To further understand the cellular and molecular events involved in IFN-γ–induced phenotype generation, we took advantage of a transgenic system developed in our laboratory in which IFN-γ effector pathways can be selectively assessed in vivo and used this system to characterize the chemokine response that is induced by IFN-γ in this setting. These studies demonstrate that IFN-γ is a potent inducer of a number of CXC and CC chemokines. Prominent on this list are the CCR5 ligands RANTES/CCL-5, MIP-1β/CCL-4, and MIP-1α/CCL-3. These effects were not specific for these moieties, as IFN-γ also stimulated the production of MCP-1/CCL-2, MCP-2/CCL-8, MCP-5/CCL-12, MIP-2/CXCL-2/3, KC/CXCL-1, ENA 78/CXCL-5, Mig/CXCL-9, IP-10/CXCL-10, I-TAC/CXCL-11, SDF-1/CXCL-12, C10/CCL6, MDC/CCL22, and TECK/CCL25. These effects were, however, at least partially specific, as eotaxin/CCL-11, TARC/CCL-17, and lungkine/CXCL-15 were not similarly regulated. Importantly, these studies also demonstrate that interventions that neutralize or abrogate CCR5 diminish the ability of IFN-γ to induce inflammatory, DNA injury, cell death, and remodeling responses in the murine lung. They also define the chemokine cascades, protease and antiprotease alterations, and apoptosis regulatory events that are dependent on CCR5 in these responses. When viewed in combination, these studies provide insights into the pathogenesis and complexity of IFN-γ–induced inflammatory and remodeling responses in the setting of type I tissue reactions.
Chemokines are small cytokines (44
) that have been subdivided into CXC, CC, C, and CXXC families based on sequence considerations. The CC and CXC chemokine groups are large and contain more than 50 identified ligands. Although in vitro characterization would suggest that there is impressive redundancy in this system, examinations in vivo have demonstrated that their production is often organized in a coordinated manner and that their effector functions can be restricted to different stages of disease development and/or pathology (44
). Thus, in vivo, a deficiency of an individual ligand or its receptor can cause striking alterations in tissue phenotype. Our studies demonstrate that, although IFN-γ is a potent stimulator of a wide variety of CC and CXC chemokines, the selective neutralization and/or ablation of CCR5 caused an impressive decrease in IFN-γ–induced inflammation and tissue remodeling responses. One could hypothesize that this previously unappreciated relationship between IFN-γ and CCR5 is the result of the amelioration of the stimulatory effects of RANTES/CCL-5, MIP-1α/CCL-3, and/or MIP-1β/CCL-4. Our studies, however, demonstrate that this is only part of the mechanism. Specifically, they demonstrate that CCR5 signaling plays a critical role in, and is required for, an optimal IFN-γ–induced chemokine response, with the induction of MCP-1/CCL-2, MIP-1α/CCL-3, MIP-1β/CCL-4, MIP-2/CXCL-2/3, RANTES/CCL-5, KC/CXCL-1, Mig/CXCL-9, SDF-1/CXCL-12, and IP-10/CXCL-10 being mediated by a CCR5-dependent mechanism. Cigarette smoke induction of RANTES/CCL-5, MIP-1α/CCL-3, and MIP-1β/CCL-4 was also shown to be CCR5 dependent. Interestingly, CCR5 signaling did not contribute to the induction by IFN-γ of a variety of other chemokines, including MCP-2/CCL-8, MCP-5/CCL-12, Mig/CXCL-9, and I-TAC/CXCL-11. These studies highlight, for the first time to our knowledge, the ability of CCR5 to contribute to the autoinduction of its own ligands, RANTES/CCL-5, MIP-1α/CCL-3, and MIP-1β/CCL-4. They also highlight, for the first time to our knowledge, the existence of a CCR5-dependent chemokine cascade that contributes to the induction, intensity, and character of IFN-γ–induced and cigarette smoke–induced tissue inflammatory and remodeling responses.
