Cell death by necrosis is typically associated with inflammation, in contrast to apoptosis. We have identified additional distinctions between the two types of death that occur at the level of dendritic cells (DCs) and which influence the induction of immunity. DCs must undergo changes termed maturation to act as potent antigen-presenting cells. Here, we investigated whether exposure to apoptotic or necrotic cells affected DC maturation. We found that immature DCs efficiently phagocytose a variety of apoptotic and necrotic tumor cells. However, only exposure to the latter induces maturation. The mature DCs express high levels of the DC-restricted markers CD83 and lysosome-associated membrane glycoprotein (DC-LAMP) and the costimulatory molecules CD40 and CD86. Furthermore, they develop into powerful stimulators of both CD4+ and CD8+ T cells. Cross-presentation of antigens to CD8+ T cells occurs after uptake of apoptotic cells. We demonstrate here that optimal cross-presentation of antigens from tumor cells requires two steps: phagocytosis of apoptotic cells by immature DCs, which provides antigenic peptides for major histocompatibility complex class I and class II presentation, and a maturation signal that is delivered by exposure to necrotic tumor cells, their supernatants, or standard maturation stimuli, e.g., monocyte-conditioned medium. Thus, DCs are able to distinguish two types of tumor cell death, with necrosis providing a control that is critical for the initiation of immunity.

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Existing drugs are slow to eradicate Mycobacterium tuberculosis (Mtb) in patients and have failed to control tuberculosis globally. One reason may be that host conditions impair Mtb's replication, reducing its sensitivity to most antiinfectives. We devised a highthroughput screen for compounds that kill Mtb when its replication has been halted by reactive nitrogen intermediates (RNIs), acid, hypoxia, and a fatty acid carbon source. At concentrations routinely achieved in human blood, oxyphenbutazone (OPB), an inexpensive anti-inflammatory drug, was selectively mycobactericidal to nonreplicating (NR) Mtb. Its cidal activity depended on mild acid and was augmented by RNIs and fatty acid. Acid and RNIs fostered OPB's 4-hydroxylation. The resultant 4-butyl-4-hydroxy-1-(4-hydroxyphenyl)-2-phenylpyrazolidine-3,5-dione (4-OH-OPB) killed both replicating and NR Mtb, including Mtb resistant to standard drugs. 4-OH-OPB depleted flavins and formed covalent adducts with N-acetyl-cysteine and mycothiol. 4-OH-OPB killed Mtb synergistically with oxidants and several antituberculosis drugs. Thus, conditions that block Mtb's replication modify OPB and enhance its cidal action. Modified OPB kills both replicating and NR Mtb and sensitizes both to host-derived and medicinal antimycobacterial agents.

Upon exposure to lysates or supernatants of necrotic transformed cell lines, human dendritic cells (DCs) undergo maturation. In contrast, DCs exposed to apoptotic transformed cell lines or necrotic lysates of primary cells remain immature. Analysis of supernatants of necrotic transformed cell lines showed them to be enriched in the heat shock proteins (hsp)70 and gp96, in contrast to supernatants of primary cells. Likewise, cells from a variety of primary human tumors contained considerably higher levels of hsp than their normal autologous tissue counterparts. Of the majority of human tumors enriched in hsps (hsp70 and/or gp96), their corresponding lysates matured DCs. The maturation effect of tumor cell lysates was abrogated by treatment with boiling, proteinase K, and geldanamycin, an inhibitor of hsps, suggesting that hsps rather than endotoxin or DNA were the responsible factors. Supporting this idea, highly purified, endotoxin-depleted hsp70, induced DC maturation similar to that seen with standard maturation stimuli LPS and monocyte conditioned medium. These results suggest that the maturation activity inherent within tumor cells and lines is mediated at least in part by hsps. The release of hsps in vivo as a result of cell injury should promote immunity through the maturation of resident DCs.

IntroductionIn the steady state, dendritic cells (DCs) capture self-antigens and maintain low expression of costimulatory molecules but nevertheless migrate to lymph nodes where they tolerize self-reactive T cells. [1][2][3][4][5][6][7] Physiologically arising apoptotic cells (ACs) are one significant source of self-antigens for DCs. 5,7 However, following their encounter with ACs, DCs can be rendered immunologically inert, immunosuppressive, or immunostimulatory. [8][9][10][11][12][13][14][15] These contradictory findings have been difficult to resolve due to the complexity of receptors on DCs and the model systems used (eg, ex vivo versus in vivo or rodent versus human sources of ACs).ACs are recognized and captured by human DCs via an array of receptors, including LOX-1, CD36, ␣v␤3, ␣v␤5, and the complement receptors (CRs) CR3 and CR4. 16 The precise contribution of individual receptors in the binding/uptake of ACs, the initiation of downstream signaling pathways, and cross-presentation of cellassociated antigens remains undefined. Blocking antibodies targeting individual receptors inhibit no more than 50% of the association with ACs, and CD36 Ϫ/Ϫ mice have no obvious defects in phagocytosis or in cross-presentation of antigens encoded within ACs, indicating a redundancy in the system and/or the incomplete characterization of AC receptors on DCs. [17][18][19][20] More recent studies have begun to evaluate the ability of specific AC receptors to modulate DC function. DCs exposed to ACs opsonized with C3bi fragments are inhibited from maturing upon stimulation with LPS or CD40L. This effect is presumably mediated through CR3 and CR4, the receptors for C3bi. 21 Studies of the scavenger receptor CD36 and the ␣v-integrin receptor CD51 have led to similar conclusions regarding their ability to modulate DC function. 12,22 However, it is not known whether such function extends to other members or components of the ␣v-integrin receptor family, particularly the well-established phagocytic receptor ␣v␤5. 17,23 Nor is it clear whether ligation of AC receptors that interfere with DC maturation consequently alters their T-cell-stimulating potential.To gain a better understanding of the phagocytic and immunomodulatory role of the various AC receptors on human DCs, we made use of an AC surrogate system 24 that permitted us to evaluate the function of individual AC receptors. We focused on ␣v␤5 and the CRs, especially CR3. We established that these different AC receptors are not equivalent in function, as might be presumed from published studies, but that there is a distinct division of labor, at least with respect to phagocytosis and tolerance induction potential. While ␣v␤5 mediates efficient phagocytosis, it does not interfere with the DC's capacity to undergo maturation or stimulate T cells. In contrast, engagement of CR3 (or CR4) inhibits the ability of DCs to undergo maturation, produce proinflammatory cytokines or M.Š . conceptualized, designed, and performed experiments; acquired, analyzed, and interpreted data; draft...