Bone Marrow Mononuclear Cell Therapy Led to Alveolar-Capillary Membrane Repair,Improving Lung Mechanics in Endotoxin-Induced Acute Lung Injury

Abstract

The aim of this study was to test the hypothesis that bone marrow mononuclear cell (BMDMC) therapy led an improvement in lung mechanics and histology in endotoxin-induced lung injury. Twenty-four C57BL/6 mice were randomly divided into four groups (n = 6 each). In the acute lung injur;y (ALI) group, Escherichia coli lipopolysaccharide (LPS) was instilled intratracheally (40 μg, IT), and control (C) mice received saline (0.05 ml, IT). One hour after the administration of saline or LPS, BMDMC (2 × 10⁷ cells) was intravenously injected. At day 28, animals were anesthetized and lung mechanics [static elastance (E_st), resistive (ΔP₁), and viscoelastic (ΔP₂) pressures] and histology (light and electron microscopy) were analyzed. Immunogold electron microscopy was used to evaluate if multinucleate cells were type II epithelial cells. BMDMC therapy prevented endotoxin-induced lung inflammation, alveolar collapse, and interstitial edema. In addition, BMDMC administration led to epithelial and endothelial repair with multinucleated type II pneumocytes. These histological changes yielded a reduction in lung E_st, ΔP₁, and ΔP₂ compared to ALI. In the present experimental ALI model, the administration of BMDMC yielded a reduction in the inflammatory process and a repair of epithelium and endothelium, reducing the amount of alveolar collapse, thus leading to an improvement in lung mechanics.

Introduction

Cell-based therapy is a promising and novel treatment for acute lung injury (ALI) and its severe form, acute respiratory distress syndrome (ARDS) (3,4,7) and other chronic and acute organ dysfunction (2,16). Experimental studies have shown that bone marrow-derived stem cells (BMDSC) contribute to lung repair in ALI (4,5), decreasing mortality rate, lung edema, alveolar epithelial cell permeability, and systemic and local inflammatory response (5). BMDSC may improve ALI due to bone marrow cell differentiation into epithelial and endothelial cells (4). Conversely, bone marrow cells decrease tissue damage and/or promote tissue repair after injury even before damage appears, suggesting a role for paracrine factors (14,15). Additionally, Herzog and colleagues demonstrated that 20–50% of bone marrow-derived epithelial cells in the lung after bone marrow transplantation are due to fusion (5). So far, however, the analysis of cell therapy on physiologic parameters as well as on the ultrastructure of lung parenchyma in ALI has not been performed.

The aim of this study was to test the hypothesis that BMDMC therapy led to alveolar-capillary membrane repair, improving lung mechanics in endotoxin-induced lung injury. For this purpose, light and electron microscopy were used, and the impact of these histological changes on lung mechanics was analyzed.

Materials and Methods

This study was approved by the Ethics Committee of the Carlos Chagas Filho Institute of Biophysics, Health Sciences Centre, Federal University of Rio de Janeiro. All animals received humane care in compliance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences, USA.

Animal Preparation and Experimental Protocol

A total of 24 C57Bl/6 female mice (20–25 g) were randomly assigned to four groups. In the control groups (C), the animals received sterile saline solution (0.9% NaCl) intratracheally (0.05 ml, IT; n = 6). In the ALI groups, mice received Escherichia coli lipopolysaccharide (LPS, O55:B5, Sigma Chemical Co., St. Louis, MO, USA) intratracheally (40 μg diluted in 0.05 ml of saline/mouse, n = 6). In the C and ALI groups, the animals received sterile saline solution (0.9% NaCl) or BMDMC (2 × 10⁷) 1 h after the administration of saline or LPS through the left jugular vein. For IT instillation, the mice were anesthetized with sevoflurane, a 1-cm-long midline cervical incision was made to expose the trachea, and LPS or saline was instilled using a bent 27-gauge tuberculin needle. The cervical incision was closed with 5.0 silk suture and the mice were returned to their cage. The animals recovered rapidly after surgery.

