hibitory C/EBP that derepress the HIV-1 LTR, whereas soluble factors produce NF- B activation that enhances HIV-1–LTR transcription. THP-1 cells were used to investigate the mechanisms underlying the contact-mediated loss of inhibitory C/EBP . THP-1 cells are a human monocytic cell line that are similar to AM when they have been differentiated with PMA and stimulated with low-dose IFN- ; both ex- press inhibitory C/EBP and repress HIV-1 replication. C/EBP sites in the HIV-1 LTR are required for HIV-1 replication in macrophages, but not in lymphocytes (9). C/EBP is the predominant C/EBP family member ex- pressed in macrophages (5, 11), and overexpression of in- hibitory 16-kD C/EBP strongly inhibits HIV-1–LTR transcription in model systems (12). TB leads to the loss of inhibitory C/EBP , which derepresses the HIV-1 LTR (11). Both Western blots of BAL cells and immunohis- tochemistry of lung sections confirm a significant reduction of C/EBP expression in lung segments involved with TB. This is particularly true of short-form C/EBP that is lo- calized in the nucleus. The expression of cytoplamic long form is maintained in a number of conditions, raising the possibility of nonnuclear functions of this protein in macro- phages. The regulation of short-form C/EBP is particu- larly important because it is a dominant-negative transcrip- tion factor (6) and a strong repressor of HIV-1 transcription in macrophages (12). Mechanisms that produce inhibitory C/EBP are not fully understood. Genetic evidence using expression con- structs suggests that inhibitory C/EBP is produced by translational initiation at an internal AUG start site (6, 19, 20). Proteolysis occurring in vivo has also been proposed as a mechanism for short-form production (21). Concern has been raised, however, that production of inhibitory C/EBP occurs during the extraction procedure and is mediated by a calpain protease (17). The protease is inhib- ited by NP-40 detergent and calpain inhibitor and is en- hanced by Ca2 (18). All of the cell extracts presented in this investigation were made with NP-40 to prevent a proteolytic artifact. The addition of calpain inhibitor to the NP-40 extraction buffer did not affect the amount of inhibitory C/EBP . The addition of Ca2 to the extrac- tion buffer increased the amount of inhibitory C/EBP , demonstrating that a Ca2 -responsive protease was present. Calpain inhibitor was effective in blunting the increase of inhibitory C/EBP after the addition of Ca2 to the ex- traction buffer, which suggests that although a calpain-like protease is present in the THP-1 macrophages extracts, it did not contribute to the amount of 16-kD C/EBP observed. These data strongly support the conclusion that under the conditions used in this investigation, the amount of 16-kD C/EBP provides a relevant measure of the inhibition of promoters with C/EBP binding sites. Adding activated lymphocytes to macrophages over- comes the transcriptional repression of the HIV-1 LTR in- duced by low-dose IFN- . This interaction is not MHC restricted and occurs with both CD4 and CD8 lympho- 503 Hoshino et al. cyte subsets. HIV-1–TB-coinfected patients have a CD8 lymphocytic alveolitis (22). Our finding that CD8 lym- phocyte subsets are capable of downregulating both C/EBP isoforms fits with the observation that in some patients no C/EBP is expressed in involved lung seg- ments (11). Lymphocytes activated by MHC incompatibil- ity, Con A, or anti-CD3 antibody abolished the expression of inhibitory C/EBP in both AM and THP-1 macro- phages. AM cocultured for 4 d with resting syngenic lym- phocytes had stable expression of both stimulatory and in- hibitory C/EBP , indicating that the changes in C/EBP produced by activated lymphocytes were not an artifact of ex vivo tissue culture. When allogenic lymphocytes from an HIV-1–negative donor are added to AM from HIV-1– infected patients, HIV-1 replication increases and inhibi- tory C/EBP is lost. The increased HIV-1 replication may be due in part to HIV-1 infection and replication in the added lymphocytes. However, the 12.5-fold increase in HIV-1–LTR transcriptional activity when lymphocytes are added to THP-1 macrophages, suggests that transcriptional activation of the LTR in macrophages significantly con- tributes to the increased viral replication observed in the ex vivo coculture experiments. When the lymphocytes and macrophages were separated by a 0.4- m insert, activated lymphocytes failed to reduce inhibitory C/EBP expression in either AM or the THP-1 cell model. Functionally, HIV-1–LTR activation in mac- rophages is reduced by 66% when macrophages are sepa- rated from activated lymphocytes. This demonstrates that direct contact between lymphocytes and macrophages is required for downregulation of inhibitory C/EBP and maximal induction of the HIV-1 LTR in macrophages. These findings are consistent with the observation that the membrane fraction of activated lymphocytes enhances HIV-1 replication in macrophages (16). HIV-1 viral load and TNF- production are strongly correlated (r2 0.95) in involved lung segments of AIDS patients with pulmonary TB (2). One explanation of these findings is that the HIV-1 LTR and the TNF- promoter are coordinately regulated in the lung during opportunistic infections. The TNF- promoter, like the HIV-1 LTR, contains C/EBP sites. One of the consequences of express- ing the inhibitory C/EBP in macrophages is that proin- flammatory cytokine production is strongly suppressed (7). Similar to the observations that maximal induction of the HIV-1 LTR requires contact, maximal induction of TNF- requires contact between activated lymphocytes and brain macrophages (23, 24). The stimulation of TNF- is due in part to lymphocyte-expressed CD-40 ligand, VLA-4, and CD-28 binding to macrophage- expressed CD-40, VCAM, and B7. Antibodies binding macrophage costimulatory receptors CD-40, VCAM, and B7 were used to mimic the effect of lymphocyte contact. To further enhance the cross-linking effect of the antibodies, the Fc portion of these antibodies was attached to protein A/G beads. Expression of inhibi- tory C/EBP is lost only when a combination of affinity- purified antibodies to CD-40, VCAM, and B7-1 or B7-2