METHODS. participated in the study. Each subject had a normal physical examination, chest

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1 METHODS Subjects Thirty-four subjects (20 men and 14 women, mean age 28.5 ± 1.2 yr) participated in the study. Each subject had a normal physical examination, chest radiograph, electrocardiogram, and laboratory tests including blood counts, coagulation profiles, and serum chemistries. None of the subjects had asthma or used tobacco products. Our institutional review board approved the study and written informed consent was obtained from each subject. After an overnight fast, the subjects were admitted to a monitored research unit and an intravenous catheter and a radial artery catheter were inserted. All were given oral sulfamethoxazole-trimethoprim (80/400 mg) or amoxacillin (500 mg) 12 h and 1 h prior to the first bronchoscopy to minimize lower airway contamination with oral bacterial flora. Oral temperatures were measured every 30 min for 8 h with a calibrated thermometer (Diatek, Inc., San Diego, CA). Endotoxin Dosing Endotoxin (E. coli O:113, lot EC-5, Center for Biologics Evaluation and Research, Food and Drug Administration, Rockville, MD) was reconstituted with sterile water USP to a final concentration of 2000 EU/ml. To establish tolerability and the time course of the response to the segmental challenge, six subjects were challenged at baseline (time 0 h) with increasing doses of endotoxin (1 ng/kg; n = 2; 2 ng/kg; n = 2; 4 ng/kg, n = 2). Bronchoalveolar lavage (BAL) was then performed at 6 h and 24 h after endotoxin instillation. After this initial study, 28 subjects were evaluated with 4 ng/kg at baseline (0 h) followed by BAL at either 2 h (n = 3), 6 h (n = 14), 24 h (n = 6), or 48 h (n = 5). Bronchoscopy and BAL After anesthetizing the nasal mucosa and oropharynx with lidocaine (1 or 2%), a fiberoptic bronchoscope (Model P30 or P200, Olympus

2 E2 Corp., Lake Success, NY) was directed through the upper airways to the carina. A balloon-tipped catheter (5F monitoring catheter, Baxter Healthcare Corp., Irvine, CA or ProBAL catheter, Milrose, Mentor, OH) was placed through the bronchoscope to occlude the challenged segment. Saline (0.9%, 10 ml) followed by 10 ml of air was instilled into the control segment (either lingula or right middle lobe). The catheter was kept in place for 30 s and then the balloon was deflated. Endotoxin (approximate volume 1-2 ml), followed by 10 ml of saline and 10 ml of air, was then instilled in the contralateral lung segment. The catheter and bronchoscope were withdrawn and the head of the bed was then raised 30. The subsequent BAL was performed by instilling 180 ml (6 30 ml aliquots) of normal saline into the control (saline-challenged) segment, followed by a similar BAL of the endotoxin challenged segment. The two pooled lavage samples from the control and endotoxin segments were placed on ice at the bedside. An aliquot was taken for cell cytospin and the lavage was centrifuged (900 * g at 4 10 C) for 10 min. The supernatant was removed and frozen at -70 C in aliquots until assayed. The total BAL leukocyte number was determined with an automated cell counter (Coulter ZM, Miami, FL) and differential cell counts were done by morphologic examination of the stained cytospin (Diff Quick, Dade Behring, Newark, DE). Monocytes were distinguished from lymphocytes and macrophages on the basis of size, morphology, and staining characteristics. The validity of these criteria was confirmed by flow cytometry (FACScan, Becton-Dickinson, San Jose, CA) based on forward and side light scatter (i.e., reflecting size and granularity- autofluorescence, respectively) and expression of CD markers. These criteria were as follows: alveolar macrophages - large,

