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Transforming Growth Factor-ß₃ Induces Pleurodesis in Rabbits and Collagen Production of Human Mesothelial Cells^*

^* From the Department of Critical Care and Pulmonary Services (Dr. Kalomenidis), Athens Medical School, Evangelismos Hospital, Athens, Greece; the Department of Allergy Pulmonary and Critical Care Medicine (Dr. Light), Vanderbilt University, Nashville, TN; the Department of Pulmonary Medicine (Drs. Lane and Hawthorne), St. Thomas Hospital and Vanderbilt University, Nashville, TN; and Department of Respiratory & Critical Care Medicine (Dr. Guo), First Affiliated Hospital of Sun Yat-sen University, Guangzhou, PR China.

Correspondence to: Ioannis Kalomenidis, MD, Department of Critical Care and Pulmonary Services, Athens Medical School, Evangelismos Hospital, 45–47 Ipsilantou, 10675, Athens, Greece; e-mail: jkalomenidis{at}hotmail.com

	Abstract

TOP Abstract Introduction Materials and Methods Results Discussion References

 
Study objective: The aim of this study was to examined whether transforming growth factor (TGF)-ß₃ induces pleurodesis in rabbits and collagen production by human pleural mesothelial cells in vitro.

Design: A combined animal and in vitro study.

Methods: TGF-ß₃ was injected intrapleurally in rabbits at the following doses: 0.167 µg, 0.5 µg, 1.67 µg, and 5 µg (five rabbits per group). The rabbits were killed 14 days following injection, and pleurodesis was graded from 1 (none) to 8 (complete symphysis). Pleural mesothelial cell cultures were established from benign pleural effusions and treated with TGF-ß₃ for 48 h at the following doses: 0 (control), 5 ng/mL, 1.5 ng/mL, 5 ng/mL, and 15 ng/mL and 50 ng/mL (two independent experiments). Collagen I messenger RNA (mRNA) expression was quantified using real-time polymerase chain reaction.

Results: The median pleurodesis score was 5 (interquartile range [IQR], 4.5) in the 0.167-µg group, 6 (IQR, 5) in the 0.5-µg group, 8 (IQR, 1) in the 1.67-µg group, and 8 (IQR, 0.5) in the 5-µg group (p = 0.012). Higher TGF-ß₃ doses induced the production of significantly more pleural fluid than the lower doses (p = 0.005). The pleural fluid induced by higher doses contained significantly lower numbers of nucleated cells (p = 0.014) and lactate dehydrogenase levels (p = 0.013) than the pleural fluid induced by lower doses of the agent. TGF-ß₃ markedly enhanced collagen I mRNA expression by the human pleural mesothelial cells. This effect peaked at 5 ng/mL TGF-ß₃. With this dose of TGF-ß₃, the collagen I mRNA expression was increased 16-fold over control levels.

Conclusion: TGF-ß₃ causes a dose-dependent pleurodesis when administered intrapleurally in rabbits, and induces collagen messenger RNA synthesis from human pleural mesothelial cells.

Key Words: collagen expression • mesothelial cells • pleurodesis • rabbits • transforming growth factor-ß₃

	Introduction

TOP Abstract Introduction Materials and Methods Results Discussion References

 
Pleurodesis is the obliteration of the pleural space with fibrous tissue, and it is commonly used to treat persistent pleural effusions and to prevent the recurrence of a spontaneous pneumothorax. Talc, tetracycline derivatives, and bleomycin, the most commonly used sclerosing agents, cause an inflammatory reaction that is followed by collagen synthesis and the local activation of the coagulation system as well as inhibition of fibrinolysis.¹ These events eventually lead to pleural fibrosis. Currently used sclerosing agents are fairly effective, but their use is associated with side effects that are probably due to the inflammation caused by these agents.²

