Figure 6. NF- B DNA binding activity in THP-1 macrophages and AM after lymphocyte addition. (A) EMSA with an oligonucleotide containing an NF- B binding site in whole cell, extracts THP-1 cells after PMA differentiation (lanes 1), the addi- tion of IFN- (lane 3), and the addition of lym- phocytes for 1 (lane 5) and 2 d (lane 7). Excess un- labeled oligonucleotide was added to the extract in the preceding lane (lanes 2, 4, 6, and 8). EMSA with nuclear extracts from an independent experi- ment 2 d after coculture of THP-1 macrophages with stimulated allogeneic lymphocytes (lane 9) su- pershifted with anti-p65 NF- B antibody (lane 10), or anti-p50 NF- B antibody (lane 11). EMSA of nuclear extracts 2 d after separation of activated lymphocytes from macrophages with a 0.4- m filter insert (lane 12), supershifted with anti-p65 NF- B antibody (lane 13), or anti-p50 NF- B antibody (lane 14). An independent experiment with whole cell extracts 2 d after addition of lymphocytes (lane 15) or separation of activated lymphocytes from macrophages by a 0.4- m filter insert (lane 16), or 2 d after antibodies to CD-40, VCAM, and B7 in the presence of protein A/G beads (lane 17). Ex- periments were repeated three or more times with similar results. (B) EMSA of whole cell extracts from AM after 1 d of culture without treatment (lane 1). Extracts from the same patient treated with 1 (lane 3) and 4 d (lane 5) of coculture with resting syngenic lymphocytes, and 1 (lane 7) and 4 d (lane 9) of coculture with Con A–activated allogeneic lymphocytes. EMSA of BAL from an HIV-1–TB- coinfected patient with uninvolved lung segment (lane 11) and involved lung segment (lane 13). Excess unlabeled oligonulceotide was added to the ex- tract (lanes 2, 4, 6, 8, 10, 12, and 14). the complex (Fig. 6 A, lanes 4, 6, and 8). Antibody to the p50 or p65 isoform of NF- B supershifts the complex (Fig. 6 A, compare lane 9 with lanes 10 and 11). When lympho- cytes are separated from the THP-1 cells with an insert, there is an increase in NF- B binding activity similar to di- rect contact of lymphocytes and macrophages (Fig. 6 A, lane 12). This complex also contains NF- B p50 and p65 (Fig. 6 A, lanes 13 and 14). These data demonstrate that lymphocyte-derived soluble factors are capable of activat- ing NF- B in this system. In an independent experiment, the soluble factors were more effective than contact in inducing NF- B (Fig. 6 A, compare lanes 15 and 16), whereas protein A/G beads with stimulatory antibodies to CD-40, VCAM, and B7, failed to induce NF- B (Fig. 6 A, lane 17). Coculture of primary AM of an HIV-1–infected patient without lung disease and without TB was used to validate THP-1 cells as a model system. In the absence of stimula- tion, NF- B was not induced in AM (Fig. 6 B, lanes 1 and 2). The addition of resting syngenic lymphocytes did not induce NF- B DNA binding activity at 1 or 4 d (Fig. 6 B, lanes 3–6). The addition of activated allogenic lym- phocytes did lead to the induction of NF- B (Fig. 6 B, lanes 7–10). The uninvolved lung segment of a patient with TB had little NF- B (Fig. 6 B, lane 11), whereas there was marked upregulation of NF- B in the involved lung segment from the same patient (Fig. 6 B, lane 13). Upregulation of NF- B was observed in the involved lung segment of two additional patients (unpublished data). Competition with excess unlabeled oligonucleotide demonstrated a nonspecific band in the resting and unin- volved AM preparations. The similarity of these results with primary and transformed cells supports the use of THP-1 cells in this system. Discussion HIV-1 replication in AM is normally suppressed with fewer than 1 in 10,000 cells harboring provirus and little or no HIV-1 present in bronchoalveolar lining fluid (1). During TB there is a marked increase in HIV-1 replication in the lung with the AM as the major source (1–3). The likely source of this virus is latently infected cells that re- side in the lung before the occurrence of TB (1, 2). TB ac- celerates AIDS mortality even when the TB is appropri- ately treated (4). The enhanced viral replication and increased viral mutation likely underlies the accelerated course of AIDS observed in TB-coinfected patients. High levels of HIV-1 replication in vivo are associated with the loss of an inhibitory C/EBP transcription factor and the increase of NF- B (11). Attempts to model this state of macrophage activation by infecting cells in vitro with M. tuberculosis did not reproduce the changes in C/EBP ex- pression nor viral replication observed in patients with pul- monary TB (14). We now report that the addition of acti- vated lymphocytes to macrophages reproduces the state of activation observed during TB. There is a loss of inhibi- tory C/EBP , an activation of NF- B, and an increase in HIV-1 replication. Investigation of this model further demonstrated that maximal transcriptional activation of the HIV-1 LTR is a two-step process that requires both con- tact and soluble factors. The contact leads to a loss of in- 502 Contact and Cytokines Are Needed for Maximal HIV Replication