In order to evaluate whether augmenting MDC levels would exacerbate lung inflammation after hemorrhage and resuscitation, recombinant murine MDC was administered intravenously after hemorrhage and prior to resuscitation. This experiment revealed that additional MDC further increased pulmonary inflammation as compared with hemorrhage and resuscitation alone (Fig. 4). Macrophage-derived chemokine administered to normal nonhemorrhaged mice did not lead to pulmonary recruitment of inflammatory cells or pulmonary inflammation. (See Supplemental Fig. 2, Supplemental Digital Content 1, at http://links.lww.com/SHK/A228, Intravenous injection of MDC does not lead to increased pulmonary cell recruitment in normal [i.e., nonhemorrhaged] mice. *P < 0.05 vs. PBS.) Together, these findings suggest MDC plays an important role in the pathogenesis of acute lung inflammation in the setting of hemorrhage and resuscitation.
Our data suggest that MDC regulates pulmonary levels of key chemokines, including KC, MIP-2, and MIP-1α (Fig. 5), and that the CCR4 receptor for MDC is present on murine bronchial epithelial cells (Fig. 6B). In order to examine the possibility that bronchial epithelial cells may directly respond to MDC stimulation and to verify that the signaling mechanisms exist in human cells, we cultured primary hBECs. In culture, these cells strongly express the CCR4 receptor (Fig. 6D). Treatment of these cells with MDC did not result in increased production of the chemokine IL-8, the human analog of KC and MIP-2 (Fig. 6E). In order to determine the possible role of MDC in IL-8 production in hBECs under proinflammatory conditions, we treated the cells with TNF-α. Tumor necrosis factor α has been previously shown to regulate pulmonary cytokine production and inflammatory lung injury following hemorrhage and resuscitation (5, 11). In our model, we found that TNF-α was elevated as early as 30 min after hemorrhage and resuscitation. (See Supplemental Figure 3 Supplemental Digital Content 1, at http://links.lww.com/SHK/A228; Serum levels of TNF-α are elevated 30 min after hemorrhage and resuscitation with LR solution. *P < 0.05 vs. sham.) Therefore, TNF-α was used as an inflammatory stimulus to evaluate the responsiveness of hBECs to MDC cultured under proinflammatory conditions. As expected, treatment of hBECs with TNF-α was associated with increased IL-8 production (Fig. 6E). Concurrent treatment of hBECs with TNF-α and MDC resulted in increased IL-8 production over treatment of TNF-α alone (Fig. 6E), suggesting that under proinflammatory conditions hBECs may produce and secrete chemotactic cytokines in response to MDC.
In the present study, we have described a novel role for MDC in mediating the proinflammatory response leading to pulmonary inflammation following hemorrhage and resuscitation. Our data demonstrate that systemic and pulmonary levels of MDC after resuscitation correlate with pulmonary infiltration of inflammatory cells and lung inflammation, a response that is attenuated by neutralization of MDC and exacerbated by the addition of recombinant MDC. Investigation into the mechanism of MDC’s role in this proinflammatory response revealed that alterations in MDC levels directly correlate with pulmonary levels of the chemoattractant cytokines KC, MIP-2, and MIP-1α. Histological evaluation revealed that CCR4 is expressed within the bronchial epithelium, and in vitro studies demonstrated that cultured hBECs express the CCR4 receptor and produce IL-8 in response to MDC treatment under proinflammatory conditions. Together, this work demonstrates a previously unidentified role for MDC in regulating lung inflammation following hemorrhage and resuscitation.
Our current and prior works indicate that hemorrhage and resuscitation result in increased systemic levels of the chemokine MDC (4); however, the source of MDC in these studies is unknown. The relatively high serum, as opposed to pulmonary, MDC concentrations suggest that the lung is not the primary source of MDC production under these conditions. Macrophage-derived chemokine is constitutively produced by macrophages and monocyte-derived dendritic cells (12). In addition, natural killer cells, monocytes, and CD4 lymphocytes are capable of producing MDC after stimulation (23). It is plausible that these peripheral blood cells are responsible for the increased systemic levels of MDC observed in our studies following hemorrhage and resuscitation. In addition, potential pulmonary sources of MDC include alveolar macrophages and smooth muscle cells (18, 22), and these cell types may also contribute to the inflammatory reaction in the lung following hemorrhage (24).
