Biological effect of tissue plasminogen activator (t-PA) and DNase intrapleural delivery in pleural infection patients

Nikolaos I Kanellakis; John M Wrightson; Rob Hallifax; Eihab O Bedawi; Rachel Mercer; Maged Hassan; Rachelle Asciak; Emma Hedley; Melissa Dobson; Tao Dong; Ioannis Psallidas; Najib M Rahman

doi:10.1136/bmjresp-2019-000440

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Pleural Disease

Biological effect of tissue plasminogen activator (t-PA) and DNase intrapleural delivery in pleural infection patients

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RE: Biological effect of tissue plasminogen activator (t-PA) and DNase intrapleural delivery in pleural infection patients
Nikolaos I. Kanellakis, John M. Wrightson, Ioannis Psallidas and Najib M. Rahman
Published on: 2 June 2020
Induction of monocyte chemotactic protein-1 by intrapleural instillation of tissue plasminogen activator
Jenette CREANEY, Hui Min CHEAH, Georgious T. STATHPOULOS, Ioannis KALOMENIDIS and Y. C. Gary Lee
Published on: 2 June 2020

Published on: 2 June 2020
RE: Biological effect of tissue plasminogen activator (t-PA) and DNase intrapleural delivery in pleural infection patients
- Nikolaos I. Kanellakis, Postdoctoral Researcher Laboratory of Pleural and Lung Cancer Translational Research, Nuffield Department of Medicine, University of Oxford, Oxford, UK
- Other Contributors:
  John M. Wrightson, Consultant
  
  Ioannis Psallidas, Associate Professor
  
  Najib M. Rahman, Clinical Lecturer
We are grateful that scientists around the world are showing interest in our research, and delighted to reply to comments from Creaney et al. with regards to our paper the “Biological effect of tissue plasminogen activator (t-PA) and DNase intrapleural delivery in pleural infection patients”.[1]
Pleural infection is a significant clinical entity, has increasing incidence worldwide and is associated with high morbidity, mortality and burden to healthcare services. Effective treatment still relies upon effective pleural fluid drainage, and thus investigation of the pathological mechanisms behind fluid formation and treatment response is key to improving care.
The MIST2 study demonstrated that intrapleural delivery of tissue plasminogen activator (t-PA) alone or t-PA plus DNase increased volume of pleural fluid drained in humans with pleural infection, in a placebo controlled double blind randomised study.[2 ] Although the exact biological mechanisms via which t-PA induces the volume increment of drained pleural fluid are unknown, the design of the MIST2 study means that we are confident in the biological observation of increased fluid production in response to t-PA administration. Lansley et al. have demonstrated that the chemokine MCP-1 (also known as CCL-2) is the key protein that upon intrapleural t-PA administration induces fluid formation in healthy mice.[3]
We designed a study to directly assess their hypothesis in human pleural infection patients fo...
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We are grateful that scientists around the world are showing interest in our research, and delighted to reply to comments from Creaney et al. with regards to our paper the “Biological effect of tissue plasminogen activator (t-PA) and DNase intrapleural delivery in pleural infection patients”.[1]
Pleural infection is a significant clinical entity, has increasing incidence worldwide and is associated with high morbidity, mortality and burden to healthcare services. Effective treatment still relies upon effective pleural fluid drainage, and thus investigation of the pathological mechanisms behind fluid formation and treatment response is key to improving care.
The MIST2 study demonstrated that intrapleural delivery of tissue plasminogen activator (t-PA) alone or t-PA plus DNase increased volume of pleural fluid drained in humans with pleural infection, in a placebo controlled double blind randomised study.[2 ] Although the exact biological mechanisms via which t-PA induces the volume increment of drained pleural fluid are unknown, the design of the MIST2 study means that we are confident in the biological observation of increased fluid production in response to t-PA administration. Lansley et al. have demonstrated that the chemokine MCP-1 (also known as CCL-2) is the key protein that upon intrapleural t-PA administration induces fluid formation in healthy mice.[3]
We designed a study to directly assess their hypothesis in human pleural infection patients following intrapleural delivery of t-PA, using pleural fluid specimens collected for MIST2.
Our data suggest that MCP-1 is not the single modulator of the increment in pleural fluid drainage volume due to intrapleural administration of t-PA in humans. Furthermore, no correlation was detected between pleural fluid MCP-1 protein concentration and the volume of drained pleural fluid or the intrapleural drug given.
These data highlight the importance of the appropriate use of laboratory models when extrapolating preclinical findings to human biology.[4-6] Lansley et al. used a single mouse strain of the same sex for experiments (female CD1), and gave a single dose of drug to mice, while MIST2 participants received daily cumulative doses. It is unclear how the dose was adjusted to mouse body surface area. The mice were healthy, and the pleural cavity was not infected with bacteria in contrast to MIST2 samples which were derived from confirmed cased of pleural infection – it is well recognised that the presence of bacteria in the pleural space drastically affects the biological microenvironment.[7, 8]
These factors could explain the differences between our and their studies. Importantly, we highlight that our data suggest that MCP-1 plays a role but is not acting in isolation. More research is needed in order to fully understand the interplay between the pleura, bacteria and t-PA – however, the suggestion that MCP-1 is the sole driver of fluid formation in the human response to intrapleural t-PA based on a healthy mouse model is overly simplistic.
With regards to methodological comments from Creaney et al. we can clarify that total protein concentration was used in order to normalise the concentration of MCP-1. We did not use the total MCP-1 expression as suggested as this would not have allowed us to investigate the correlation between volume of pleural fluid drained and concentration of MCP-1 in pleural fluid. Their suggestion that adjusting for the total volume of pleural fluid present is required is not in our view rational, and makes the assumption that the concentration of MCP-1 is somehow different in the retained fluid in the thorax than that collected, and we see no justification for this assumption. The major pleural fluid parameters used in daily clinical practice (total protein, glucose, LDH) are all measured using concentrations – do they suggest that these parameters are not reliable unless the entire pleural space is drained, or unless total amount is measured? We are also somewhat surprised at their suggestion of the primacy of total (as opposed to concentration) of MCP-1, given that many papers routinely report the concentration of cytokines in pleural fluid (and not total amount) including papers published by the authors of the e-letter to which we here respond, including in their own studies of MCP-1.[9-13] Their suggestion that DNase may have interfered with the analysis is not based on any published data we can find - DNase is an enzyme that cleaves the phosphodiester linkages in DNA molecules and no interaction with chemokines has been reported. We acknowledge that the residual pleural fluid is a limitation of the study. However, even in animal models there is loss of fluid during aspiration.
There is no doubt that laboratory animal models of human disease are of importance and have significantly contributed to major scientific discoveries. However, preclinical disease models must be used with care to ensure that they faithfully and appropriately reflect the properties of any studied disease, and their purposes (which is to study the human disease condition) should not be forgotten. Creaney et al. conclude by suggesting that “Clinical observations are useful in generating hypotheses”. The MIST2 study was not a “clinical observation” and remains the only placebo controlled double blind study in humans with pleural infection treated with t-PA and DNase, and as such, translational discoveries from this dataset are likely to most closely represent the underlying pathobiology in human pleural infection.