Since cytokines are involved in the process of producing the pleurodesis after the pleura is injured by the currently used sclerosing agents, our group hypothesized that the intrapleural injection of a cytokine could produce a pleurodesis. This approach would circumvent the need for producing a pleural injury and would be expected to be associated with fewer side effects. Transforming growth factor (TGF)-ß was an attractive candidate since it is both a potent profibrotic and an antiinflammatory cytokine.³ It stimulates extracellular matrix production and inhibits matrix degradation, promoting fibrosis and tissue repair under normal conditions and in certain disease states.³⁴ In humans, TGF-ß consists of three isoforms (ß₁, ß₂, and ß₃), all of which may be involved in the production of fibrous tissue.⁴ Our group has previously shown that the intrapleural injection of TGF-ß₂ causes effective pleurodesis faster than talc or doxycycline.⁵⁶ Additionally, the pleural fluid produced after the intrapleural injection of the TGF-ß₂ is less inflammatory than that produced after the administration of talc or doxycycline.⁵⁶

While all the three TGF-ß isoforms are involved in fibrinogenesis, the role of each one of them has not been clearly defined, and there is evidence that different isoforms are not equally effective in causing collagen production.⁴ The purpose of the present study was to evaluate whether TGF-ß₃ would produce pleurodesis in rabbits and stimulate human pleural mesothelial cells to express collagen messenger RNA (mRNA). We hypothesized that the intrapleural injection of TGF-ß₃ would produce pleurodesis in a dose-dependent fashion, would be associated with production of pleural fluid with a low WBC count and lactate dehydrogenase (LDH), and that TGF-ß₃ would induce collagen mRNA expression by human pleural mesothelial cells.

	Materials and Methods

TOP Abstract Introduction Materials and Methods Results Discussion References

 
TGF-ß₃
Recombinant human TGF-ß₃ was provided by GroPep (Adelaide, Australia). It was diluted with rabbit plasma (Del-Freez Biological; Little Rock, AR) to create a stock (1 mg/mL) that was further diluted with saline solution in order to create the desired concentration.

Animal Study
The study protocol was approved by the Vanderbilt University Institutional Animal Care and Use Committee. The method used was similar to that described in our previous studies.⁵⁶ New Zealand white rabbits weighing 1.5 to 2.0 kg were anesthetized with an IM injection of 35 mg/kg ketamine hydrochloride (Fort Dodge Animal Health Laboratories; Fort Dodge, IA) and 5 mg/kg xylazine hydrochloride (Fermenta; Kansas City, MO). The chest was shaved, and the skin was sterilized with 10% povidone iodine (Baxter; Deerfield, IL). The rabbit was placed in the lateral decubitus position, and a small (< 3 cm) skin incision was made midway between the tip of the scapula and the sternum approximately 2 cm above the costal margin. A chest tube (silicone tube, 0.062-inch internal diameter and 0.125-inch outer diameter; Braintree Scientific; Braintree, MA) was made with three extra openings near the distal end of the tube to enhance drainage. The chest tube was inserted by blunt dissection into the right pleural cavity. The chest tube was secured at the muscle layers with purse-string sutures. The proximal end of the chest tube was then tunneled underneath the skin and drawn out through the skin posteriorly and superiorly between the two scapulae. A three-way stopcock was attached to the end of the chest tube via an adapter through which any pleural air was evacuated from the pleural space. The three-way stopcock was then removed from the chest tube and the exterior end of the chest tube was sealed with a one-way valve with a cap (Medexinc; Hilliard, OH) via the adapter and sutured to the skin. Pleural fluid could be aspirated through the adaptor. The left pleural cavity was used for control.

