Serum pulmonary and activation-regulated chemokineCCL18 levels in patients with systemic sclerosisA sensitive indicator of active pulmonary fibrosis.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 52, No. 9, September 2005, pp 2889–2896 DOI 10.1002/art.21257 © 2005, American College of Rheumatology Serum Pulmonary and Activation-Regulated Chemokine/CCL18 Levels in Patients With Systemic Sclerosis A Sensitive Indicator of Active Pulmonary Fibrosis Masanari Kodera,1 Minoru Hasegawa,1 Kazuhiro Komura,1 Koichi Yanaba,1 Kazuhiko Takehara,1 and Shinichi Sato2 Objective. To clarify the clinical significance of serum levels of pulmonary and activation-regulated chemokine (PARC) in the diagnosis and monitoring of pulmonary fibrosis (PF) in patients with systemic sclerosis (SSc) and to compare PARC levels with KL-6 antigen or surfactant protein D (SP-D) levels. Methods. Serum PARC levels were determined by enzyme-linked immunosorbent assay in 123 SSc patients. In a retrospective longitudinal study, correlation of serum PARC levels with the activity of PF was assessed in 21 SSc patients with active PF. Results. PARC levels at the first visit were higher in patients with SSc than in patients with systemic lupus erythematosus (SLE) or healthy controls. Increased serum PARC levels were associated with involvement of PF, decreased diffusing capacity for carbon monoxide, and decreased vital capacity in SSc patients. In the longitudinal study, serum PARC levels were significantly decreased in SSc patients with inactive PF compared with those with active PF. Conclusion. Elevated serum PARC levels correlated with PF and more sensitively reflected the PF activity than did serum KL-6 or SP-D levels in SSc. Serum PARC levels may be a useful new serum marker for active PF in SSc. Systemic sclerosis (SSc) is a connective tissue disease characterized by sclerotic changes in the skin and internal organs. Pulmonary fibrosis (PF) develops in more than 50% of SSc patients and is the major cause of death (1). To assess the activity of PF, previous studies have identified several important signs, including patchy areas with a ground-glass or reticular appearance on high-resolution computed tomography (HRCT) and neutrophilic alveolitis on analysis of bronchoalveolar lavage (BAL) fluid (1). However, easier, less-invasive, and lung-specific serologic markers would be helpful for closely monitoring the activity of PF in SSc patients. KL-6 and surfactant protein D (SP-D) may be the most reliable serum markers at present. KL-6 antigen is expressed mainly on type II pneumocytes in alveoli and respiratory bronchiolar epithelial cells (2), whereas SP-D is produced and secreted by alveolar type II pneumocytes in alveoli and Clara cells (3). Levels of KL-6 and SP-D are elevated in the sera of patients with interstitial lung diseases, including PF related to SSc (2,4). Recent studies have shown that serum levels of KL-6 and SP-D are a serologic marker of the severity and activity of PF in SSc (5–7). However, in some SSc patients with active PF, we sometimes found discrepancies in the serum levels of these 2 markers, as well as elevated KL-6 and SP-D levels despite improvements in clinical symptoms, HRCT findings, and pulmonary function test results after treatment with immunosuppressive agents. Therefore, another serum marker that more closely reflects PF activity is needed. Pulmonary and activation-regulated chemokine (PARC) might be a candidate marker. Also known as 1 Masanari Kodera, MD, Minoru Hasegawa, MD, PhD, Kazuhiro Komura, MD, PhD, Koichi Yanaba, MD, PhD, Kazuhiko Takehara, MD, PhD: Kanazawa University Graduate School of Medical Science, Kanazawa, Japan; 2Shinichi Sato, MD, PhD: Kanazawa University Graduate School of Medical Science, Kanazawa, Japan, and Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan. Address correspondence and reprint requests to Shinichi Sato, MD, PhD, Department of Dermatology, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki 852-8501, Japan. E-mail: firstname.lastname@example.org. Submitted for publication October 7, 2004; accepted in revised form June 3, 2005. 