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* From the Medical Research Service, Seattle VA Medical Center, and the Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of Washington School of Medicine, Seattle, WA. Supported in part by grants HL30542, A129103, and GM37696 from the National Institutes of Health, and the Medical Research Service of the Department of Veterans Affairs.
Correspondence to: Thomas R. Martin, MD, FCCP, Pulmonary Research Labs, 151L, Seattle VA Medical Center, 1660 S Columbian Way, Seattle, WA 98108; e-mail: trmartin{at}u.washington.edu
The cellular and molecular basis for ARDS remains uncertain > 30 years after the original description of the syndrome. With the explosion of information about the involvement of cells and cytokines in inflammation, there has been intense interest in understanding the involvement of cytokines in the pathogenesis of ARDS. Cytokines are low-molecular-weight soluble proteins (generally < 30 kda) that transmit signals between cells. It is now clear that cytokine production is not limited to lymphoid and myeloid cells, and that cytokines produced by epithelial and mesenchymal cells amplify inflammatory responses in the lungs and other organs. Cytokines are produced in "cascades" in which the initial cytokine signals are amplified many-fold by target cells, such as epithelial cells, fibroblasts, and endothelial cells. Cytokines function in "networks" in which feedback occurs at many points to coordinate and regulate cytokine and cellular responses.
There are three major reasons to study the involvement of cytokines in patients with ARDS: first, to understand the pathogenesis of the disease; second, to identify markers that might be used to determine which patients are at highest risk for developing ARDS; and third, to identify markers that can predict outcome, including survival and/or long-term disability.1 Studies of single cytokines have shown that no single cytokine consistently predicts either the onset or the outcome of ARDS, despite promising early results. Instead, it is now recognized that a balance of proinflammatory and anti-inflammatory factors influences the net inflammatory response in the lungs.2 Current efforts are directed at defining the cytokine balance that exists in the lungs at the onset of ARDS, and how this balance changes over time.
Sampling and Measurement Considerations
The study of cytokine balance in the lungs is difficult because of sampling and measurement issues. Experimental studies suggest that cytokine responses normally are compartmentalized in the lungs, and that the study of blood specimens provides an incomplete reflection of inflammatory events in the lungs.3 However, compartmentalization is lost to some extent during severe inflammatory responses.4 Thus, measurements of cytokines in the lungs are likely to be more valuable than measurements in plasma or serum. Sampling cytokines in the lungs is difficult, because cytokines function not only in the alveolar compartment, where they may exist as soluble constituents of alveolar fluids, but also in the tissue compartment, where they bind to components of the extracellular matrix. Thus, sampling alveolar fluids by direct aspiration of the distal airways or by BAL may provide incomplete information about the concentration and function of specific cytokines in the lungs. However, at the present time, to my knowledge, methods do not exist to sample lung tissue repeatedly in patients with ARDS, and studies of alveolar fluids provide the best current assessment of cytokine concentrations in the alveolar spaces.
Two methods have been used to sample lung fluids in patients with ARDS: BAL and direct aspiration of edema fluid using a suction catheter.1 Direct aspiration of edema fluid is advantageous because the fluid is not diluted; however, fluid is usually obtainable only very early in the course of lung injury.5 The BAL method is safe in critically ill patients, but requires fiberoptic bronchoscopy and dilutes the alveolar fluids.6 Although a comparison of these two methods has not been performed in the same patients, studies performed using the same assays on BAL and edema fluid obtained from different patients at similar times in their course suggest that the BAL method dilutes total proteins by about 50-fold, and cytokines by about 100-fold.7 Thus, cytokine concentrations may be low or undetectable in BAL because of the dilution factor.
