Intrapleural delivery of MSCs attenuates acute lung injury by paracrine/endocrine mechanism

Abstract

Two different repair mechanisms of mesenchymal stem cells (MSCs) are suggested to participate in the repair of acute lung injury (ALI): (i) Cell engraftment mechanism, (ii) Paracrine/endocrine mechanism. However, the exact roles they play in the repair remain unclear. The aim of the study was to evaluate the role of paracrine/endocrine mechanism using a novel intrapleural delivery method of MSCs. Either 1 × 10(6) MSCs in 300 μl of PBS or 300 μl PBS alone were intrapleurally injected into rats with endotoxin-induced ALI. On days 1, 3 or 7 after injections, samples of lung tissues and bronchoalveolar lavage fluid (BALF) were collected from each rat for assessment of lung injury, biochemical analysis and histology. The distribution of MSCs was also traced by labelling the cells with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI). MSCs intrapleural injection significantly improved LPS-induced lung histopathology compared with PBS-treated group at day 3. There was also a significant decrease in total cell counts and protein concentration in BALF at day 7 in the MSCs -treated rats compared to PBS control group. Tracking the DAPI-marked MSCs showed that there were no exotic MSCs in the lung parenchyma. MSCs administration resulted in a down-regulation of pro-inflammatory response to endotoxin by reducing TNF-α both in the BALF and in the lung, while up-regulating the anti-inflammatory cytokine IL-10 in the lung. In conclusion, treatment with intrapleural MSCs administration markedly attenuates the severity of endotoxin-induced ALI. This role is mediated by paracrine/endocrine repair mechanism of MSCs rather than by the cell engraftment mechanism.

Fig. 1

No MSCs were found in the lung parenchyma of rats with LPS-induced ALI after cells were injected intrapleurally. (A) Surface of right lung. (B) Surface of mediastinal pleura. (C, D) Frozen sections of lung tissue. DAPI-marked MSCs masses were found on the surfaces of pleurae and the lung, whereas no fluorescent cells were found in the lung parenchyma (white arrows). All images, ×200 magnification.

Fig. 2

Intrapleural transplantation of MSCs improved lung injury as assessed by histological method. LPS-induced increase in lung injury score was significantly reduced at day 3 in the MSCs treatment group, and the peak of lung injury found at this time-point in the PBS treatment control group disappeared. Data are expressed as mean ± SD, n = 6 per group per time-point. **P < 0.01 versus the PBS treatment group at the same time-point. Data were analysed using non-paired t-test.

Fig. 3

Haematoxylin and eosin staining of lung sections demonstrated attenuated lung injury in the MSCs treatment group at day 3 after instillation of endotoxin. (A, B) PBS-treated group. (C, D) MSCs-treated group. (E, F) Normal control. Histopathological changes in the lungs are shown at low (×100 for A, C, E) and high (×400 for B, D, F) magnification of the black boxed area on the left.

Fig. 4

Macroscopic observation of the whole right lung specimens showed a decreased LPS-induced lung injury at day 3 in the MSCs treatment group. (A–F) Six specimens collected from PBS treatment group at day 3. (G–L) Six specimens collected from MSCs treatment group at day 3. The degree of haemorrhage showed on the surfaces of three lung specimens (B, C and F) collected in the PBS control group were more severe than that of any six specimens collected in MSCs treatment group.

Fig. 5

MSCs significantly improved endothelial/epithelial permeability of the lung. (A) BALF protein was significantly reduced in the MSCs treatment group compared with the PBS treatment group at day 7. (B) Similarly, total cell count in BALF was also significantly reduced in the MSCs treatment group at day 7.(C, D) Both neutrophil count in BALF and W/D of lung were not significantly different between MSCs- and PBS- treatment groups at the three time-points. Data are expressed as mean ± SD, n = 6 per group per time-point. *P < 0.05 versus the PBS treatment group at the same time-point, ^#P < 0.05 versus normal group, ^##P < 0.01 versus normal group. Data were assessed using one-way analysis of variance with SNK-q post hoc tests.

Fig. 6

Macroscopic observation of the BALF suggested an attenuated haemorrhage induced by LPS at day 3 in the MSCs treatment group. (A) Six specimens collected from PBS treatment group at day 3. (B) Six specimens collected from MSCs treatment group at day 3. Obviously, the two reddest BALF samples (black arrows) were from the PBS control group rather than from the MSCs treatment group.

Fig. 7

Intrapleural administration of MSCs did not affect the lung MPO activity after endotoxin-induced ALI, which is an index of neutrophil sequestration. No significant difference was detected between MSCs- and PBS- treatment groups at day 1, 3 or 7 after injury (n = 6 per group per time-point). Data are expressed as mean ± SD. ^#P < 0.05 versus normal group. Data were assessed using one-way analysis of variance with SNK-q post hoc tests.

Fig. 8

Levels of pro- and anti-inflammatory cytokines. LPS-induced increase of TNF-α in BALF was significantly reduced in the MSCs treatment group at day 3, whereas IL-10 in lung tissue was significantly increased at day 1 at both protein and at mRNA levels with MSCs therapy. (A, C, E) Levels of TNF-α in BALF, lung tissue and in mRNA respectively. (B, D, F) Levels of IL-10 in BALF, lung tissue and in mRNA respectively. Data are expressed as mean ± SD, n = 6 per group per time-point. *P < 0.05 versus the PBS treatment group at the same time-point, ^#P < 0.05 versus normal group, ^##P < 0.01 versus normal group. Data were assessed using one-way analysis of variance with SNK-q post hoc tests.

Fig. 9

Immunostaining levels of TNF-α and IL-4. (A) and (C) LPS induced high levels of TNF-α staining in bronchial ciliated columnar epithelial cells (red arrows) as well as in the tunica adventitia of small vessels (green arrows). MSCs treatment showed a trend towards reduction in TNF-α staining at day 3 and day 7 compared with PBS treatment group, though the difference was not significant between the two groups. (B) and (D) LPS challenge did not change the staining level of IL-4 in bronchial ciliated columnar epithelial cells (black arrows) at day 1. However, it increased significantly from day 3 to day 7. The levels of IL-4 showed no difference between the MSCs group and the PBS group. All images, ×200 magnification. Data are expressed as mean ± SD, n = 6 per group per time-point. ^#P < 0.05 versus normal group, ^##P < 0.01 versus normal group. Data were assessed using one-way analysis of variance with SNK-q post hoc tests.