2889 2890 KODERA ET AL macrophage inflammatory protein 4 (MIP-4), alternative macrophage activation-associated CC chemokine 1, dendritic cell chemokine 1, CC chemokine ligand 18, small secreted cytokine A-18, and chemokine ␤7, PARC is structurally most closely related to MIP-1␣ (8,9), a chemotactic factor for T lymphocytes. PARC is constitutively expressed at high levels in the lung (9), particularly by alveolar macrophages (10,11). A recent study has shown that PARC stimulates collagen messenger RNA (mRNA) and protein production by dermal and lung fibroblasts (12). Furthermore, levels of PARC protein in BAL fluid were found to be highly increased in SSc patients with active PF compared with those without PF and compared with healthy controls (12,13). PARC protein and mRNA levels are also elevated in the lungs of patients with interstitial lung diseases (10,14,15). These findings suggest that PARC is an important cause of immune-mediated fibrotic lung diseases. Therefore, PARC secreted mainly from alveolar macrophages in the lung may be detectable in serum and, if so, could become a new lung-specific serologic marker in SSc. To test this possibility, we evaluated serum levels of PARC and examined their correlation with clinical features in patients with SSc. MATERIALS AND METHODS Serum samples. Serum samples were obtained from 123 Japanese patients with SSc (106 women and 17 men). All patients fulfilled the criteria proposed by the American College of Rheumatology (ACR; formerly, the American Rheumatism Association) (16). Patients were grouped according to the degree of skin involvement, based on the classification system proposed by LeRoy et al (17): 70 patients (67 women and 3 men) had limited cutaneous SSc (lcSSc), and 53 patients (39 women and 14 men) had diffuse cutaneous SSc (dcSSc). The age of the SSc patients was 51 ⫾ 14 years (mean ⫾ SD). The disease duration in those with lcSSc was 8.8 ⫾ 9.8 (mean ⫾ SD) and the duration in those with dcSSc was 4.4 ⫾ 6.6 years. None of the SSc patients received any treatment, including corticosteroids, D-penicillamine, or other immunosuppressive therapy at their first visit. Antinuclear antibody was determined by indirect immunofluorescence using HEp-2 cells as the substrate. Autoantibody specificities were further assessed by enzyme-linked immunosorbent assay (ELISA) and immunoprecipitation. Anticentromere antibody was positive in 50 patients, anti– topoisomerase I antibody was positive in 42, anti–U1 RNP antibody was positive in 9, anti–U3 RNP antibody was positive in 2, anti–RNA polymerases I and III antibody were positive in 4, anti-Th/To antibody was positive in 3, and antinuclear antibody of unknown specificity was positive in 7. Six patients were negative for autoantibodies. Twenty-one patients with systemic lupus erythematosus (SLE) (19 women and 2 men; mean ⫾ SD age 34 ⫾ 9 years) who fulfilled the ACR criteria (18) were also evaluated as disease controls. In addition, 37 age- and sex-matched healthy Japanese persons (32 women and 5 men; mean ⫾ SD age 49 ⫾ 10 years) served as normal controls. Samples of venous blood were drawn, allowed to clot, and centrifuged shortly after clot formation. Sera were removed, and all samples were stored at –70°C prior to use. Clinical assessments. Complete medical histories, physical examinations, and laboratory tests were conducted