Results

Intrapleural injection of TGFβ₂ in doses of both 5.0 μg and 1.7 μg produced effective pleurodesis in rabbits with mean pleurodesis scores of 7.3 (1.3) and 6.9 (1.2), respectively. There was no difference in the pleurodesis scores between rabbits that received TGFβ₂ and systemic corticosteroids and rabbits that received TGFβ₂ alone (7.2 (1.3) and 7.1 (1.2), respectively, p=NS, Mann-Whitney rank sum test). The results of the rabbits in each subgroup are presented in table 1.

The injection of TGFβ₂ stimulated the production of a large quantity of pleural fluid in all rabbits. The effusion produced was relatively non-inflammatory as evidenced by the low protein and LDH levels and total leucocyte count (table 2). The production of the fluid was transient and did not hinder the development of effective pleurodesis.

Rabbits that received TGFβ₂ and corticosteroids showed no significant difference from those given only TGFβ₂ in the amount of effusion produced and the biochemical analysis of the pleural fluid by the Student's t test (table 2). The only exception was the effusion protein level which was higher in the steroid group (mean 3.1 mg/dl v2.9 mg/dl, p=0.02), although the difference is unlikely to be of clinical significance. There was no difference in the other markers of inflammation (LDH level and total leucocyte count) between the two groups. The inflammatory indices of the pleural fluid from both groups of rabbits were significantly lower than previously published results of pleural fluids induced by intrapleural injections of talc, doxycycline, and mitoxantrone (table 3).

There was no difference in the effusion protein, LDH, and total leucocyte counts between rabbits given 5.0 μg TGFβ₂and those given 1.7 μg. Interestingly, although the mean volumes of effusion at 24 hours were not different (26.6 ml in the 5.0 μg group and 28.9 ml in the 1.7 μg group), rabbits that received the higher dose of TGFβ₂ had a significantly lower amount of total effusion drained (44.0 ml v 65.6 ml; p=0.02, Student's t test).

There were no significant differences in the microscopic grading scores for pleural fibrosis, pleural inflammation (fig 1), or in pleural thickness (fig 2) and collagen deposition (fig 3) between pleural tissues of the rabbits that received TGFβ₂ and steroid and the tissues of rabbits given only TGFβ₂ (Mann-Whitney rank sum test). The degree of alveolar inflammation and alveolar fibrosis was minimal in both groups (fig 1).

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Figure 1

Comparison of histological changes in rabbits given TGFβ₂ + corticosteroids v rabbits given TGFβ₂ only.

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Figure 2

Pleural thickness of rabbits given TGFβ₂+ corticosteroids v rabbits given TGFβ₂ only.

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Figure 3

Collagen deposition in rabbits given TGFβ₂ + corticosteroids v rabbits given TGFβ₂ only.

Four rabbits died and were replaced. Three of them died from causes related to surgery such as pneumothorax or pulmonary contusion during or within 48 hours of chest tube insertion. The fourth rabbit had a blocked chest tube and died from a tension hydrothorax 3 days after the injection of TGFβ₂ (5.0 μg). Six rabbits in both groups had evidence of a haemothorax at the time of necropsy, although most of them were mild. The median (25–75% range) score of haemothorax was 0 (0.0–2.7) in rabbits given TGFβ₂ + corticosteroids and 0 (0.0–2.0) in rabbits given TGFβ₂only. None of the rabbits had evidence of empyema at necroscopic examination. The weight gain was comparable in rabbits that received corticosteroids and those that did not (0.18 (0.16) kgv 0.22 (0.19) kg; p=NS, Student'st test). Rabbits that received the lower dose gained more weight than rabbits that received the higher dose of TGFβ₂ (0.14 (0.16) kg v 0.34 (0.12) kg; p=0.001, Student's t test). Three rabbits (all in 5.0 μg group; two had corticosteroids) required gentamicin for skin wound infections. One rabbit (in the no steroid group) required treatment with supplementary subcutaneous fluid.

Discussion

This study shows that TGFβ₂ is effective in producing pleurodesis in a validated rabbit model. Systemic corticosteroids, at a dose that inhibits talc pleurodesis, did not affect the efficacy of TGFβ₂ induced pleurodesis nor the inflammatory indices in the effusion fluid. The study provides further evidence that TGFβ₂ induces pleurodesis via a pathway that involves minimal inflammation of the pleura and thus remains effective in the presence of high dose parenteral corticosteroids.