Twenty rabbits were put into four groups of 5 rabbits each: TGF-ß₃ in 2 mL 0.9% sodium chloride was injected intrapleurally after chest tube placement in the following doses: 0.167 µg, 0.5 µg, 1.67 µg, and 5 µg. Injection of the TGF-ß₃ was followed with an injection of 1 mL of 0.9% sodium chloride solution to clear the dead space of the chest tube. After the intrapleural injection, the chest tube was aspirated at 24-h intervals for any pleural fluid. The volume of the pleural fluid collected every day and the total volume of the pleural fluid were recorded. The protein and LDH levels were determined with an automated analyzer (Johnson & Johnson; Rochester, NY). The upper limit for serum with this LDH method is 618 IU/L. The total leukocyte count was measured using an automated counter (Coulter Electronics; Luton, UK) that was calibrated daily. The first reading was ignored, and the mean of the next three readings was recorded. The chest tube was removed under light sedation when the pleural fluid drainage was < 3 mL over the preceding 24 h.

The rabbits were killed at day 14 by CO₂ euthanasia following sedation. The thorax was removed en bloc. The lungs were expanded by the injection of 50 mL of 10% neutral-buffered formalin into the exposed trachea via a plastic catheter (6 mm in diameter). The trachea was then ligated and the entire thorax submerged into 10% neutral-buffered formalin solution for at least 48 h. To assess the pleurodesis, the pleural cavity was carefully exposed as previously described.⁵⁶ A consensus grading was reached by two blinded investigators (K.B.L. and R.W.L.) on the degree of macroscopic pleurodesis using a semiquantitative scheme. The degree of pleurodesis was graded using the following scale: 1 = no adhesions between the visceral and parietal pleura; 2 = rare adhesions between the visceral and parietal pleura with no symphysis; 3 = a few scattered adhesions between the visceral and parietal pleura with no symphysis; 4 = many adhesions between the visceral and parietal pleura with no symphysis; 5 = many adhesions between the visceral and parietal pleura with symphysis involving < 5% of the hemithorax; 6 = many adhesions between the visceral and parietal pleura with symphysis involving 5 to 25% of the hemithorax; 7 = many adhesions between the visceral and parietal pleura with symphysis involving 25 to 50% of the hemithorax; and 8 = many adhesions between the visceral and parietal pleura with symphysis involving > 50% of the hemithorax. Adhesions were defined as fibrous connections between the visceral and parietal pleura. Symphysis was present if the visceral and parietal pleura were difficult to separate as a result of adhesions.

Human Mesothelial Cell Cultures
Human pleural mesothelial cells were cultured from benign pleural effusions as previously described.⁷ Briefly, pleural fluid was centrifuged at 1,500 revolutions per minute for 5 min, and cell pellets were washed three times with RPMI 1640 medium (Cellgro; Herndon, VA) [complete medium]. The pellets were resuspended in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U/mL of penicillin G, and 100 µg/mL of streptomycin (GIBCO Laboratories; Grand Island, NY) and 10 mmol/L hydroxylethyl piperazine-ethanesulfonic acid (GIBCO Laboratories). The cells were cultured in 100-mm Petri dishes in a humidified atmosphere of 5% CO₂ at 37°C. The dishes were washed with warmed saline solution to remove nonadherent cells, and fresh complete medium was added every 2 to 3 days for 2 to 3 weeks. A small number of cells was immunostained with DC-10 anti-keratin 8/18 monoclonal antibody (Monosan; Uden, Holland), V-9 anti-vimentin monoclonal antibody (Immuno Thermo Shandon; Pittsburgh, PA), and PGM1 anti-CD68 monoclonal antibody (Dako; Golstrup, Denmark). The cultured mesothelial cells were vimentin-positive and keratin-positive and CD68-negative.