on all patients. The degree of skin involvement was determined according to the modified Rodnan skin thickness score, as described elsewhere (19). Organ system involvement was defined as described previously (6). Abnormal values for vital capacity (VC) and diffusing capacity for carbon monoxide (DLCO) were considered to be ⬍80% and ⬍75%, respectively, of the predicted normal values. The activity of the PF was initially determined by HRCT of the chest, pulmonary function testing, and BAL fluid analysis. Specifically, PF was considered to be active when the following 3 criteria were met: a ground-glass appearance or reticular pattern on HRCT of the chest (20), ⬎10% change in VC or ⬎15% change in the DLCO within 1 year (21), and ⬎3.0% neutrophils or 2.2% eosinophils on BAL fluid analysis (22). PF activity was monitored by serial HRCT scans of the chest (findings scored according to the HRCT scoring system ) and by pulmonary function testing, as previously described (24–26). In the HRCT scoring system (23), each lobe of the lung was scored separately for the extent of ground-glass opacity (ground-glass appearance) and reticular opacities and honeycombing (fibrosis) using a 0–5 scale, where 0 ⫽ absent, 1 ⫽ ⬍5%, 2 ⫽ 5–25%, 3 ⫽ 25–50%, 4 ⫽ 50–75%, and 5 ⫽ ⬎75%. A fibrotic score and a ground-glass score were calculated for each patient by determining the mean value for each feature in all lobes. The study protocol was approved by the Kanazawa University Graduate School of Medical Science and the Kanazawa University Hospital. Informed consent was obtained from all study participants. Determination of PARC levels. Serum levels of PARC were measured with a specific ELISA kit (R&D Systems, Minneapolis, MN), according to the manufacturer’s protocol. Briefly, 96-well plates were coated overnight at 25°C with mouse anti-human PARC monoclonal antibodies and were then blocked with phosphate buffered saline containing 1% bovine serum albumin and 5% sucrose. Serum samples diluted to 1:100 were added in duplicate, and the plates were incubated for 2 hours at 20°C. After washing, color was developed with biotinylated goat anti-human PARC monoclonal antibodies and streptavidin–peroxidase. The detection limit of this assay was 10 pg/ml. Determination of KL-6 and SP-D levels. Serum levels of KL-6 and SP-D were measured with specific ELISA kits (Eitest KL-6 from Eisai, Tokyo, Japan; SP-D kit from Yamasa, Chiba, Japan), according to the manufacturers’ protocols. Briefly, 96-well plates were coated with monoclonal antibodies to KL-6 or SP-D, and the diluted serum samples were added to duplicate wells. After washing, the bound antibodies were detected with peroxidase-conjugated monoclonal antibodies against KL-6 or SP-D. The cutoff values for positivity were 500 units/ml for KL-6 and 110 ng/ml for SP-D (27,28). SERUM PARC LEVELS IN SSC 2891 Statistical analysis. Statistical analysis was performed using the Mann-Whitney U test and Wilcoxon’s signed rank test for comparison of sample means, Fisher’s exact probability test for comparison of frequencies, and Bonferroni’s test for multiple comparisons. Spearman’s rank correlation coefficient was used to examine the relationship between 2 continuous variables. P values less than 0.05 were considered statistically significant. All values are reported as the mean ⫾ SD. RESULTS Serum levels of PARC in SSc patients at the first visit. The 123 SSc patients had significantly higher serum PARC levels at the first visit (mean ⫾ SD 78.2 ⫾ 34.9 ng/ml) compared with the levels in the normal control subjects (35.9 ⫾ 17.2) (P ⬍ 0.0001) and in the SLE control patients (51.4 ⫾ 17.8) (P ⬍ 0.002) (Figure 1). PARC levels in the sera of dcSSc patients (90.2 ⫾ 36.9) were significantly increased