Blocking RAGE expression after injury reduces inflammation in mouse model of acute lung injury.

ABSTRACT

BACKGROUND: Receptor for Advanced Glycated Endproducts (RAGE) plays a major role in the inflammatory response to infectious and toxin induced acute lung injury. We tested the hypothesis that a RAGE blocking antibody when administered after the onset of injury can reduce lung inflammation compared to control antibody. METHODS: Male and female C57BL/6 (WT) mice were used. Forty-six received lipopolysaccharide (LPS) and 26 PBS by nasal instillation on day one, repeated on day three. On day 2, 36 mice receiving LPS were divided into two groups of 18, one treated with 200 µg of non-immune isotype control IgG and the second group treated with 200 µg of anti-RAGE Ab, each dose divided between IV and IP. Ten of the 46 were not treated. On day 4, before euthanasia, mice were injected with fluorescein isothiocyanate (FITC) labelled albumen. BALF and serum samples were collected as well as lung tissue for immunohistochemistry (IHC). BALF was analyzed for cell (leukocyte) counts, for FITC BALF/serum ratios indicating pulmonary vascular leak, and for cytokines/chemokines using bead based multiplex assays. Quantitative IHC was performed for MPO and RAGE. RESULTS: Ten LPS mice showed minimal inflammation by all measures indicating poor delivery of LPS and were excluded from analysis leaving n = 11 in the LPS + IgG group and n = 12 in the LPS + anti-RAGE group. BALF cell counts were low in the PBS administered mice (4.9 ± 2.1 × 105/ml) and high in the LPS injured untreated mice (109 ± 34) and in the LPS + IgG mice (91 ± 54) while in comparison, LPS + anti-RAGE ab mice counts were significantly lower (51.3 ± 18 vs. LPS + IgG, P = 0.03). The BALF/serum FITC ratios were lower for the LPS + anti-RAGE mice than for the LPS + IgG mice indicating less capillary leakiness. Quantitative IHC RAGE staining was lower in the LPS + anti-RAGE ab mice than in the LPS + IgG treated mice (P = 0.02). CONCLUSIONS: These results describe a four-day LPS protocol to sustain lung injury and allow for treatment and suggests that treatment aimed at blocking RAGE when given after onset of injury can reduce lung inflammation.