Kinetic profiling of in vivo lung cellular inflammatory responses to
mechanical ventilation
Samantha J. Woods, Alicia A.C. Waite, Kieran P. O’Dea, Paul Halford, Masao Takata,
Michael R. Wilson
Section of Anaesthetics, Pain Medicine and Intensive Care, Faculty of Medicine, Imperial
College London, Chelsea and Westminster Hospital, London, SW10 9NH, UK
Corresponding author name and address:
Michael Wilson, Ph.D., Anaesthetics, Pain Medicine and Intensive Care, Faculty of Medicine,
Imperial College London, Chelsea and Westminster Hospital, 369 Fulham Road, London,
SW10 9NH, UK. E-mail: [email protected]
Author contributions:
Conception and design of the study: AW, KO, MT, MW; Data acquisition, analysis and
interpretation: SW, AW, KO, PH, MW; writing and revising the article: SW, MT, MW
Sources of support:
Work was supported by grants from the British Journal of Anaesthesia/Royal College of
Anaesthetists and the Wellcome Trust (# 081208).
Running Title: Cellular activation during VILI
1
ABSTRACT
Mechanical ventilation, through over-distension of the lung, induces substantial inflammation
that is thought to increase mortality among critically ill patients. The mechanotransduction
processes involved in converting lung distension into inflammation during this ventilatorinduced lung injury (VILI) remain unclear, though many cell types have been shown to be
involved in its pathogenesis. This study aimed to identify the profile of in vivo lung cellular
activation that occurs during the initiation of VILI. This was achieved using a flow cytometrybased method to quantify the phosphorylation of several markers (p38, ERK1/2, MK2 and
NF-κB) of inflammatory pathway activation within individual cell types.
Anesthetized
C57BL/6 mice were ventilated with low (7ml/kg), intermediate (30ml/kg) or high (40ml/kg)
tidal volumes for 1, 5 or 15 minutes followed by immediate fixing and processing of the
lungs. Surprisingly, the pulmonary endothelium was the cell type most responsive to in vivo
high tidal volume ventilation, demonstrating activation within just 1 minute, followed by the
alveolar epithelium.
Alveolar macrophages were the slowest to respond, though still
demonstrated activation within 5 minutes. This order of activation was specific to VILI, as
intratracheal lipopolysaccharide induced a very different pattern. These results suggest that
alveolar macrophages may become activated via a secondary mechanism which occurs
subsequent to activation of the parenchyma, and that the lung cellular activation mechanism
may be different between VILI and lipopolysaccharide. Our data also demonstrates that
even very short periods of high stretch can promote inflammatory activation, and importantly,
this injury may be immediately manifested within the pulmonary vasculature.
Keywords: LPS; MAPK; NF-κB; inflammation; ventilator-induced lung injury
Abbreviations used in this article: AECs, alveolar epithelial cells; AMs, alveolar
macrophages; ARDS, acute respiratory distress syndrome; MK2, MAPK-activated protein
kinase 2; VILI, ventilator-induced lung injury.
2
INTRODUCTION
Mechanical ventilation is an integral tool in intensive care, but evidence shows that
excessive tidal volumes can lead to ventilator-induced lung injury (VILI) (43) and increase
mortality (2, 16). Patients with pre-existing lung injury such as acute respiratory distress
syndrome (ARDS) (30) are especially at risk since even low tidal volumes are enough to
overstretch the reduced volume of aeratable lung (17, 46). Over-distension induces
substantial inflammation (48, 55) and is ultimately thought to promote multiple-system organ
failure (37, 44), the most common cause of death in ARDS patients (50). Furthermore,
recent studies show that even patients with healthy lungs undergoing mechanical ventilation
during surgery for relatively short lengths of time are susceptible to VILI.
The use of
ventilation strategies employing lower tidal volumes in the operating room has been shown
to reduce the incidence of post-operative pulmonary complications and inflammation (41,
61). This stretch-induced ‘biotrauma’ is therefore considered to be an important therapeutic
target, but the risk of general immunosuppression means that ‘stretch-specific’ processes
must be identified.