compared with those in the normal controls and in the SLE patients (P ⬍ 0.0001 for both comparisons). Serum PARC levels in the lcSSc patients (69.2 ⫾ 30.7) were also significantly elevated relative to those in the normal controls (P ⬍ 0.0001) and the SLE patients (P ⬍ 0.01). Patients with dcSSc had higher serum PARC levels than did patients with lcSSc (P ⬍ 0.01). PARC levels in the SLE control patients were also significantly increased compared with those in the normal control subjects (P ⬍ 0.05). Frequency of elevated serum PARC levels and correlation with clinical features in SSc. The upper limit of the normal range of serum PARC levels was determined as the mean ⫹ 2 SD of the levels in the healthy control subjects (70.3 ng/ml). Elevated serum PARC levels were observed in 56% of the SSc patients (69 of 123), 66% of the dcSSc patients (35 of 53), and 49% of lcSSc patients (34 of 70). The levels were not elevated in the normal controls and were increased in only 14% of the SLE patient controls (3 of 21) (Figure 1). With regard to the disease stage, 57% of the SSc patients with normal levels of PARC (31 of 54 patients) and 70% of those with elevated levels (48 of 69 patients) had early disease (disease duration ⬍5 years) (Table 1). With regard to the disease pattern, 31% of the SSc patients with normal levels of PARC (17 of 54 patients) and 52% of those with elevated levels (36 of 69 patients) had dcSSc. SSc patients with elevated PARC levels had a significantly increased prevalence of PF and significantly decreased VC and DLCO values (% predicted) relative to those in SSc patients with normal PARC levels (P ⬍ 0.005 for all comparisons). Anti– topoisomerase I antibody was more frequent in SSc patients with elevated levels of PARC than in those with Figure 1. Levels of pulmonary and activation-regulated chemokine (PARC) in serum samples from patients with diffuse cutaneous systemic sclerosis (dcSSc), limited cutaneous SSc (lcSSc), and systemic lupus erythematosus (SLE) as well as healthy controls (CTL). Serum PARC levels were determined by enzyme-linked immunosorbent assay. Broken line indicates the mean ⫹ 2 SD level of PARC in sera from normal controls. Bars show the group means. normal levels (P ⬍ 0.005), whereas anticentromere antibody was less frequent in SSc patients with elevated levels of PARC than in those with normal levels (P ⬍ 0.05). Furthermore, serum levels of PARC correlated inversely with DLCO (r ⫽ –0.326, P ⬍ 0.001) and with VC (r ⫽ –0.244, P ⬍ 0.01) values (Figure 2). Correlation between serum PARC levels and PF activity. To determine whether the changes in serum PARC levels correlated with the activity of PF, we analyzed serum samples obtained at the time of active 2892 KODERA ET AL Table 1. Clinical and laboratory features of SSc patients, grouped according to serum PARC levels* Sex, no. males/females Age, mean ⫾ SD years Disease duration, mean ⫾ SD years Disease stage, no. with early/late disease Disease pattern, no. with dcSSc/lcSSc Clinical features MRSS, mean ⫾ SD Digital pitting scars or ulcers Contracture of phalanges Diffuse pigmentation Organ involvement Lungs Pulmonary fibrosis VC, mean ⫾ SD % predicted DLCO, mean ⫾ SD % predicted Pulmonary hypertension Esophagus Heart Kidneys Joints Muscles Laboratory findings Anti–topoisomerase I antibodies Anticentromere antibodies Elevated ESR Elevated CRP Elevated IgG Elevated PARC (n ⫽ 69) Normal PARC (n ⫽ 54) 10/59 46.4 ⫾ 16.4 6.8 ⫾ 9.4 48/21 36/33† 7/47 43.3 ⫾ 14.4 7.0 ⫾ 8.0 31/23 17/37 12.0 ⫾ 10.6 41 45 51 9.5 ⫾ 8.1 28 42 42 59‡ 90.0 ⫾ 21.6‡ 54.8 ⫾ 14.6‡ 11 49 20 23 23 14 27 102.7 ⫾ 24.7 65.6 ⫾ 16.2 11 49 9 9 28 17 42‡ 31† 25 19 46 18 57 36 15 30 * Except where indicated otherwise, values are percentages. SSc ⫽ systemic