of the parameters contributing to the disease activity or the parameters distinguishing among the three subgroups, multivariate stepwise logistic regression was performed using a procedure (STEPDISC) with statistical software programs (SAS). The correlation between variables was examined by calculating Pearson's product correlation coefficient. A p value of less than 0.05 was considered significant for all statistical tests.
Bronchoalveolar Lavage Cellularity, CD4 / CD8 Ratio, and Chemical Substances in Patients With IPF, PF-CVD, and Sarcoidosis and Healthy Controls The total number of recovered cells per milliliter of BALF in all patient groups was higher than that in the control group (Table 2). In the differential cell count, the lymphocyte count was significantly increased in all patient groups compared with that in the healthy controls, and the highest mean value was seen in the PF -CVD group. The neutrophil count in the IPF and PF -CVD groups was significantly increased compared with that in the sarcoidosis and control group (all, p<0.05). Eosinophils were rarely detected in BALF from controls and did not exceed 0.3% in any control subject. In contrast, the eosinophil count was> 1% of the total cells recovered in 17 (63%) of the 27 patients with IPF, 6 (32%) of the 19 patients with PF-CVD, and 5 (21%) of the 24 patients with sarcoidosis; mean percent eosinophils was 3.6% (range, 0 to 13.0%), 1.7% (0 to 9.5%), and 1.2% (0 to 12.8%), respectively. Both the percent eosinophils and absolute eosinophil count in the patients with IPF were significantly higher than those in the controls (p<0.01), and a significant increase in the percent eosinophils in the IPF group compared with that in the sarcoidosis group was also observed (p<0.05). The percent and number of eosinophils in BALF were significantly correlated with the eosinophil count per high-power field in tissue obtained at open lung biopsy in the 16 patients with IPF and PF-CVD (r=0.64, p<0.01) (Fig 1). The CD4/ CD8 ratio in the patients with IPF was significantly lower than that in the patients with sarcoidosis who showed slightly elevated ratio compared with the healthy controls. All patient groups showed elevated concentrations Clinical Investigations
(ng/L)
800
II.
600
=
400
..J c( ID
~
u
.. + 0
0
0
w 200
0 0 0
IPF
0
PF-CVD Sarcoldoala Control
FIGURE 2. The ECP concentrations in BALF in individual patients with idiopathic pulmonary fibrosis (IPF, n=27), pulmonary fibrosis associated with a collagen vascular disorder (PF-CVD, n=l9), and sarcoidosis (n=24), and in healthy control subjects (n= 13). The horizontal bar represents the mean value in each group. Asterisk=p<0.05; two asterisks=p
of TP, albumin, and LDH in BALF. The PF-CVD group showed significantly increased TP (p<0.01) and albumin (p<0.05) concentrations compared with the healthy controls. The mean BALF ECP concentration was 106.2±35.8 ng/ L (range, <10 to 767.8 . ng/ L) in the patients with IPF and 63.5 ±18.8 ng/ L (range, <10 to 620.0 ng/ L) in those with PF-CVD (Fig 2) . The ECP concentration in both groups was higher than that in the controls (12.3 ± 1.7 ng/ L;
range, <10 to 31.0 ng/ L) and patients with sarcoidosis (29.3±9.3 ng/ L; range, <10 to 210.4 ng/ L), and the IPF group showed significant difference from the values in controls (p<0.01) and patients with sarcoidosis (p<0.05). The patients with sarcoidosis showed a relative increase in ECP concentration compared with that in the controls, but the difference was not significant. The median plllp concentration in BALF was 47.1 ± 25.0 ng/ L (range, <0.5 to 463.0 ng/ L) in the patients with IPF, 63.1 ±44.9 ng/ L (range, <0.5 to 826.4 ng / L) in those with PF-CVD , and 13.4±5.1 ng/ L (range, <0.5 to 85.5 ng/ L) in those with sarcoidosis; the piiip level was significantly higher than that in the controls (0.5 ± 0.1 ng/ L; range, <0.5 to 1.68 ng/ L).