It is therefore essential to understand how overstretch is sensed within the lung, and which
cell types may be responsible for initiating the consequent inflammatory cascade, but as yet
this remains unclear.
Parenchymal (epithelial/endothelial) cells and resident leukocytes
(alveolar macrophages/lung-marginated monocytes) have all been demonstrated to be
involved in VILI in some way, be it production of inflammatory mediators or changes in
barrier function (12, 13, 22, 57). In vitro, many pulmonary cell types respond to mechanical
deformation, including alveolar epithelial cells (AECs) (3, 33, 51, 59, 60), endothelial cells
(20, 25) and alveolar macrophages (AMs) (38).
It is uncertain though, precisely how
relevant the in vitro conditions are to in vivo ventilation; for example, it seems unlikely that
AMs would be exposed to the same stretching forces as structural cells in vivo.
The
simplest paradigm to explain the initiation of ventilator-induced inflammation would
incorporate an initial stretch ‘sensor’ followed by amplification and propagation of the
3
inflammatory response, although this remains speculation and there is little hard evidence to
support such a theory. We have therefore tested the hypothesis that during high stretch
mechanical ventilation in vivo, alveolar epithelial cells play the role of initial sensor, and thus
display activation of inflammatory pathways earlier than alveolar macrophages, which we
suggest would be the likely amplifiers of inflammation.
To explore this, we evaluated cellular inflammatory pathway activation in terms of
phosphorylation of the mitogen-activated protein (MAP) kinases p38 and ERK1/2, p38’s
immediate downstream substrate MK2, and the transcription factor NF-κB, during the first
few minutes of VILI. p38 responds to a range of cell stresses, and through activation of MK2
promotes the translation of pro-inflammatory mediators (32), whilst ERK1/2 is commonly
associated with responses such as cell proliferation and differentiation (26).
NF-κB is
present in the cytoplasm of quiescent cells and upon phosphorylation rapidly translocates
into the nucleus where it regulates many inflammatory genes (28).
Activation of these
markers has been frequently detected following high tidal volume ventilation in vivo (9, 29,
34, 49), and their inhibition attenuates aspects of VILI including cytokine release (1, 8, 19,
20, 33, 42). However, these previous studies have utilised techniques such as Western
blotting and immunohistochemistry, which are either not truly quantitative or do not allow
localisation of cellular activation. Furthermore, the earliest time point previously utilised to
investigate in vivo MAP kinase activation is 30 minutes (49), which cannot be considered as
related to the initiation of VILI. To circumvent these issues we developed a flow cytometrybased method to quantify MAP kinase and NF-κB phosphorylation in whole lungs. This
methodology has the combined advantages of high detection sensitivity and the ability to
localise activation to specific cell types following in vivo interventions.
The current study examined the kinetic profile of individual cell types within the first 15
minutes of exposure to high tidal volume ventilation in vivo and found that the pattern of
inflammatory pathway activation was specific to VILI, as compared to intratracheal
lipopolysaccharide (LPS). Surprisingly, our data suggest that pulmonary endothelial cells
4
initiate inflammatory signalling pathways at least as fast as AECs, and may be the most
sensitive cell type to stretch of those tested, indicating that VILI directly induces pulmonary
vascular inflammation from the start of its mechanotransduction process. AMs appeared to
respond the slowest (though still within just 5 minutes) suggesting they may be activated by
a secondary mechanism, albeit an extremely rapid one.
METHODS
In vivo models
Protocols were approved by the UK Home Office in accordance with the Animals (Scientific
Procedures) Act 1986, UK. A total of 96 male C57BL/6 mice (Charles River, Margate, UK)
aged 10.9±3.1 weeks (mean±SD), weighing 26.8±3.0g, were anesthetised by intraperitoneal
injection of ketamine (90mg/kg) and xylazine (10mg/kg) before use as controls or exposure
to either intratracheal LPS or VILI.