sclerosis; PARC ⫽ pulmonary and activation-regulated chemokine; dcSSc ⫽ diffuse cutaneous SSc; lcSSc ⫽ limited cutaneous SSc; MRSS ⫽ modified Rodnan skin thickness score; VC ⫽ vital capacity; DLCO ⫽ diffusing capacity for carbon monoxide; ESR ⫽ erythrocyte sedimentation rate; CRP ⫽ C-reactive protein. † P ⬍ 0.05. ‡ P ⬍ 0.005. Figure 2. Correlation of serum levels of pulmonary and activation-regulated chemokine (PARC) with vital capacity (VC) and with diffusing capacity for carbon monoxide (DLCO) (% predicted) in patients with systemic sclerosis (SSc). Serum PARC levels were determined by enzyme-linked immunosorbent assay. SERUM PARC LEVELS IN SSC 2893 Figure 3. Changes in serum levels of pulmonary and activation-regulated chemokine (PARC), KL-6 antigen, and surfactant protein D (SP-D) during active and inactive pulmonary fibrosis (PF) in patients with systemic sclerosis (SSc). Serum samples were obtained during an active phase and an inactive phase of PF in 21 SSc patients who had anti–topoisomerase I antibodies. PARC, KL-6, and SP-D levels were determined by enzyme-linked immunosorbent assay. Broken lines indicate cutoff values for normal levels. and inactive phases of PF in 21 SSc patients who had anti–topoisomerase I antibody (Figure 3). These patients initially exhibited active PF by HRCT (mean ⫾ SD fibrosis score 1.9 ⫾ 0.9 and mean ⫾ SD ground-glass score 2.1 ⫾ 0.8), pulmonary function testing, and BAL fluid analysis. The disease duration in 17 of these 21 patients was ⬍5 years (mean 2.1 years), and the mean followup period was 4.9 years (range 2–9 years). Changes in serum PARC levels in these 21 patients were also compared with changes in serum KL-6 and SP-D levels. Serum levels of PARC, KL-6, and SP-D decreased significantly in parallel with an improvement in PF activity (P ⬍ 0.0001, P ⬍ 0.05, and P ⬍ 0.001, respectively). However, serum PARC levels increased in only 1 of the 21 patients during inactive PF (fibrosis score 1.7 ⫾ 0.9; ground-glass score 1.5 ⫾ 1.3), whereas 7 patients and 5 patients had increased KL-6 and SP-D levels, respectively, during inactive PF. Two patients (patients 1 and 2) exhibited a drastic decrease in serum PARC levels in parallel with a significant improvement in the PF activity. Patient 1 showed a slight increase in KL-6 and SP-D levels in the presence of mild PF on HRCT of the chest at the first visit (Figure 4A). Seven months after the first visit, KL-6 and SP-D levels increased in parallel with a subacute deterioration of the PF activity. On HRCT of the chest, the ground-glass appearance and reticular shadow were increased. Like the KL-6 and SP-D levels, the serum PARC level gradually increased to 83 ng/ml (from 71 ng/ml at the first visit). Intravenous pulse cyclophosphamide treatment was started, followed by an increase in the oral prednisolone dosage and initiation of oral cyclosporine. The PF activity stabilized, and the serum PARC level rapidly decreased to the normal range. The levels of KL-6 and SP-D, however, increased during the 5 months following the last cyclophosphamide pulse treatment and finally began to decrease 6 months later. Patient 2 had high levels of KL-6 and SP-D, a ground-glass appearance on HRCT of the chest, and decreased DLCO and VC values at the first visit (Figure 4B). Pulse therapy with cyclophosphamide and oral prednisolone were initiated. Levels of KL-6 and SP-D gradually began to decrease, but at 8 months after cyclophosphamide treatment, when progression of PF activity had ceased, neither was below the normal range. 