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Acute respiratory distress syndrome (ARDS) involves an intense inflammatory response in the lungs, with accumulation of both pro-
and antiinflammatory cytokines in bronchoalveolar lavage fluid
(BALF). Our goal was to determine how the balance between pro-
and antiinflammatory mediators in the lungs changes before and
after the onset of ARDS. We identified 23 patients at risk for ARDS
and 46 with established ARDS and performed serial bronchoalveolar lavage (BAL). We used immunoassays to measure tumor necrosis factor 
(TNF-) and soluble TNF- receptors I and II; interleukin 1
(IL-1), IL-1 receptor antagonist, and soluble IL-1 receptor II; IL-6 and soluble IL-6 receptor; and IL-10. We used sensitive bioassays to measure net TNF-, IL-1, and IL-6 activity. Although individual cytokines increased before and after onset of ARDS, greater
increases occurred in cognate receptors and/or antagonists, so that
molar ratios of agonists/antagonists declined dramatically at the
onset of ARDS. The molar ratios remained low for 7 d or longer,
limiting the activity of soluble IL-1 and TNF- in the lungs at the
onset of ARDS. This significant antiinflammatory response early in
ARDS may provide a key mechanism for limiting the net inflammatory response in the lungs.
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Keywords: acute respiratory distress syndrome; cytokine receptors; cytokines; IL-1; TNF
The acute respiratory distress syndrome (ARDS) is characterized by an acute inflammatory response in the lung parenchyma that is associated with severe injury to the epithelial
and endothelial barriers (1). Cytokines play a critical role as
signaling molecules that initiate, amplify, and perpetuate inflammatory responses on a local and systemic basis. Two of
the most important early response cytokines are tumor necrosis factor (TNF-) and interleukin 1 (IL-1). Both TNF-
and IL-1 are present in the bronchoalveolar lavage fluid
(BALF) of patients at risk for ARDS and with established ARDS (2). The highest concentrations of TNF- and IL-1
occur in the BALF from patients with sustained ARDS. The
ratios of BALF to serum cytokine concentrations are typically
elevated, suggesting a pulmonary origin (5, 6). Despite key
roles for both TNF- and IL-1 in the development and progression of inflammation, clinical trials directed at neutralizing
TNF- and IL-1 in patients with sepsis have not shown a significant improvement in outcome or the onset of lung injury
(6, 7). In addition, single cytokine measurements in serum and
BAL do not consistently predict either the onset or the outcome of ARDS (8). The failure of these efforts underscores an
incomplete understanding of the pathophysiology of ARDS.
Specific and nonspecific cytokine antagonists were described
soon after the initial discovery of cytokines and provide mechanisms for limiting the biologic effects of proinflammatory cytokines. These discoveries increased the understanding of the
complexity of cytokine signaling pathways and inflammatory
cascades. Examples of nonspecific antagonists include 2-macroglobulin and antiinflammatory cytokines such as IL-10, whereas
specific antagonists include IL-1 receptor antagonist (IL-1ra),
soluble IL-1 receptor II (sIL-1RII), soluble TNF receptor I (sTNF-RI) and soluble TNF receptor II (sTNF-RII). In contrast, the
soluble IL-6 receptor (sIL-6R) is a specific agonist (9). Significant concentrations of sTNF-RI and sTNF-RII have been
found in BALF from patients with ARDS (4). In addition, low
concentrations of IL-10 and IL-1ra in BALF from patients with ARDS were found to be associated with increased mortality,
suggesting an important role for antiinflammatory mediators in
counter balancing the proinflammatory response (10).
Because the net inflammatory balance is of greater physiological and clinical importance than individual cytokine concentrations, ratios between proinflammatory and antiinflammatory mediators have been used to measure net pro- and
antiinflammatory balance (11). For example, in inflammatory bowel disease an imbalance between pro- and antiinflammatory mediators has been suggested as a mechanism underlying persistent disease, and an elevation in the ratio between
IL-1 and IL-1ra is an effective marker of active disease (11).
Although pro- and antiinflammatory mediators have been
found in the BALF of patients with ARDS, the relative production of pro- and antiinflammatory mediators, their net biological effects in BALF, and the relationships between ratios
of pro- and antiinflammatory mediators and clinical parameters have not been examined in patients with ARDS.