References
1. Kanellakis NI, Wrightson JM, Hallifax R, et al. Biological effect of tissue plasminogen activator (t-PA) and DNase intrapleural delivery in pleural infection patients. BMJ Open Respir Res 2019;6(1):e000440. doi: 10.1136/bmjresp-2019-000440 [published Online First: 2019/11/02]
2. Rahman NM, Maskell NA, West A, et al. Intrapleural use of tissue plasminogen activator and DNase in pleural infection. N Engl J Med 2011;365(6):518-26. doi: 10.1056/NEJMoa1012740 [published Online First: 2011/08/13]
3. Lansley SM, Cheah HM, Varano Della Vergiliana JF, et al. Tissue plasminogen activator potently stimulates pleural effusion via a monocyte chemotactic protein-1-dependent mechanism. Am J Respir Cell Mol Biol 2015;53(1):105-12. doi: 10.1165/rcmb.2014-0017OC [published Online First: 2014/12/05]
4. Hackam DG, Redelmeier DA. Translation of research evidence from animals to humans. JAMA 2006;296(14):1731-2. doi: 10.1001/jama.296.14.1731 [published Online First: 2006/10/13]
5. Knight A. Systematic reviews of animal experiments demonstrate poor contributions toward human healthcare. Rev Recent Clin Trials 2008;3(2):89-96. doi: 10.2174/157488708784223844 [published Online First: 2008/05/14]
6. Roberts I, Kwan I, Evans P, et al. Does animal experimentation inform human healthcare? Observations from a systematic review of international animal experiments on fluid resuscitation. BMJ 2002;324(7335):474-6. doi: 10.1136/bmj.324.7335.474 [published Online First: 2002/02/23]
7. Corcoran JP, Wrightson JM, Belcher E, et al. Pleural infection: past, present, and future directions. Lancet Respir Med 2015;3(7):563-77. doi: 10.1016/S2213-2600(15)00185-X [published Online First: 2015/07/15]
8. Lee YC, Idell S, Stathopoulos GT. Translational Research in Pleural Infection and Beyond. Chest 2016;150(6):1361-70. doi: 10.1016/j.chest.2016.07.030 [published Online First: 2016/08/16]
9. Thomas R, Cheah HM, Creaney J, et al. Longitudinal Measurement of Pleural Fluid Biochemistry and Cytokines in Malignant Pleural Effusions. Chest 2016;149(6):1494-500. doi: 10.1016/j.chest.2016.01.001 [published Online First: 2016/02/03]
10. Bielsa S, Davies HE, Davies RJ, et al. Reproducibility and reliability of pleural fluid cytokine measurements. Eur Respir J 2009;34(4):1001-3. doi: 10.1183/09031936.00088409 [published Online First: 2009/10/03]
11. Jin HY, Lee KS, Jin SM, et al. Vascular endothelial growth factor correlates with matrix metalloproteinase-9 in the pleural effusion. Respir Med 2004;98(2):115-22. doi: 10.1016/j.rmed.2003.09.002 [published Online First: 2004/02/20]
12. Rovina N, Dima E, Psallidas I, et al. Interleukin-18 is up-regulated in infectious pleural effusions. Cytokine 2013;63(2):166-71. doi: 10.1016/j.cyto.2013.04.017 [published Online First: 2013/05/11]
13. Lansley SM, Cheah HM, Lee YC. Role of MCP-1 in pleural effusion development in a carrageenan-induced murine model of pleurisy. Respirology 2017;22(4):758-63. doi: 10.1111/resp.12951 [published Online First: 2016/11/24]
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Conflict of Interest:
None declared.
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Published on: 2 June 2020
Induction of monocyte chemotactic protein-1 by intrapleural instillation of tissue plasminogen activator
- Jenette CREANEY, Professor University of Western Australia, Perth, Australia
- Other Contributors:
  Hui Min CHEAH, Research Assistant
  
