CHARACTERISTICS OF STUDY POPULATION

Ten patients with WG were studied, all of whom met the criteria of the American College of Rheumatology13 and the Chapel Hill Consensus Conference definition for WG.14 The disease extension and vasculitis activity were described by the DEI and Birmingham vasculitis activity score (BVAS) as outlined elsewhere.15 ,16 In brief, the DEI is the equivalent of current organ involvement in WG,15 whereas the BVAS considers clinical features and laboratory data to give a measure of vasculitis activity.16 Partial remission of WG was defined as improvement in vasculitis activity, although clinical or serological signs of inflammation were still detectable. Respiratory tract involvement was diagnosed when pulmonary infiltrates were seen on the chest radiograph, an increase in neutrophils and/or lymphocytes was found in the BAL fluid, and transbronchial biopsy specimens and/or biopsy specimens from the nasal mucosa supported the diagnosis.

Six patients with acute sarcoidosis served as disease controls. The term “acute” sarcoidosis refers to the rapid onset of disease. Löfgren's syndrome is one well known form of acute sarcoidosis with a classical triad of active disease manifestations (bilateral hilar lymphadenopathy (BHL), oligoarthritis, and erythema nodosum). Chronic sarcoidosis is the insidious form of the disease in which a Th2 cytokine pattern may be seen.11 Patients with chronic sarcoidosis were not therefore used as disease controls. The diagnosis of sarcoidosis was based on clinical symptoms such as new or progressive dyspnoea, cough, active uveitis, new radiographic abnormalities on the chest radiograph, an increase in lymphocytes in BAL fluid with an increased CD4/CD8 ratio of lymphocytes and raised levels of serum angiotensin converting enzyme. The diagnosis was supported by transbronchial lung biopsy specimens and biopsy specimens from other affected organs, in line with recently published guidelines.17

The fraction of CD28– T cells in blood was also determined in 15 healthy controls for comparison. The erythrocyte sedimentation rate (ESR, normal range <10 mm/h), C reactive protein (CRP, <5 mg/l), complete blood count (including leucocytes: 4–10 × 10³/mm³, thrombocytes 150–300 × 10³/mm³), and serum creatinine levels (6–12 mg/l) were routinely determined. Normal values for other routinely determined parameters are: CD4/CD8 ratio in blood, 0.6–3.2; BAL fluid leucocyte count, <20 × 10⁶; BAL fluid lymphocyte count, <15%, <3 × 10⁶; CD4/CD8 ratio in BAL fluid, 1–3. All patients provided written informed consent.

BRONCHOSCOPY AND BAL CELL PREPARATION

Fibreoptic bronchoscopy was performed as described previously.18 In brief, local anaesthesia with lidocaine and premedication with atropine and pethidine were given prior to the procedure. BAL was preferentially performed in radiographical abnormal lung segments in the patients with WG and in the middle lobe or the lingula in patients with sarcoidosis. Twelve fractions of 20 ml sterile saline 0.9% were instilled and aspirated in a single segment. The fluid recovery was at least 60% and all materials were placed immediately on ice. The first two fractions were separated and the third to 12th fractions were pooled and further analysed. Aliquots of the material were routinely examined for conventional bacterial pathogens, acid fast bacteria, Legionellaspecies, Chlamydia species,Mycoplasma species, andPneumocystis carinii. Any infected material was omitted from further study.

The lavage material was filtered through surgical gauze and centrifuged at 4°C. The cell pellet was resuspended in RPMI 1640 medium (GIBCO, Eggenstein, Germany) containing 0.01% human serum albumin (Sigma, München, Germany) to a density of 10⁶ cells/ml.