Two approaches have been used to measure cytokines in biological
fluids: biological assays using responsive target cells, and
enzyme-linked immunosorbent assays using polyclonal or monoclonal
antibodies. Important measurement issues exist, as cytokines in
biological fluids function in a complex milieu of inhibitors and other
molecules that modify cytokine activity. Biological assays have an
advantage, as they measure the net biological activity of a given
cytokine if the assays are specific. This is an important issue, as
many biological assays using target cell lines often are affected by
more than one cytokine. Specificity is established by using specific
inhibitory antibodies against the cytokine in question to show the
proportion of the biological activity that is blocked in the assay. An
example of this is the "proinflammatory activity" assay used by
Pugin et al8
to measure interleukin-1ß (IL-1ß)
activity in ARDS BAL fluid. Although this assay is sensitive to IL-1ß
and tumor necrosis factor-alpha (TNF-
), studies with inhibitory
antibodies showed that the activity measured in BAL fluids was due to
IL-1ß, and not TNF-.
Enzyme-linked immunosorbent assays use combinations of monoclonal
and/or polyclonal antibodies to capture and detect cytokines in
solution. This is advantageous, as it is possible to get an accurate
measurement of the amount of a cytokine ligand in solution. Problems
arise when inhibitors complex with the target cytokine and hide
epitopes that normally would be detected by the assay. In addition,
target epitopes may be shared by unexpected molecules that interfere
with the specificity of the measurement. Shielding of reactive epitopes
is often a particular problem with assays using monoclonal antibodies,
as there may be few specific epitopes on a target molecule. This
concept is germane for measurements of IL-8, which forms complexes in
plasma and BAL with IgG molecules and
1-antitrypsin.9
10
11
IL-8 concentrations are
underestimated by some commercially available kits. We have found that
IL-8 concentrations rise in samples that are repeatedly frozen and
thawed, perhaps reflecting release of IL-8 from inhibitory complexes.
There is no reliable a priori method to determine either the accuracy or the specificity of cytokine measurements in complex biological fluids. This must be worked out in advance for each assay by using specific inhibitory antibodies for bioassays, and by "spiking" experiments, in which known amounts of the target cytokine are added to specific samples from normal subjects and patients.
Cytokines and the Pathophysiology of ARDS
What have we learned about the pathophysiology of ARDS from studies of cytokines in BAL fluids? The hallmark lesion in ARDS, described carefully by Bachofen and Weibel,12 13 is widespread destruction of the alveolar epithelium and flooding of the alveolar spaces with proteinaceous exudates containing large numbers of neutrophils (polymorphonuclear leukocytes [PMNs]). Vascular lesions also are present, but the vascular destruction often is not as prominent as the damage to the alveolar epithelium.14 Because of laboratory evidence linking PMNs and lung injury, and the critical involvement of cytokines in the recruitment of PMNs into tissues, it was hoped that the study of cytokines in BAL would provide clues about the mechanisms that regulate injury in the lungs.15 16 17 Although most studies have focused on the potential importance of single cytokines, it is now recognized that a complex balance exists between proinflammatory and anti-inflammatory cytokines, and that "cytokine balance" is a key concept in understanding the biological activity of cytokines in biological fluids.2
Cellular Recognition Pathways
A major advance in the study of cytokine production in the lungs
came with the recognition that the CD14 antigen is a pattern
recognition receptor on the surface of monocytes and macrophages that
mediates responses to lipopolysaccharide (LPS) and other Gram-negative
and Gram-positive cell wall products.18
19
20
A soluble form
of CD14 (sCD14) is released from cellular membranes and mediates
LPS-dependent responses of CD14-negative cells, such as endothelial and
epithelial cells.21
CD14 is present on alveolar
macrophages, and the cytokines IL-4 and IL-6 modulate CD14 expression
and shedding from the macrophage surface.22
23
The
concentration of sCD14 increases in lung fluids before and after the
onset of ARDS, and is significantly related to the number of PMNs in
alveolar fluids, suggesting that sCD14 marks a pathway that is
important in stimulating PMN migration in the lungs.24
The
biological importance of the CD14-dependent recognition pathway has
been shown in animal models of LPS-induced shock, in which inhibition
of CD14 with specific monoclonal antibodies blocked systemic cytokine
responses, reduced lung protein leak, and improved
survival.25
26
TNF- and IL-1ß
Interest in the roles of the early response cytokines TNF- and
IL-1ß was stimulated by the recognition that these cytokines
stimulate cytokine production by lung epithelial and mesenchymal cells
that do not respond directly to bacteria and their
products.27
28
29
30
Suter et al31
found
significant levels of TNF- in lung fluids of patients at the onset
of ARDS using a small-volume lavage (5.0 mL). Investigators using
larger-volume lavages have found low concentrations of TNF-, and we
have found very low levels in BAL fluid by immunoassay.32
The effects of TNF- are modulated by two TNF- receptors (TNFRI,
p55, and TNFRII, p75) that are shed from the surface of macrophages and
other cells.33
We have found that the concentrations of
TNFRI and TNFRII exceed the concentration of immunoreactive TNF- in
BAL at all times during the course of ARDS, and that the ratios of
TNF- to its receptors are lowest on days 1 and 3 of
ARDS.34
This suggests that the activity of TNF- is
effectively inhibited in the aqueous phase of lung fluids of patients
with ARDS.