3 E3 highly autofluorescent, CD14+, CD49d+ cells; monocytes - moderate size with moderate autofluorescence, CD49d+ and brightly positive for CD14+; lymphocytes small size with low autofluorescence, CD14-, CD49d + cells; neutrophils similar in size to monocytes, mildly autofluorescent with CD16b +, dim CD14+, and CD49d- cells. Assays All assays were performed on nonconcentrated BAL. Tumor necrosis factor-α (TNF-) bioactivity was assayed as previously described with minor modifications (E1). The assay is based on the killing of a TNF-sensitive mouse fibrosarcoma cell line (WEHI 164, clone 13) and has a detection limit of approximately 1 pg/ml. Each BAL sample was assayed in triplicate and compared with a standard curve of recombinant human TNF-α in saline (R&D Systems, Minneapolis, MN). Cell survival was determined colorimetrically. Confirmation of TNF-α bioactivity was done by repeat assays using a neutralizing polyclonal anti-tnf-α antibody (Genzyme, Cambridge, MA). Proinflammatory activity was quantified by direct ELISA as previously described using BAL fluid to induce the upregulation of intercellular adhesion molecule (ICAM)-1 on cultured human alveolar type II-like A549 cells (E2). Proinflammatory activity (1 unit/ml) is equal to the level of ICAM-1 upregulation induced by 1 pg/ml of recombinant interleukin-1iβ (ΙL-1β). Total BAL protein (BCA protein assay, Pierce, Rockford, IL) and albumin (albumin reagent - BCG, Sigma Diagnostics, St. Louis, MO) were quantified. Levels of mediators and inflammatory markers in the BAL were measured in duplicate by quantitative immunoassays. Standard curves were generated using saline or calibrated diluent from the kit. The limits of detection of the ELISA were TNF-α (R&D Systems) 3.9 pg/ml, soluble TNF receptors (TNFR) type 1 (p55) and type 2 (p75) (R&D

4 E4 Systems) both 7.8 pg/ml, interleukin (IL)-1 receptor antagonist (IL-1ra) (R&D Systems) 31.3 pg/ml, IL-6 (R&D Systems) 3.1 pg/ml, granulocyte - colony stimulating factor (G- CSF) (R&D Systems) 19.5 pg/ml, IL-8 (R&D Systems) 31.3 pg/ml, growth-regulated peptide-α (GRO-α) (R&D Systems) 7.8 pg/ml, epithelial neutrophil-activating protein 78 (ENA-78) (R&D Systems) 15.6 pg/ml, macrophage inflammatory protein (MIP)-1α and MIP-1β (R&D Systems) both 7.8 pg/ml, monocyte chemotactic protein (MCP)-1 (R&D Systems) 7.8 pg/ml, IL-10 (R&D Systems) 7.8 pg/ml, IL-1β (Cistron Biotechnology, Pinebrook, NJ) 2 pg/ml, soluble L-selectin (R&D Systems) 2.8 ng/ml, neutrophil myeloperoxidase (R&D Systems) 1.6 ng/ml, and lactoferrin (R&D Systems) 1.6 ng/ml. Gelatinase-B (pro-matrix metalloproteinase-9) was measured by gelatin zymography as previously described (E3). Because of the absence of IL-10 in the inflammatory lavage, we tested the recovery of IL-10 in the BAL using the above ELISA. Recombinant IL-10 (R&D Systems) was added to lavage from subjects undergoing BAL at 6 h (n = 2) and 24 h (n = 2). Recovery of recombinant human IL-10 (50 pg/ml) from BAL (four endotoxin and four saline-challenged segments) was 90 ± 5%. Serum samples were assayed for C-reactive protein (The Binding Site, San Diego, CA) (0.52 mg/l limit of sensitivity) and serum amyloid A (Biosource International, Camarillo, CA, 9.4 ng/ml limit of sensitivity) by radial immunodiffusion assay and immunoassay, respectively. Total peripheral blood leukocyte with differential cell counts and serum levels of TNF-α, IL-6, G-CSF, and IL-1ra (all R&D Systems) were performed at 0, 2, 5, 6, 8, and 24 h as previously described (E1).

5 E5 Symptom Assessment Symptoms were quantified by each subject using an hourly questionnaire during the initial 8 h study period regarding the presence of systemic (malaise, headache, myalgias) or pulmonary symptoms (dyspnea, cough, wheezing, chest pain, and secretions), which were rated by severity (0 none, 1 minimal, 2 moderate, and 3 severe). Statistical Methods Data were analyzed using analysis of variance ANOVA (SAS System 6.09, SAS Institute, Cary, NC) (E4). For lavage data, analysis included main effects for time point of lavage, patient (nested within time point), and an indicator of endotoxin exposure (e.g., whether the lavage came from the site that received endotoxin or not). In addition, an interaction between time and endotoxin was included in the model to determine if a significantly different time course existed for those segments challenged with endotoxin or saline (e.g., endotoxin time interaction). All remaining interactions were pooled to form the error term in the ANOVA. Remaining systemic data could not isolate local effects of endotoxin. These ANOVA models include main effects for timing of lavage, patient (nested within timing of lavage), and time postendotoxin administration. An interaction between timing of lavage and time postendotoxin administration was also included in the model (e.g., group time interaction), with remaining interactions pooled to form the error term. For all ANOVA models, normality of residuals was assessed using a Shapiro-Wilk test, and, if necessary, data were transformed to improve the distribution of the residuals. Data are presented as mean ± SEM.

