Biotechnology, Inc.) for 60 min at 4 C. These were then added to culture medium and incubated for 48 h. Cell Culture and Cytokines. THP-1 cells (TIB-202; Ameri- can Type Culture Collection) or BF24 cells (AIDS Research and Reference Reagent Program #1296) were cultured in RPMI 1640 with 10% FCS. Cells were differentiated with 20 ng/ml PMA (Sigma-Aldrich) for 24 h and incubated with IFN- (Bio- source International) at 1 U/ml for 48 h after PMA treatment. Where noted, activated T lymphocytes and AM or THP-1 cells were separated by 0.4 m pore cell culture insert (Millipore). IL- 1 , IL-6, and TNF- in cell culture supernatants from insert ex- periments were measured by ELISA (R&D Systems). Cloned and purified IL-1 , IL-6, and TNF- (R&D Systems) were added to BF-24 cells for 48 h before chloramphenicol acetyltransferase (CAT) measurement. Cell Extract Preparation. Cells were washed twice in PBS (Bio-Whittaker). Whole cell extracts were prepared for immuno- blot analysis by incubation in NP-40 buffer (0.5% NP-40, 10% glycerol, 0.1 mM EDTA, 20 mM Hepes [pH 7.9], 10 mM NaF 10 mM NaPpi, 300 mM NaCl, 3 g/ml aprotinin, 2 g/ml leu- peptin, 2 g/ml pepstatin, 1 mM DTT, 1 mM PMSF, and 1 mM Na3VO4) for 30 min on ice with vigorous shaking. Nuclear ex- tracts were prepared by NP-40 lysis (buffer A: 10 mM Hepes- KOH [pH 7.8], 10 mM KCl, 0.1 mM EDTA [pH 8.0], 0.1% NP-40, 3 g/ml aprotinin, 2 g/ml leupeptin, 2 g/ml pepsta- tin, 1 mM DTT, 1 mM PMSF, and 1 mM Na3VO4) and incuba- tion of recovered nuclei in high salt buffer (buffer C: 10 mM Hepes-KOH [pH 7.8], 420 mM KCl, 0.1 mM EDTA [pH 8.0], 5 mM MgCl2, 2% glycerol, 3 g/ml aprotinin, 2 g/ml leupeptin, 2 g/ml pepstatin, 1 mM DTT, 1 mM PMSF, and 1 mM Na3VO4). Where indicated, we added 20 g/ml calpain inhibitor (Sigma-Aldrich) into NP-40 buffer, buffer A, or buffer C before cell extraction. Pierce BCA reagents were used to determine ex- tract protein concentrations. Protein extracts for chloramphenicol acetyl transferase CAT ELISA (Roche Molecular Biochemicals) were processed according to manufacturer’s instructions. Immunoblots. Proteins were separated by SDS-PAGE (Bio- Rad Laboratories) as described previously (11), and then probed with antibodies against anti-C/EBP , followed by visualization with anti–rabbit HRP antibodies (Santa Cruz Biotechnology, Inc.) and ECL plus (Amersham Pharmacia Biotech). Electrophoretic Mobility Shift Assays (EMSA). The DNA probe used for C/EBP EMSA is the HIV-1 LTR NRE (11). The NF- B probe was TGGGCTGGGGAATCCCGCTAA with bold letters denoting the NF- B binding domain. The DNA probe was labeled with [ 32P]ATP using T4 polynucleotide ki- nase in an end-labeling reaction. Full-length reaction products were isolated and 105 cpm-labeled DNA mixed with 10 g of protein extract, 2.5 g poly dI/dC, and gel mobility shift buffer. For supershift experiments, 1–2 g of antibody was added to the reaction (anti-C/EBP , anti–NF- B p65, or anti–NF- B p50 antibodies; Santa Cruz Biotechnology, Inc.). Within the experi- ments, each binding reaction included a constant amount of ex- tract protein. The DNA–protein complexes were electrophoresed on a 6% polyacrylamide (Bio-Rad Laboratories) gel at 4 C with 20 mM Tris-borate, pH 8.3, and 0.4 mM EDTA buffer. Images were produced by a PhosphorImager (Molecular Dynamics). Results Transcription Factor Expression and HIV-1 Replication in Pulmonary TB. To test if BAL accurately reflects what is occurring in the lung, immunohistochemistry using anti- 497 Hoshino et al. bodies to C/EBP , NF- B p65, and HIV-1 p24 antigen was performed on lung sections obtained from normal and TB patients, with and without HIV-1 infection. Lung sec- tions from TB patients included regions involved and unin- volved with TB. C/EBP was strongly expressed in the nucleus and cy- toplasm of macrophages of normal lung, as demonstrated by immunoperoxidase staining of macrophages (Fig. 1 A). Scoring of 154 AM from normal lung demonstrated 64 (41%) C/EBP with strong nuclear staining. The unin- volved lung segments of an HIV-1–TB-coinfected patient also showed strong C/EBP expression in AM (Fig. 1 B). Scoring of 116 AM from uninvolved lung demonstrated 39 (33%) C/EBP with strong nuclear staining. Near sites of granulomatus inflammation, AM lost nuclear expression of C/EBP expression. Only 3 out of 116 (3%) of AM from patient 1 (Fig. 1 C), and 1 out of 21 (5%) AM from patient 2 (Fig. 1 D), expressed nuclear C/EBP . There was cytoplasmic C/EBP staining in AM from the in- volved lung segment (Fig. 2 C) and in type II pneu- mocytes (unpublished data). NF- B p65 had a markedly different pattern of expres- sion. In normal and uninvolved lung segments of patients with TB, there was little or no binding of antibody to the lung (Fig. 1 E). In normal lung, 0 out of 43 (0%) AM ex- pressed NF- B, whereas only 3 out of 104 (3%) AM from an uninvolved lung expressed NF- B. Lung segments in- volved with TB had increased nuclear NF- B p65 staining in both lymphocytes and macrophages (Fig. 1 F). In in- volved lung segments, 105 out of 192 cells (55%) from pa- tient 1, and 123 out of 181 cells (68%) from patient 2, ex- pressed NF- B. The identity of AM was confirmed by anti-CD 68 staining in serial sections. The presence of my- cobacteria was confirmed by numerous acid-fast bacilli in the alveolar space (unpublished data). HIV-1 p24 was expressed in and around areas of granu- lomatous inflammation (Fig. 1 G). Only epithelioid mac- rophages (Fig. 1 H, arrows) demonstrated positive im- munostaining. Multinucleated cells, dendritic cells, and lymphocytes, had no detectable staining for p24 (unpub- lished data). The blood lymphocytes in areas of pulmonary hemorrhage and normal pulmonary parenchyma from two patients did not stain with anti-p24 antibody. The distribu- tion of anti-p24 immunostaining was markedly different in the lymph node samples where dendritic cells were the predominant cell type, staining with anti-p24 antibody (unpublished data). There was no background staining in either the lung or lymph node. Lymphocyte Contact Is Required for Loss of Inhibitory C/EBP in AM. Similar to our previously reported results (11), BAL cells obtained from an uninvolved lobe of an HIV-1–infected patient with TB, strongly expressed in- hibitory 16-kD C/EBP (Fig. 2 A, lane 1), whereas BAL cells from involved lung segments did not have inhibitory 16-kD C/EBP expression (Fig. 2 A, lane 7). The addi- tion of allogenic lymphocytes stimulated with Con A to AM preparations from the uninvolved lung, abolished in- hibitory C/EBP expression over 4 d (Fig. 2 A, lane 3).