The major goal of this study was to determine the relationships between early response cytokines in the lungs and their
naturally occurring modulators to understand how these relationships change net biological activity in the lungs over time.
Our second goal was to try to determine the relationship between molar ratios of agonist and antagonists and parameters
of lung injury and outcome of patients at risk for ARDS and
with established ARDS. We studied TNF- and IL-1 because of their prominent roles as immediate response cytokines. We also studied IL-6 because TNF- and IL-1 induce
its production and because it has both pro- and antiinflammatory effects (17).
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Patient Population and Bronchoalveolar Lavage Protocol
Patients with sepsis or trauma who were at risk for ARDS and patients with established ARDS from any cause were identified by prospective screening of all patients admitted to the Medical and Surgical Intensive Care Units of Harborview Medical Center (Seattle, WA) between February 14, 1994 and July 13, 1997. The specific criteria for sepsis and trauma risks and for ARDS have been described in prior studies from the Seattle ARDS research program (18). All patients with ARDS met the American European Consensus Conference definition of ARDS (21). The BAL techniques and handling of specimens also have been reported (18). The protocol was approved by the University of Washington Investigational Review Board. Either the patient or a responsible relative gave written informed consent.
Measurements of Physiologic Parameters
All patients at risk for ARDS and with ongoing ARDS had daily measurements of vital signs and physiologic parameters according to standardized protocols, including hypoxemia, expressed as the PaO2/FIO2 ratio, tidal volume, and static compliance.
Cytokine Measurements in BALF
Immunoassay and protein analysis
TNF-, sTNF-RI, sTNF-RII, IL-1, IL-1ra, sIL-1RII, IL-10, IL-6, and sIL-6R measurements were performed by enzyme immunoassay using commercially available reagents (R&D Systems, Minneapolis, MN). The assay sensitivities were < 20-50 pg/ml for all measured proteins. The concentration of total
protein was measured by the bicinchoninic acid method (Pierce,
Rockford, IL).
TNF--specific bioassay.
TNF- bioactivity in BALF was measured with L929 cells as described previously with minor modifications (22, 23). A standard curve was created from dilutions of recombinant human TNF- (R&D Systems) and concentrations of TNF-
were determined from the linear range of the standard curve. The
specificity of the assay for TNF- was confirmed by neutralization of
the measured bioactivity with a murine anti-human TNF- antibody
(R&D Systems).
IL-1-specific bioassay
To assess the net biological activity of IL-1, we measured the upregulation of intercellular adhesion molecule
1 (ICAM-1) on the human epithelial lung adenocarcinoma A549 cell
line (American Type Culture Collection, Rockville, MD) as described
(24). The IL-1 activity was calculated using results from the linear
portion of the standard curve. The specificity of the assay for IL-1 in
BALF was determined with an anti-human TNF- antibody or a recombinant human IL-1 receptor antagonist as described (24).
IL-6-specific bioassay IL-6 bioactivity in BALF was assayed by measuring the proliferation of the IL-6-dependent murine hybridoma cell line B9 (courtesy of P. Lansdorp, BC Cancer Research Center, Vancouver, British Columbia, Canada), as described (25, 26). The IL-6 bioactivity in each well was calculated from the linear portion of the standard curve.
Statistical Analysis
For normally distributed data, the Student two-tailed t test was used for comparisons between two groups. The Mann-Whitney test was used for nonnormally distributed data. Correlations between cytokines, bioassays, and physiological parameters were calculated with Spearman correlation coefficients. The mortality associations were determined by Wilcoxon analysis. Multivariate analysis of the influence of IL-6 and sIL-6R was conducted by linear regression analysis.
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Patient Population
We prospectively identified 46 patients with ARDS and 23 patients at risk for ARDS between January 1994 and November 1997. Seven of the at risk patients developed ARDS and were included in the ARDS data. The BALF from six normal volunteers was included for comparison. The clinical characteristics of the patient groups are summarized in Table 1. The at risk and ARDS populations were closely matched for age, sex, and APACHE II score. The number of patients with sepsis as a primary risk was similar in both groups, but there was a significantly higher percentage of patients with trauma in the at risk population (p < 0.05). The mortality of patients with ARDS was higher than the mortality of patients at risk for ARDS (23.9 versus 12.5%). The variation in the number of patients on different study days reflects changes in clinical status, including successful extubation or death, as well as the enrollment of some patients on Day 3 of ARDS.