Extraction of Mononuclear Bone Marrow-Derived Cells

Bone marrow cells from the male C57Bl/6 mice (n = 12, 6–10 weeks old) were aspirated from the femur and tibia by flushing the bone marrow cavity with Dulbecco's modified Eagle's medium (DMEM) (Life Technologies, Grand Island, NY, USA). After a homogeneous cell suspension was achieved, the cells were centrifuged (400 x g for 10 min), resuspended in DMEM, added to Ficoll-Hypaque (Histopaque 1083, Sigma Chemical Co.), and again centrifuged and supplemented with sterile phosphate-buffered saline (PBS). Cells were counted in a Neubauer chamber with trypan blue for evaluation of viability. For the administration of saline or BMDMC, mice were anesthetized with sevoflurane, and the cells were injected using a bent 27-gauge tuberculin needle. All substances and surgical material were sterilized.

Lung Mechanics

Twenty-eight days after saline or E. coli LPS administration, 24 mice (n = 6/group) were sedated (diazepam 1 mg, IP), anesthetized (thiopental sodium 20 mg/kg, IP), tracheotomized, paralyzed (vecuronium bromide, 0.005 mg/kg, IV), and ventilated with a constant flow ventilator (Samay VR15; Universidad de la Republica, Montevideo, Uruguay) with the following parameters: frequency of 100 breaths/min, tidal volume of 0.2 ml, and fraction of inspired oxygen of 0.21. A positive end-expiratory pressure (PEEP) of 2 cmH₂O was applied and the anterior chest wall surgically removed. After a 10-min ventilation period, lung mechanics were computed.

A pneumotachograph was connected to the tracheal cannula for the measurements of airflow. V_T was calculated by digital integration of the flow signal. The flow resistance of the equipment (R_eq), including the tracheal cannula, amounted to 0.12 cmH₂O/ml/s. The resistive pressure of the equipment (R_eqV') was subtracted from the pulmonary resistive pressure so that the results represented intrinsic values. Tracheal pressure (P_tr) was measured with a differential pressure transducer SCIREQ (SC-24, Montreal, Canada). All signals were filtered (100 Hz) and amplified in a four-channel conditioner (SC-24, SCIREQ). Flow and pressure signals were then sampled at 200 Hz with a 12-bit analog-to-digital converter (DT2801A, Data Translation, Marlboro, MA, USA), and stored on a microcomputer. All data were collected using LABDAT software (RHT-InfoData, Inc., Montreal, Quebec, Canada).

Lung mechanics were measured by the end-inflation occlusion method (1). In an open chest preparation, P_tr reflects transpulmonary pressure (P_L). Briefly, after end-inspiratory occlusion, there is an initial fast drop in P_L (ΔP₁) from the preocclusion value down to an inflection point (P_i), followed by a slow pressure decay (ΔP₂), until a plateau is reached. This plateau corresponds to the elastic recoil pressure of the lung (P_el). ΔP₁ selectively reflects the pressure used to overcome the airway resistance. ΔP₂ reproduces the pressure spent by stress relaxation, or viscoelastic properties of the lung, together with a small contribution of pendelluft. Total pressure drop (ΔP_tot) is equal to the sum of ΔP₁ and ΔP₂. Lung static elastance (E_st) was determined dividing P_el by V_T. Lung mechanics were performed 10 times for each animal. All data were analyzed using ANADAT data analysis software (RHT-InfoData).

Histology

Light Microscopy.

A laparotomy was done immediately after the determination of the lung mechanics and heparin (1000IU) was injected intravenously in the vena cava. The trachea was clamped at end expiration, and the abdominal aorta and vena cava were sectioned, yielding a massive hemorrhage that quickly killed the animals. Then, the lungs were removed en bloc in all groups to avoid distortion of lung morphometry. The right lung was fixed with Carnoy's solution and embedded in paraffin. Slices (4 μm thick) were cut and stained with hematoxylin/eosin.

