Figure 2. C/EBP expression and HIV-1 viral loads in AM after lym- phocyte addition. (A) Western blot of whole cell extract probed with C/EBP polyclonal antibody. AM from an uninvolved lung segment of an HIV-1–TB-coinfected patient (lane 1). Con A–stimulated allogenic lymphocytes were added to the AM for 1 (lane 2) or 4 d of coculture (lane 3). Resting syngenic lymphocytes were added for 1 (lane 4) or 4 d (lane 5). Con A–activated lymphocytes are separated from AM by a 0.4- m filter insert (lane 6). AM from a lung segment involved with TB (lane 7). (B) AM from two HIV-1–infected patients with normal chest radio- graphs after 1 d of culture (lanes 1 and 5). Activated allogenic lympho- cytes were added for 1 (lanes 2 and 6), 4 (lanes 3 and 7), or 6 d (lane 4). The HIV-1 viral copy number released into the cell culture supernatant for each condition is shown in the boxes below the Western lanes. vivo and therefore contained variable amounts of HIV-1. The addition of allogenic lymphocytes from an HIV-1– negative donor enhanced HIV-1 replication in AM prepa- rations from two HIV-1–infected patients (Fig. 2 B, com- pare lane 1 with lanes 2–4, and lane 5 with lanes 6 and 7). Increasing levels of HIV-1 production correlated with the loss of inhibitory C/EBP expression in this system. We have observed that THP-1 cells differentiated with PMA and treated with IFN- are similar to AM, and we have used this model system to investigate the mechanisms controlling HIV-1 replication in macrophages (11, 14). We used EMSA with the HIV-1 LTR NRE to measure C/EBP binding activity in the THP-1 extracts. In differen- tiated THP-1 cells there is a single DNA protein complex that competes with excess unlabeled NRE oligonucleotide (Fig. 3 A, compare lanes 1 and 2). The addition of IFN- leads to the induction of another specific, rapidly migrating NRE–protein complex (Fig. 3 A, lanes 3 and 4). Con A–activated lymphocytes produce loss of the rapidly mi- grating NRE–protein complex over 2 d (Fig. 3 A, lanes 5–8). Supershift with antibody to C/EBP demonstrates that both the NRE–protein complexes contain C/EBP (Fig. 3 A, lane 10). A minor rapidly migrating NRE–protein com- 499 Hoshino et al. plex is unmasked by the supershift reaction, whereas C/EBP contributes over 90% of the NRE binding activ- ity in this system. Western blotting assessed expression of both C/EBP isoforms. Activated lymphocytes down-regulate inhibitory C/EBP expression in THP-1 macrophages (Fig. 3 B, lanes 1–3). When the activated lymphocytes and macro- phages are separated by a 0.4- m insert, inhibitory C/EBP expression is increased (Fig. 3 B, lanes 4–6). These data support using THP-1 cells treated with PMA and IFN- as a model for AM, and demonstrate that contact between lymphocytes and macrophages is required for loss of inhib- itory C/EBP in this model system. We then tested the ability of purified CD4 and CD8 lymphocyte subsets to down-regulate inhibitory C/EBP . Surprisingly, both acti- vated CD4 and CD8 lymphocytes were able to down- regulate inhibitory C/EBP (Fig. 3 C, lanes 2 and 4). In addition, the CD 8 lymphocytes abolished stimulatory C/EBP expression (Fig. 3 C, lane 4). To address the pos- sibility that production of the inhibitory 16 kD is due to calpain-mediated proteolytic cleavage (17), we repeated these experiments with a calpain inhibitor (18). The addi- tion of calpain inhibitor did not lead to significant change in the expression of 16-kD C/EBP in whole cell extracts (Fig. 3 D, compare lanes 1 and 2) or nuclear extracts (Fig. 3 D, compare lanes 5 and 6). Ca2 , a known stimulator of calpain-mediated proteolysis, increased the amount of 16- kD C/EBP (Fig. 3 D, compare lanes 2 and 3); calpain in- hibitor blocked this increase (Fig. 3 D, lanes 3 and 4). The 16-kD C/EBP measured by Western blot analysis was present in the nuclear fraction (Fig. 3 D, lane 5), whereas a majority of the 37-kD C/EBP was in the cytoplasmic fraction (Fig. 3 D, lane 7). As a result, the ratio of inhibi- tory to stimulatory C/EBP in whole cell extracts is an un- derestimate of the state of transcriptional inhibition present in the nucleus. The Effect of Lymphocyte Contact is Mimicked by Cross- linking Costimulatory Molecules CD-40, VCAM, and B7. Because macrophage-expressed costimulatory molecules CD40, VCAM, and B7 are important mediators of lym- phocyte–macrophage interaction, we tested whether or not antibodies against CD-40, VCAM, and B7 would alter C/EBP expression. There was no change in C/EBP ex- pression when a combination of antibodies against CD-40, VCAM, and B7 (stimulating antibodies) were added in the absence of protein A/G beads (Fig. 4 A, lanes 1, 3, and 5). When a combination of stimulating antibodies were at- tached to a solid substrate by incubating them with agarose protein A/G beads, macrophages markedly down-regulate C/EBP expression after 2 d (Fig. 4 A, lanes 4 and 6). Goat IgG isotype control did not alter C/EBP expression with or without protein A/G beads (Fig. 4 A, lanes 1 and 2). The beads by themselves did not alter C/EBP expres- sion (unpublished data). The expression of inhibitory C/EBP was not significantly changed when only two of these antibodies were used in combination (Fig. 4 B, com- pare lane 1 with lanes 3, 5, and 7). The addition of protein A/G beads did, however, lead to the downregulation of