For LPS experiments, mice (n=16) were exposed to intratracheal LPS for either 1, 5 or 15
minutes. For experiments lasting 5 or 15 minutes, 20µg ‘Ultrapure’ LPS (E. coli O111:B4;
InVivoGen, San Diego, CA) in a final volume of 50µl saline, was instilled intratracheally via a
fine catheter briefly passed through the vocal chords, which were visualised using a
microscope and external light source (36, 56). Animals were maintained spontaneously
breathing under anesthesia and kept warm before sacrifice. One minute before termination
of the experiments, an endotracheal tube was inserted via tracheostomy to facilitate timely
instillation of inhibitors as described below. To ensure accurate timing, in LPS experiments
lasting only 1 minute the endotracheal tube was inserted first, before LPS (at the same dose
and volume as above) was administered by passing the fine catheter through the
endotracheal tube.
Untreated mice (n=11) were used as controls as pilot experiments
showed that administration of saline vehicle had no effect on cellular activation markers.
5
For VILI experiments, mice (n=69) were connected to a custom-made ventilator through an
endotracheal tube inserted via tracheostomy (55). Animals were initially ventilated with low
stretch settings: tidal volume (VT) 7-8ml/kg, 120 breaths per minute (bpm) and positive endexpiratory pressure (PEEP) of 3cmH2O, using 100% oxygen. Two recruitment manoeuvres
were performed (sustained inflation of 35cmH2O for 5 seconds) to standardise lung volume
history before starting specific ventilation strategies. No further recruitments were performed
during the timed ventilation exposure periods (as the longest of these was only 15 minutes).
Mice were then randomly allocated to receive either injurious ventilation or to remain on the
low stretch settings.
Initial experiments (26 mice) explored high stretch ventilation (VT
40ml/kg, 80bpm, 3cmH2O PEEP using O2 + 4% CO2) for 5 or 15 minutes, with 5 minutes of
low stretch acting as controls. Subsequent experiments (14 mice) were carried out for 1
minute with both high and low stretch settings. In a separate set of experiments (21 mice)
lasting 5 minutes, ventilation with low stretch was compared to an intermediate stretch
strategy (30ml/kg, 80bpm, 3cmH2O PEEP using O2 + 4% CO2).
All experiments were terminated by exsanguination followed by immediate instillation of
500µl cell-permeant phosphatase inhibitor cocktail (Calbiochem, Darmstadt, Germany) into
the lungs via the endotracheal tube, to limit deterioration of the phospho-proteins. Lungs
were rapidly removed within 2 minutes of termination and mechanically disrupted in warm
(37°C) fixation/permeabilisation buffer (Cytofix/Cytoperm, BD Biosciences, Oxford, UK)
using a GentleMACS dissociator (Miltenyi Biotec, Surrey, UK).
Samples were then
incubated at 37°C for a further 10 minutes, following which fixation was stopped by addition
of ice-cold permeabilising buffer (phosphate-buffered saline, 0.2% saponin, 2% fetal calf
serum, 0.1% sodium azide). Samples were filtered through 40µm sieves, and finally
washed/re-suspended in permeabilising buffer (to ensure access of antibodies to the
cytoplasmic phospho-proteins) to yield a fixed single cell suspension of the whole lung as
previously described (31).
Phospho-flow cytometry
6
Lung cell suspensions were stained for 30 minutes at room temperature with fluorophoreconjugated anti-mouse antibodies to identify pulmonary cell populations.
Epithelial cell
populations were identified using antibodies against CD45-PerCP (clone 30-F11; BioLegend,
San Diego, CA), CD31-FITC (MEC 13.3; BioLegend), EpCAM-PE (G8.8; eBioscience, San
Diego, CA) and T1-alpha-PeCy7 (8.1.1; BioLegend) (15, 35). In addition, type I and type II
AECs were further differentiated based on positive or negative staining for CD54 (ICAM-1)FITC (3E2; BD Biosciences, San Jose, CA).
Endothelial cells were identified using
antibodies against CD45-PerCP and CD31-FITC (4), and were also confirmed as staining
positive for CD105-PE (MJ7/18; eBioscience), and negative for the platelet marker CD41FITC (MWReg,30; Serotec, Oxford, UK).
