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. 2014 Dec;42(6):525-31.
doi: 10.1097/SHK.0000000000000253.

Macrophage-derived chemokine (CCL22) is a novel mediator of lung inflammation following hemorrhage and resuscitation

Jillian R Richter  1 , Jeffrey M Sutton, Ritha M Belizaire, Lou Ann Friend, Rebecca M Schuster, Taylor A Johannigman, Steven G Miller, Alex B Lentsch, Timothy A Pritts
Affiliations

Macrophage-derived chemokine (CCL22) is a novel mediator of lung inflammation following hemorrhage and resuscitation

Jillian R Richter et al. Shock. 2014 Dec.

Abstract

Resuscitation of patients after hemorrhage often results in pulmonary inflammation and places them at risk for the development of acute respiratory distress syndrome. Our previous data indicate that macrophage-derived chemokine (MDC/CCL22) is elevated after resuscitation, but its direct role in this inflammatory response is unknown. Macrophage-derived chemokine signaling through the C-C chemokine receptor type 4 (CCR4) is implicated in other pulmonary proinflammatory conditions, leading us to hypothesize that MDC may also play a role in the pathogenesis of lung inflammation following hemorrhage and resuscitation. To test this, C57BL/6 mice underwent pressure-controlled hemorrhage followed by resuscitation with lactated Ringer's solution. Pulmonary inflammation and inflammatory cell recruitment were analyzed with histological staining, and serum- and tissue-level cytokines were measured by enzyme-linked immunosorbent assay. Pulmonary inflammation and cell recruitment following hemorrhage and resuscitation were associated with systemic MDC levels. Inhibition of MDC via injection of a specific neutralizing antibody prior to hemorrhage and resuscitation significantly reduced pulmonary levels of the chemotactic cytokines keratinocyte-derived chemokine and macrophage inflammatory proteins 2 and 1α, as well as inflammatory cell recruitment to the lungs. Intravenous administration of recombinant MDC prior to resuscitation augmented pulmonary inflammation and cell recruitment. Histological evaluation revealed the expression of CCR4 within the bronchial epithelium, and in vitro treatment of activated bronchial epithelial cells with MDC resulted in production and secretion of neutrophil chemokines. The present study identifies MDC as a novel mediator of lung inflammation after hemorrhage and resuscitation. Macrophage-derived chemokine neutralization may provide a therapeutic strategy to mitigate this inflammatory response.

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Figures

Figure 1
Figure 1. Serum MDC levels are significantly elevated following hemorrhage and resuscitation (H/R)
Compared to Sham animals, elevated MDC levels were detected in the serum of mice immediately following resuscitation with lactated Ringer’s solution and for 4h post-resuscitation. Dashed line represents the baseline level of serum MDC in naive (untreated) mice. *p<0.05 and +p<0.01 versus corresponding Sham time point.
Figure 2
Figure 2. Neutralization of MDC reduces (A) systemic and (B) pulmonary levels of MDC following hemorrhage and resuscitation (H/R)
Mice treated with an IgG control antibody prior to hemorrhage and resuscitation (IgG+H/R) demonstrated elevated levels of MDC 4h after resuscitation. Pre-treatment with a specific MDC neutralizing antibody (anti-MDC+H/R) resulted in post-resuscitation MDC levels equivalent to Sham levels. Data are presented as amount of MDC per mL of serum (A) and amount of MDC per microgram of tissue protein (B). *p<0.05 versus all groups.
Figure 3
Figure 3. Neutralization of MDC reduces pulmonary inflammation and inflammatory cell recruitment following hemorrhage and resuscitation (H/R)
Representative micrographs of lung tissue stained with hematoxylin and eosin (A-C) and anti-mouse Ly-6B.2 (D-F) for inflammatory cell identification. Sham mice (A, D) and mice injected with an anti-MDC neutralizing antibody prior to hemorrhage (C, F) demonstrated less pulmonary edema and cell infiltration than mice receiving the IgG antibody control (B, E). (G) Quantification of positively-stained cells confirmed decreased pulmonary inflammatory cell recruitment following neutralization of MDC. **p<0.05 versus all groups and *p<0.05 versus Sham.
Figure 4
Figure 4. Recombinant mouse MDC (rmMDC) exacerbates lung inflammation following hemorrhage and resuscitation (H/R)
Representative micrographs of lung tissue stained with hematoxylin and eosin (A-C) and anti-mouse Ly-6B.2 (D-F) for inflammatory cell identification. The lungs from mice injected with rmMDC prior to resuscitation (C,F) had substantial interstitial tissue edema and cell infiltration as compared to sham mice (A,D) and mice resuscitated with lactated Ringer’s solution only (B,E). (G) Quantification of positively-stained cells confirmed the effect of rmMDC on increased inflammatory cell recruitment. **p<0.001 versus all groups and *p<0.001 versus Sham
Figure 5
Figure 5. Neutralization of MDC results in decreased pulmonary levels of the chemoattractant cytokines (A) keratinocyte-derived chemokine (KC), (B) macrophage inflammatory protein 2 (MIP-2), and (C) MIP-1α 2h after hemorrhage and resuscitation (H/R)
Mice pre-treated with MDC neutralizing antibody (anti-MDC+H/R) prior to resuscitation had attenuated levels of chemokines compared to mice receiving the IgG antibody control (IgG+H/R). Data are presented as amount of cytokine per microgram of tissue protein. *p<0.05 versus all groups.
Figure 6
Figure 6. Bronchial epithelial cells express the MDC receptor CCR4 and produce IL-8 under proinflammatory conditions in response to MDC
(A) Minimal background staining was observed in paraffin-embedded sections of mouse lung tissue treated with the IgG control antibody. (B) The receptor CCR4 was identified in lung tissue stained with an anti-mouse polyclonal CCR4 antibody and DAB staining. Scale bars represent 200μm. (C) No staining was observed in cultured human bronchial epithelial cells with the IgG control antibody. (D) CCR4 was identified on human bronchial epithelial cells (hBEC) using an anti-human CCR4 primary antibody and an AlexaFluor®-555 secondary antibody. (E) In culture, hBEC did not increase production of IL-8 in response to MDC treatment alone. Treatment of hBEC with the proinflammatory mediator TNF-α increased IL-8 expression levels above untreated (media) controls, and concurrent treatment with TNF-α and MDC resulted in significantly more IL-8 production as compared to TNF-α alone. *p<0.001 versus Media and MDC and **p<0.01 versus TNF-α.
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