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TNF- and Soluble TNF Receptors
TNF- was detectable by immunoassay in a minority of the
BALF samples from patients at risk for ARDS (10.5% of
BALF on Day 1 of at risk period, 7.7% of BALF on Day 3 of
at risk period) (see Figure E1 in the online data supplement).
TNF- was also detectable in a minority of BALF specimens
from patients with established ARDS from Days 1 to 7 (detectable in 28.6% of BALF on Day 1 of ARDS, 12.5% of
BALF on Day 3, 6.9% of BALF on Day 7). TNF- was not
present in measurable concentrations in the BALF from normal volunteers. The measurable TNF- concentrations were
highest in the BALF on Day 1 of established ARDS and declined progressively after the onset of ARDS to undetectable
levels after Day 7 of ARDS.
In contrast to TNF-, sTNF-RI and sTNF-RII were detectable in nearly all of the BALF from patients at risk for ARDS
and patients with established ARDS for up to 21 d after the
onset of ARDS (Table 2). TNF receptors were also present in
all of the normal BALF specimens. The median sTNF-RI and
sTNF-RII concentrations increased more than 10-fold at the
onset of ARDS (p < 0.05) and then declined during the course
of ARDS (see Figures E2 and E3 in the online data supplement).
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The soluble TNF- receptors, sTNF-RI and sTNF-RII,
neutralize TNF- with 1:1 stoichiometry. The molar TNF-/
sTNF-RI and TNF-/sTNF-RII ratios in patients at risk for
ARDS were significantly lower on Days 1 and 2 than in normal volunteers (Figures 1 and 2). These ratios were even
lower at the onset of ARDS, and on Days 1 and 3 were less
than 15% of the values measured in normal volunteers (p < 0.005). In patients with sustained ARDS, the TNF-/sTNF-RI and TNF-/sTNF-RII ratios increased with time, reaching
near normal values on Days 14 and 21 of ARDS.
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A bioassay for TNF- activity was used to measure the net
balance between TNF- and the soluble TNF- receptors in
BALF (Figure 3). Net TNF- bioactivity was measured in
BALF from 6 normal volunteers, 10 patients on Day 1 of
ARDS, and 9 patients on Day 7 of ARDS. Net TNF- bioactivity was not detectable in five of six normal volunteers. TNF-
bioactivity was detectable in all of the BALF samples from patients on Day 1 of ARDS (19.1 ± 25.1 pg/ml) and on Day 7 of
ARDS (53.5 ± 39.3 pg/ml). The median TNF bioactivity increased significantly between Days 1 and 7 of ARDS (p = 0.034), which paralleled the increase in the median TNF-/
sTNF-RI and TNF-/sTNF-RII ratios from Day 1 to Day 7 of
ARDS. Despite this similar pattern, there was no significant
correlation between the results of the TNF- bioassay for individual patients and the individual measurements of immunoreactive TNF-, sTNF-RI, and sTNF-RII, or the ratios between TNF- and either soluble receptor.
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IL-1 and IL-1 Antagonists
Interleukin 1 immunoreactivity was detectable in the BALF
from normal volunteers, patients at risk for ARDS, and patients with established ARDS for up to 21 d after the onset of
ARDS (Table 2). The concentrations of IL-1 in BALF from
patients at risk for ARDS (at-risk Days 1 and 3) were elevated
above those measured in BALF from normal volunteers. The
IL-1 concentration in patients with established ARDS was
highest at the onset of ARDS on Day 1 of ARDS (p < 0.05)
(see Figure E4 in the online data supplement). Thereafter, the
IL-1 concentration declined with time but remained elevated
for up to 21 d in patients with sustained ARDS. Like the soluble TNF receptors, IL-1ra and sIL-1RII were detectable in the
BALF from normal volunteers, patients at risk for ARDS, and patients with established ARDS (Table 2). The median
concentrations of IL-1ra and sIL-1RII increased more than 5- and 100-fold, respectively, at the onset of ARDS and remained
elevated for the first 3 d of ARDS (p < 0.005). Thereafter, the
median concentrations of IL-1ra and sIL-1RII declined (see
Figures E5 and E6 in the online data supplement).