Alveolar macrophages were identified using
antibodies against CD45-PerCP, CD11b-PE-CF954 (M1/70; BD Horizon), F4/80-FITC (BM8;
BioLegend), and CD11c-Ax780 (N418; eBioscience) (5).
Using a previously published technique (24, 31), lung cell suspensions were also stained
with AF-647 conjugated antibodies against intracellular phospho-p38 (Thr180/182; clone
28B10), phospho-MK2 (Thr334; 27B7), phospho-ERK1/2 (Thr202/Tyr204; D13.14.4E) and
phospho-NF-κB p65 (Ser536; 93H1) (Cell Signaling, Danvers, MA). To validate the findings,
in a separate set of experiments 8 animals were ventilated with the high stretch or low
stretch settings for 5 minutes, and levels of both phosphorylated and non-phosphorylated
ERK1/2 (137F5; Cell Signaling) were determined within the same lungs. Samples were
analysed with a CyAn ADP flow cytometer (Beckman Coulter, High Wycombe, UK) and
FlowJo software V10 (TreeStar, Ashland, OR). Signals were determined as geometric mean
of fluorescence intensity (gMFI).
Statistical analysis
Shapiro-Wilk normality and Levene’s homogeneity of variance tests were conducted on all
data.
Where possible, non-parametric data were normalised using square root
transformation. Comparisons between two datasets were performed using T-tests or Mann
7
Whitney U-tests.
Comparisons between three or more datasets were performed using
ANOVA with Tukey HSD, Welch ANOVA with Games-Howell for parametric data with
unequal variance, or Kruskal Wallis with Dunn’s test for non-parametric data. Statistical
significance was defined as p<0.05. Data were analysed and graphed using IBM SPSS
(V20) and Prism software (V6).
RESULTS
Intratracheal LPS model
Initial experiments (for both the LPS and VILI models) were carried out over 5 and 15 minute
periods. Figure 1 illustrates the gating strategies used to identify the different pulmonary cell
types, and for each a representative overlay of histograms shows the increase in phosphoMK2 after 15 minutes of LPS compared to an untreated control. Intratracheal LPS induced
significant activation in each of the cell types studied in this experiment, with the clearest
response exhibited by the AMs (figure 2). In the AMs, a >5-fold increase in the mean
fluorescence intensity (gMFI) of phospho-p38 was detected at both 5 and 15 minutes after
LPS instillation. MK2 also displayed sustained activation in response to LPS, with a 7-9 fold
increase in phosphorylation after 5 and 15 minutes.
In contrast, ERK1/2 and NF-κB
demonstrated a more transient activation, which peaked at 5 minutes. Phosphorylation of
both these markers decreased considerably by 15 minutes, though were still significantly
increased compared to their respective baseline levels.
In comparison to the AMs, the type I and II AECs both showed clear activation of NF-κB
within 5 minutes, though most of the MAP kinases only reached significant levels of
activation after 15 minutes.
The endothelial response was less apparent than that of
epithelial cells (figure 2), with no significant activation of any marker at 5 minutes. NF-κB
took 15 minutes to become significantly activated and was accompanied by activation of p38
8
and MK2 (1.5 fold increase in signal), though ERK1/2 activation failed to reach significance
at all.
VILI model
In comparison to LPS, high stretch ventilation induced a markedly different pattern of cellular
activation, with all the cell types demonstrating inflammatory signalling pathway activation
within just 5 minutes (figure 3). The AMs displayed significantly increased phosphorylation
of MK2 at 5 minutes, albeit transiently.
The type I AECs displayed transient ERK1/2
activation which peaked at 5 minutes, whilst MK2 displayed a trend for increased
phosphorylation at 5 minutes that became significant at 15 minutes. The type II AECs
exhibited similar responses to the TI AECs, with transient ERK1/2 activation peaking at 5
minutes and increased MK2 phosphorylation at both time points. As with all the other cell
types, the endothelial cells displayed a transient increase in ERK1/2 phosphorylation that
peaked at 5 minutes. MK2 was also significantly activated by 5 minutes and demonstrated
sustained activation at 15 minutes.