Although 18 million people in the United States and millions more worldwide suffer from emphysema, the mechanisms of emphysematous alveolar remodeling and destruction are poorly understood. In keeping with the belief that Tc1 responses contribute to the pathogenesis of pulmonary emphysema, a CD8 cell–, mononuclear cell–, and neutrophil-rich inflammatory response has been noted in tissues from patients with COPD (18
), and previous studies from our laboratory demonstrated that the prototypic Th1/Tc1 cytokine IFN-γ induces pulmonary emphysema when expressed in the murine lung (19
). In the present studies, we add to this understanding by demonstrating that CCR5 plays a key role in Th1-induced and cigarette smoke–induced remodeling responses in the lung. By demonstrating that interventions that neutralize and or ablate CCR5 only partially abrogate these responses, we also highlight the CCR5-dependent and -independent mechanisms that are operative in these settings. Last, we provide insights into the multiple mechanisms that CCR5 may use in this setting. Specifically, the protease-antiprotease hypothesis suggests that alveolar destruction is induced in the lung by an increase in proteases and/or a decrease in antiproteases (20
). In accordance with this concept, our studies demonstrate that the decrease in IFN-γ–induced remodeling that is seen after CCR5 neutralization and/or ablation is associated with a decrease in the expression of MMP-9 and an increase in the expression of the antiprotease SLPI. Most recently, structural cell apoptosis has been proposed to contribute to the pathogenesis of pulmonary emphysema (24
). In accordance with this concept, our studies demonstrate that transgenic IFN-γ is a potent inducer of epithelial cell apoptosis and that this response is mediated via a mechanism that is, at least in part, CCR5 dependent. Recent studies have also demonstrated that CCR5 ligands are impressively potent stimulators of CD8 cell migration (52
). In combination, these studies demonstrate that CCR5 signaling contributes to IFN-γ–induced inflammatory, proteolytic, antiprotease, DNA injury, and cell death responses in the lung. Our demonstration that cigarette smoke causes emphysema via an IFN-γ–dependent mechanism and that null mutations of CCR5 diminish the inflammatory and remodeling alterations that are induced by chronic cigarette smoke inhalation further support the relevance of our transgenic studies and our CCR5-based investigations to human COPD. These studies provide what we believe to be the first mechanistic link between inflammation and apoptosis in COPD and, when combined with our transgenic studies, demonstrate that IFN-γ is necessary and sufficient to induce pulmonary emphysema. When viewed in combination, they also suggest that interventions that alter CCR5-ligand binding and/or CCR5 signaling may be therapeutically useful in the treatment of this often-times devastating disorder.
Although cell death can be triggered by a vast array of stimuli and mediated via an increasingly complex series of pathways, the vast majority of signals engage the cell death machinery at the level of the cell membrane or at the level of the mitochondria. The membrane (“extrinsic”) pathway triggers cell surface “death receptors” such as Fas, which bind FasL, and TNF receptor 1 (TNFR1), which binds TNF and lymphotoxin and subsequently activate caspase-8. Other stimuli use mitochondrial dysfunction to signal death responses. In this “intrinsic” response, BH3 domain–only family members such as Bid are activated to tBid and interact with Bax-type proteins to form or interact with mitochondrial pores, release cytochrome c
, activate caspase-9, and induce cell death (53
). Thus, to further understand the mechanism(s) by which CCR5 regulates IFN-γ–induced cell death responses, we characterized the intrinsic and extrinsic pathways in mice treated with anti-CCR5 or control Ig and mice with wild-type and null CCR5
loci. These studies demonstrate that IFN-γ activates both the intrinsic and extrinsic cell death pathways. They also demonstrate that CCR5 contributes to these responses in a variety of ways, including regulating the levels of mRNA encoding Fas, FasL, TNF, TNFR1, TNFR2, caspases, Bid, and Bax; the levels of TNF protein; and the activation and bioactivity of caspase-3, -8, and -9 and Bid. These are the first studies to our knowledge to demonstrate a prominent role for CCR5 in the regulation of IFN-γ–induced cell death responses and the first to demonstrate a prominent role of CCR5 in structural cell apoptosis. These observations, however, are not without precedent, as CCR5 has been shown to be a coreceptor for HIV, where it activates Fas and caspase-8 and induces CD4 cell death (42
). They also agree with the recent demonstration that, in contrast to Th2 responses, Th1 granulomatous responses are preferentially associated with the induction of genes that are involved in tissue injury and apoptosis (4
). It is clear from these studies that CCR5 is a multifunctional regulator of IFN-γ–induced apoptotic and necrotic responses in the lung. Our demonstration that CCR5
-null mice are protected from the DNA injury and cell death induced by cigarette smoke further substantiates the disease relevance of these findings in Th1/Tc1 responses and oxidant injury states.
In summary, these studies demonstrate that IFN-γ is a potent stimulator of a variety of CC and CXC cytokines, including the CCR5 ligands RANTES/CCL-5, MIP-1α/CCL-3, and MIP-1β/CCL-4. They also demonstrate that CCR5 plays a critical role in the generation of IFN-γ–induced inflammation, DNA injury, cell death, and tissue remodeling and illustrate important CCR5-dependent pathways that IFN-γ uses to stimulate chemokines, proteases, antiproteases, and the intrinsic and extrinsic cell death pathways. Dysregulated Th1 inflammation and exaggerated IFN-γ production are prominent findings in emphysema and a wide variety of other disorders, including diabetes, atherosclerosis (2
), Crohn disease (3
), coeliac disease (5
), rheumatoid arthritis (6
) periodontitis (7
), Bechet disease (8
), aphthous ulcers (9
), autoimmune gastritis (10
), and uveoretinitis (11
). Our studies suggest that IFN-γ contributes to the genesis of the inflammation and tissue destruction that are seen in these settings and that these responses are mediated, at least in part, by a CCR5-dependent pathway. These studies also suggest that interventions that block the activation and/or signaling of CCR5 may be therapeutic in these disorders. Additional investigation of the roles of CCR5 in the pathogenesis of these diseases and the utility of CCR5-based interventions and therapeutics in their treatment is warranted.