In vitro TGF-ß₃ Treatment and Real-time Polymerase Chain Reaction
Established mesothelial cells were grown to confluence and then transferred to 12-well culture plates (for the survival studies) or to 6-well culture plates (for RNA isolation). After 48 h, the complete medium was aspirated, the wells were carefully washed with warm saline solution, and RPMI 1640 with 0.1% heat-inactivated fetal bovine serum, 100 U/mL of penicillin G, 100 µg/mL of streptomycin and 10 mmol/L hydroxylethyl piperazine-ethanesulfonic acid was added to the wells. Ascorbic acid (400 mmol/L) was added in every well at the start of the experiment and after 24 h. Cells at confluence were treated with the following TGF-ß₃ doses (two wells per dose): 0 (control), 0.5 ng/mL, 1.5 ng/mL, 5 ng/mL, 15 ng/mL and 50 ng/mL (two independent experiments). At 48 h, the cells were harvested and the percentage of surviving cells were determined by trypan blue exclusion. Total RNA was extracted using a Qiagen RNeasy Mini-kit (Qiagen; Valencia, CA) according to the instructions of the manufacturer. Complementary DNA was made from RNA with the use of Access RT-PCR System (Promega; Madison, WI) following the instructions of the manufacturer. The synthesized complementary DNA was quantitated by real-time polymerase chain reaction (PCR) using a cycler (SmartCycler SC1000–1; Cepheid; Kingwood, TX). The primers for precollagen-I and the housekeeping gene, ß-actin, were purchased from IDT (Coralville, IA). DNA from 3T3 cells (American Type Culture Collection; Manassas, VA) was used as positive control. The conditions for real-time PCR consisted of 12.5 µL of PCR master mix (Promega), 2.5 mmol/L MgCl₂, 1x SYBR green I, 2 µL of the product of the complementary DNA reaction, and 300 nmol/L of the specific forward and reverse primers for the target gene in a total of 25 µL. The real-time quantitative assessment of the complementary DNA was done by monitoring the incorporation of SYBR green dye (Sigma; St. Louis, MO) into the product. The amount of collagen complementary DNA was compared to the ß-actin to give a relative value of collagen RNA present in the starting material. The amplicons were also assessed using gel electrophoresis of the PCR products on a 1.5% agarose gel at 100 V for 30 min.

Statistical Analysis
Data were expressed as mean ± SD when normally distributed and as median (interquartile range [IQR]) when they were not normally distributed. The Kruskal-Wallis test was used to compare medians (for nonparametric data), and the analysis of variance was used to compare means (for parametric data) between subgroups. All data were analyzed using software (SPSS 11.0; SPSS; Chicago, IL); p < 0.05 was considered significant.

	Results

TOP Abstract Introduction Materials and Methods Results Discussion References

 
Intrapleural Injection of TGF-ß₃
TGF-ß₃ was very effective at producing pleurodesis, particularly at the higher doses. The median pleurodesis score was significantly higher in the treated hemithorax as compared to the left hemithorax: 8 (IQR, 2) and 1 (IQR, 0.75), respectively (p = 0.03). The pleurodesis scores were dose dependent: 5 (IQR, 4.5) in the 0.167-µg group, 6 (IQR, 5) in the 0.5-µg group, 8 (IQR, 1) in the 1.67-µg group, and 8 (IQR, 0.5) in the 5-µg group (p = 0.012) [Fig 1 ]. Higher TGF-ß₃ doses induced the production of significantly more pleural fluid than the lower doses. The difference was significant when the volume of the pleural fluid produced during the first 24 h after the injection (p = 0.004) and the total volume of the pleural fluid were examined (p = 0.005) [Table 1 ]. The pleural fluid induced by higher doses contained significantly lower numbers of nucleated cells (p = 0.014) and LDH levels (p = 0.013) than the pleural fluid induced by lower doses (Table 1). Rabbits with large pleural effusions had different degrees of respiratory distress but survived. Their respiratory rate was increased, and they stood with a distended neck and head. The distress of the rabbits was rapidly relieved after the aspiration of the pleural fluid.

View larger version (15K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Pleurodesis scores in rabbits after the intrapleural injections of four TGF-ß3 doses.