The molar ratios of IL-1/IL-1ra and IL-1/sIL-1RII paralleled the decline in the molar ratios of TNF- and its soluble receptors in patients at risk for ARDS and early after the onset of
ARDS (Figures 4 and 5). The IL-1/IL-1ra and IL-1/sIL-1RII
ratios on Days 1 and 3 of the at risk period were lower than the
ratios measured in normal volunteers. These ratios were even
lower in patients on Days 1 and 3 of ARDS (p < 0.05). The ratios increased by Days 14 and 21 of sustained ARDS, returning
to values that were similar to those in normal volunteers.
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The net bioactivity of IL-1 was measured in BALF from
six normal patients, and in all patients at risk for ARDS or
with established ARDS. The IL-1 bioactivity increased at the
onset of ARDS and declined with time in patients with sustained ARDS (Figure 6). There was a significant relationship
between immunoreactive IL-1 and IL-1 bioactivity in BALF
on Day 1 of ARDS (r = 0.403, p = 0.018). This relationship
was strongest on Day 3 of ARDS (r = 0.849, p < 0.001). When
the results from all patients at all times were pooled, there was
a strong and significant relationship between immunoreactive
IL-1 and IL-1 bioactivity (r = 0.534, p < 0.001) (see Figure
E7 in the online data supplement). There was no significant relationship between IL-1 bioactivity and either IL-1ra or
sIL-1RII. There was also no relationship between IL-1 bioactivity and the ratios of IL-1 and either IL-1ra or sIL-1RII.
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IL-6 and sIL-6R
Immunoreactive IL-6 and sIL-6R were detectable in the BALF of normal volunteers, patients at risk for ARDS, and patients with established ARDS for up to 21 d after the onset of ARDS (Table 2). In the Day 1 and 3 BALF from patients at risk for ARDS, the median IL-6 concentrations detected by enzyme-linked immunosorbent assay (ELISA) were increased by 50-fold or more as compared with BALF from normal volunteers (p < 0.05 on Day 1 of at risk period). On Days 1 and 3 of established ARDS, the median IL-6 concentrations were approximately 5-fold higher than in BALF from patients at risk of ARDS, and 100-fold higher than in normal subjects (p < 0.005). In BALF from patients with sustained ARDS, the IL-6 concentration declined with time, but remained above the normal range for up to 21 d in patients with persistent ARDS (see Figure E8 in the online data supplement). The sIL-6R concentration was also increased in the BALF from patients at risk of and with established ARDS. Like IL-6, the sIL-6R concentrations were elevated on Days 1 and 3 in the BALF from patients at risk for ARDS (p < 0.05). The BALF sIL-6R concentration was increased on Days 1 and 3 of ARDS (p < 0.005), and fell with time in patients with persistent ARDS (see Figure E9 in the online data supplement).
In contrast to the low ratios of the respective soluble receptors or antagonists for TNF- and IL-1, the molar ratio of IL-6 to sIL-6R increased more than 10-fold in the BALF from patients at risk for ARDS (p < 0.05 on Day 1 of at risk period)
(Figure 7). In patients with established ARDS, the IL-6/sIL-6R
ratio was approximately 100-fold higher than in normal subjects (p < 0.005), and 3-fold higher than in patients at risk. The
IL-6/sIL-6R ratio declined with time but remained significantly
elevated at all times as compared with normal volunteers.
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The IL-6 bioactivity was detectable in BALF from patients at risk of and with established ARDS (Figure 8). Net IL-6 bioactivity was measured in BALF from 3 normal volunteers, and subgroups of 13 patients on Day 1 of the at risk period for ARDS and 16 patients on Day 1 of ARDS. There was a significant relationship between the IL-6 bioactivity and immunoreactive IL-6 in BALF from patients at risk on Day 1 (r = 0.929, p < 0.001) as well as in the BALF of patients on Day 1 of established ARDS (r = 0.938, p < 0.001). When the data from all patients at all times were pooled, there was a strong linear relationship between IL-6 concentration and IL-6 bioactivity (r = 0.995, p < 0.001) (see Figure E10 in the online data supplement). There also was a significant relationship between IL-6 bioactivity and IL-6 soluble receptor in BALF from patients at risk on Day 1 (r = 0.9, p < 0.001), as well as in BALF from patients with established ARDS on Day 1 (r = 0.644, p = 0.007).