  Georgious T. STATHPOULOS, Professor
  
  Ioannis KALOMENIDIS, Assistant Professor and Consultant
  
  Y. C. Gary Lee, Professor
Monocyte chemotactic protein (MCP)-1 has raised interests concerning its role in pleural
fluid formation. Recent preclinical studies have found that antagonists against MCP-1
reduces formation of malignant pleural effusions from lung cancer1 and mesothelioma as
well as from benign (carrageenan-induced) pleuritis2 in murine models. In humans,
longitudinal collection of malignant effusions via indwelling pleural catheters also showed a
rise in MCP-1 level over time.3

In humans and animals, pleural instillation of fibrinolytics such as tissue plasminogen
activator (tPA) consistently generates large volume of pleural fluid formation in healthy as
well as in various pleural disease states4, 5. In mice, MCP-1 antagonists also decrease tPA-induced
fluid formation.6

We therefore read with interest the work by Kanellakis et al7 on measuring MCP-1
concentration in pleural fluid samples collected from patients in the MIST (Multicentre
Intrapleural Sepsis Trial)-24 who were given tPA or placebo.

The study by Kanellakis et al7 highlights the challenges and limitations of using clinical
samples/data to decipher biological signals. They reported that following tPA installation,
MCP-1 level in pleural fluid increased. However, the pleural fluid MCP-1 levels were similar,
and not significantly higher, in the tPA-treated patients compared with those who did not
receive tPA.

We sug...
Show More

Monocyte chemotactic protein (MCP)-1 has raised interests concerning its role in pleural
fluid formation. Recent preclinical studies have found that antagonists against MCP-1
reduces formation of malignant pleural effusions from lung cancer1 and mesothelioma as
well as from benign (carrageenan-induced) pleuritis2 in murine models. In humans,
longitudinal collection of malignant effusions via indwelling pleural catheters also showed a
rise in MCP-1 level over time.3

In humans and animals, pleural instillation of fibrinolytics such as tissue plasminogen
activator (tPA) consistently generates large volume of pleural fluid formation in healthy as
well as in various pleural disease states4, 5. In mice, MCP-1 antagonists also decrease tPA-induced
fluid formation.6

We therefore read with interest the work by Kanellakis et al7 on measuring MCP-1
concentration in pleural fluid samples collected from patients in the MIST (Multicentre
Intrapleural Sepsis Trial)-24 who were given tPA or placebo.

The study by Kanellakis et al7 highlights the challenges and limitations of using clinical
samples/data to decipher biological signals. They reported that following tPA installation,
MCP-1 level in pleural fluid increased. However, the pleural fluid MCP-1 levels were similar,
and not significantly higher, in the tPA-treated patients compared with those who did not
receive tPA.