ANTIBODIES, REAGENTS AND FACS IMMUNOPHENOTYPING

Antibodies for FACS analysis were fluorescein (FITC) conjugated anti-CD4 and FITC conjugated anti-CD8, phycoerythrin (PE) conjugated anti-CD28, and peridin-chlorophyll (PerCP) conjugated anti-CD45 which were obtained from Becton-Dickinson (Heidelberg, Germany). FITC conjugated IgG1 and PE conjugated IgG2a (also from Becton-Dickinson) were used for negative controls. Antibodies for immunohistochemistry used in this study were rabbit anti-CD3 antiserum and APAAP-complex from Dako (Glostrup, Denmark), monoclonal mouse anti-CD28 antibody (Biosource International, Nivelles, Belgium), anti-CD8 antibody (clone Leu2a, BD Biosciences, Heidelberg, Germany), Cy-3 conjugated goat anti-mouse IgG, and FITC conjugated donkey anti-rabbit IgG from Dianova (Hamburg, Germany).

Identical double staining protocols were used for BAL cells and heparinised peripheral venous blood. 1 × 10⁶ cells per sample were incubated on ice with previously determined optimal concentrations of FITC or PE labelled antibodies for 30 minutes. After staining the cells were washed and fixed with PBS containing 1.5% paraformaldehyde. Flow cytometric analysis was performed using a FACSCalibur flow cytometer (Becton-Dickinson). Data were acquired withcell-quest software (Becton-Dickinson). Lymphocytes were gated for analysis based on light scattering properties and on CD45 and CD4 or CD8 staining. Data on 1.5 × 10³ lymphocytes were collected. Positively and negatively stained populations were calculated by quadrant dot plot analysis determined by isotype controls.

IMMUNOHISTOLOGY

Nasal biopsy specimens of patients with WG obtained for diagnostic purposes were submerged in 0.9% NaCl and snap frozen in liquid nitrogen and stored until use at –80°C. Cryostat frozen sections were fixed in acetone for 30 minutes, followed by fixation in chloroform for 30 minutes. Incubation with the primary antibody was performed for 30 minutes and immunostaining was undertaken according to the APAAP (alkaline phosphatase anti alkaline phosphatase) method with New Fuchsin development. Finally, slides were counterstained with haematoxylin and mounted. Immunostaining was controlled by implementing the secondary reagents alone in order to confirm specificity or enzyme development alone to rule out endogenous enzyme activities. Double immunofluorescence staining was performed after a blocking step with 10% bovine serum albumin in Tris buffered saline (TBS, 50 mM Tris-HCl, 150 mM NaCl, pH 7.5) with the primary antibodies for 30 minutes. After washing in TBS the secondary antibodies were added and incubated for 30 minutes. Specimens were mounted in DABCO anti-fading solution (2.5% DABCO (1,4-diazabicyclo(2,2,2)-octane) in 90% glycerol, pH 8.6) and viewed under a fluorescence microscope. Stainings were controlled by using only the secondary antibodies to detect non-specific and background fluorescence. Images were obtained using a Leica TCS SP confocal microscope (Bensheim, Germany). The single fluorescence intensities were adjusted using identically stained tonsillar tissue as a measure. Subsequent images of patient tissues stained under the same procedure were acquired using the same instrument settings as for tonsil tissue.

STATISTICAL ANALYSIS

Data are expressed as mean (SE). Data referring to CD4+/CD28– and CD8+/CD28– T cells indicate the fraction of CD28– cells of the respective CD4+ and CD8+ T cell subsets stained for CD28 expression. We also determined total numbers of leucocytes, lymphocytes, CD4+ T cells, CD8+ T cells, CD4/CD8 ratio, and CD4+/CD28– and CD8+/CD28– T cells to test the possibility that the percentage of CD4+/CD28– and CD8+/CD28– T cells in a compartment (blood, BAL fluid) may be different in WG and sarcoidosis, whereas the total number of recruited CD28– T cells may be similar because of different CD4/CD8 ratios in blood or BAL fluid in the two diseases.

The non-parametric Mann-Whitney U test was used to compare CD4+/CD28– and CD8+/CD28– T cell populations in patients with WG and sarcoidosis. p values of <0.05 were considered significant. Spearman rank order correlation coefficients were determined to assess associations of clinical and laboratory parameters. Analysis was performed applyingspss software package.