6 E6 References E1. Suffredini AF, Reda D, Banks SM, Tropea M, Agosti JM, Miller R. Effects of recombinant dimeric TNF receptor on human inflammatory responses following intravenous endotoxin administration. J Immunol 1995; 155: E2. Pugin J, Ricou B, Steinberg KP, Suter PM, Martin TR. Proinflammatory activity in bronchoalveolar lavage fluids from patients with ARDS, a prominent role for interleukin-1. Am J Respir Crit Care Med 1996; 153: E3. Pugin J, Widmer MC, Kossodo S, Liang CM, Preas HL, Suffredini AF. Human neutrophils secrete gelatinase B in vitro and in vivo in response to endotoxin and proinflammatory mediators. Am J Respir Cell Mol Biol 1999; 20: E4. Scheffe H. The analysis of variance. New York: John Wiley & Sons;

7 TABLE E1. CHANGES IN BAL CELL NUMBER AND DIFFERENTIAL IN SIX SUBJECTS AT 6 h AND 24 h AFTER ENDOBRONCHIAL INSTILLATION OF SALINE (CONTROL) OR ENDOTOXIN.* 6 h (n = 6) 24 h (n = 5) Control Endotoxin Control Endotoxin Total cell count, * 10 4 cells/ml ± ± ± ± Macrophages, % 71 ± 7 11 ± 6 62 ± 6 40 ± 11, Lymphocytes, % 19 ± 4 13 ± 2 20 ± 6 12 ± 1 Monocytes, % 1 ± 1 8 ± 2 2 ± 1 11 ± 2 Neutrophils, % 9 ± 9 66 ± 7 15 ± 4 35 ± 10, Definition of abbreviation: BAL = bronchoalveolar lavage. * Endotoxin dose instilled 1, 2, or 4 ng/kg. Endotoxin effect (p < 0.02). endotoxin - time interaction (p < 0.02).

8 E8 TABLE E2. CHANGES IN BAL CELL NUMBER AND DIFFERENTIAL IN 28 SUBJECTS AT 2, 6, 24, or 48 h AFTER ENDOBRONCHIAL INSTILLATION OF SALINE (CONTROL) OR ENDOTOXIN.*, 2 h (n = 3) 6 h (n = 14) Control Endotoxin Control Endotoxin Total cell count, * 10 4 cells/ml ± ± ± ± Macrophages, % 91 ± 6 84 ± 7 85 ± 2 33 ± 6 Lymphocytes, % 5 ± 2 2 ± 2 10 ± 2 9 ± 2 Monocytes, % 0.3 ± ± ± 1 7 ± 2 Neutrophils, % 4 ± 3 12 ± 6 3 ± 1 51 ± 6 24 h (n = 6) 48 h (n = 5) Control Endotoxin Control Endotoxin Total cell count, * 10 4 cells/ml ± ± ± ± Macrophages, % 83 ± ± 8 87 ± 5 70 ± 7 Lymphocytes, % 4 ± 1 8 ± 2 6 ± 3 8 ± 3 Monocytes, % 4 ± 4 18 ± 5 2 ± 1 14 ± 4 Neutrophils, % 10 ± 8 43 ± 5 5 ± 2 6 ± 2 Definition of abbreviation: BAL = bronchoalveolar lavage. * Endotoxin dose instilled 4 ng/kg.

9 E9 p values for endotoxin effect and endotoxin - time interaction: total cell count (p = and p = NS), % macrophages (p = and p = 0.007), % lymphocytes (p = NS), % monocytes (p = and p = 0.01), and % neutrophils (p = and p = ), respectively.