2894 KODERA ET AL Figure 4. Serial changes in serum levels of pulmonary and activation-regulated chemokine (PARC), KL-6 antigen, and surfactant protein D (SP-D) during the followup period in 2 patients with diffuse cutaneous systemic sclerosis. PARC, KL-6, and SP-D levels were determined by enzyme-linked immunosorbent assay. %VC ⫽ vital capacity (% predicted); %DLCO ⫽ diffusing capacity for carbon monoxide (% predicted); HRCT ⫽ high-resolution computed tomography (of the chest; see Patients and Methods for the derivation of the fibrosis and ground-glass scores). However, the serum PARC level was increased at 108 ng/ml at the first visit, and after the first cyclophosphamide pulse, it rapidly decreased to the normal range. DISCUSSION In the present study, serum PARC levels were elevated in association with PF activity in patients with SSc. This study is the first to evaluate serum levels of PARC in a large number of SSc patients. Recent studies have shown that levels of PARC protein are increased in BAL fluid from SSc patients with lung inflammation, as compared with levels in patients without lung inflammation and in healthy controls (12,13). Although the mechanism of PF in SSc remains unknown, several chemokines are involved in the fibrotic process of PF associated with SSc by indirectly promoting fibrosis through attracting and activating the profibrotic activities of inflammatory cells, including alveolar macrophages (29). PARC is produced abundantly by alveolar macrophages (10,11,13) and slightly by peripheral blood monocytes, tissue macrophages, and dendritic cells (10,11). Therefore, the increased levels of PARC in the serum of SSc patients with PF may be derived from alveolar macrophages. Since PARC has a chemotactic effect on lymphocytes and monocyte/macrophages (30), it may mediate the recruitment of lymphocytes and monocyte/macrophages into the lung tissues during the development of PF in SSc. The current longitudinal study showed that serum PARC levels correlated with the activity of PF. Many studies have indicated that serum KL-6 and SP-D levels are useful, sensitive diagnostic markers and indicators of disease activity in patients with pulmonary interstitial diseases (4–7). Our recent study (5) has suggested that the combined use of KL-6 and SP-D measurements is more helpful in the diagnosis and monitoring of the activity of PF in SSc patients than is the use of either marker alone. However, the fluctuations in SP-D and KL-6 concentrations are not parallel and are sometimes still elevated even after treatment results in an improvement in the PF activity. In this study, serum levels of PARC, KL-6, and SP-D were all significantly decreased in parallel with improvement in PF activity. However, the serum PARC levels most closely reflected the activity of the PF, since all but 1 of SERUM PARC LEVELS IN SSC the patients exhibited a decrease in serum PARC levels after progression of the PF activity had ceased. Moreover, the serum PARC levels decreased sooner than the serum KL-6 and SP-D levels after PF progression had ceased in 2 patients who were treated with intravenous pulse cyclophosphamide. Although studies with larger numbers of SSc patients will be needed to confirm our findings, serum PARC levels may be a new and useful marker of the activity of PF in SSc. Serum PARC levels, which probably derive from activated alveolar macrophages, may reflect the lung inflammation that precedes fibrotic changes. With serum SP-D levels, however, elevations are induced by the destruction of the alveolar endothelium, followed by spillover of SP-D into the bloodstream, so they may reflect the extent of injury of the alveolar endothelium (27). KL-6 is abundantly expressed on regenerating and proliferating type II pneumocytes in patients with pulmonary interstitial diseases (2). With an increase in epithelial and vascular permeability, KL-6 may flow into blood vessels in a soluble form. Thus, the increase