Our data demonstrate that the MDC produced during hemorrhage and resuscitation is associated with an influx of inflammatory cells into the lung, but the exact identity of these cells is unknown at present. The antibody that we utilized in our experiments (Ly-6B.2, clone 7/4) has been shown to be specific for neutrophils (25), but a recent study has indicated that it also stains for inflammatory monocytes and some activated macrophages (26). Thus, in the setting of hemorrhage and resuscitation, MDC-mediated inflammatory cell chemotaxis may represent the direct pulmonary recruitment of CCR4+ monocytes and macrophages and/or the extravasation of neutrophils as an indirect response to MDC-mediated production of chemotactic cytokines by pulmonary epithelial cells (as implied by the data presented in Figs. 5 and 6). Macrophage-derived chemokine/CCR4 signaling in systemic inflammation has been previously established by a previous study (19), but the current work does not rule out the potential for intrapulmonary MDC/CCR4 signaling that may also contribute to inflammatory cell recruitment and lung inflammation.
Our data indicate that cultured hBECs produce the chemokine IL-8 following stimulation with MDC and TNF-α, an established regulator of pulmonary cytokine production and inflammatory lung injury following hemorrhage and resuscitation (11). These data suggest that hBECs are responsive to MDC in the proinflammatory conditions generated by TNF-α and that the cellular mechanisms responsible for our in vivo murine observations are also present in human cells. Data from other laboratories have also demonstrated the role of pulmonary epithelial cells in inflammatory cell recruitment during the development of lung inflammatory injury (2, 27–29). In a rat model of hemorrhagic shock, Hierholzer et al. (2) demonstrated the involvement of bronchoepithelial cells in the development of pulmonary injury via the production of granulocyte colony-stimulating factor, a potent cytokine involved in neutrophil chemotaxis and activation. Human bronchial epithelial cells have also been shown to produce inflammatory mediators in response to particulate matter exposure such as that present in ambient air pollution (27, 28). In addition, alveolar type II epithelial cells were shown to produce the chemokines KC and MIP-2 in response to alveolar macrophage-produced TNF-α (29). Our data confirm the presence of CCR4 within the bronchial epithelium (Fig. 6B) and support the notion that MDC may target pulmonary epithelial cells and mediate neutrophil chemotaxis via the production of chemokines such as KC, MIP-1α, and MIP-2 (Fig. 6E). Our data, however, do not exclude the involvement of other cell types, including type I or type II pneumocytes, in mediating pulmonary cytokine production and/or lung inflammatory injury following hemorrhage and resuscitation, and future work is needed to evaluate these cell types in terms of CCR4 expression and responsiveness to MDC. Furthermore, there is some evidence to suggest that MDC may serve as a ligand for receptors other than CCR4 because MDC modified to block its interaction with CCR4 still showed appreciable chemotactic activity for monocytes (30, 31). Ultimately, however, the identities of alternative receptors for MDC have not been defined, and further research is needed to consider whether CCR4-independent signaling mechanisms are involved in MDC’s mediation of pulmonary inflammation following hemorrhage and resuscitation.
Previously, our group and others have demonstrated an integral relationship between resuscitation strategy and clinical outcome following hemorrhagic shock (4, 32–34). These works provide evidence that resuscitative fluids may differentially modulate the systemic inflammatory response syndrome that ensues following hemorrhage and subsequent resuscitation. The resuscitative approach for the current work involved the use of LR solution based on current Advanced Trauma Life Support guidelines and common practice among trauma/surgical centers worldwide. The outcomes of this work highlight MDC as a mediator of pulmonary inflammation, but these conclusions are limited to the physiologic setting that is present following hemorrhage and subsequent resuscitation with LR solution. Given the significance of the findings and potential for therapeutic intervention, additional investigations are warranted to determine the role of MDC in modulating inflammation following the use of other resuscitative approaches, such as those utilizing colloidal solutions or blood-based products.
In summary, our data provide evidence for a novel role of MDC in regulating the proinflammatory response in lung following hemorrhage and resuscitation. We demonstrate for the first time that MDC is upregulated in response to fluid resuscitation and is capable of mediating inflammatory cell trafficking and inflammation, at least in part through regulation of chemoattractant cytokines. Although the pathogenesis of lung inflammation is complex, our data suggest that interventions to neutralize MDC are a potential method to attenuate lung inflammation after hemorrhage and resuscitation.
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