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Comparative Study
. 2005 Nov;11(11):1180-7.
doi: 10.1038/nm1303. Epub 2005 Oct 2.

CCL5-CCR5 interaction provides antiapoptotic signals for macrophage survival during viral infection

Jeffrey W Tyner  1 , Osamu Uchida, Naohiro Kajiwara, Edy Y Kim, Anand C Patel, Mary P O'Sullivan, Michael J Walter, Reto A Schwendener, Donald N Cook, Theodore M Danoff, Michael J Holtzman
Affiliations
Comparative Study

CCL5-CCR5 interaction provides antiapoptotic signals for macrophage survival during viral infection

Jeffrey W Tyner et al. Nat Med. 2005 Nov.

Abstract

Host defense against viruses probably depends on targeted death of infected host cells and then clearance of cellular corpses by macrophages. For this process to be effective, the macrophage must presumably avoid its own virus-induced death. Here we identify one such mechanism. We show that mice lacking the chemokine Ccl5 are immune compromised to the point of delayed viral clearance, excessive airway inflammation and respiratory death after mouse parainfluenza or human influenza virus infection. Virus-inducible levels of Ccl5 are required to prevent apoptosis of virus-infected mouse macrophages in vivo and mouse and human macrophages ex vivo. The protective effect of Ccl5 requires activation of the Ccr5 chemokine receptor and consequent bilateral activation of G(alphai)-PI3K-AKT and G(alphai)-MEK-ERK signaling pathways. The antiapoptotic action of chemokine signaling may therefore allow scavengers to finally stop the host cell-to-cell infectious process.