in serum SP-D is due to destruction of the alveolar endothelium, whereas the increase in serum KL-6 reflects the regeneration and proliferation of pneumocytes. In the process of PF stabilization, infiltration of inflammatory cells, including activated macrophages, into the lung may decrease first, and then the permeability of the air– blood barrier and destruction of alveolar endothelium may be suppressed. Therefore, it is possible for serum levels of PARC to decrease more rapidly, in parallel with a decrease in lung inflammation, since they are derived from activated alveolar macrophages than for serum levels of SP-D or KL-6 to decrease, since they are released by injury of the alveoli or by the regenerative proliferation of pneumocytes. Although PARC was more sensitive for assessing PF activity than was SP-D or KL-6, correlations between PARC levels and the VC or the DLCO were not strong, which is also the case for SP-D and KL-6 levels (5). This is a limitation when evaluating PF activity with the use of serum markers. Nonetheless, it does not diminish the clinical significance of serum markers, since they represent simple, easy, and noninvasive techniques and can be assessed repeatedly. However, it should be noted that PARC levels must be interpreted in combination with the findings of other laboratory tests and radiologic assessments to precisely evaluate the activity of PF. REFERENCES 1. Silver RM. Clinical problems: the lungs. Rheum Dis Clin North Am 1996;22:825–40. 2895 2. Kohno N, Kyoizumi S, Awaya Y, Fukuhara H, Yamakido M, Akiyama M. New serum indicator of interstitial pneumonitis activity: sialylated carbohydrate antigen KL-6. Chest 1989;96: 68–73. 3. Hermans C, Bernard A. Lung epithelium-specific proteins: characteristics and potential applications as markers. Am J Respir Crit Care Med 1999;159:646–78. 4. Greene KE, King TE Jr, Kuroki Y, Bucher-Bartelson B, Hunninghake GW, Newman LS, et al. Serum surfactant proteins-A and -D as biomarkers in idiopathic pulmonary fibrosis. Eur Respir J 2002;19:439–46. 5. Yanaba K, Hasegawa M, Takehara K, Sato S. Comparative study of serum surfactant protein-D and KL-6 concentrations in patients with systemic sclerosis as markers for monitoring the activity of pulmonary fibrosis. J Rheumatol 2004;31:1112–20. 6. Sato S, Nagaoka T, Hasegawa M, Nishijima C, Takehara K. Elevated serum KL-6 levels in patients with systemic sclerosis: association with the severity of pulmonary fibrosis. Dermatology 2000;200:196–201. 7. Vesely R, Vargova V, Ravelli A, Massa M, Oleksak E, d’Alterio R, et al. Serum level of KL-6 as a marker of interstitial lung disease in patients with juvenile systemic sclerosis. J Rheumatol 2004;31: 795–800. 8. Guan P, Burghes AH, Cunningham A, Lira P, Brissette WH, Neote K, et al. Genomic organization and biological characterization of the novel human CC chemokine DC-CK-1/PARC/MIP-4/ SCYA18. Genomics 1999;56:296–302. 9. Hieshima K, Imai T, Baba M, Shoudai K, Ishizuka K, Nakagawa T, et al. A novel human CC chemokine PARC that is most homologous to macrophage-inflammatory protein-1␣/LD78␣ and chemotactic for T lymphocytes, but not for monocytes. J Immunol 1997;159:1140–9. 10. Pardo A, Smith KM, Abrams J, Coffman R, Bustos M, McClanahan TK, et al. CCL18/DC-CK-1/PARC up-regulation in hypersensitivity pneumonitis. J Leukoc Biol 2001;70:610–6. 11. Kodelja V, Muller C, Politz O, Hakij N, Orfanos CE, Goerdt S. Alternative macrophage activation-associated CC-chemokine-1, a novel structural homologue of macrophage inflammatory protein-1␣ with a Th2-associated expression pattern. J Immunol 1998;160:1411–8. 12. Atamas SP, Luzina IG, Choi J, Tsymbalyuk N, Carbonetti NH, Singh IS, et al. Pulmonary and activation-regulated chemokine stimulates collagen production in lung fibroblasts. Am J Respir Cell Mol Biol 2003;29:743–9. 