Interestingly, unlike the situation following LPS
stimulation, there was very little evidence of NF-κB activation in response to high stretch,
with only type I AECs showing increased NF-κB phosphorylation, after 15 minutes of stretch
(figure 3).
To further validate this discovery of widespread MAP kinase phosphorylation in response to
stretch, in particular given that it was more subtle than LPS-induced activation, we carried
out a separate set of experiments to quantify the levels of both phosphorylated and total
ERK1/2 protein within the same lungs after 5 minutes of high or low stretch ventilation.
ERK1/2 was chosen for these experiments as it was consistently upregulated at 5 minutes
within most cell types, and there is no commercially available, flow cytometry validated total
MK2 antibody. These experiments once again demonstrated significantly increased
phosphorylation of ERK1/2 in response to high stretch ventilation, whilst the total levels of
ERK1/2 protein were not altered (figure 4).
9
Parenchymal cells are the initial ‘inflammatory responders’
As all cell types examined were found to demonstrate activation following as little as 5
minutes of high stretch mechanical ventilation, it was not possible to draw any conclusions
regarding which cell types may be the initiating cells, responsible for starting the
inflammatory cascade. To address this, we carried out two further sets of experiments: i) an
investigation of an even earlier time point for both LPS and VILI experiments (1 minute), and
ii) an investigation of lower tidal volumes for the VILI model.
Despite clear activation of alveolar macrophages after 5 minutes of exposure to intratracheal
LPS, there was no significant activation at 1 minute (figure 5). The type I and II AECs, which
both demonstrated NF-κB phosphorylation after 5 minutes of LPS, showed no evidence of
activation after 1 minute, and neither (unsurprisingly) did the endothelium (figure 5). In
contrast, just 1 minute of high stretch ventilation induced significant cellular activation within
the lung (figure 6). Both type I and II AECs displayed significantly increased p38 activation,
while somewhat surprisingly, the endothelial cells demonstrated the clearest signs of
significant activation, with p38, MK2 and ERK1/2 all showing ~2-fold increases in
phosphorylation (figure 6). In contrast, AMs showed no significant activation, suggesting
that they were responding less rapidly than other cell types (figure 6). Finally, experiments
conducted at a lower tidal volume of 30ml/kg for 5 minutes only induced significant MAP
kinase (MK2) activation in endothelial cells (figure 7), further indicating that they may be the
most sensitive to stretch during ventilation.
DISCUSSION
This study was designed primarily to investigate which pulmonary cell types initiate the
inflammatory sequelae following over-distension during high tidal volume ventilation. Until
now, research has focused on the downstream consequences of VILI such as cytokine
release, but very little is known about the in vivo mechanotransduction and signal
10
propagation processes underlying the initiation of inflammation.
We have investigated
intracellular inflammatory signalling events within the first 15 minutes after initiating high tidal
volume ventilation in vivo, and compared this to intratracheal LPS administration to see
whether we could identify a stretch-specific pattern. We used a flow cytometric technique
previously established within our group (31) to quantitatively assess the phosphorylation
status of intracellular MAP kinases, which provides the advantages of measuring an
upstream signal transduction event, with cell-specific localisation of the signal.
This novel approach has revealed differential responses of pulmonary cells in vivo to a
bacterial and mechanical insult. The bacterial endotoxin LPS induced activation of multiple
inflammatory pathways just 5 minutes after intratracheal instillation into the lungs. This
rapidity of response is consistent with previous observations of MK2 phosphorylation in the
whole lung (40), but our data allow us to clarify the cells responsible. The data show a
distinct pattern of activation within cell types, with alveolar macrophages showing activation
of MAP kinases and NF-κB at 5 minutes, while epithelial cells primarily showed activation of
NF-κB at this early time point. These data are mainly consistent with previous in vitro
studies indicating that LPS induces cytokine secretion in macrophages through activation of
ERK1/2 and p38/MK2 pathways, while epithelial cell cytokine production is not mediated
through these MAP kinases (47). Interestingly, these in vitro studies showed no ERK1/2 or
p38 activation in epithelial cells for up to 4 hours following LPS, while we found
phosphorylation of these within 15 minutes.