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IL-10
IL-10 was detectable by ELISA in low concentrations (< 20 pg/ml) in the BALF of patients at risk for ARDS and patients with established ARDS, but not in the BALF of normal volunteers (Table 2). The BALF IL-10 was present in low concentrations on Days 1 and 3 of at risk patients. On Days 1 and 3 of ARDS, however, the concentration of IL-10 was significantly higher (p < 0.005). The median concentration of IL-10 in BALF on Day 1 of ARDS was approximately 2.5-fold higher than the median concentration in BALF on either Day 1 or 3 of patients at risk for ARDS (see Figure E11 in the online data supplement). In BALF from patients with sustained ARDS, the concentration of IL-10 declined to nearly undetectable levels by Day 21 of ARDS.
The molar ratio of TNF-/IL-10 declined like the molar ratios of TNF- and its soluble receptors before and after the
onset of ARDS (see Figure E12 in the online data supplement); however, the decline in the molar ratio of TNF-/IL-10
was less dramatic. The ratios of TNF-/IL-10 on Days 1 and 3 of the at risk period were 3-fold lower than the ratios measured in normal volunteers, but this change was not significant. The ratio was at least 4-fold lower in BALF from patients on Days 1 and 3 of ARDS compared with BALF from
normal volunteers but this also was not significant. By Days 14 and 21 of sustained ARDS the ratios increased gradually but
remained 2- to 3-fold lower than the ratios in BALF from normal volunteers.
Relationships between Cytokines, Inhibitors, and Clinical Parameters
To determine whether measures of cytokine balance were
associated with clinical or physiological outcomes, we investigated the relationships between the ratios of proinflammatory/
antiinflammatory mediators and mortality, lung dysfunction,
and lung injury. We used measures of lung compliance and hypoxemia expressed as the PaO2/FIO2 ratio as measures of lung
dysfunction and the BALF total protein concentration as a
measure of lung injury. In univariate analyses, on Day 7 of
ARDS the concentrations of immunoreactive IL-1 and IL-6
and the ratio of IL-6 to sIL-6R were significantly related to
subsequent mortality (p < 0.05). This was not true for measurements in BALF from at risk patients or in BALF immediately after the onset of ARDS (Days 1 and 3). There were significant positive relationships between the PaO2/FIO2 ratio and
the TNF/sTNF-RII ratio on Day 1 of the at risk period, and
with IL-1/IL-1ra on Day 7 of ARDS (p < 0.05). There were
significant positive relationships between lung compliance and
the individual of IL-1/IL-1ra, IL-1/sIL-1RII, IL-6/sIL-6R,
TNF/sTNF-RI, and TNF/sTNF-RII ratios on Days 1 and 3 of
ARDS (see Table E1 in the online data supplement). The total protein concentration in BALF from patients at risk for
ARDS and with established ARDS was significantly related
to each of the individual soluble receptors and receptor antagonists. These relationships were present on all days studied,
before and after the onset of ARDS (Table E1).
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DISCUSSION
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The main goal of this study was to determine the immunological and biological balance between cytokine agonists and antagonists in the BALF of patients at risk for ARDS and with
established ARDS. We measured pro- and antiinflammatory
cytokines (IL-1, TNF-, IL-6, and IL-10) and specific agonists and antagonists (IL-1ra, sIL-1RII, sTNF-RI and -RII,
and sIL-6R) in patients at risk for ARDS and in patients with
ARDS. We found a major antiinflammatory response that
peaked early after the onset of ARDS, and exceeded the
proinflammatory response. The antiinflammatory mediators
included IL-1ra, IL-1RII, sTNF-RI, sTNF-RII, sIL-6R, and
IL-10. Each of these reached maximal concentrations in BALF
on Days 1 and 3 of ARDS, and returned to near normal values
by Day 14 of ARDS. This prominent antiinflammatory response in the lungs provides a mechanism for limiting the intensity of the inflammatory response in the soluble phase of
lung fluids before and after the onset of ARDS.
To evaluate the balance of pro- and antiinflammatory mediators, we calculated the molar ratios of cytokines and their
respective soluble receptors and receptor antagonists. The use
of molar ratios to reflect net biological activity is based on the
known biochemical interactions of TNF-, IL-1, and IL-6
with their respective antagonists or agonists, each of which interacts on a 1:1 stoichiometric basis (27). Findings from
both infectious and noninfectious inflammatory diseases suggest that the molar balance between pro- and antiinflammatory mediators may be a better measure of the net biological
effects of cytokines and may have greater clinical relevance. In
studies of rheumatoid arthritis, for example, mononuclear
cells from patients with active rheumatic disease release significantly higher ratios of IL-1 to IL-1ra than do those from
normal control subjects (14, 15). In addition, in patients with
inflammatory bowel disease, the IL-1/IL-1ra ratio is significantly higher than in normal subjects and is closely related to
disease severity (11, 33).
