This study investigated the role of CXCR2 receptor on neutrophils in acute cigarette smoke-induced inflammation. We have developed a cigarette smoke exposure protocol that results in acute lung inflammation characterized by massive increases in BAL neutrophils (up to 50% of total BAL cells), tissue neutrophils, and production of inflammatory cytokines, prostaglandins, and neutrophil chemokines. SCH-N, a CXCR2 antagonist, inhibited smoke-induced lung neutrophilia by 50%, with reductions in BAL neutrophils and β-glucuronidase, an enzyme associated with tissue breakdown. Perivascular inflammatory infiltrates were smaller, less frequent and contained fewer neutrophils, and fewer neutrophils were observed adherent to blood vessel endothelium. SCH-N did not alter the number of inflammatory lymphocytes or eosinophils recruited by smoke, and did not alter the general proinflammatory milieu, as indicated by measurement of IL-6 and PGE2
in the BAL. It should be noted that while TNF-α has been shown by some to be a key initiator of inflammation following cigarette smoke exposure (7
), we did not observe changes in TNF-α with either smoke exposure or drug treatment (). Since we determined that neutrophilic inflammation of the lungs peaks 24 hours after the final smoke exposure in this model, the mice were sacrificed at this time point for all analyses. However, TNF-α levels peak 2–4 hours after smoke exposure (data not shown), so it is not surprising that we did not observe changes in TNF-α in these mice. However, since IL-6 and PGE2
levels are elevated by smoke exposure and unaffected by SCH-N, we expect that TNF-α would follow the same pattern if mice were sacrificed at an earlier time point.
It was interesting that SCH-N, a CXCR2 antagonist, increased the levels of the CXC chemokines MIP-2 and KC 2–3 fold over smoke alone. SCH-N should be acting against CXCR2 on circulating neutrophils, while MIP-2 and KC are probably produced by lung resident cells exposed to smoke. One possible explanation is that MIP-2 and KC are regulated in part by a negative feedback mechanism, and that the arrival of neutrophils at the site of inflammation sends a signal that downregulates further chemokine production. Because fewer neutrophils enter the lung with SCH-N treatment, this downregulatory signal is not sent. It is also possible that an autocrine negative feedback loop exists in which cells that produce MIP-2 and KC modulate chemokine production via their own CXCR2 receptors. It has recently been reported that mouse alveolar type 2 cells express both MIP-2 and CXCR2 (38
), so it is possible that blockade of CXCR2 blocks an autocrine regulatory mechanism resulting in increased production of MIP-2.
SCH-N is a potent CXCR2 antagonist, inhibiting mouse neutrophil chemotaxis in vitro
with an IC50
of 3 nM (data not shown), while inhibiting the neutrophilic inflammatory response to cigarette smoke by 50%. It should be noted that in mice, neutrophil migration can also be induced by the CC chemokine MIP-1α acting via the CCR1 receptor (34
). Cigarette smoke induces MIP-1α in rat alveolar macrophages in vitro
), so it is likely that the partial inhibition seen in this study is due to redundancy in neutrophil chemotaxis in mice (28
Neutrophilic inflammation is a key factor in chronic bronchitis and emphysema. Disease progression is associated with a switch from a T-cell mediated inflammation in healthy smokers and patients with mild COPD to neutrophilic inflammation in severely ill patients (10
), who also have higher levels of the human neutrophil CXC chemokine IL-8 (25
). Neutrophils have also been shown to be a key effector cell of matrix breakdown in an animal model (8
). Due to the fact that neutrophil recruitment in humans requires activation of the CXCR1 or CXCR2 receptors by CXC chemokines, these interactions have been explored as potential therapeutic targets (2
). In mouse models of inflammation, neutrophil recruitment can be blocked by treatment with antibodies to MIP-2 and KC, CXCR2 antibodies (mice lack CXCR1), and CXCR2 antagonist peptides derived from CXCR2 ligands (14
). This study demonstrates that CXCR2 plays a key role in cigarette smoke-induced inflammation, and that inhibiting chemokine binding to CXCR2 can significantly reduce neutrophilic infiltration and tissue damage resulting from acute exposure to cigarette smoke. Further experiments will be needed to determine whether long-term blockade of CXCR2 can reduce the severity of emphysema-like changes in mouse models of chronic cigarette smoke exposure.