Like TNF-, IL-1ß is present in BAL at the onset of ARDS. Jacobs et
al35
were the first to show that alveolar macrophages from
patients with ARDS spontaneously released IL-1ß, suggesting that
these macrophages had been activated in the alveolar spaces. Pugin et
al8
investigated proinflammatory activity in ARDS BAL,
using an assay that measured upregulation of the adhesion molecule
ICAM-1 on the surface of A549 epithelial cells. Although TNF- and
IL-1ß each caused upregulation of ICAM-1 in this assay, studies with
inhibitory antibodies showed that the proinflammatory activity in ARDS
BAL was due to IL-1ß and not to TNF-. In patients with sustained
ARDS, the IL-1ß concentration in BAL on day 7 correlates with
survival, with higher concentrations in patients who
die.36
This is also true in our more recent studies of
patients with ARDS, in which IL-1ß measurements were made using a
different immunoassay. IL-1ß is antagonized by IL-1 receptor
antagonist protein (IL-1RA protein), and by a circulating IL-1 receptor
(IL-1R2) that does not signal when it is in the cellular
membrane.37
IL-1RA protein competes with IL-1ß for the
IL-1 signaling receptor, whereas IL-1/IL-1R2 complexes are inactive. We
have found that the molar concentrations of IL-1RA protein and IL-1R2
exceed the concentration of IL-1ß at the onset of ARDS, when the
inflammatory response is greatest in the lungs. The observation that
very few molecules of IL-1ß are needed to trigger target cells
probably explains why bioactive IL-1ß is detectable in the
proinflammatory assay, despite the apparent excess of inhibitors over
free IL-1ß.37
Chemokines
Leukocyte migration is directed to a large extent by chemokines
(chemotactic cytokines). The two major classes
include the -chemokines, which recruit PMNs, and ß-chemokines,
which recruit monocytes and lymphocytes.38
The
-chemokines include IL-8, GRO (melanoma growth stimulating
activity), and ENA-78 (epithelial cell neutrophil activating factor).
