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* From the Division of Medical Intensive Care, University Hospital of Geneva, Geneva, Switzerland. Supported by grants from the Swiss National Foundation for Scientific Research 32-40344.94 and 32-50764.97, the 3R Research Foundation, Switzerland, grant No. 46-96, the Lancardis, and the Prof. Dr. Max Cloëtta Foundations (J.P.), and by a biomedical and natural science research grant, Switzerland (I.D.).
Correspondence to Jérôme Pugin, MD, Division of Medical Intensive Care, Department of Medicine, University Hospital of Geneva, 1211 Geneva 14, Switzerland; e-mail: pugin{at}cmu.unige.ch
Ventilator-induced lung injury (VILI) is the result of a complex interplay among various mechanical forces acting on alveoli during mechanical ventilation. Data from animal models strongly suggest that the two main determinants of VILI are alveolar overdistention and the repeated collapse and reopening of alveoli during the ventilatory cycle.1 2 Studies exploring the pathophysiologic mechanisms underlying VILI suggest that these forces probably exert their damaging effects through the initiation of a localized inflammatory response, the latter being responsible for ongoing lung injury.3 It is conceivable that injurious ventilatory regimens participate in lung injury and alveolar inflammation such as that observed during the ARDS. However, direct proinflammatory effects of the physical stress generated by positive pressure mechanical ventilation on lung cells have not been shown. In this work, we identify the lung macrophage as a critical mechanosensor cell capable of triggering lung inflammation in response to pressure/stretching mechanical forces.
Materials and Methods
An in vitro model has been developed in which
various isolated lung cell types can be submitted to a prolonged (8 to
36 h) cyclic pressure/stretching strain resembling that of
conventional mechanical ventilation.4 Primary human cells
or cell lines were cultured on collagen I-coated Silastic membranes
(BioFlex; Flexcell International Corp; Hillsborough, NC) that
formed the bottom of wells of a 12-well plate; a plexiglass lid
was adapted to the plate, bolted down in an airtight manner, and
connected to an adult ventilator (Evita 2; Drägerwerk;
Lübeck, Germany). Cells were submitted to mechanical ventilation
at a frequency of 20 cycles/min in a pressure-limited controlled mode,
with a pressure (approximately 70 cm H2O) inducing a
vertical Silastic membrane excursion of 6 mm, which corresponded to a
mean surface cell stretching of 12%, with a 37°C heated, 100% humid
atmosphere containing 74% N2, 21% O2, and 5%
CO2. Controls included cells cultured on the same Silastic
membranes but not submitted to mechanical ventilation, and cells
ventilated in the presence of an agonist for these cells (Table 1)
.
The following human cell types were tested in the "plastic lung":
freshly isolated primary alveolar macrophages, monocyte-derived
macrophages, promonocytic THP-1 cell line (ATCC) rendered adherent by
phorbol ester treatment, type II-like A549 and bronchial BET-1A
epithelial cell lines (ATCC), endothelial ECV (ATCC) and EA ·
hy926 cell lines (gift from C.J. Edgell), and primary lung fibroblasts
(Clonetics; San Diego, CA). Conditioned supernatants were sampled at
various times (8, 24, and 32 h) and assayed for the presence of
interleukin (IL)-8 in all cell types, and in some cell types for tumor
necrosis factor-
(TNF), IL-6, IL-10, surface expression of
intercellular adhesion molecule-1 (ICAM-1) using enzyme-linked
immunosorbent assay techniques, and gelatinases using gelatin
zymography. Ventilated and control cells were collected in some
experiments, and activation of the transcription factor nuclear
factor-kappa B (NF-
B) in nuclear extracts was assayed using a
classical electromobility shift assay.
|
Using this in vitro model, we tested whether
human cells of the monocyte/macrophage, epithelial, endothelial, and
fibroblastic lineages secreted IL-8 in response to a
pressure/stretching strain resembling that of mechanical ventilation.
Measurement of this neutrophil chemokine was particularly relevant
since it was found that neutrophil recruitment to the lung was a
hallmark of VILI.4
5
Among the various cells tested, the
macrophages appeared clearly as the main cellular source for IL-8
(Table 1) . Except for type II-like A549 cells that produced small
amounts of IL-8, all the other cell types failed to secrete this
chemokine in response to mechanical ventilation (Table 1)
. Ventilated
macrophages also produced matrix metalloproteinase-9 (gelatinase B),
which might be of importance in the remodeling aspect of the injured
lung. In addition, activation of "proinflammatory" gene
transcription was demonstrated by the findings of the nuclear
translocation of activated NF-B in macrophages submitted to the
pressure/stretching load. Although mechanical ventilation did not
per se induce macrophage secretion of TNF and IL-6, the
cyclic pressure/stretching strain greatly enhanced the secretion of
these mediators when induced by endotoxin (lipopolysaccharide [LPS])
(Table 1)
. These results suggest that positive pressure mechanical
ventilation may not be too deleterious when applied to normal lungs
(only IL-8 secretion and neutrophil recruitment), but may greatly
enhance lung inflammation in injured or infected lungs, with production
of extremely potent proinflammatory mediators such as TNF and
IL-1ß.6
These macrophage-derived cytokines, which have
been found in lungs from rodents submitted ex vivo to
injurious ventilatory regimens,2
7
may in turn activate
other alveolar cell types such as epithelial cells, endothelial cells,
and fibroblasts to amplify and propagate the inflammation locally and
systemically.8
Of note is the fact that a similar cytokine
profile was found in supernatants from macrophages submitted to
ventilation and in edema fluid or BAL fluid from patients with
ARDS.9
10
It remains unclear which lung macrophage cell
type is responsible for the response to mechanical ventilation within
the lung parenchyma. Because of its adherence to the interstitial
matrix, the interstitial macrophage may represent a better candidate
than the loosely adherent alveolar macrophage.
In conclusion, the data presented herein provide the cellular and molecular basis for VILI. They highlight the central role of the lung macrophage in modulating lung inflammation in response to the mechanical stress induced by positive pressure ventilation. Our in vitro model is of particular value to further explore pressure/stretching-induced signaling pathways, as well as for testing the effects of novel ventilatory strategies or adjunctive substances aimed at modulating cell activation induced by mechanical ventilation.
Acknowledgements
The authors thank the group of Dr. L.P. Nicod for the generous gift of alveolar macrophages, and Drs. P. Jolliet and J.-Cl. Chevrolet for stimulating discussions.
References
This article has been cited by other articles:
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  C. C. Dos Santos and A. S. Slutsky Cellular Responses to Mechanical Stress: Invited Review: Mechanisms of ventilator-induced lung injury: a perspective J Appl Physiol, October 1, 2000; 89(4): 1645 - 1655. [Abstract] [Full Text] [PDF]
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