Bronchoalveolar Lavage Cellularity, CD4 / CD8 Ratio, and Chemical Substances in the Three Subgroups of Patients With IPF and PF-CVD In the analysis according to the progression of pulmonary dysfunction in the patients with IPF and PF-CVD (Table 3), those in the acute progressive subgroup showed significantly increased counts of total cells (p<0.05), lymphocytes (p<0.01), and eosinophils (p<0.05) , and significantly decreased CD4/ CD8 ratio (p<0.05) compared with both the chronic stable and progressive subgroups. However, there were no significant differences between the chronic stable and progressive subgroups in any cell population or the CD4/ CD8 ratio. The acute progressive subgroup showed the high-
Table 3-Comparison of BALF Findings in the Three Subgroups of Patients With IPF and PF-CVD Classified
According to the Progression of Pulmonary Dysfunction*
Cell No. Total cells, X103 / mL Macrophage, X103 / mL (%) Lymphocyte, X103 / mL (%) Neutrophil, X103 / mL (%) Eosinophil, X103 / mL (%) CD4/ CD8 ratio Total protein, mg/ dl Albumin, !Lg/mL LDH, U/ L ECP, ng/L piiip, ng/ L
Chronic Stable (n=18)
Progressive (n=21 )
Acute Progressive (n =7)
284.6±65.2 214.3±46.6 (78.7±3.0) 51.9± 19.6 (15.9±2.8) 12.9±8.6 (3.0± 1.0) 5.6±2.2 (2.3 ±0 7) 2.6± 0.7 21.7±2.3 84.4± 11.3 36.9±8.6 24.1 ±5.6 1.7±0.5
214.9± 29.6 155.8±28.9 (72.1±4.5) 41.2±12.0 (189±4.0) 12.6±6.3 (6.2 ±2.5) 4.6± 1.3 (2.5± 0.7) 4.3 ±2.2 24.7±4. 1 82.4 ±9.3 58.4± 14.4 94.2±32.01 3.7 ± l.l
521.7±116.811 200.5± 17.9 (44.9± 5.7§11) 275.3 ± 121.3§11 (44.6±7.5§11) 18.6±8.4 (4.7 ±2.4) 23.7 ± 10.911 (4.9±2.0) 0.7±0.211 131.0 ± 42.5§11 603.2±243.9§11 101.0±31.7 237.6 ± 93.5H 337.7 ± 105.2§11
*Chronic Stable=VC~l5 % and % Dco~ 20% at 3 and 6 months; progressive=VC>I5% or %Dco >20% at 6 months but not at 3 months; acute progressive=VC> 15% or %Dco>20% at orwithin 3 months. Values are mean± SE. lp<0.05 vs chronic s table. lp<0.05 vs progressive. §p
51
(ng/L)
•
0
BOO
u.
e
Chronic Stable
Progr .. alve
•
Acute Progre .. lve
0
BOO
..J ~
Ill
.:
400
a.
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0
r•0.62, p<0.001
•
•
oc!' ~
0
Q.o• 0
0
20
40
80
Eosinophil Count in BALF
80
3
100
(x10 cello/ml)
FIGURE 3. Relationship between the concentration of ECP and absolute recovered eosinophil count in individual patients with IPF and PF-CVD classified into the three subgroups of chronic stable (triangles), :::::15% VC and :::::20% Dco for 6 months; progressive (open circles),> 15% VC or >20% Dco for 6 months; and acute progressive (closed circles),> 15% VC or >20% Dco for <3 months.
est level of ECP as well as piiip, TP, albumin, and LDH concentration in BALF, corresponding to the disease activity. The acute progressive subgroup showed significantly elevated TP, albumin, ECP, and piiip in comparison with the chronic stable subgroup (all, p<0.01) and significantly increased TP (p<0.01), albumin (p<0.01) , ECP (p<0.05), and piiip (p<0.01) in comparison with the progressive subgroup. The latter subgroup also showed significantly increased ECP level compared with the chronic stable subgroup (p<0.05). Multivariate stepwise logistic regression revealed that , of all variables, including recovered cell analysis variables, only ECP and piiip significantly contributed to discrimination among the three subgroups (p=O.Oll and p<0.001, respectively), and piiip made the greatest contribution to discrimination according to the disease activity class. In patients with IPF and PF -CVD, there was a significant correlation of the BALF ECP level with the recovered eosinophil number (r=0.62; p<0.001) (Fig 3), the eosinophil count in tissue per high-powerfield (r=0.52, p<0.05) (Fig 1), the CD4/ CD8 ratio (r=-0.40, p<0.01), and the LDH (r=0.59, p<0.01) and piiip (r=0.45, p<0.01) (Fig 4) levels in BALF. The plllp values in patients with IPF and PF -CVD were significantly correlated with total recovered cells (r=0.54; p
Our finding of increased eosinophil number in 52
BALF in patients with pulmonary fibrosis, especially IPF , is in agreement with previous studies.l- 4 The recovered eosinophil count was significantly correlated with the eosinophil infiltration in lung tissue in the 16 open lung biopsy specimens. The role of these accumulated eosinophils in the lungs in patients with pulmonary fibrosis , however, has not been clearly elucidated. It was recently suggested that eosinophilia in BALF may be a marker of progressive lung disease in patients with IPF and PF-CVD 1-3 and can be used to identify patients with poor response to therapy. 1•2 Peterson et al 2 have reported that BAL eosinophilia, but not increased number of polymorphonuclear leukocytes in BAL, predicted a greater than 15% reduction in VC over a 6-month period in patients with IPF and PF -CVD. In this study, the activity of disease of IPF and PF-CVD was defined in accordance with the changes in VC and Dco at 3-month intervals over a 6-month period in patients with no immunosuppressive or steroid therapy. In the patients who showed acute progression of pulmonary dysfunction, the recovered eosinophil number and lymphocyte count in BALF were significantly higher than those in the chronic stable and progressive subgroups. However, there was no difference between the chronic stable and progressive subgroups in either the recovered eosinophil or lymphocyte count, and the eosinophil count did not contribute to discrimination between the two subgroups. The eosinophil represents a potent arsenal of highly cytotoxic products that may be involved in the development of tissue damage.5- 9 Eosinophil cationic protein , a specific constituent of the eosinophil granulocyte, is one of these cytotoxic components.l 0
ra0.45, p<0.01
(log(ng/L)) • Chronic Stobie 0 Progreaalve
3
e
Acute Prograaalva
II. ..J
0
••
~
Ill
.: ii:' u !!