Lung morphometric analysis was performed with an integrating eyepiece with a coherent system consisting of a grid with 100 points and 50 lines (known length) coupled to a conventional light microscope (Olympus BX51, Olympus Latin America-Inc., Brazil). The volume fraction of the lung occupied by collapsed alveoli or normal pulmonary areas were determined by the point-counting technique at a magnification of 200x across 10 random, noncoincident microscopic fields (12).

Transmission Electron Microscopy

Three slices (2 × 2 × 2 mm) were cut from three different segments of the left lung and fixed (2.5% glutaraldehyde and phosphate buffer 0.1 M, pH 7.4) for electron microscopy (JEOL 1010 transmission electron microscope, Tokyo, Japan) analysis. For each electron microscopy image (15/animal), the following structural damage was analyzed: (a) alveolar capillary membrane; (b) type II epithelial cells; and (c) endothelial cells. The pathologic findings were graded according to a 5-point semiquantitative severity-based scoring system: 0, normal lung parenchyma; 1, changes in 1–25%; 2, changes in 26–50%; 3, changes in 51–75%; and 4, changes in 76–100% of tissues examined.

Immunogold

Lung samples were fixed in 4% paraformaldehyde and embedded in Lowicryl HM-20 resin using a substitution Automat (AFS-Leica). Ultrathin sections, collected on collodion-coated Ni grids, were treated with 0.1 M glycine, and then with 10% BSA to block nonspecific binding sites. Sections were incubated overnight at 4°C with the monoclonal antibody CK7 directed against pneumocyte receptors, and thereafter with rabbit anti-mouse immunoglobulin (Sigma). They were then treated with a 15-nm protein G–gold complex, rinsed, postfixed with 2% glutaraldehyde and 1% osmic acid, and stained. Negative controls were performed with sections labeled in the absence of the first antibody 9A7g. Sections were examined with a JEOL transmission electron microscope at 60 kV.

Statistical Analysis

The normality of the data (Kolmogorov-Smirnov test with Lilliefors' correction) and the homogeneity of variances (Levene median test) were tested. If both conditions were satisfied, the effects of saline or BMDMC in the C and ALI groups were analyzed using two-way ANOVA followed by Tukey's test. Otherwise, two-way ANOVA on ranks followed by Dunn's post hoc test were selected. The significance level was always set at 5%. The parametric data were expressed as the mean ± SEM, and the nonparametric data were expressed as the median (interquartile range). All tests were performed using SigmaStat 3.0 (Jandel Corporation, San Raphael, CA, USA).

Results

In pilot studies we determined that this LPS model of ALI resulted in an approximate 88% survival rate at 28 days. A single intravenous BMDMC therapy significantly increased (p < 0.05) survival (100%) at 28 days (ALI-Cell).

The area of alveolar collapse and polymorphonuclear cells were increased in the ALI group compared to control C-Sal group (19.7 and 27.1 folds, respectively, n = 6, p < 0.05) and the use of BMDMC minimized these histological changes (to 7.5- and 5.6-fold, respectively, n = 6, p < 0.05). The control animal that received BMDMC (C-Cell group) also presented an increase in alveolar collapse (7.2-folds, n = 6, p < 0.05) compared to the control C-Sal group (Table 1, Fig. 1) with no significant changes in polymorphonuclear cells (PMN) cells.

Figure 1.

Representative photomicrographs of lung parenchyma. ALI was induced by intratracheal instillation of E. coli LPS. In the C group saline was instilled intratracheally; after 1 h, the C and ALI groups were treated with saline (Sal) or BMDMC (2 × 10⁷, Cell). Note the areas of alveolar collapse (arrows). Original magnification: 200x.

Table 1.