The ß-chemokines include the monocyte chemotactic peptides
(MCP-1,2,3,4) and RANTES (regulated on
activation, T-cell
expressed and secreted). IL-8, GRO, and ENA-78
are detectable in the BAL of patients at risk for ARDS and during the
course of established ARDS.7
36
39
40
Alveolar macrophages
are a major source of chemokines in the airspaces, and produce IL-8,
GRO-related peptides, and ENA-78. Alveolar macrophages respond directly
to bacterial products such as bacterial LPS and Gram-positive cell wall
products such as leipoteichoic acids. On a quantitative basis, IL-8 is
the most abundant product following LPS stimulation.41
Other cells of the alveolar environment also produce - and
ß-chemokines, but do so in response to the proinflammatory cytokines
TNF- and IL-1ß, and not directly in response to bacterial products
such as LPS.27
28
29
30
The - and ß-chemokines are present in the lungs of patients with
ARDS.42
IL-8, GRO, ENA-78, and MCP-1 all have been found
in BAL fluid of patients at risk for and with established
ARDS.7
36
39
40
Although other potent leukocyte
chemoattractants also exist, including the complement component C5a and
the low-molecular-weight lipids leukotriene B4 and platelet
activating factor, the neutrophil chemotactic activity in BAL is due
predominantly to IL-8, and not to C5a.36
43
Nevertheless,
correlations between IL-8 and total PMN in BAL at the onset of ARDS are
poor in most studies.42
We found that the relationship
between IL-8 and PMN actually grows stronger with time in patients with
persistent ARDS.36
Other chemokines in ARDS BAL also are
likely to contribute to PMN recruitment. Villard et al40
found that the concentration of GRO was higher than that of IL-8 in
patients with ARDS. We have found that the concentrations of GRO and
ENA-78 exceed the concentration of IL-8 in BAL throughout most of the
course of ARDS, despite the fact that depletion studies with antibodies
to IL-8 suggested that IL-8 was the dominant PMN chemoattractant in the
fluids studied.36
MCP-1, which regulates monocyte
recruitment, is detectable in ARDS BAL at the onset of ARDS and
persists in the lungs of patients with sustained ARDS.36
The concentrations of chemokines in BAL are likely to be inexact
reflections of the quantity and activity of chemokines in the alveolar
environment for several reasons. First, the -chemokines all contain
heparin-binding domains at the C-terminus that enhance binding to
interstitial matrix.44
Chemokines in the aqueous phase of
alveolar fluids are in equilibrium with chemokines bound to tissue
matrix. At present, there is no way to estimate the size of the
tissue-bound pool. Second, the biological activity of chemokines is
modulated by immunoglobulins and other proteins that move from the
plasma into the lungs. The biological activity of IL-8 is inhibited by
IgG molecules in blood and alveolar fluid, which block biological
activity,9
10
and by 2-macroglobulin, which
binds IL-8 and other cytokines and prevents proteolytic
degradation.11
Interleukin-6
IL-6 is a cytokine that was originally identified as a B-cell
growth factor.45
IL-6 is produced by activated macrophages
and stimulates acute-phase responses in the liver. IL-6 production is
induced in part by TNF- and IL-1ß, and it has been proposed that
IL-6 "integrates" signals produced early in the inflammatory
response. IL-6 measurements in peripheral blood have been used to
stratify patients in clinical trials of new treatments for
sepsis.46
47
We have found that IL-6 concentrations are
very high in the BAL of patients at risk for ARDS and that they remain
elevated throughout the course of established ARDS. IL-6 concentrations
do not predict either the onset or outcome of ARDS. The IL-6 receptor
(IL-6R) is released from the surface of cell membranes and circulates.
Unlike the receptors for TNF- and IL-1ß, the soluble IL-6R is an
agonist rather than an antagonist. IL-6R promotes IL-6 signaling when
it binds soluble IL-6 and engages a second protein, known as gp130,
that is widely distributed on cell membranes. We have found that the
concentration of soluble IL-6R is elevated in the BAL of patients at
risk, and throughout the course of ARDS. Thus, cellular reactions
mediated by IL-6 should be highly favored during the course of ARDS.
More information is needed about IL-6-dependent pathways in the lungs
of patients with ARDS.
Interleukin-10
IL-10 is a counterregulatory cytokine that inhibits cytokine
production by stimulated macrophages.48
49
IL-10 is
detectable in ARDS BAL fluids, but the concentrations are very low as
compared with other cytokines (10 to 20 pg/mL range). Low
concentrations of IL-10 favor cytokine production in the alveolar
environment. The effects of IL-10 depend on the experimental system,
and more information is needed about the function of IL-10 in the lungs
of humans. In experimental endotoxemia or peritonitis, IV treatment
with IL-10 protects animals from death.50
51
However, in
animals with bacterial pneumonia, IL-10 treatment impairs bacterial
clearance and worsens survival.52
53
Donnelly et
al54
found that patients who died with ARDS had low
concentrations of IL-10 in BAL fluid at the onset of ARDS, suggesting
inadequate dampening of lung inflammatory responses.