0
0
• • 8 • ~ • •
2'
•
0
••
0
0
0
0
0
01
0 ..J
0
•
•
0
0
0
1
. .~~~~r-~~~~~~~~
+-~~~
-1
2
0
Log (plllp) in BALF
3
(log(ng/L))
FIGURE 4. Relationship of the concentration of ECP to piiip in BALF in individual patients with IPF and PF-CVD classified into the three subgroups as defined in the previous figure of chronic stable (triangles), progressive (open circles), and acute progressive (closed circles). Clinical Investigations
In this study, the significantly increased BALF ECP level in patients with IPF and PF -CVD was correlated with eosinophilia in BALF. Hallgren et aF 5 reported that increased ECP level in BALF was correlated with the eosinophil number in BALF in patients with IPF, and a significant correlation was found between BALF ECP and BALF myeloperoxide (MPO) or percent Dco. Although our study did not demonstrate any correlation of BALF ECP with percent VC (r=O.l3, NS), FEY 1% (r=O.OO, NS), or percent Dco (r=O.l9, NS) in patients with IPF and PF -CVD at the time that BALF was obtained, the ECP level showed a close association with the progression of pulmonary dysfunction . The patients who showed acute deterioration of pulmonary dysfunction showed the highest BALF ECP level, which was significantly increased compared with those in the chronic stable and progressive subgroups. The ECP level in the progressive subgroup was also significantly higher than that in the chronic stable subgroup. These findings are supported by the findings reported by Pohl et al, 16 who observed that the changes in ECP level paralleled the clinical course and that successful treatment resulted in a significant decrease of BALF ECP concentration, while clinical deterioration or treatment failure was associated with an increase of ECP level. We also evaluated the concentration of total protein, albumin, LDH, and piiip in BALF . Pilip, produced mainly by fibroblasts,l7,1B has been postulated to be a potential marker of activated fibroblasts or an expanded fibroblast mass associated with pulmonary fibrosis 11 ,1 2 early in the disease process. 19 It has been demonstrated that the levels of piiip in BALF increase in respect to the disease activity.l 2·20 A significantly larger amount of piiip was recovered in the patients with IPF, PF -CVD, and sarcoidosis compared with the healthy control subjects. In the patients with IPF and PF-CVD, the level of piiip increased in relation to the disease activity; significantly increased piiip, TP , and albumin levels, compared with the chronic stable and progressive subgroups, were observed in the acute progressive subgroup. Correlations of the ECP levels with LDH and with piiip and of the piiip levels with TP and albumin in BALF were also observed. Multivariate stepwise logistic regression analysis revealed that, of all the parameters, only BALF ECP and piiip levels significantly contributed to discrimination among the three subgroups. These findings suggest that the ECP and piiip levels in BALF are important indices of disease activity in patients with IPF and PF -CVD . The increased ECP levels in BALF reflect degranulation from activated eosinophils in the lung. These findings indicate that eosinophils are activated in the lung in patients with IPF and PF -CVD and that cy-
totoxic granule proteins, as reflected by ECP, are released from the activated eosinophils. Eosinophil infiltration and extracellular eosinophil granule deposition have been observed in fibrous tissue in various fibrotic disorders, including pulmonary fibrosis.6,7,9,15 These substances may cause tissue injury and participate in the development of pulmonary fibrosis. Eosinophil extracts may stimulate fibroblast proliferation21 and eosinophil-derived neurotoxin, an eosinophil granule protein, can also stimulate fibroblast proliferation.22 ECP has no direct action on fibroblast proliferation , but can alter proteoglycan metabolism , which may contribute to fibrosis in concert with other growth factors. 23 However, human lung fibroblast-derived granulocyte macrophage colony-stimulating factor mediates the in vitro survival of the human eosinophil. 24 There are apparent interactions between fibroblasts and eosinophils. Our study demonstrated the close association between elevated ECP levels in BALF and progression of pulmonary dysfunction, decreased CD4/ CD8 ratio, and elevated BALF LDH and piiip levels in IPF and PF -CVD , suggesting that eosinophils are involved in the inflammatory process in pulmonary fibrosis and that the released ECP and other cytotoxic eosinophil products may contribute to the lung injury and development of fibrosis. REFERENCES
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