Pneumonia from gram-negative bacteria is a leading cause of mortality from infectious diseases in the U.S.(Armstrong et al., 1999
; Mizgerd, 2006
). Neutrophil influx is a crucial component of innate immunity against bacterial infection, but excess neutrophil influx and activation may initiate acute lung injury, necessitating exquisite control over inflammatory cell recruitment and activation. One of the components of this regulatory system is the family of ELR+
CXC chemokines (CXCL1, 2, 5, and 15), which play critical roles in the inflammatory responses in rodents and humans. Despite considerable homology between these family members, it is unclear whether they are functionally redundant or capable of distinct actions. In humans, CXCL5 was found to be most strongly and consistently correlated with neutrophil numbers in the lung fluids of patients with acute respiratory distress syndrome (ARDS)(Goodman et al., 1996
), highlighting the potential importance of CXCL5 in regulation of lung inflammation and injury.
In attempting to define genetically the role of CXCL5 in pulmonary inflammation and host defense against gram-negative bacterial pneumonia, we have uncovered unique aspects of CXCL5 biology. With in vitro and in vivo studies, we have demonstrated that CXCL5 inhibits chemokine scavenging at least in part through its homeostatic and high-affinity binding with erythrocyte DARC. Hence in the absence of CXCL5, DARC scavenges proinflammatory chemokines, thus contributing to re-shaping the chemokine gradients for neutrophil influx to the lung during severe E.coli pneumonia. In contrast, in response to LPS inhalation, a self-limited inflammatory response in the lung, deletion of CXCL5 markedly impairs neutrophil accumulation in the airspace, thus revealing critical non-redundant roles for this chemokine in lung inflammation in these two models. Furthermore, CXCL5 demonstrates features of both homeostatic chemokines (detected in plasma and bound to erythrocytes of normal mice) and inflammatory chemokines (enhanced expression during inflammation). Perhaps most surprising of all, our data indicate that the source of circulating CXCL5 in the basal homeostatic condition is the platelet, while lung resident cells (principally AE II cells, we suggest) are the source of CXCL5 in the lung and (to a large extent) blood during lung inflammation.
Based on our observations, we propose a model in which during homeostatic conditions, platelet-derived CXCL5 is loaded onto erythrocyte DARC. During self-limited inflammation, CXCL5 itself, perhaps by virtue of its prolonged expression by AE II cells in the lung, is necessary for optimal neutrophil accumulation. CXCL5 in this scenario has little effect on local concentrations of CXCL1 or 2, which are only transiently (albeit significantly) induced and are but minimally detected in plasma. During a severe inflammatory response, such as that accompanying E.coli pneumonia, further expression of CXCL5 by AE II cells inhibits the chemokine scavenging capability of DARC, at a time when the production of CXCL1 and CXCL2 increases dramatically, resulting in marked increases in circulating plasma concentrations of these chemokines, with adverse consequences for the efficient accumulation of neutrophils. Our studies support the “chemokine sink” function of erythrocyte DARC, while suggesting that under normal circumstances it is inhibited by endogenous CXCL5 from platelets, and further impaired by AE II cell-derived CXCL5 during severe E.coli pneumonia. The phenotypes of WT mice we observed in this severe E.coli pneumonia model resemble that of “cytokine storm (hypercytokinemia)” in influenza pneumonia. Deletion of CXCL5 in our model decreased large circulating amounts of chemokines CXCL1 and 2 and improved survival by permitting effective neutrophil accumulation and bacterial killing.
DARC has previously been reported to bind many pro-inflammatory chemokines, but not homeostatic chemokines (Allen et al., 2007
; Borroni et al., 2008
; Gardner et al., 2004
). Here we have demonstrated that CXCL5 is also a homeostatic chemokine, derived from platelets, which modulates neutrophil homeostasis in naïve mice. Thus the binding of homeostatic chemokines to DARC, and the attendant consequences, are novel findings of this work that may alter our view of DARC function. Our analysis, however, has focused on erythrocyte DARC binding chemokines and functioning as sink (and reservoir). DARC is also expressed on endothelial cells, where it exerts additional actions that promote migration. Whether endothelial DARC contributes to the scavenging functions described here will require further investigation.