Unstimulated human pleural mesothelial cells produced a small amount of collagen I mRNA. TGF-ß₃ markedly enhanced collagen I mRNA expression by the human pleural mesothelial cells. This effect peaked at 5 ng/mL TGF-ß₃ (Fig 2 ). With this dose of TGF-ß₃, collagen I mRNA expression was increased 16-fold over control levels: the average collagen I/actin mRNA levels ratio was 0.09 in control cells and 1.35 in cells treated with 5 ng/mL TGF-ß₃. At higher doses, TGF-ß₃ caused a less prominent up-regulation of collagen mRNA expression: tenfold increase over the control levels in cells treated with 5 ng/mL TGF-ß₃ and sixfold increase in cells treated with 50 ng/mL TGF-ß₃.

View larger version (18K): [in this window] [in a new window] [Download PPT slide]   Figure 2. Collagen I mRNA expression by human pleural mesothelial cells after treatment with different doses of TGF-ß3 (two independent experiments).

	Discussion

TOP Abstract Introduction Materials and Methods Results Discussion References

Our group has recently challenged the idea that pleural inflammation is required for the initiation of pleurodesis by showing that the intrapleural injection of TGF-ß₂ in rabbits leads to an excellent pleurodesis, while the pleural fluid produced after the injection is less inflammatory than the pleural fluid produced after the intrapleural injection of talc or doxycycline.⁵⁶ Moreover, intrapleural injections of talc and doxycycline induced the production while those of TGF-ß₂ inhibited the production of the inflammatory cytokine interleukin-8 in the pleural cavity in rabbits.⁸ In the present study, we show that another TGF-ß isoform, TGF-ß₃, is also effective in producing pleurodesis in the same rabbit model. The dose-response curve for pleurodesis following TGF-ß₂ and TGF-ß₃ appears to be very comparable.⁵⁶ TGF-ß₃ doses induced significantly more pleural fluid than the lower doses, suggesting that TGF-ß₃ induces pleural fluid production. This phenomenon was previously observed after the intrapleural injection of TGF-ß₂ in rabbits.⁵⁶ The mechanisms of TGF-ß–induced pleural fluid accumulation are not clear. It has been suggested that TGF-ß induces the production of vascular endothelial growth factor (VEGF) by pleural mesothelial cells, which in turn causes increased permeability of the endothelial and mesothelial membrane with subsequent fluid exudation.⁹ A study examining the effect of VEGF inhibition on TGF-ß–induced pleural fluid formation would show whether VEGF is really involved in this process. The pleural fluid induced by higher doses had significantly fewer leukocytes and lower LDH levels than the pleural fluid induced by lower doses of the agent. This observation supports the concept of an antiinflammatory effect of the agent in the pleural cavity, although there is the possibility that the lower leukocyte count and LDH levels could be due to dilution.

The pleural fluid characteristics are comparable with those reported previously when TGF-ß₂ was used in the same animal model of pleurodesis.⁵ Intrapleural injection of TGF-ß₂ in doses of 0.5 to 5 µg had caused a dose-dependent increase of the pleural fluid production and a dose-dependent decrease of the number pleural fluid cells and the pleural fluid LDH levels. However, although the number of the pleural fluid cells in this study is similar with the number of the cells reported in the previous study, the pleural fluid LDH levels are somewhat lower.

The role of TGF-ß₃ in other models of in vivo fibrinogenesis is not clear, and the results are conflicting. In rats, peritoneal adhesions induced by surgical abrasion contained increased TGF-ß₃ mRNA levels in comparison to uninjured and normally healed peritoneum.¹⁰ This finding suggests that TGF-ß₃ promotes fibrosis in the peritoneal cavity, as it does in the pleural cavity. Interestingly, both peritoneal and pleural cavities are covered by mesothelial cells. However, injection of TGF-ß₃ at the site of a dermal wound in mice decreased scarring in contrast to what happened after the injection of TGF-ß₁ and TGF-ß₂.¹¹ The conflicting findings concerning the involvement of TGF-ß₃ in different animal models of fibrosis may be due to the use of different doses of the agent but may also denote different regulation of fibrinogenesis in different tissues.⁴