Conventional pleurodesing agents such as talc and tetracycline derivatives generate pleurodesis by creating acute pleural injury with subsequent inflammation and fibrosis.1 The intense inflammation is likely to be the cause of pain and fever which commonly complicate pleurodesis. The inflammatory process is, however, essential to the pleurodesis they produce. We have previously shown that the concurrent use of corticosteroids markedly decreases the inflammatory reaction and inhibits the development of pleurodesis after the intrapleural injection of talc.2

TGFβ is a ubiquitous cytokine notable for its potent profibrotic and anti-inflammatory properties, both of which make TGFβ an attractive option as a novel class of pleurodesing agent. Of the known growth factors, TGFβ is the most potent in stimulating extracellular matrix production and also readily inhibits its degradation.4Increased production and accumulation of TGFβ is an important feature in fibrotic diseases including pulmonary fibrosis and glomerulosclerosis.3 At the same time, TGFβ is a potent immunomodulatory cytokine. It can inhibit the production of TNFα, interleukin(IL)-1,3 and IL-813 and is more potent than cyclosporin as an inhibitor of lymphocyte functions.4

In the present study we have confirmed that direct administration of TGFβ₂ to the pleura creates excellent pleurodesis. The doses of TGFβ₂ we used were the lowest effective doses that stimulated pleurodesis in our previous studies.5 ,6We found that the pleurodesis induced by 5.0 μg TGFβ₂was not inhibited by systemic corticosteroids at a dose that blocks talc pleurodesis. We also showed that corticosteroids did not inhibit the TGFβ₂ induced pleurodesis even when we applied a lower dose of TGFβ₂ (1.7 μg) which produced lower pleurodesis scores. The results of this study provide further evidence that TGFβ₂ induces pleurodesis without necessitating pleural injury and inflammation. This is also confirmed by the low inflammatory indices (protein, LDH, and leucocyte counts) in the pleural fluids induced after TGFβ₂ injection compared with pleural fluids induced after the intrapleural administration of conventional pleurodesing agents such as talc, doxycycline, or mitoxantrone (table 3).

Interestingly, the intrapleural injection of TGFβ₂induced the production of large volumes of effusion. TGFβ can alter the morphology of mesothelial cells in vitro and increase the permeability of the mesothelial cell monolayer.14 It is also a potent stimulator for the release of vascular endothelial growth factor (VEGF) which, in turn, increases vascular permeability.15 We found that concurrent administration of corticosteroids made no difference to the amount of fluid produced, indicating that the production of fluid after TGFβ injection is unlikely to be due to inflammation. The total amount of effusion drained was significantly higher in rabbits receiving lower dose of TGFβ₂ than in those receiving the higher dose, although there was no difference in the amount drained at 24 hours. We speculate that higher doses of TGFβ₂ may induce more fibrosis and loculations in the first few days which impair pleural fluid drainage.

In our study rabbits that received 5 μg TGFβ₂ gained less weight than those given 1.7 μg. Terrell et al 16 observed that rabbits treated with a single intravenous bolus of mid or high dose human recombinant TGFβ₁ had a transient reduction in food consumption and weight. There was an associated slight decrease in albumin and total protein on day 3 which was reversed by 2 weeks. Similar findings were observed in rats given repeated injections of high doses of intravenous TGFβ₁ 16 or intraperitoneal TGFβ₂.17 Increased serum levels of TGFβ and reduced levels of insulin-like growth factor 1 have recently been shown in mice with poor food intake and weight loss.18 The relationship between high doses of TGFβ and weight loss requires further investigation.

TGFβ is multifunctional and its actions on target cells are critically dependent on the cell type, its state of differentiation, and the particular set of growth factors and hormones acting on the cell.19 Its interactions with corticosteroids are complex. While TGFβ can reduce cortisol20 and mineralocorticoid21 synthesis, glucocorticoids in turn are potent regulators of the expression of TGFβ isoforms.22These regulations are again cell and stimulus specific.22 ,23 For example, glucocorticoids enhance accumulation of TGFβ mRNA in some cell lines including fibroblasts,24 osteoblasts,25 and T lymphocytes26 ,27 but inhibit the production of TGFβ in others.28 ,29

Our study is the first to examine the effects of corticosteroids on the action of TGFβ in pleural tissues. Previous studies of the in vivo effects of corticosteroids and TGFβ are limited and yielded conflicting results. In one study dexamethasone blocked the profibrotic effect of TGFβ in the abdominal wounds of rats.30 In another study, using a rat model of bleomycin induced pulmonary fibrosis, corticosteroids reduced the influx of inflammatory cells but the alveolar macrophage production of TGFβ was not inhibited by the corticosteroids.31 Conversely, other authors have also demonstrated that both local32 ,33 and systemic34 administrations of TGFβ could reverse the steroid induced impairment in wound healing. Little, however, is known of the in vivo interactions between corticosteroids and TGFβ in other organ systems.

In summary, TGFβ₂ induces pleurodesis through a novel pathway whereby it induces pleural fibrosis without provoking excessive inflammation. We confirmed this by demonstrating that corticosteroids, at the same dose which blocks talc pleurodesis, did not impair the effectiveness of TGFβ₂ in inducing pleurodesis. If these data can be extrapolated to humans, then TGFβ₂ (as a non-inflammatory pleurodesing agent) may be less likely to induce pain and fever than talc and will remain effective in patients receiving corticosteroids.