We were surprised to find that the molar ratios of TNF-
and IL-1 and their soluble receptors and antagonists declined by one to two orders of magnitude at the onset of
ARDS whereas the molar ratio of IL-6 to sIL-6R increased.
Although IL-6 has mixed pro- and antiinflammatory effects,
its effects are predominantly antiinflammatory (17). Thus, unlike other inflammatory diseases, the balance between TNF-,
IL-1, and IL-6 with their respective antagonists varies
throughout the course of ARDS in a pattern that suggests a
net antiinflammatory effect in the soluble phase sampled by
BAL at the onset of the disease.
We also found that IL-10 peaked on Day 1 of ARDS and
slowly declined to undetectable levels by Day 21. IL-10 is an
antiinflammatory cytokine and because TNF- expression
and IL-10 expression are closely related (34), it has been
hypothesized that the ratio of IL-10 to TNF- is important in
determining risk for ARDS and outcome (38). We found that
the molar ratio of TNF- to IL-10 decreased at the onset of
ARDS and rose to near normal levels as ARDS progressed.
Taken together, the observations about the IL-10 concentration and TNF-/IL-10 ratio suggest significant antiinflammatory effects in the airspaces at the onset of ARDS. These results are consistent with those of Donnelly and coworkers, who found elevated concentrations of IL-10 and IL-1ra in the
BALF of 28 patients with ARDS (10). However, our results
differ from those of Armstrong and Millar, who found significantly lower concentrations of IL-10 and an increase in the
TNF-/IL-10 ratio in a small group of patients with ARDS (n = 10), as compared with patients at risk for ARDS (38). Overall,
our measurements of IL-10, IL-6/sIL-6R, and the ratios of
TNF- and IL-1 with their respective antagonists consistently support a predominantly antiinflammatory profile in
the soluble environment in the lungs of patients at the onset of ARDS.
Although the ratios of cytokines and their soluble receptors and antagonists suggest an antiinflammatory balance in
the lungs, ARDS is characterized by an explosive acute inflammatory response associated with infection, trauma, and
other clinical risks. To further examine the relationships between pro- and antiinflammatory cytokines and to assess the
validity of the cytokine agonist:antagonist ratios, we used specific bioassays to measure the net biological activity attributable to TNF-, IL-1, and IL-6. The results of these bioassays
differed for each cytokine. The TNF- bioassay showed low
values of TNF- bioactivity on Day 1 of ARDS that were significantly higher on Day 7 of ARDS, when the ratio of TNF- to its inhibitors increased. The IL-1 bioassay showed measurable IL-1 activity at the onset of ARDS, and correlated most
strongly with immunologically measured IL-1. The IL-6 bioassay was strongly related to the measured IL-6 and sIL-6R
values as well as to the IL-6/sIL-6R ratio.
The parallel rise between TNF- bioactivity from Day 1 to
Day 7 of ARDS and the rising ratios of TNF- to its soluble
receptors suggests that the molar ratio may be a more accurate measure of net TNF- activity. Measurements of TNF-
concentration by immunological assays have several potential
limitations. Specifically, the TNF- immunoassays could detect biologically inactive precursors or be unable to detect active, protein-bound TNF-. Most importantly, the immunoassays do not measure either the presence or the biological effects of the soluble TNF- receptors.
In contrast to the findings with TNF-, there was a significant relationship between the IL-1 bioassay and the immunologic measurement of IL-1. The lack of a significant correlation between IL-1ra or sIL-1RII and the net biological
activity of IL-1 is not surprising. IL-1 receptor I (IL-1RI) is
the primary signal transducing receptor for IL-1 and is the
site of inhibition by IL-1ra (28). Although not measured in
this study, soluble IL-1RI is released during inflammation and
maintains its ability to bind IL-1ra, significantly reducing the
availability of IL-1ra and dampening its effect on IL-1 activity (29). In addition, the cell-bound IL-1RI receptor is extremely sensitive to IL-1 and requires only low concentrations of IL-1 to initiate transmembrane signaling because of
postreceptor amplification through multiple phosphorylation
steps (39). Thus, our results support the conclusion that despite the presence of significant concentrations of specific IL-1
antagonists, the concentration of free IL-1 remains the best
immunologic measure of net IL-1 bioactivity in lung fluids.