Collagen synthesis is a sine qua non event of pleurodesis.¹² However, the cellular source of collagen in the pleural cavity is still not well defined. It has been believed that following the inflammatory reaction caused by the intrapleural administration of the traditional sclerosing agents, fibroblasts invade the pleural space. There they are activated by locally produced fibrogenic cytokines, including TGF-ß, produce collagen and other extracellular matrix proteins, and cause pleural fibrosis.¹ In this article we show that human pleural mesothelial cells can also produce collagen mRNA when treated with TGF-ß₃ in vitro. This is the first report showing that a TGF-ß isoform activates human pleural mesothelial cells to express collagen mRNA. These results agree with a previous observation⁸ that TGF-ß₂, talc, and doxycycline up-regulate collagen mRNA in rabbit mesothelial cells.

In our experiments, TGF-ß₃ up-regulated collagen mRNA expression that peaked at 5 ng/mL of agent. With this dose of TGF-ß₃, collagen I mRNA expression was increased 16-fold over control levels. It is not clear why higher doses induce lower levels of collagen mRNA expression. This phenomenon may be related to the fact that different isoforms of the cytokine induce the production of other isoforms and interact with each other to regulate the production of collagen.³ In this connection, Murata and associates¹³ have shown that TGF-ß₃ induces collagen synthesis through both TGF-ß₁–dependent and –independent mechanisms. However, while lower doses of TGF-ß₃ amplified the effect of TGF-ß₁, high doses of TGF-ß₃ attenuated TGF-ß₁–induced collagen production by human fibroblasts. This finding suggests that in high doses, TGF-ß₃ failed to activate or even suppress the TGF-ß₁–dependent mechanism that may lead to a weaker stimulation of collagen production by TGF-ß₃. Interestingly, Razzaque and Ahmed¹⁴ reported that the TGF-ß₁ effect on human fibroblasts was similar to the effect of TGF-ß₃ we observed on human pleural mesothelial cells: the maximum levels of collagen mRNA expression occurred when cells were treated with intermediate doses and not with the higher doses of the agent. An alternative explanation of our in vitro findings may be cellular toxicity with higher doses of TGF-ß₃. However, we believe that this possibility is unlikely since cell survival was not affected by the dose of the cytokine.

In conclusion, TGF-ß₃ causes a dose-dependent pleurodesis when administered intrapleurally in rabbits. The pleural fluid produced after the administration of higher doses of the agent had fewer cells and lower LDH levels than that produced by lower doses. Additionally, TGF-ß₃ induces collagen mRNA expression by human pleural mesothelial cells. All the above suggest that TGF-ß₃ may be useful in human pleurodesis.

	Footnotes

 
Abbreviations: IQR = interquartile range; LDH = lactate dehydrogenase; mRNA = messenger RNA; PCR = polymerase chain reaction; TGF = transforming growth factor; VEGF = vascular endothelial growth factor

Supported in part by Saint Thomas Foundation, Nashville, TN, and Cumberland Pharmaceuticals, Nashville, TN.

Received for publication April 13, 2004. Accepted for publication October 14, 2004.

	References

TOP Abstract Introduction Materials and Methods Results Discussion References

 

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11. Shah, M, Foreman, DM, Ferguson, MW Neutralization of TGF-ß1 and TGF-ß2 or exogenous addition of TGF-ß3 to cutaneous rat wounds reduces scarring. J Cell Sci 1995;108,985-1002[Abstract]
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This Article Abstract Full Text (PDF) Free Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Article Archive Download to citation manager Citing Articles Citing Articles via ISI Web of Science (2) Citing Articles via Google Scholar Google Scholar Articles by Kalomenidis, I. Articles by Light, R. W. Search for Related Content PubMed PubMed Citation Articles by Kalomenidis, I. Articles by Light, R. W.