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Figures

Figure 1
Figure 1
Excessive airway inflammation and respiratory failure after paramyxoviral infection in Ccl5−/− mice. (a) Ccl5−/− and wild-type mouse lungs from day 5 after inoculation with SeV or UV-inactivated SeV (SeV-UV) were subjected to in situ hybridization with 35S-labeled Ccl5 cRNA or immunostained with Ccl5-specific monoclonal antibody or SeV-specific antibody. (b) Ccl5−/−, Ccr5−/− and corresponding control mice were inoculated as in a and monitored for survival (n = 29 mice per group). * P < 0.05. (c) Mice were inoculated as in a, and lung sections were subjected to immunofluorescence microscopy using Ccr5-specific monoclonal antibody and FITC-conjugated secondary antibody. (d) Mice were inoculated as in a, and BAL fluid cells from day 9 after inoculation were subjected to immunofluorescence microscopy using Ccr5-specific monoclonal antibody and Mac-3–specific antibody (top row) or SeV-specific antibody and FITC-conjugated secondary antibody and CD68-specific monoclonal antibody (bottom row) as well as corresponding FITC- or Cy3-conjugated secondary antibody and Hoescht dye for nuclei. For a,c and d, controls for immunostaining (nonimmune IgG) and in situ hybridization (sense probe) gave no signal over background. Scale bars, 20 μm.
Figure 2
Figure 2
Excessive macrophage apoptosis after paramyxoviral infection in Ccl5−/− mice. (a) Ccl5−/− and wild-type control mice were inoculated with SeV, and BAL fluid was obtained for total and differential cell counts. Values represent mean ± s.e.m. (n = 3). (b) Lung tissue and BAL fluid cells from day 12 after inoculation were used to monitor levels of CD8+ and SeV+ specific T cells by flow cytometry using tetrameric MHC-Sev peptide. Control analysis with tetrameric MHC-ova peptide was negative (data not shown). (c) Serial lung sections from day 9 after inoculation were subjected to immunostaining for SeV-specific and Mac-3–specific monoclonal antibodies and TUNEL. Scale bars, 20 μm. (d) BAL fluid cells from day 9 after inoculation were subjected to immunofluorescence microscopy using CD68-specific monoclonal antibody with Cy3-conjugated secondary antibody and activated caspase 3–specific monoclonal antibody with FITC-conjugated secondary antibody. Similar results were obtained for lung tissue cells (data not shown). (e) Quantitative analysis of results from d. *P < 0.05.
Figure 3
Figure 3
Ccl5- and Ccr5-dependent protection from virus-induced apoptosis in isolated macrophages. (a) Wild-type mouse macrophages were infected with SeV (MOI 20) or SeV-UV, and 4 d later, cellular mRNA was subjected to oligonucleotide microarray analysis. Red circles indicate statistically significant and blue circles indicate no significant difference in SeV- versus SeV-UV–inoculated cells. (b) Macrophages from indicated mice were infected as in a, and cell supernatants were subjected to ELISA for Ccl3, Ccl4 and Ccl5 at indicated times after inoculation. (c) Wild-type mouse macrophages were infected as in a and subjected to DAPI stain and immunofluorescent stain for Ccr1, Ccr3 and Ccr5. (d) Macrophages from indicated mice were infected as in a and subjected to TUNEL at day 4 after infection. Ccl5−/− cells were also incubated with Ccl5 for 1 h before inoculation, and wild-type cells were incubated with Ccr5-specific antibody (Ccr5 Ab) from day 0 to day 4. (e) Human macrophages were incubated with or without CCR5-specific antibody (CCR5 Ab) and infected with SeV or respiratory syncytial virus (MOI 20) and subjected to TUNEL, or infected with influenza virus and subjected to immunostaining for active caspase 3 on day 4 after inoculation. For mouse and human cells, the same patterns were observed at days 1, 2 and 8 after infection and MOI 1–100. Control inoculation with SeV-UV gave no detectable signal for viral staining or TUNEL reaction. For b–e, values represent mean ± s.e.m. (n = 5). *P < 0.05. (f) Macrophages from Ccl5−/− and wild-type mice were infected as in a, and cell supernatants were analyzed by real-time quantitative PCR for viral copy number. Ccl5−/− macrophages were also treated with vehicle or Ccl5 (10 nM) for 12 h before inoculation.
Figure 4
Figure 4
CCL5 signals to Gαi-PI3K-Akt and MEK-ERK pathways that block virus-induced apoptosis. (a) Macrophages from Ccr5−/− or wild-type control mice were incubated with Ccl5, and cell lysates were subjected to western blotting for phosphorylated Erk1/2, phosphorylated Akt or β-actin. (b) Human macrophages were incubated with CCL5 without or with preincubation with pertussis toxin, LY294002, PD98059, herbimycin A or PP2. Cell lysates were subjected to western blotting as in a. (c) Quantitative analysis of western blotting of human macrophages incubated with CCL5 as described in b. (d) Macrophages from wild-type or Ccl5−/− mice were inoculated with SeV or SeV-UV along with vehicle, LY294002 or PD98059 treatment, and then subjected to TUNEL 4 d later. For c and d, values represent mean ± s.e.m. *P < 0.05.
Figure 5
Figure 5
Decreased activation of Akt and Erk1/2 in Ccl5−/− mice after viral infection. (a) BAL fluid from Ccl5−/− and wild-type mice after SeV and SeV-UV inoculation were used to prepare cell lysates that were analyzed by western blotting for phosphorylated Erk1/2, phosphorylated Akt and β-actin. (b) BAL fluid cells from day 5 after inoculation were also subjected to immunostaining for phosphorylated Erk1/2, phosphorylated Akt and Mac-3. (c) Quantitative analysis of results from b. (d) Lung sections from day 5 after inoculation were immunostained for phosphorylated Akt. Arrows indicate macrophages that immunostained positive for phosphorylated Akt. Scale bar, 20 μm. (e) Lung sections obtained from day 8 after inoculation were immunostained for c-fos family members. Arrows indicate macrophages that immunostained positive. Scale bar, 50 μm. (f) Quantification of results from e. Values represent mean ± s.e.m. *P < 0.01.
Figure 6
Figure 6
Effects of macrophage depletion on viral infection in wild-type and Ccl5−/− mice. (a) Wild-type mice were treated with control (−) or clodronate-containing (+) liposomes. Lung sections were subjected to immunostaining for Mac-3, SeV or active caspase-3 at indicated times. (b) Wild-type mice were treated as in a and BAL fluid and lung tissue cells were obtained for total and differential cell counts. (c) Wild-type mice were treated as in a, and lung tissue samples were analyzed by realtime quantitative PCR for viral copy number. (d) Quantitative analysis of results in a for active caspase 3 immunostaining. (e) Ccl5−/− and corresponding control mice were inoculated and treated as in a and monitored for survival (n = 18–38 mice per group). *P < 0.05.
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