Prognosis of nonspecific interstitial pneumonia correlates with perivascular CD4+ T lymphocyte infiltration of the lung

Between April 2003 and December 2011, 97 patients from Peking Union Medical College Hospital (PUMCH) were diagnosed with NSIP according to analysis of lung biopsies. Fifty-five patients who completed follow-up by undergoing tests of pulmonary function tests and computed tomography (CT) of the chest were included in this study. NSIP was diagnosed according to the American Thoracic Society (ATS)/European Respiratory Society consensus classification [ 8, 9]. Patients were not treated with corticosteroids or other immunosuppressants before undergoing a lung biopsy. Connective tissue disease (CTD) was diagnosed according to the criteria of the American College of Rheumatology as follows: autoantibodies against the nucleus (ANA titer >1:320), rheumatoid factor, Sjögren’s-syndrome-related antigens SSA or SSB, Scl-70, Sm, anti-Jo-1, ribonucleoprotein, or cyclic citrullinated peptide [ 10– 13]. Patients with NSIP were classified into the groups as follows: (1) Members of the CTD-NSIP group met the criteria of the American College of Rheumatology for CTD. (2) Members of the NSIP-Ab + group had at least one positive serologic test. (3) Members of the NSIP-Ab– group were autoantibody negative. We extracted clinical characteristics documented at the time of a patient’s first visit as follows: age, ethnicity, sex, symptoms (cough, dyspnea, or wheeze) at the time of lung biopsy, symptoms or signs of CTD, smoking status, physical examination findings, pulmonary function results, serologic results, and chest CT scan.

Informed consent to use medical records was obtained from every patient, their guardian, or both when the patient was admitted to the hospital. The PUMCH Institutional Review Board approved this study (reference number for ethics approval: 2012–10–312).

Spirometry, total lung capacity determined using plethysmography, forced vital capacity (FVC), and diffusing capacity of the lung for carbon monoxide were measured according to ATS recommendations [ 14– 16], and the results are expressed as the percentage of predicted normal values. Prognosis was described according to FVC 1 year after steroid therapy. We classified patients as follows: FVC-group 1, improvement of FVC >10 %; FVC-group 2, improvement of FVC between 10 % and −10 %; and FVC-group 3, FVC reduced greater than −10 % [ 17– 20].

All patients underwent high-resolution CT (HRCT) of the chest upon initial evaluation. A radiologist who specializes in diffuse parenchymal lung disease reviewed the CT scans. The extent and prevalence of abnormalities were measured in the areas as follows: (1) from the lung apex to the carina, (2) from the carina to the upper pulmonary vein, and (3) from the upper pulmonary vein to the base of the lung. HRCT images were assessed to determine the extent of parenchymal abnormalities, including ground-glass opacity (GGO), reticulation, honeycombing, consolidation, and emphysema. The extent of these abnormalities was determined using a 10 % scale for six zones [ 21]. Prognosis is described according to the manifestations detected using CT 1 year after steroid therapy. Patients with NSIP were classified as follows: CT-group 1, improvement of GGO and reticular opacity >50 %; group, CT-group 2, lesions reduced between 30 % and 50 %, and CT-group 3, lesions reduced by <30 % [ 21– 23].

Two experienced pathologists reviewed independently the lung biopsy specimens, and the histologic pattern was assigned according to the consensus opinion. The pathologists were unaware of the patients’ clinical information. The histological patterns of the 55 patients were classified further, according to criteria proposed by Katzenstein and Fiorelli [ 24], as cellular, mixed, or fibrotic. Small airways were defined as those with an internal diameter <2 mm without cartilage in the airway walls [ 25]. Small blood vessels were defined as those with an internal diameter ≤100 μm [ 26].

Lung biopsy specimens were fixed in 10 % neutral-buffered formalin, cut into slices, embedded in paraffin, and cut into 4-μm-thick sections. Paraffin sections were reacted anti-CD4 (clone SP35, Zeta), and anti-CD8 (clone EP1150, Zeta) antibodies using the labeled-streptavidin-biotin complex method. The sections were deparaffinized, rehydrated with Tris-Buffered Saline (TBS) (0.005 M Tris, 0.15 M NaCl, pH 7.6) for 10 min, treated with 3 % hydrogen peroxide for 5 min to inhibit endogenous peroxidase activity, washed in TBS, and incubated with primary antibodies for 1 h. Immunohistochemical analyses of CD4 and CD8 expression were performed using the same tissue sections.

The numbers of dark-brown cells were determined using a NanoZoomer 2.0-RS Slide Scanning System (Hamamatsu Photonics KK; Japan Tokyo) and an Anymicro DSS Pro Image Analysis System (Yu Tian Shi Ji Wei Ye INC; Beijing, China). The follicles as well as perivascular, interstitial, and peribronchial regions were analyzed. The images of CD4+ and CD8+ T lymphocytes were obtained from the same region of the slide. At least six high-power fields (magnification × 200; analysis area, approximately 0.162 mm ²) were randomly selected for each region and used to count the stained cells (Fig.  1).

All patients received a standard initial course of oral prednisone, starting at 0.5 mg/kg/d for 1 month that was tapered every 3 weeks to 5–7.5 mg/d (10 % decrease of the initial dose every 3 weeks), and maintained at 5–7.5 mg/d. Patients were treated for 12–18 months. Cyclophosphamide was administered concurrently with steroid therapy at an oral dose of 100 mg/d administered for 3–6 months to patients diagnosed with CTD-NSIP. Patients underwent routine examinations every 3–6 months after lung biopsy and then once each year at the Interstitial Lung Disease Clinic of PUMCH.

All values are expressed as the mean ± standard deviation (SD). Normally distributed data were evaluated using the t test. The Wilcoxon and Kruskal–Wallis tests were used to compare two or more non-normally distributed values, respectively. Fisher’s exact test was used to determine differences between groups. Correlation coefficients were calculated using the Spearman rank method. Cox multivariate regression analysis was used to evaluate risk factors of survival. The times to overall survival were calculated using the Kaplan–Meier method. The probability value was obtained using 2-sided tests, and statistical significance was defined as p < 0.05. SPSS 15.0 (SPSS for Windows, version 15.0; SPSS Inc., Chicago, IL, USA) was used for statistical analysis.