As befits a chemokine with homeostatic functions, CXCL5 is found circulating in normal mice, almost all of it bound to erythrocytes. Our studies demonstrated this circulating CXCL5 is derived from platelets. Given the potential role of platelets in a variety of inflammatory diseases (Gear and Camerini, 2003
), such as atherosclerosis and acute lung injury (Bozza et al., 2009
), it is tempting to speculate that platelet-derived CXCL5 may be involved in establishing conditions that alter a subsequent inflammatory response. Indeed, we have demonstrated that the homeostatic platelet-derived CXCL5 significantly inhibits chemokine scavenging both in vitro
and in vivo
. In striking contrast, however, lung cells but not platelets provided most of the inflammation-induced increases in CXCL5 and the attendant inhibition of chemokine scavenging after E.coli challenge. Furthermore, most CXCL5 in platelets or secreted from them after thrombin stimulation is the less active CXCL5-93 form. Previous studies have indicated that many cellular forms of CXCL5 can be detected, including the most potent CXCL5-70 and less potent CXCL5-93 in response to stimulation (Wuyts et al., 1999
). Our analysis showed many forms released from platelets, but most was CXCL5-93. Whether platelet-secreted CXCL5-93 can be cleaved into potent CXCL5-70 by matrix metalloproteinases during inflammation (Tester et al., 2007
), and what forms of CXCL5 are released by AE II cells, or detected in plasma or bound to erythrocytes in both homeostatic and inflammatory conditions, are the subject of on-going study. Platelets also contain CXCL7 (β-thromboglobulin), another ELR+
CXC chemokine in mice, but it has not yet been determined whether murine CXCL7 is processed to a neutrophil-chemoattractant form similar to human NAP-2(Smith et al., 2002
While increases in circulating CXCL1 and 2 appear to be a consequence of impaired scavenging, the mechanism by which elevation of circulating plasma chemokines attenuates neutrophil accumulation to the lung and other organs remains obscure. Here we propose two mechanisms. First we suggest that the chemokine gradients (the relative ratio of BALF versus plasma concentrations of CXCL1 and CXCL2) are important especially considered in light of the dilution consequent to lavage, but we also suggest that the absolute value of the plasma chemokines may also be relevant. Ligand-induced desensitization of CXCR2, the receptor for CXCL1, 2, 5 and 15 occurs normally during chemotaxis in vitro
, and migration in vivo
, and is thus a physiologic process. Here we have documented desensitization of neutrophils even before their entry into the circulation in WT mice after E.coli exposure. Isolation of BM-derived neutrophils demonstrated loss of surface CXCR2 and non-responsiveness to CXCL1, both of which were improved in Cxcl5−/−
mice. Since CXCR2 is important for mobilization of neutrophils from marrow, as well as migration into tissues, these data suggest that large absolute amounts of CXCL1 and CXCL2 may impair accumulation of neutrophils through effects at several levels. Further studies will be required to distinguish the impact of these mechanisms in vivo
. Furthermore, CXCR2 deficiency leads to neutrophil dysplasia (over 90% Gr-1+
cells in the BM)(Cacalano et al., 1994
), indicating that CXCR2 signaling contributes to homeostatic control of neutrophil numbers in the BM, but also indicates that CXCR2 ligands other than CXCL5 play a role in neutrophil homeostasis. Additionally, since endothelial CXCR2 plays an important role in regulating neutrophil influx in LPS-induced lung inflammation (Reutershan et al., 2006
) and lung epithelial cells may express CXCR2 as well (Vanderbilt et al., 2003
), CXCL5 may modulate neutrophil transepithelial and transendothelial migration.
In summary, CXCL5 exhibits non-redundant properties with respect to other ELR+
CXC chemokines that exerts profound effects on the inflammatory response. Considering the involvement of CXCL5 in a variety of human inflammatory diseases (Goodman et al., 1996
; Kwon et al., 2005
; Qiu et al., 2003
; Qiu et al., 2007
; Zineh et al., 2008
mice and the mechanisms revealed here may be useful to study the role of CXCL5 in the pathogenesis and therapeutics of inflammatory diseases.