This is likely to suggest that this in vivo
response is indicative of a secondary activation via a paracrine mediator, for example TNF.
In contrast to alveolar macrophages and epithelial cells, endothelium showed no signs of
activation until 15 minutes. The fact that this activation included not only NF-κB but also
MAP kinases, along with the delay in response, may suggest that this is also secondary
mediator-induced activation.
Compared to LPS, injurious mechanical ventilation induced an entirely different sequence of
events. Each of the individual cell types studied showed signs of significant inflammatory
11
pathway activation within 5 minutes, although interestingly this did not include NF-κB, which
was the most consistently activated marker 5 minutes after LPS. The rapidity of this
response to ventilation led us to explore even earlier time points, finding that the endothelial
cells displayed activation of all MAP kinase markers after 1 minute of high stretch, while the
AECs displayed some signs (increased phosphorylation of p38) of becoming activated at this
time point. AMs however displayed no significant increases in any marker at 1 minute.
Taken together these data indicate that parenchymal cells are likely to be the cells within the
lungs that initiate the inflammatory response to stretch, followed by communication to other
cell types, which can then propagate the cascade. Within the alveolar space, this would
seemingly take the form of stretched epithelial cells sending a signal to alveolar
macrophages. Many in vivo studies have localised MAP kinase and NF-κB activation to the
alveolar epithelium using immunohistochemistry, though only after longer periods of high
tidal volume ventilation (1, 29, 49) than used presently. One previous in vivo study indicated
that alveolar macrophages could be activated by soluble signals contained within
bronchoalveolar lavage fluid taken from animals ventilated with high stretch for 20 minutes
(13), although it was not clarified whether said mediator(s) actually came from epithelial cells
within that study. The nature of this signal is therefore not yet clear, but according to our
data is limited to mechanisms that could stimulate macrophage responses within 5 minutes.
Possible options include i) soluble mediators such as prostaglandins, which have been
shown to be rapidly released from AECs stretched in vitro within a 10 minute time frame (8),
ii) direct communication between epithelial cells and macrophages through gap junction
channels (54), or iii) indirect sensing of epithelial cell deformation mediated through adhesive
contacts between macrophages and the epithelium.
Intriguingly, our data indicate that endothelial cells were potentially not only the first cell type
to activate inflammatory pathways during high stretch, but also the most sensitive to lower
levels of stretch. Indeed, Dreyfuss et al showed nearly 20 years ago that just 5 minutes of
high pressure ventilation induced endothelial cell blebbing (11), and increased p38 and
12
ERK1/2 activation have been detected after 5 minutes in pulmonary endothelial cells
stretched in vitro (1, 20, 23). To the best of our knowledge, our study is the first report of
such rapid endothelial MAP kinase activation following in vivo ventilation.
One of the
fundamental functions of endothelial cells is to control vascular tone in response to
mechanical forces applied onto them, i.e. shear stress by blood flow and circumferential
stretch force generated by vascular transmural pressure.
During high tidal volume
ventilation, both the shear stress and circumferential stretch would likely decrease due to
somewhat reduced pulmonary flow and capillary compression (in a direction perpendicular to
the alveolar wall) by positive alveolar pressure. However, at the same time, because of the
physical nature of the capillary network as a mesh surrounding alveoli, firmly attached to the
alveolar wall via the basement membrane, lung inflation would produce significant
‘longitudinal’ stretch of the capillary wall, as proposed by West as part of his theory of
capillary stress failure (14, 53). The rapidity of the endothelial response in our experiments
supports the likelihood of such longitudinal capillary deformation, indicating that these
endothelial cells are indeed responding directly to stretch rather than a released secondary
mediator. Our results would seem to be consistent with the hypothesis that pulmonary
capillary endothelial cells may be by nature more responsive than alveolar epithelial cells to
such longitudinal stretch of the alveolar wall during lung inflation, and suggest a potentially
central role for endothelial cells in initiating VILI.
