the 37-kD C/EBP in this set of experiments. EMSA with the HIV-1 LTR NRE shows similar results. Stimulating antibodies on a solid substrate markedly reduced NRE– protein complexes when compared with antibodies added in solution (Fig. 4 C, compare lanes 1 and 3 with lanes 2 and 4). When single antibodies to CD-40, VCAM, B7-1, or B7-2 were added to the THP-1 model, there was no change in NRE–protein complexes. This was true whether or not protein A/G beads were added (Fig. 4 C, compare lane 5 with lanes 6–13). These data suggest that the cross-linking of multiple macrophage-expressed co- stimulatory molecules is required to down-regulate inhibi- tory C/EBP expression. CD40 ligand, VLA-4, and CD-28 are the lymphocyte- expressed ligands that activate macrophage-expressed CD- 40, VCAM, and B7 (16, 17). FACS® analysis showed that CD-40 ligand, VLA-4, and CD-28 were strongly expressed on Con A–activated lymphocytes, and that both CD-40 ligand and CD-28 were upregulated by activation (unpub- lished data). A combination of antibodies to CD-40 ligand, VLA-4, and CD-28 blocked the downregulation of macro- phage 16-kD C/EBP (Fig. 4 D, compare lane 3 with lanes 2 and 4). This was true in both the THP-1 model and in primary AM. Blocking occurred when lymphocytes were activated by MHC incompatibility (Fig. 4 D, lanes 1–4) or by antibody to CD-3 (unpublished data). Maximal HIV-1–LTR Induction Requires both Lymphocyte– Macrophage Contact and Lymphocyte-derived Cytokines. To investigate the functional effects of macrophage–lympho- cyte interaction on HIV-1 replication we used BF-24 cells, which are THP-1 cells with an integrated HIV-1 LTR CAT reporter construct. As shown in Fig. 5, LTR activity increased 12.5- 1.6-fold (mean SEM) when Con A–activated lymphocytes were mixed with BF-24 cells. LTR activity increased only 5.1- 1.5-fold when the acti- vated lymphocytes were separated from the BF-24 cells with a 0.4- m pore-size insert (P 0.01 Student’s t test when compared with contact with Con A–stimulated lym- phocytes), in spite of a marked elevation of IL-1 (350 pg/ ml), IL-6 (11,600 pg/ml), and TNF- (1,000 pg/ml) in the cell culture supernatant. Similarly, LTR activity increased only 3.3- 1.1-fold when a combination of cross-linking antibodies to CD-40, VCAM, and B7 were added in the presence of protein A/G beads (P 0.01 when compared with Con A–lymphocyte contact). To combine the contribution of soluble factors and con- tact, activated lymphocytes were added to the upper cham- ber of an insert well to provide both lymphocyte-derived soluble factors, and stimulatory antibodies on a solid sub- strate were added to BF-24 cells in the lower chamber to provide contact-mediated stimuli. Maximal LTR activa- tion was restored (14.6- 0.7-fold increase) when BF-24 cells were exposed to lymphocyte-soluble factors and con- tact with antibodies to CD40, VCAM, and B7 on protein A/G beads. When IL-1 (350 pg/ml), IL-6 (11,600 pg/ ml), and TNF- (1,000 pg/ml) were added to BF-24 cells with cross-linking antibodies to CD40, VCAM, and B7, HIV-1–LTR activity was markedly increased (8.1- 1-fold). This demonstrates that both contact and soluble factors are necessary and sufficient to produce the level of LTR stimu- lation observed when lymphocytes and macrophages are cocultured. The NF- B transcription factors are excellent candidates to mediate the effects of soluble factor(s) released by lym- phocytes. We used EMSA to measure NF- B DNA bind- ing activity in cocultured cells. THP-1 cells that differenti- ated with PMA have no specific NF- B DNA binding activity (Fig. 6 A, lane 1). The addition of IFN- produces a slight increase in the amount of NF- B DNA binding ac- tivity (Fig. 6 A, lane 3). The addition of activated lympho- cytes produced a marked increase in NF- B DNA binding activity after 1 or 2 d of coculture (Fig. 6 A, lanes 5 and 7). This DNA binding activity is specific for the NF- B site because excess, unlabeled oligonucleotide competes with 501 Hoshino et al. Figure 5. HIV-1 LTR promoter in IFN- –treated THP-1 macrophages. From left to right, HIV-1–LTR CAT production 2 d after the addition of: Con A–activated allo- genic lymphocytes; activated lymphocytes separated from macrophages with a 0.4- m filter; antibodies to CD-40, VCAM, and B7 in the presence of protein A/G beads; acti- vated lymphocytes added to the upper chamber of an insert well and stimulatory antibodies with protein A/G beads added to the lower chamber; and stimulatory antibodies at- tached to protein A/G beads in addition to IL-1 (350 pg/ ml), IL-6 (11,600 pg/ml), and TNF- (1,000 pg/ml).