Macrophages are the resident professional phagocytes in noninflamed tissues and survive in situ for extended periods of time (1
). In keeping with this phenotype, macrophages exhibit inherent resistance to apoptosis and maintain viability in the presence of proapoptotic factors (2
). Bacterial infection is a potent stimulus for apoptosis, and microbial virulence factors inhibit macrophage viability to impede antibacterial defenses (3
). Surprisingly, macro-phage apoptosis can also facilitate immune responses by limiting bacterial replication (4
). This apparent paradox suggests that optimal macrophage antibacterial responses require transient maintenance of cell viability in the face of proapoptotic stimuli, followed by apoptosis induction for maximal bacterial clearance and minimal immune-mediated tissue injury. The control of this switch in macrophage phenotype is therefore an essential regulator of innate host defense.
The molecular basis of macrophage resistance to apoptosis includes expression of antiapoptotic Bcl-2 family members, among which Mcl-1 predominates in differentiated human macrophages (2
). Mcl-1 was first identified as a molecule that is upregulated during differentiation of immature myeloid cells along the monocyte/macrophage pathway (5
). It is unique among antiapoptotic members of the Bcl-2 family in that it is an early-response gene that can be rapidly induced and turned over (6
). Mcl-1 is expressed during critical transitions in cell phenotype and protects against apoptosis during the initial steps of differentiation, exposure to growth stimuli, or the rapid response to potentially cytotoxic stimuli including chemotherapeutic agents, UV irradiation, and calcium ionophores (5
). The importance of Mcl-1 to macrophage survival has been confirmed in Mcl-1 transgenic mice and by antisense inhibition in human macrophages (2
Antiapoptotic Bcl-2 family members exert their effect in part by maintaining mitochondrial homeostasis and inhibiting mitochondrial membrane permeabilization (MMP) (11
). Mcl-1 is found in mitochondrial-enriched cell fractions and can heterodimerize with proapoptotic Bcl-2 family members, such as Bax and Bak, to prevent MMP (8
). Loss of Mcl-1 is an apical event in the apoptotic cascade, as illustrated by its downregulation during UV-induced or adenoviral protein E1A–induced apoptosis (12
). Mcl-1 is subject to differential splicing, resulting in the generation of proapoptotic splice variants (14
). Mcl-1 is, therefore, an ideal regulator of cell viability during rapidly evolving conditions of cell stress and could allow the dynamic changes in cell viability required during bacterial infection.
The most extensively studied model of host-mediated macro-phage apoptosis involves the chronic intracellular pathogen Mycobacterium tuberculosis
, in which apoptosis limits bacterial replication and, conversely, intracellular replication is prolonged by inhibiting apoptosis (4
). Mcl-1 expression is upregulated in macrophages by M. tuberculosis
and inhibits host-mediated apoptosis, hence promoting mycobacterial survival and chronic intracellular persistence (16
). We have characterized a different model of host-mediated macrophage apoptosis involving Streptococcus pneumoniae
(the pneumococcus). This model does not exhibit chronic intracellular persistence of bacteria (17
). Instead, bacteria are effectively eliminated through a response that proceeds in 2 phases: an initial period of macrophage viability and intracellular bacterial killing, followed after 14–16 hours by apoptosis induction and clearance of bacteria. Inhibition of the latter phase of apoptosis induction reduces bacterial clearance in vitro and in vivo (17
). This model therefore allows analysis of both phases of the macrophage response to bacterial infection.
We used a model of pneumococcal-associated host-mediated macrophage apoptosis to test the hypothesis that Mcl-1 is critical to both initial maintenance of cell viability and subsequent induction of apoptosis. Mcl-1 was upregulated in association with the retention of cell viability after initial bacterial phagocytosis. Mcl-1 was subsequently downregulated, and a protein of approximately 34 kDa with an electrophoretic mobility corresponding to a novel proapoptotic BH3-only splice variant Mcl-1Exon-1 was upregulated, during the initiation of MMP and induction of apoptosis. These changes in Mcl-1 expression have a role in the response to infection, as macrophages from transgenic mice overexpressing Mcl-1 demonstrated delayed MMP and impaired bacterial clearance in vitro. Mcl-1 transgenic mice demonstrated less effective clearance of bacteria from the lung in vivo. These findings demonstrate the importance of an Mcl-1–regulated switch from macrophage viability to apoptosis for optimal host defense.