Lung Morphometric Parameters and Cellularity in Lung Parenchyma

Groups	Normal Area (%)	Alveolar Collapse (%)	PMN (%)	MN (%)
C-Sal	97.8 ± 1.2	2.2 ± 0.8	0.12 ± 0.04	26.90 ± 1.77
C-Cell	84.1 ± 0.7 ∗	15.9 ± 0.7 ∗	0.39 ± 0.15	25.29 ± 0.15
ALI-Sal	56.6 ± 6.6 ∗	43.4 ± 6.6 ∗ †	3.26 ± 0.43 ∗	24.24 ± 0.62
ALI-Cell	83.4 ± 4.4 ∗ †	16.6 ± 4.4 ∗ †	0.68 ± 0.24	26.46 ± 1.92

Values are means ± SEM of six animals in each group. All values were computed in 10 random noncoincident fields per mice. The volume fraction of the lung occupied by normal pulmonary areas and/or collapsed alveoli were measured. ALI was induced by intratracheal instillation of E. coli LPS. In the C group saline was instilled intratracheally, after 1 h, the C and ALI groups were treated with saline (Sal) or BMDMC (2 × 10⁷, Cell). PMN, polymorphonuclear cells; MN, mononuclear cells.

∗

Significantly different from C-Sal (p < 0.05).

†

Significantly different from ALI-Sal (p < 0.05).

In ALI animals it was shown, by photomicrographs of electron microscopy, cytoplasmic degeneration of lamellar bodies in type II pneumocytes, increased amounts of extracellular matrix elements, such as collagen fibers, and endothelial lesions (Fig. 2). BMDMC therapy led to an unusual multinucleated cell observation in lung parenchyma with no phenotypic characterization (Fig. 2) and a repair of both epithelium and endothelium (Table 2). Immunogold labeling (monoclonal CK7 antibody) depicted perinuclear filaments in the cytoplasm of these cells, suggesting that the multinucleated cells represent type II pneumocyte (Fig. 2).

Figure 2.

Ultrastructural immunocytochemical localization of type II cells in ALI-Cell group lung tissue fixed in 4% paraformaldehyde and embedded in Lowicryl HM-20 resin. Immunogold electron microscopy was performed in ultrathin mice lung sections fixed in 4% paraformaldehyde, embedded in Lowicryl HM-20 resin and labeled with monoclonal antibody CK-7. Small gold particles stained in black (arrows) indicate immunoreactivity of perinuclear filaments of keratin in the cytoplasm. Pictures are representative of 16 to 20 grids. Note the presence of multinucleated (N) cells.

Table 2.

Semiquantitative Analysis of Electron Microscopy

Groups	Alveolar Capillary Membrane	Type II Epithelial Cells	Endothelial Cells
C-Sal	0 (0–0)	0 (0–0)	0 (0–0)
C-Cell	0 (0–0)	0 (0–0)	0 (0–0)
ALI-Sal	3 (2.5–3) ∗	2 (2–2.5) ∗	2 (1–2.5) ∗
ALI-Cell	1 (1–1.5) ∗ †	1 (0–1)	1 (0–1)

Lung tissue score was done independently by two different investigators. The pathologic findings were graded according to a 5-point semiquantitative severity-based scoring system: 0, normal lung parenchyma; 1, changes in 1–25%; 2, 26–50%; 3, 51–75%; and 4, 76–100% of the examined tissue. Electron microscopy of lung parenchyma in the C and ALI groups. ALI was induced by intratracheal instillation of E. coli LPS. In the C group saline was instilled intratracheally; after 1 h, the C and ALI groups were treated with saline (Sal) or BMDMC (2 × 107, Cell).

∗

Significantly different from C-Sal (p < 0.05).

†

Significantly different from ALI-Sal (p < 0.05).

Lung static elastance, resistive (ΔP₁) and viscoelastic (ΔP₂) pressures were also increased (1.44-, 1.84-, and 1.32 fold, respectively, n = 6, p < 0.05) in the ALI-Sal group. The use of BMDMC successfully avoided these mechanical changes (Fig. 3).

Figure 3.