13. Luzina IG, Atamas SP, Wise R, Wigley FM, Xiao HQ, White B. Gene expression in bronchoalveolar lavage cells from scleroderma patients. Am J Respir Cell Mol Biol 2002;26:549–57. 14. Zou J, Young S, Zhu F, Gheyas F, Skeans S, Wan Y, et al. Microarray profile of differentially expressed genes in a monkey model of allergic asthma. Genome Biol 2002;3:research0020.1–13. 15. Mrazek F, Sekerova V, Drabek J, Kolek V, du Bois RM, Petrek M. Expression of the chemokine PARC mRNA in bronchoalveolar cells of patients with sarcoidosis. Immunol Lett 2002;84:17–22. 16. Subcommittee for Scleroderma Criteria of the American Rheumatism Association Diagnostic and Therapeutic Criteria Committee. Preliminary criteria for the classification of systemic sclerosis (scleroderma). Arthritis Rheum 1980;23:581–90. 17. LeRoy EC, Black C, Fleischmajer R, Jablonska S, Krieg T, Medsger TA Jr, et al. Scleroderma (systemic sclerosis): classification, subsets, and pathogenesis. J Rheumatol 1988;15:202–5. 18. Tan EM, Cohen AS, Fries JF, Masi AT, McShane DJ, Rothfield NF, et al. The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 1982;25:1271–7. 19. Clements PJ, Lachenbruch PA, Seibold JR, Zee B, Steen VD, Brennan P, et al. Skin thickness score in systemic sclerosis: an assessment of interobserver variability in 3 independent studies. J Rheumatol 1993;20:1892–6. 2896 20. Wells AU, Hansell DM, Corrin B, Harrison NK, Goldstraw P, Black CM, et al. High resolution computed tomography as a predictor of lung histology in systemic sclerosis. Thorax 1992;47: 738–42. 21. Bouros D, Wells AU, Nicholson AG, Colby TV, Polychronopoulos V, Pantelidis P, et al. Histopathologic subsets of fibrosing alveolitis in patients with systemic sclerosis and their relationship to outcome. Am J Respir Crit Care Med 2002;165:1581–6. 22. White B, Moore WC, Wigley FM, Xiao HQ, Wise RA. Cyclophosphamide is associated with pulmonary function and survival benefit in patients with scleroderma and alveolitis. Ann Intern Med 2000;132:947–54. 23. Kazerooni EA, Martinez FJ, Flint A, Jamadar DA, Gross BH, Spizarny DL, et al. Thin-section CT obtained at 10-mm increments versus limited three-level thin-section CT for idiopathic pulmonary fibrosis: correlation with pathologic scoring. AJR Am J Roentgenol 1997;169:977–83. 24. Taylor ML, Noble PW, White B, Wise R, Liu MC, Bochner BS. Extensive surface phenotyping of alveolar macrophages in interstitial lung disease. Clin Immunol 2000;94:33–41. 25. Schnabel A, Reuter M, Gross WL. Intravenous pulse cyclo- KODERA ET AL 26. 27. 28. 29. 30. phosphamide in the treatment of interstitial lung disease due to collagen vascular diseases. Arthritis Rheum 1998;41:1215–20. Giacomelli R, Valentini G, Salsano F, Cipriani P, Sambo P, Conforti ML, et al. Cyclophosphamide pulse regimen in the treatment of alveolitis in systemic sclerosis. J Rheumatol 2002;29: 731–6. Nagae H, Takahashi H, Kuroki Y, Honda Y, Nagata A, Ogasawara Y, et al. Enzyme-linked immunosorbent assay using F(ab⬘)2 fragment for the detection of human pulmonary surfactant protein D in sera. Clin Chim Acta 1997;266:157–71. Kobayashi J, Itoh Y, Kitamura S, Kawai T. Establishment of reference intervals and cut-off value by an enzyme immunoassay for KL-6 antigen, a new marker for interstitial pneumonia. Jpn J Clin Pathol 1996;44:653–8. Atamas SP, White B. Cytokine regulation of pulmonary fibrosis in scleroderma. Cytokine Growth Factor Rev 2003;14:537–50. Schraufstatter I, Takamori H, Sikora L, Sriramarao P, di Scipio RG. Eosinophils and monocytes produce pulmonary and activation-regulated chemokine, which activates cultured monocytes/ macrophages. Am J Physiol Lung Cell Mol Physiol 2004;286: L494–501.