Delayed resolution of lung inflammation in Il-1rn-/- mice reflects elevated IL-17A/granulocyte colony-stimulating factor expression

Abstract

IL-1 has been associated with acute lung injury (ALI) in both humans and animal models, but further investigation of the precise mechanisms involved is needed, and may identify novel therapeutic targets. To discover the IL-1 mediators essential to the initiation and resolution phases of acute lung inflammation, knockout mice (with targeted deletions for either the IL-1 receptor-1, i.e., Il-1r1(-/-), or the IL-1 receptor antagonist, i.e., Il-1rn(-/-)) were exposed to aerosolized LPS, and indices of lung and systemic inflammation were examined over the subsequent 48 hours. The resultant cell counts, histology, protein, and RNA expression of key cytokines were measured. Il-1r1(-/-) mice exhibited decreased neutrophil influx, particularly at 4 and 48 hours after exposure to LPS, as well as reduced bronchoalveolar lavage (BAL) expression of chemokines and granulocyte colony-stimulating factor (G-CSF). On the contrary, Il-1rn(-/-) mice demonstrated increased BAL neutrophil counts, increased BAL total protein, and greater evidence of histologic injury, all most notably 2 days after LPS exposure. Il-1rn(-/-) mice also exhibited higher peripheral neutrophil counts and greater numbers of granulocyte receptor-1 cells in their bone marrow, potentially reflecting their elevated plasma G-CSF concentrations. Furthermore, IL-17A expression was increased in the BAL and lungs of Il-1rn(-/-) mice after exposure to LPS, likely because of increased numbers of γδ T cells in the Il-1rn(-/-) lungs. Blockade with IL-17A monoclonal antibody before LPS exposure decreased the resultant BAL neutrophil counts and lung G-CSF expression in Il-1rn(-/-) mice, 48 hours after exposure to LPS. In conclusion, Il-1rn(-/-) mice exhibit delayed resolution in acute lung inflammation after exposure to LPS, a process that appears to be mediated via the G-CSF/IL-17A axis.

Figures

Figure 1.

Bronchoalveolar lavage (BAL) cell counts after LPS exposure. (A) Overall (analyzed by two-way ANOVA, significance indicated by P value at top of graph) total leukocytes (WBC), at 4 hours (analyzed by Bonferroni post hoc test, *P < 0.01) and at 48 hours (*P < 0.001), as well as (B) neutrophil counts overall in Il-1r1−/− mice (with targeted deletions for IL-1 receptor–1), at 4 hours (*P < 0.05) and at 48 hours (*P < 0.01), compared with C57BL/6 control mice after exposure to LPS (n = 10–12 mice per time point). (C) Total WBC at 36 hours (*P < 0.0001) as well as 48 hours (*P < 0.01) and (D) total neutrophil counts in Il-1rn−/− mice (with targeted deletions for the IL-1 receptor antagonist), at 36 hours (*P < 0.001) and at 48 hours (*P < 0.01), compared with littermate controls (Il-1rn+/+ mice) after LPS exposure (n = 9–19 mice per time point).

Figure 2.

BAL total protein and lung histology after exposure to LPS. Hematoxylin and eosin–stained sections of lungs fixed 48 hours after LPS exposure at ×10 show (A) minimal inflammatory cells present in the alveolar space or interstitium of C57BL/6 murine lungs, but (B) substantial cellular infiltrates in Il-1rn−/− murine lungs. (C) Semiquantitative analysis of the number of neutrophils counted per alveolus in Il-1rn−/− mice 48 hours after exposure to LPS, compared with C57BL/6 mice (five mice per genotype, 10 fields counted per mouse). (D) BAL total protein in Il-1r1−/− mice (n = 10–12 mice per time point). (E) BAL total protein in Il-1rn−/− mice was increased exclusively at 48 hours (P < 0.05) after exposure to LPS (n = 9–16 mice per time point).

Figure 3.

Cytokine concentration in the BAL of Il-1r1−/− mice after exposure to LPS, compared with C57BL/6 control mice. (A) CXCL1 at 4 hours (*P < 0.0001). (B) CXCL2 at 4 hours (*P < 0.0001). (C) CXCL5 at 4 hours (*P < 0.01) and 48 hours (*P < 0.05). (D) Granulocyte colony–stimulating factor (G-CSF) expression at 8 hours (*P < 0.01; n = 10–12 mice per time point).

Figure 4.

BAL cytokine expression in Il-1rn−/− mice after exposure to LPS. (A) CXCL1 was exclusively elevated at 24 hours (*P < 0.05) in Il-1rn−/− mice, as opposed to littermate control mice (Il-1rn+/+). (B) CXCL2 at 4 hours (*P < 0.001). (C) CXCL5 at 4 hours (*P < 0.001). (D) G-CSF expression at 24 hours (*P < 0.05), 36 hours (*P < 0.05), and 48 hours (*P < 0.01) after exposure to LPS (n = 10–19 mice per time point).

Figure 5.

Differences in peripheral and bone marrow cell counts in Il-1rn−/− mice after exposure to LPS. (A) Total peripheral WBC and (B) peripheral neutrophil counts in Il-1rn−/− mice, compared with littermate control mice (Il-1rn+/+). Peripheral neutrophil counts at 36 hours (*P < 0.05) in Il-1rn−/− mice (n = 9–17 mice per time point). (C) G-CSF protein expression in Il-1rn−/− mice 48 hours (*P < 0.05) after exposure to LPS, compared with C57BL/6 mice (n = 5–7 mice at each time point). (D) Representative dot plots illustrate the relative percentage of GR1+ cells in the unstimulated marrow of C57BL/6 mice, compared with (E) marrow in Il-1rn−/− mice. APC, allophycocyanin; FSC, forward scatter. (F) Absolute number of GR1+ cells in Il-1rn−/− mice versus C57BL/6 mice, as well as GR1+ cells in the bone marrow of Il-1r1−/− versus C57BL/6 mice (n = 3–4 mice per genotype).

Figure 6.

Cytokine and cell count response in Il-1rn−/− mice pretreated with recombinant human (rh) IL-1RA before exposure to LPS. Forty-eight hours after exposure to LPS, (A) the BAL expression of CXCL1 and (B) plasma concentration of G-CSF in Il-1rn−/− mice that had been pretreated 2 hours before LPS exposure with 10 μg rhIL-1RA intratracheally were compared with those in mice pretreated with PBS intratracheally (C) Neutrophil numbers in BAL after pretreatment with rhIL-1RA (n = 3–5 mice per treatment).

Figure 7.

IL-17A expression and IL-17A blockade in Il-1rn−/− mice. (A) Relative expression of Il-17a mRNA (measured by real-time PCR) in Il-1rn−/− mice overall, and at 24 hours (*P < 0.05) and 48 hours (*P < 0.01) after exposure to LPS, compared with littermate control mice (Il-1rn+/+). RQ, relative quantity. (B) Representative dot plots illustrate the number of γδ T cells positive for IL-17A in the lungs of Il-1rn−/− mice, relative to (C) C57BL/6 mice, 48 hours after LPS (n = 4–6 mice per time point). (D) BAL neutrophil counts in Il-1rn−/− mice pretreated 24 hours before exposure to LPS with 100 μg intraperitoneal IL-17A monoclonal antibody (mAb). (E) Lung mRNA G-csf concentrations, 48 hours after exposure to LPS in Il-1rn−/− mice pretreated with 100 μg intraperitoneal isotype control antibody (Ab) (n = 6 mice per time point).