are attached to agarose beads and presented to macrophages. The specific effect of the stimulating antibody–agarose bead combination is demonstrated by the stable expression of C/EBP after the addition of goat IgG isotype-control an- tibody, either in the presence or absence of protein A/G agarose beads. These findings show that lymphocyte- derived soluble factors are not required to abolish in- hibitory C/EBP expression, and antibodies to CD-40, VCAM, and B7 on a solid substrate, are able to substitute for lymphocyte contact. C/EBP expression is unchanged when single antibod- ies to CD-40, VCAM, and B7-1 or B7-2 are attached to agarose beads and presented to macrophages. This demon- strates that multiple costimulatory receptors must be cross- linked before the signal to reduce C/EBP expression is transduced. This suggests that when antibodies are oriented on a solid substrate they are more effective in producing signal transduction, possibly because all costimulatory mol- ecules are cross-linked at the point of contact between the bead and the macrophage. Lymphocyte-expressed CD-40 ligand, VLA-4, and CD-28 bind macrophage-expressed CD-40, VCAM, and B7. Activated lymphocytes expressed all three ligands. An- tibodies directed against lymphocyte-expressed ligands were tested for the ability to block downregulation of in- hibitory C/EBP expression. A mixture of antibodies to lymphocyte-expressed CD-40 ligand, VLA-4, and CD-28 blocked the downregulation of C/EBP in both AM and THP-1 macrophages. These antibodies were capable of blocking the effect of activated lymphocytes. These data support a model in which multiple lymphocyte-costimula- tory molecules must interact with multiple macrophage- costimulatory molecules in order to down-regulate inhibi- tory C/EBP (Fig. 7). Whereas lymphocyte-derived soluble factors are unable to down-regulate inhibitory C/EBP , they are able to par- tially activate the HIV-1 LTR. There are many cytokines released in this system including high levels of IL-1 , IL-6, and TNF- . All of these cytokines are capable of activating NF- B, and the binding of NF- B to the HIV-1 LTR strongly stimulates the HIV-1 LTR (12, 13). EMSA dem- onstrated that NF- B p50 and p65 were activated in cocul- ture experiments. In addition to activation of NF- B, lymphocyte-derived soluble factors alter macrophage mor- phology and activate other DNA binding activities (unpub- lished data). The activation of NF- B, and possibly other transcription factors by soluble factors, accounts for the par- tial activation of the HIV-1 LTR observed in experiments in which activated lymphocytes are separated from macro- phages by an insert. The combination of lymphocyte-derived soluble factors with contact-mediated stimuli provided by antibodies to CD-40, VCAM, and B7-1 or B7-2, restored maximal HIV-1–LTR activity. The addition of IL-1 , IL-6, and TNF- at concentrations found in coculture experi- ments to THP-1 macrophages with cross-linked CD-40, VCAM, and B7, also markedly increased HIV-1–LTR ac- tivity. This leads to a model in which two steps are re- Figure 7. A model of lymphocyte–macrophage interaction during the cellular immune response leading to increased HIV-1 replication. Contact leads to the loss of inhibitory C/EBP , derepressing the 5 HIV-1 LTR whereas soluble factors activate NF- B, which enhances HIV-1 replication. quired for full activation of the HIV-1 LTR. Contact leads to the loss of inhibitory C/EBP derepressing the HIV-1 LTR, whereas soluble factors leads to the activation of NF- B. Neither stimulus is sufficient for full induction of the HIV-1 LTR. Upregulation of the HIV-1 LTR in macrophage by acti- vated lymphocytes represents another example of HIV-1 usurping normal immune regulation in order to enhance its replication. Granulomatus inflammation is highly destruc- tive to lung architecture. Many of the macrophage media- tors of cellular immunity have C/EBP sites in their pro- moters, and inhibition of these proinflammatory pathways might be important for maintaining lung homeostasis in the absence of infection. The requirement for lymphocyte contact to produce the state of activation seen in TB, likely limits the tissue destruction observed in granulomatous in- flammation to areas where lymphocytes are activated by the presence of antigen stimulation. We would like to thank Richard Pine (Public Health Research In- stitute, New York, NY), Nobukazu Watanabe, and Tetsuya Naka- mura (University of Tokyo, Department of Infectious Diseases, In- stitute of Medical Science, Tokyo, Japan) for technical advice. This work is supported by MO1 RR00096, HL57879, HL59832, HL62055 American Lung Association, New York Uni- versity Center for AIDS Research, and the Japanese Foundation for AIDS Prevention. Submitted: 20 September 2001 Revised: 13 December 2001 Accepted: 8 January 2002 504 Contact and Cytokines Are Needed for Maximal HIV Replication