Macrophage Inhibitory Factor
Recently, the cytokine macrophage migration inhibitory factor
(MIF) was identified in BAL fluid from patients studied on the first
day of ARDS.55
MIF, which was the first cytokine
discovered, was identified as a factor in fluid from cutaneous
delayed-type hypersensitivity reactions that inhibited monocyte
migration.56
MIF is produced by several different types of
cells, including cells of the anterior pituitary gland, activated
macrophages, and possibly the airway epithelium. Immunoreactive MIF is
detectable in macrophages recovered from the airspaces of patients with
ARDS and antagonizes the suppressive effects of cortisol on cytokine
production by alveolar macrophages.55
This suggests that
MIF may act to sustain inflammation in the alveolar spaces. We have
found that MIF is detectable in BAL fluid of patients at risk for ARDS,
and that the concentration of MIF increases in the lungs of patients
with sustained ARDS.57
The role of MIF in the lungs of
patients with ARDS is unclear, and more information is needed about
this cytokine.
Cytokines and Apoptosis
Cytokines also modify the life span of leukocytes that migrate
into tissues. PMN accumulation in the lungs is a characteristic feature
of acute lung injury. Although PMNs typically have a short life span in
tissue, we have found that PMN survival in the airspace is prolonged by
leukocyte colony-stimulating factors such as granulocyte
colony-stimulating factor and granulocyte-macrophage colony-stimulating
factor, which inhibit PMN apoptosis.58
59
While this might
favor sustained neutrophil-dependent lung injury, it also favors
effective antibacterial defenses in the airspaces. The relationship
between cytokine concentrations and apoptosis of other cells in the
alveolar environment is uncertain.
Cytokines and Epithelial Injury
One of the major challenges is to try to relate the cytokine
responses in the lungs of patients with ARDS to the injury to the
alveolar epithelial and endothelial barriers of the lungs. Markers of
endothelial injury such as the von Willebrand's factor antigen have
been used to reflect systemic endothelial cell activation and/or damage
in patients with sepsis, but the findings have varied and better
markers of endothelial and epithelial cell injury are
needed.60
61
In rats, a type I cell antigen increases with
lung injury, and this marker needs to be tested in humans with
ARDS.62
Surfactant-associated proteins have been used as
markers of type II pneumocyte function, and the concentrations of the
surfactant-associated proteins (SP-A and SP-B) are low at the onset of
ARDS.63
Studies in vitro suggest that SP-A and
SP-B are subject to regulation by inflammatory cytokines, so
alterations of SP-A and SP-B may not be specific for type II pneumocyte
"injury."64
65
SP-D is not affected by inflammatory
cytokines in vitro, so it is possible that SP-D might
provide different information about the status of type II pneumocytes
in the lungs.65
Studies linking cytokine variables,
cellular injury variables, and clinical variables will need to involve
large numbers of patients, because of the number of comparisons that
need to be tested and the need to categorize patients with ARDS by the
underlying disease and outcome. Data sets involving > 100 patients
all studied in the same way will probably be needed.
Cytokines and Repair
Cytokines also stimulate collagen production and repair.
Procollagen peptide III (PCPIII) is a marker of collagen synthesis that
is detectable in BAL at the onset of ARDS.66
67
High
concentrations of PCPIII in BAL are associated with an increased risk
of death, suggesting more severe lung injury.66
Transforming growth factor-alpha (TGF-) increases fibroblast
collagen production and may contribute to the fibrosis that occurs in
patients with sustained ARDS.68
TGF- is detectable in
BAL for prolonged periods after the onset of ARDS, and high
concentrations of TGF- in BAL on day 7 are associated with an
increased risk of death in patients with sustained ARDS.69
TGF- also stimulates epithelial cell proliferation in
vitro.70
Other growth factors that stimulate
epithelial cell proliferation, including keratinocyte growth factor and
hepatocyte growth factor, have been detected in ARDS
BAL.71
Cytokines and Prediction
What have we learned about the prediction of either the onset or
the outcome of ARDS from studies of cytokines in BAL fluid? Initial
studies focused on IL-8, because neutrophils (PMNs) are abundant in the
BAL of patients with ARDS, and experimental studies have linked PMNs
with lung injury.15
16
In two small studies, Miller et
al,7
found that IL-8 in BAL at the beginning of ARDS was
highest in patients who died, and Donnelly et al39
found
that IL-8 was highest in patients at risk for ARDS who later developed
ARDS. These studies provided hope that IL-8 could be used to predict
the onset of ARDS, and the clinical outcome once it begins.