These data may have a further important implication in respect to the systemic propagation
of inflammation. Lung-derived mediators produced during VILI have been proposed to play
significant roles in causing injury to non-pulmonary organs, but while studies have attempted
to identify the nature of such mediators (21), the cell source has received relatively little
attention. The ‘decompartmentalisation’ hypothesis suggests a leak of mediators from the
alveolar space to the circulation, but the very rapid activation of inflammatory pathways in
endothelial cells in our study suggests that other mechanisms are likely to be involved. We
have previously shown that lung-marginated monocytes play an important role in VILI in vivo
13
(57), and in systemic cytokine release during in situ ventilation (52). Taken together, these
data could suggest that in a process analogous to that proposed in the alveolar space,
endothelial cells initially respond to deformation and then pass signals to leukocytes
marginated within the capillaries to amplify the inflammatory signal systemically.
The tidal volumes used in this study are much higher than those used clinically. However,
they do reflect the degree of stretch experienced by heterogeneously injured human lungs
(17, 46, 58), which are inherently less compliant than those of mice (45). Furthermore, tidal
volumes clinically relevant to human ARDS are not necessarily always ideal for experimental
purposes in mice.
Although many examples exist of mouse models demonstrating the
development of deteriorated lung function with more ‘clinically consistent’ tidal volumes (18,
27), it is often not clear to what extent stretch per se, rather than other confounding factors
such as atelectasis or a preceding lung insult, is the primary cause of injury (39, 58).
Indeed, we have previously demonstrated that tidal volumes up to 20ml/kg are unlikely to
induce substantial stretch within the lungs of healthy mice (58). The current study was
designed to address the cellular in vivo responses to stretch specifically, and therefore the
most appropriate model was deemed to be one in which stretch would predominate. While
we did not determine blood pressure or blood gases directly in this study (to minimise
surgical instrumentation and potential extra inflammatory insult) we routinely use the same
ventilator settings in our mouse VILI experiments and do not observe any deterioration in
blood pressure or gases until well after the 15 minute time-frame of the current study (10, 55,
58). Furthermore, as there was virtually no NF-κB response to ventilation, the possibility that
activation was due to contamination by LPS appears minimal. Overall, we therefore believe
that it is highly likely that the activation measured in the current study is a direct
consequence of lung overstretch.
Finally, it is important to clarify that in the current study we were attempting specifically to
explore the initiation of inflammatory cascades within the lungs, rather than identify which
cells may be ‘responsive’ per se to stretch. For example, one large breath is capable of
14
mobilising intracellular calcium and promoting surfactant secretion from epithelia (60).
However, such responses do not necessarily have immediate relevance to the way in which
inflammatory cascades and inter-cellular communication begin.
It remains possible that
inflammatory pathways other than the ones we have measured are upregulated; however
the markers used are involved in three separate signalling pathways that promote
inflammatory responses to myriad stress stimuli. Many of the markers displayed transient
activation in response to both LPS and high stretch ventilation, a pattern which seems to be
characteristic of the MAP kinases and NF-κB. Indeed, many in vitro studies have also
detected transient patterns of activation in response to both stretch and LPS in a variety of
cell types (1, 6, 7, 40). Exploring the mechanisms behind this transience was outside the
scope of the current study, but may include processes such as translocation into the nucleus
for NF-κB (a compartment inaccessible to the antibodies under the current protocols) and
enhanced activity of phosphatases as part of an auto-regulation process. Regardless, p38,
ERK1/2, and NF-κB have been frequently implicated in inflammatory signalling initiated by
VILI, and their inhibition has been shown to attenuate symptoms of VILI including cytokine
release (19, 20, 33), suggesting that even very transient activation is sufficient to induce an
inflammatory cascade.
In summary, our data demonstrate a substantially differing cellular activation profile between
mechanical stretch and intratracheal LPS.
By studying the immediate responses to
challenge we have identified likely ‘initial responders’, and the cells which become activated
later, potentially in response to secondary signals.
We propose that blocking lines of
communication between cell types or amplification of signals has the potential to be a safer,
more targeted approach to patient treatment for VILI. Our data suggests that the nature of
mediators / lines of communication are likely to be different between mechanical ventilation
and bacterial challenge. The protocols developed here will allow us to explore in detail the
processes responsible, and when these are identified it may be possible in the future to
15
attenuate the ventilation-induced exacerbation of local and systemic inflammation, whilst
retaining immune responses to infection intact.
