Critical role of endothelial CXCR2 in LPS-induced neutrophil migration into the lung

Abstract

In models of acute lung injury, CXC chemokine receptor 2 (CXCR2) mediates migration of polymorphonuclear leukocytes (PMNs) into the lung. Since CXCR2 ligands, including CXCL1 and CXCL2/3, are chemotactic for PMNs, CXCR2 is thought to recruit PMNs by inducing chemotactic migration. In a model of PMN recruitment to the lung, aerosolized bacterial LPS inhalation induced PMN recruitment to the lung in wild-type mice, but not in littermate CXCR2-/- mice. Surprisingly, lethally irradiated wild-type mice reconstituted with CXCR2-/- BM still showed about 50% PMN recruitment into bronchoalveolar lavage fluid and into lung interstitium, but CXCR2-/- mice reconstituted with CXCR2-/- BM showed no PMN recruitment. Conversely, CXCR2-/- mice reconstituted with wild-type BM showed a surprisingly large defect in PMN recruitment, inconsistent with a role of CXCR2 on PMNs alone. Cell culture, immunohistochemistry, flow cytometry, and real-time RT-PCR were used to show expression of CXCR2 on pulmonary endothelial and bronchial epithelial cells. The LPS-induced increase in lung microvascular permeability as measured by Evans blue extravasation required CXCR2 on nonhematopoietic cells. Our data revealed what we believe to be a previously unrecognized role of endothelial and epithelial CXCR2 in LPS-induced PMN recruitment and lung injury.

Figure 1

LPS inhalation caused a significant recruitment of PMN into the BAL. In CXCR2 ^{–/ –} mice, PMN recruitment was completely abolished. In mice heterozygous for the CXCR2 allele (CXCR2^{+/ –}), a reduction of ∼50% was observed. Data are mean ± SD of n = 6 mice in each group. Cytospins of LPS-exposed BAL in CXCR2^+/+ and CXCR2 ^{–/ –} mice are shown above the corresponding bars. *P < 0.05 versus CXCR2^+/+; ^#P < 0.05 versus no LPS.

Figure 2

Flow cytometry of lung homogenate. (A) PMNs were identified by their typical appearance in the forward scatter (FSC) and side scatter (SSC) and their expression of CD45 and CD11b (data not shown). GR-1 was injected intravenously to distinguish between intravascular and extravascular (i.e., interstitial after removal of BAL) PMNs. (B) In control animals (saline inhalation), 88% of PMNs were found intravascularly. (C) LPS caused transendothelial migration into the interstitium, as demonstrated by the appearance of CD11⁺GR-1^– PMNs. (D) In contrast, transendothelial migration was suppressed in CXCR2 ^{–/ –} mice.

Figure 3

Chemotactic activity of LPS-exposed BAL from CXCR2^+/+ mice and the 2 critical CXCR2 ligands, CXCL1 and CXCL2/3, was assessed in an in vitro transmigration assay. PMNs isolated from CXCR2^+/+ mice showed significant migration toward LPS-exposed BAL, CXCL1, and CXCL2/3. PMNs from CXCR2 ^{–/ –} mice did not migrate toward the CXCR2 ligands, but still migrated toward LPS-exposed BAL from CXCR2 ^{–/ –} mice. Data are mean ± SD of 3 experiments. **P < 0.001 versus negative control (medium only).

Figure 4

LPS-induced PMN migration into different lung compartments. (A) Complete reconstitution of hematopoietic cells after BMT was confirmed by RT-PCR (whole blood). Solid lines, positive (CXCR2^+/+) and negative (CXCR2 ^{–/ –}) controls; lines with diamonds, CXCR2^+/+ mice reconstituted with BM from CXCR2 ^{–/ –} mice. (B) When CXCR2^+/+ mice were reconstituted with BM from CXCR2 ^{–/ –} mice, interstitial (gray bars) and BAL (white bars) PMN content was reduced by 40% and 50%, respectively. When CXCR2 ^{–/ –} mice were reconstituted with BM from CXCR2^+/+ mice, the reduction was by 50% and 60%, respectively. LPS-induced accumulation of PMN in the pulmonary vasculature (black bars) did not differ among the groups. CXCR2^+/+ mice reconstituted with BM from CXCR2^+/+ and CXCR2 ^{–/ –} mice reconstituted with BM from CXCR2 ^{–/ –} served as positive and negative controls. Data are mean ± SD of n = 4 mice. *P < 0.05 versus positive control.

Figure 5

CXCR2 mRNA expression in pulmonary endothelial cells (PECs) and BAL. Isolated pulmonary endothelial cells exhibited readily detectable amounts of CXCR2 mRNA. BAL from CXCR2^+/+ also showed CXCR2 mRNA expression, but negligible CXCR2 mRNA was found in the BAL of CXCR2 ^{–/ –} mice. In CXCR2^+/+ mice reconstituted with BM from CXCR2 ^{–/ –} mice, low levels of CXCR2 mRNA were detected, most likely derived from contamination with epithelial cells. CXCR2 mRNA expression was almost normal when CXCR2 ^{–/ –} mice were reconstituted with BM from CXCR2^+/+ mice. CXCR2 mRNA levels were normalized to GAPDH and presented as mean ± SD of n = 3 samples.

Figure 6

CXCR2 protein expression shown by immunohistochemistry. (A) Lungs from CXCR2^+/+ mice demonstrated CXCR2 expression among large vessels, small vessels (insert), and alveolar septal walls (arrow) and among the epithelial layer of large airways (C). No CXCR2 was detected in lungs from CXCR2 ^{–/ –} mice (B and D).

Figure 7

CXCR2 expression in lung homogenate. (B) Leukocytes (CD45⁺) and nonleukocytes (CD45^–) were analyzed separately for their expression of CXCR2 (shown in C and A, respectively). (A) Among all CD45^– cells, endothelial cells were identified by their expression of CD31. The majority of endothelial cells (∼67%) expressed CXCR2. In addition, CXCR2 was found in a large population of CD31^– cells. (C) Among all leukocytes (CD45⁺), only PMNs (GR-1^high) exhibited CXCR2 surface expression. Neither monocytes/macrophages (Mφs; GR-1^intermediate) nor most lymphocytes (Lys; GR-1^negative) showed detectable CXCR2 expression.

Figure 8

Pulmonary microvascular permeability was assessed in CXCR2^+/+, CXCR2 ^{–/ –}, and chimeric mice. In CXCR2 ^{–/ –} mice and in CXCR2 ^{–/ –} mice reconstituted with BM from CXCR2^+/+ mice, LPS-induced protein leakage was significantly reduced compared with CXCR2^+/+ mice. In contrast, no protection was observed in CXCR2^+/+ mice reconstituted with BM from CXCR2 ^{–/ –}. *P < 0.05 versus CXCR2^+/+ mice. Data are mean ± SD of n = 4 mice.

Figure 9

Endothelial cell response to CXCR2 activation was investigated by F-actin localization. Isolated pulmonary endothelial cells were treated with 10 or 100 ng/ml of recombinant CXCL1, and F-actin was localized by phalloidin staining. CXCL1 induced an increase in F-actin formation over time. Stress fibers appeared rapidly and were most pronounced at cell-cell borders. After 120 minutes, most endothelial cells appeared in a polygonal, retracted shape. Images are representative of 3 experiments with similar results.