We suggest that this paradox can easily be explained by the way the authors chose to
analyse the data. If the hypothesis is that tPA induces MCP-1 formation within the pleural
cavity which in turn drives fluid formation, it should be the total amount of MCP-1, rather than
its concentration in the fluid after tPA installation, that should be captured. This is critical as
MCP-1 concentration is directly confounded by the amount of fluid generated. The more
potent MCP-1 induces fluid, the lower the pleural fluid MCP-1 concentration will become. It
is important to consider the great differences in the volume drained in the tPA treated groups
(mean 1700 and 1730 mL) compared with non-tPA groups (mean 699 and 725 mL). We
anticipate that the total amount of MCP-1 induced (concentration in fluid multiplied by its
volume) would be significantly higher in the tPA-treated groups. We urge the authors to reanalyse
the data as it is likely to change the conclusion of the paper.

In figure 27, MCP-1 results were presented as 'pg/mg protein'. No information was given on
what protein concentration the MCP-1 level was adjusted to, how protein was measured and
the rationale justifying these choices. It is common in cell culture studies to adjust cytokine
levels to total protein as a partial way to adjust for variations in cell numbers within each well.
However, it is unusual (and probably incorrect) to adjust cytokine levels to pleural fluid
proteins especially given the variations in protein concentration of patients' pleural fluid.

This paper highlights also the importance of vigorously examining confounding factors
before assigning biological meanings to clinical trial samples, especially when the trial was
not designed to test the particular biological concept. In MIST-2, the primary endpoint data
showed that radiographic improvement was only in the range of ~17% (in non-tPA treated
groups) to 30% (in the tPA/DNase group). Residual fluid was thus common and potentially in
significant volumes. The fluid drained may therefore only represent part of the entire pleural
fluid volume. Correlation of the fluid volume drained with cytokine levels is difficult to
interpret and can probably be misleading. Volume drained would also be significantly
affected by chest tube blockage or misplacement, which the authors have shown to affect up
to 42% of patients who had small bore tubes in a malignant effusion trial8. Baseline pleural
fluid and MCP-1 levels also varied among patients. The stability of MCP-1 in the presence of
other proteinases, including DNase, is not known.

Formation of pleural fluid is without a doubt a complex interplay of numerous mediators. We
agree with the authors that MCP-1 is only part of the pathogenic milieu. This was reflected in
all the animal model papers on MCP-1 where its antagonists decrease, but not completely
stop, fluid formation1, 2, 6. Future work will require continual use of information gained from
both laboratory and clinical research. Clinical observations are useful in generating
hypotheses. We must however caution against extrapolating/extending data from clinical
studies, with its many confounders, as a test of disease biology unless the clinical study is
designed, and properly controlled, for evaluating a pathobiological hypothesis.

References
1. Stathopoulos GT, Psallidas I, Moustaki A, et al. A central role for tumor-derived
monocyte chemoattractant protein-1 in malignant pleural effusion. J Natl Cancer Inst.
2008;100(20):1464-76.
2. Lansley SM, Cheah HM, Lee YC. Role of MCP-1 in pleural effusion development in a
carrageenan-induced murine model of pleurisy. Respirology. 2017;22(4):758-63.
3. Thomas R, Cheah HM, Creaney J, Turlach BA, Lee YC. Longitudinal Measurement
of Pleural Fluid Biochemistry and Cytokines in Malignant Pleural Effusions. Chest.
2016;149(6):1494-500.
4. Rahman NM, Maskell NA, West A, et al. Intrapleural use of tissue plasminogen
activator and DNase in pleural infection. N Engl J Med. 2011;365(6):518-26.
5. Thomas R, Piccolo F, Miller D, et al. Intrapleural Fibrinolysis for the Treatment of
Indwelling Pleural Catheter-Related Symptomatic Loculations: A Multicenter Observational
Study. Chest. 2015;148(3):746-51.
6. Lansley SM, Cheah HM, Varano Della Vergiliana JF, Chakera A, Lee YC. Tissue
plasminogen activator potently stimulates pleural effusion via a monocyte chemotactic
protein-1-dependent mechanism. Am J Respir Cell Mol Biol. 2015;53(1):105-12.
7. Kanellakis NI, Wrightson JM, Hallifax R, et al. Biological effect of tissue plasminogen
activator (t-PA) and DNase intrapleural delivery in pleural infection patients. BMJ Open
Respir Res. 2019;6(1):e000440.
8. Rahman NM, Pepperell J, Rehal S, et al. Effect of Opioids vs NSAIDs and Larger vs
Smaller Chest Tube Size on Pain Control and Pleurodesis Efficacy Among Patients With
Malignant Pleural Effusion: The TIME1 Randomized Clinical Trial. Jama.
2015;314(24):2641-53.
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Conflict of Interest:
None declared.
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