16
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FIGURE LEGENDS
Fig. 1. Gating strategies for identifying specific cell types from a whole lung single cell
suspension using flow cytometry. Alveolar macrophages were identified as CD45+, CD11b-,
F4/80+ and CD11c+. Endothelial cells were identified as CD45-, CD31+, and were also
confirmed as staining positive for CD105, and negative for the platelet marker CD41. Type I
AECs were identified as CD45-, CD31-, EpCAM+, T1-α+ and type II AECs as CD45-, CD31, EpCAM+, T1-α-. In addition, type I and type II AECs were further differentiated based on
positive or negative staining for CD54 (ICAM-1). Representative histograms display the
increase in geometric mean fluorescence intensity (gMFI) of phospho-MK2 after 15 minutes
of exposure to intratracheal LPS (grey) versus untreated (white).
Fig. 2.
Phosphorylation levels (gMFI) of the activation markers for all cell types after
exposure to intratracheal LPS for 5 or 15 minutes compared to untreated controls. AMs and
epithelial cells displayed responses to LPS within just 5 minutes. N=5, *p<0.05, **p<0.01,
***p<0.001.
Data were analysed by ANOVA with Tukey HSD, or Welch ANOVA with
Games-Howell tests for parametric data with unequal variance. Data are displayed as mean
± SD.
Fig. 3. Phosphorylation levels (gMFI) of the activation markers for all cell types after
exposure to 5 or 15 minutes of high stretch ventilation compared to 5 minutes of low stretch.
The parenchymal cells appeared more responsive to stretch than the AMs. N=5-8, *p<0.05,
**p<0.01, ***p<0.001. Data were analysed by ANOVA with Tukey HSD and are displayed as
mean ± SD, or geometric mean with 95% confidence intervals (lower, upper bounds) for
transformed data. Non-parametric data were analysed by Kruskal-Wallis with Dunns and
are displayed as box-whisker plots with error bars extending to minimum and maximum
values.
Fig. 4. Levels of phosphorylated and total ERK1/2 protein within lungs subjected to 5
minutes of low or high stretch ventilation. Consistent with findings from figure 3, levels of
23
phospho-ERK1/2 clearly increased with high stretch ventilation, whilst levels of nonphosphorylated ERK1/2 within the same lungs were unaltered. N=4, *p<0.05, **p<0.01.
Data were analysed by t-test and are displayed as mean ± SD.
Fig. 5.
Phosphorylation levels (gMFI) of the activation markers for all cell types after
exposure to intratracheal LPS for 1 minute compared to untreated controls. No significant
signs of activation could be detected after 1 minute of LPS. N=4-6. Data were analysed by ttest and are displayed as mean ± SD, or geometric mean with 95% confidence intervals
(lower, upper bounds) for transformed data. Non-parametric data were analysed by Mann
Whitney U and are displayed as box-whisker plots with error bars extending to minimum and
maximum values.
Fig. 6. Phosphorylation levels (gMFI) of the activation markers for all cell types after
exposure to 1 minute of low or high stretch ventilation. The endothelial cells displayed
activation of the most markers after just 1 minute of high stretch ventilation. N=5-7, *p<0.05,
**p<0.01. Data were analysed by t-test and are displayed as mean ± SD.
Fig. 7. Phosphorylation levels (gMFI) of the activation markers for all cell types after
exposure to 5 minutes of ventilation at 30ml/kg compared to 5 minutes of low stretch
ventilation.
Only the endothelial cells demonstrated sensitivity to the intermediate tidal
volume of 30ml/kg. N=7-11, *p<0.05. Data were analysed by t-test and are displayed as
mean ± SD, or geometric mean with 95% confidence intervals (lower, upper bounds) for
transformed data.
24
FIGURES
Fig. 1.
25
Fig. 2.
26
Fig. 3.
27
Fig. 4.
28
Fig. 5.
29
Fig. 6.
30
Fig. 7.
31