Unfortunately, subsequent studies in several centers have found that
IL-8 does not predict outcome either at the outset or during the course
of ARDS.36
In addition, we have found that IL-8 does not
predict the onset of ARDS in patients at risk who are studied within
the first 24 h after the onset of risk. In ongoing studies in
Seattle that include > 45 patients studied serially throughout the
course of ARDS, we have found that measurements of TNF-, IL-1ß,
IL-6, IL-8, GRO-, and MCP-1 in patients at risk or on day 1 of ARDS
are not reliable predictors of either the onset or the clinical outcome
of ARDS. Meduri et al72
found that all cytokines measured
remained high during the course of ARDS in patients who died. We have
not found that this is the case in a more diverse patient population in
Seattle, although we do find that IL-1ß and MCP-1 are higher on day 7
in patients who later die.36
Our ongoing studies suggest
that patients with persistent elevation of BAL cytokines are more
likely to have pulmonary dysfunction if they survive. The importance of
considering anti-inflammatory constituents of BAL is shown by the work
of Donnelly et al,54
who found that patients with ARDS who
died had significantly lower initial concentrations of the inhibitors
IL-1RA and IL-10 in BAL than patients who lived.
Why then are cytokine measurements poor predictors of outcome? The answer to this question lies in the complexity of the inflammatory response, because it is unlikely that one single factor drives the injury to the epithelial and endothelial barriers that occur in ARDS. Complex interactions between cells and cytokines are likely, and multivariate analyses will be needed to determine whether groups of variables are better predictors of outcome than single cytokine measurements. This approach will require studies of larger numbers of patients. In addition, the inflammatory response changes with time during the course of ARDS, as we have found that cytokine values on day 7 are more useful predictors of mortality than cytokine values on day 1.36 69 It is also possible that cytokines are the wrong molecules to use in predictive equations. Markers of collagen synthesis such as PCPIII and/or markers of epithelial and endothelial injury may be better variables to use, because they may better reflect the structural injury that causes persistent lung dysfunction and determine clinical outcome.66 67
ARDS is a diverse illness, and variations in the composition of patient populations may account for some of the variability in the published studies. Prospective studies are needed in which larger numbers of patients are studied throughout the course of the illness. Population sizes need to be large enough to permit stratification of the population by the risk factor associated with ARDS, and the outcome. In Seattle, approximately one third of patients develop ARDS because of severe sepsis, one third have trauma, and one third have a mixture of other risks, including gastric aspiration and mixed drug overdoses.36 73 Thus, to stratify an experimental sample by three clinical factors and two outcome variables would require a sample size of 120 patients in order to have 20 patients in each of six groups. This is a formidable challenge, as identifying patients and gathering lung fluid samples are complex tasks.
Conclusions
In summary, cytokine measurements in BAL fluid of patients before and after the onset of ARDS have provided valuable insights about the complexity of the inflammatory response that occurs in the lungs. Initial hopes that single cytokines would be useful predictors of onset or outcome have given way to the realization that the complexity of the inflammatory response prevents generalizations from measurements of single cytokines. The complex balance between cytokines and their naturally occurring inhibitors or antagonists requires that the evaluation of inflammatory pathways take into account the proinflammatory and anti-inflammatory factors that affect each pathway. ARDS is a disease in which biopsies of the affected tissue are not feasible, and BAL measurements may or may not represent the activity of individual cytokines in tissue. The major challenge now is to determine whether groups of cytokine variables can be used to predict more accurately either the onset or the outcome of the injury that occurs in the lungs. To do this, better markers of the state of the alveolar epithelial and endothelial barriers are needed, so that the relationship between the alveolar inflammatory response and the integrity of these important structural barriers can be assessed.
References
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