Values are means ± SEM of six animals in each group (10 determinations per animal). (A) Lung static elastance. (B) Stacked bar chart: the data in each white bar represent the lung viscous pressure (ΔP ₁) and the gray bars are the viscoelastic/inhomogeneous (ΔP₂) pressure dissipations. The whole column represents the total pressure (ΔP_tot) variation in each group. ALI was induced by intratracheal instillation of E. coli LPS. In the C group saline was instilled intratracheally; after 1 h, the C and ALI groups were treated with saline (Sal) or BMDMC (2 × 10⁷, Cell). ∗Significantly different from C-Sal (p < 0.05).

Discussion

In the present study, ALI led to alveolar collapse, interstitial edema, epithelium and endothelium lung injury, and an increased amount of collagen fiber, resulting in lung mechanics impairment. BMDMC yielded: 1) a reduction in mortality rate, an improvement in lung static elastance, resistive and viscoelastic pressures, and a decrease in the fraction area of alveolar collapse as well as lung tissue cellularity, and 2) beneficial effects on the repair of basement membrane, epithelium, and endothelium.

Acute lung injury experimental model was induced by intratracheal instillation of E. coli LPS, which triggers a network of inflammatory responses leading to alveolar epithelium lesions (8,9,11). The use of this model is advantageous because the moment of the analysis can be precisely controlled, and the animals breathe spontaneously, avoiding the side effects resulting from mechanical ventilation.

BMDMC were chosen because they express several genes involved in inflammatory response and chemotaxis, while mesenchymal stem cells preferentially express a large number of genes involved in development, morphogenesis, cell adhesion, and cell proliferation (10). In addition, ALI/ARDS needs to be treated as soon as possible, and using BMDMC the therapy can be performed on the same day because the cells do not need to be expanded in vitro. Finally, the technique is easier and the costs are lower. We analyzed the effects of BMDMC therapy on day 28. To our knowledge, this is the first study showing the late effects of BMDMC in a model of direct insult, because most studies were performed early in the course of lung injury up to day 14 (4–6,13–15). Thus, we cannot be sure that the beneficial effects reported at the early phase persist late in the course of lung injury. In our study, we were able to demonstrate not only the epithelial/endothelium lesions but also the repair induced by BMDMC, using a semiquantitative analysis of electron microscopy (Table 2).

Semiquantitative analysis of electron microscopy showed extensive injury of alveolar epithelium and endothelium in ALI-Sal group (Table 2). Cell therapy led to a repair in endothelium and epithelium as well as unusual multinucleated cells with no phenotypic characterization. To clarify the etiology of these cells, immunogold electron microscopy was performed and multinucleated cells were identified as immunoreactive type II pneumocytes.

Recently, some authors described that intravenous (14) or intrapulmonary (15) instillation of MSCs after intratracheal LPS-induced ALI reduced the inflammatory response through paracrine effects. In this line, we found that BMDMC therapy yielded a reduction in polymorphonuclear cells in lung tissue. However, these authors did not analyze lung function.

In our study, lung static elastance (E_st), resistive (ΔP₁), and viscoelastic/inhomogeneous (ΔP₂) pressures were computed 28 days after intratracheal instillation of LPS. All mechanical parameters were increased in ALI-Sal group. The changes in ΔP₁ could be attributed to active airway constriction caused by the release of endogenous mediators following endotoxemia; the increase in E_st and ΔP₂ may have resulted from the same amount of alveolar collapse. These findings are in accordance with the previous description of LPS-induced alteration of alveolar surfactant, which could favor alveolar collapse (11). The C-Cell and ALI-Cell groups presented an increase in alveolar collapse compared to the C-Sal group, although these histological changes were not enough to alter lung mechanics. Atelectasis may be related to the presence of cell infiltration in the alveolar septa. In our study, the intravenous injection of BMDMC minimized elastic, resistive, and viscoelastic parameters, histology, inflammatory process, and apoptotic epithelial cells in ALI.

Our data should not be directly extrapolated to the clinical scenario. More studies are required to address the optimal timing, number, and route of cell administration.

In conclusion, in the experimental ALI model, the administration of BMDMC yielded a reduction in the inflammatory process and a repair of epithelium and endothelium, reducing the amount of alveolar collapse, thus leading to an improvement in lung mechanics.