Role of CCL5 (RANTES) in viral lung disease

Abstract

CCL5/RANTES is a key proinflammatory chemokine produced by virus-infected epithelial cells and present in respiratory secretions of asthmatics. To examine the role of CCL5 in viral lung disease, we measured its production during primary respiratory syncytial virus (RSV) infection and during secondary infection after sensitizing vaccination that induces Th2-mediated eosinophilia. A first peak of CCL5 mRNA and protein production was seen at 18 to 24 h of RSV infection, before significant lymphocyte recruitment occurred. Treatment in vivo with Met-RANTES (a competitive chemokine receptor blocker) throughout primary infection decreased CD4+ and CD8+ cell recruitment and increased viral replication. In RSV-infected, sensitized mice with eosinophilic disease, CCL5 production was further augmented; Met-RANTES treatment again reduced inflammatory cell recruitment and local cytokine production. A second wave of CCL5 production occurred on day 7, attributable to newly recruited T cells. Paradoxically, mice treated with Met-RANTES during primary infection demonstrated increased cellular infiltration during reinfection. We therefore show that RSV induces CCL5 production in the lung and this causes the recruitment of RSV-specific cells, including those making additional CCL5. If this action is blocked with Met-RANTES, inflammation decreases and viral clearance is delayed. However, the exact effects of chemokine modulation depend critically on time of administration, a factor that may potentially complicate the use of chemokine blockers in inflammatory diseases.

FIG. 1.

CCL5 mRNA and protein levels after RSV infection. BALB/c mice were scarified with recombinant vaccinia viruses expressing the G (□) protein of RSV or a control construct (rVV-βgal [○]). Two weeks later, they were infected intranasally with RSV. (A) CCL5 mRNA levels in whole, snap-frozen lung, determined by RNase protection assay. (B) CCL5 protein concentration in the BAL measured by enzyme-linked immunosorbent assay; data points represent means (n > 4) ± standard errors. **, P < 0.01. This graph shows one of three similar independent experiments.

FIG. 2.

Cellular CCL5 production during RSV infection. BALB/c mice were scarified with rVV-G. Two weeks later, they were infected intranasally with RSV. (A) CCL5 production after in vitro live RSV stimulation (open symbols) or mock stimulation (HEp-2 cell lysate [filled symbols]) from isolated cells measured by ELISPOT; results are means (n = 5 mice) ± standard errors. *, P < 0.05; circles, uninfected mice; triangles, rVV-G-primed mice. (B and C) Intracellular staining for CCL5 in lung CD4⁺ (B) or CD8 + (C) lymphocytes isolated on day 7 after infection from rVV-G primed, RSV infected mice and analyzed by flow cytometry, gated on lymphocytes by forward/side scatter.

FIG. 3.

Met-RANTES treatment during primary RSV infection. Mice were treated with Met-RANTES daily throughout the first 7 days of a primary RSV infection. (A and B) Met-RANTES treatment (⧫) reduced CD4⁺ and CD8⁺ lymphocyte accumulation in BAL, compared to results for untreated mice (⋄); data points represent means (n = 5 per group) ± standard errors. (C) RSV PFU were measured in whole homogenized lung taken 4 days after RSV infection; results are means (n = 5) ± standard errors. (D) IFN-γ-secreting lung cells 7 days after i.n. RSV infection measured by ELISPOT; data points represent means (n = 4 mice per group) ± standard errors. *, P < 0.05. (Open bars, mock stimulation; filled bars, live RSV stimulation in vitro.).

FIG. 4.

Met-RANTES treatment during RSV infection following Th2 disease exacerbation. Mice were primed with rVV-G and infected with RSV 2 weeks later. Met-RANTES treatment was given on days 5 and 6 after RSV infection. Mice were harvested on day 7. (A) Total cells isolated from the BAL and lung following Met-RANTES treatment. (B) Effect of Met-RANTES treatment on CD4⁺ and CD8⁺ populations in BAL; memory, CD44^hi/CD62L^lo. Effect of late Met-RANTES treatment on number of IFN-γ-secreting (C) or IL-5-secreting (D) cells in the lung. Data points represent means (n = 5 mice per group) ± standard errors. *, P < 0.05. (Open bars, untreated mice; filled bars, Met-RANTES-treated mice). Memory phenotype cells were defined as CD62L^lo/CD44^hi.

FIG. 5.

Effect of Met-RANTES treatment on cell recruitment during subsequent RSV challenge. Mice were infected i.n. with RSV on day 0 and rechallenged on day 28. During days 0 to 5 of primary RSV infection, some mice were treated with Met-RANTES. (A) Total cells isolated from the BAL and lung following RSV rechallenge. (B) Effect of Met-RANTES treatment during primary infection on CD4 and CD8 memory (CD44^hi/CD62L^lo) populations after RSV rechallenge. (C) Effect of Met-RANTES treatment on number of IFN-γ or IL-5 secreting cells in the lung; data points represent means (n = 5 mice per group) ± standard errors. *, P < 0.05; **, P < 0.01. (Open bars, untreated mice; filled bars, Met-RANTES-treated mice; gray bars, primary RSV infection).