The IL-6 bioassay had a strong relationship with both the soluble ligand and its soluble receptor as well as the IL-6/sIL-6R ratio. Linear regression analysis, however, did not identify a significant independent contribution of sIL-6R to the net IL-6 bioactivity, as measured in the B9 hybridoma assay. This result might seem surprising, as previous work demonstrated the sensitivity of B9 cells to the agonist effects of sIL-6R at concentrations as low as 5 ng/ml (40). This effect diminishes, however, at concentrations of IL-6 above 10 pg/ml (41). In BALF of patients at risk for ARDS and with established ARDS, the concentration of IL-6 was always higher than 10 pg/ml. Thus, our results support the conclusion that despite significant concentrations of sIL-6R in BALF, the contribution of sIL-6R to the net bioactivity of IL-6 in BALF from patients with ARDS is small, most likely because of the high IL-6 concentrations.
A second goal of this study was to determine whether relationships existed between the presence or activity of the inflammatory mediator systems and either the clinical severity
or outcome of ARDS. We found significant relationships between lung compliance and the IL-1/sIL-1RII, IL-6/sIL-6R,
TNF/sTNF-RI, and TNF/sTNF-RII ratios on Days 1 and 3 of
ARDS. There were also significant relationships between the
degree of hypoxemia and TNF/sTNF-RII on Day 1 of the at
risk period and with IL-1/IL-1ra on Day 7 of ARDS. This
may be a function of endothelial/epithelial capillary leak, as
suggested by the strong relationship between soluble receptors and the total protein concentration measured in the BAL
of patients at risk of ARDS and with established ARDS. This
could reflect either local production in the lungs or movement
from the plasma into the lungs when significant injury occurs
to the endothelial-epithelial barrier (42).
The IL-6/sIL-6R ratio was the only molar ratio that was significantly related to outcome, with a higher ratio associated
with a higher risk of death. As in prior studies, we found a significant relationship between IL-1, IL-6, and subsequent
mortality on Day 7 of ARDS (2, 5, 6). This finding is consistent with the central role of IL-1 in ARDS and is additional
evidence that sustained inflammation in the lungs is associated
with a worse outcome.
Several limitations of this study should be noted. First, the
BAL method samples only soluble constituents in the airspaces. Biologically active tissue and membrane-bound cytokines and
other ligands are not measured by this technique. Examples include membrane-bound TNF- (43), soluble IL-1RI and IL-1R
accessory protein (44), and soluble autoantibodies to IL-6 as
well as a soluble form of signal-transducing gp130 protein (9,
45). From a clinical standpoint, the study population does
not include patients with ARDS who improved rapidly and
were extubated, or patients who were the most severely ill and
were excluded for safety reasons. However, the patient population does provide an accurate representation of patients with
ARDS who remain intubated and mechanically ventilated.
In summary, we have found that at the onset of ARDS, high concentrations of antiinflammatory cytokines produce markedly lower molar ratios of proinflammatory to antiinflammatory mediators in the lungs. This provides a mechanism for dampening the otherwise intense inflammatory response in the airspaces. These results highlight the complexity of the inflammatory response in the lungs of patients with ARDS and show that individual cytokine measurements cannot be considered in isolation but must be understood in the context of the balance between agonistic and antagonistic responses in the lungs.
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Footnotes |
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Correspondence and requests for reprints should be addressed to Thomas R. Martin, M.D., Pulmonary Research Laboratories, 151L, Seattle VA Medical Center, 1660 S. Columbian Way, Seattle, WA 98108. E-mail: trmartin{at}u.washington.edu
(Received in original form April 3, 2001 and accepted in revised form August 21, 2001).
This article has an online data supplement, which is accessible from this issue's table of contents online at www.atsjournals.orgAcknowledgments: The authors thank Ellen Caldwell and Todd Freudenberger for advice about the statistical analysis of the data.
Supported in part by grants HL30542 and AI29103 from the National Institutes of Health and the Medical Research Service of the Department of Veterans Affairs.
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References |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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