Departments of 1 Internal Medicine, 2 Integrative Biology, and 3 Surgery, Trauma Research Center, University of Texas Health Science Center, Houston, Texas 77030
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ABSTRACT |
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Gastrointestinal stasis during sepsis may be
associated with gastrointestinal smooth muscle dysfunction. Endotoxin
[lipopolysaccharide (LPS)] impairs smooth muscle
contraction, in part through inducible nitric oxide synthase (NOS II)
and enhanced nitric oxide production. We studied the roles of tumor
necrosis factor- (TNF) and interleukin-1 (IL-1) in this process by
using TNF binding protein (TNFbp) and IL-1 receptor antagonist
(IL-1ra). Rats were treated with TNFbp and IL-1ra, or their vehicles, 1 h before receiving LPS or saline. At 5 h after LPS, contractility was
measured in strips of ileal longitudinal smooth muscle, and NOS II
activity was measured in full-thickness segments of ileum. LPS
decreased maximum stress (mean ± SE) from 508 ± 55 (control) to
355 ± 33 g/cm2
(P < 0.05). Pretreatment with TNFbp
plus IL-1ra prevented the LPS-induced decrease. Separate studies of
TNFbp alone or IL-1ra alone indicated that, at the doses and timing
used, TNFbp was more effective. LPS also increased NOS II activity by
>10-fold (P < 0.01) over control.
This increase was prevented by TNFbp plus IL-1ra
(P = not significant vs. control). We
conclude that the LPS-induced increase in NOS II activity and the
decrease in ileal muscle contractility are mediated by TNF and IL-1.
tumor necrosis factor; interleukin-1; tumor necrosis factor binding protein; interleukin-1 receptor antagonist; soluble tumor necrosis factor receptors; sepsis; nitric oxide; inducible nitric oxide synthase; nitric oxide synthase activity; intestinal transit; endotoxin; ileal smooth muscle
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INTRODUCTION |
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ALTHOUGH SEPSIS is the most common cause of death in
medical intensive care units, with a mortality of ~50%, its
pathophysiology is only incompletely understood. The syndrome of sepsis
is a systemic inflammatory response to invading microorganisms. This
response is now believed to be the result of a cascade of mediators:
microbial toxins, such as lipopolysaccharide (LPS) endotoxin of
gram-negative bacteria, cause the release by the host of potent
proinflammatory mediators, which, in turn, are largely responsible for
the cardiovascular derangement, shock, multiple organ failure, and
death (26, 28). The proinflammatory cytokines interleukin-1 (IL-1) and
tumor necrosis factor- (TNF) are principal among the host mediators.
Gastrointestinal stasis, or ileus, commonly accompanies sepsis and multiorgan dysfunction in critically ill patients and may contribute to their pathophysiology. The mechanism of this impairment of transit during sepsis is unclear but may be associated with impaired contractile function of gastrointestinal smooth muscle. The administration of LPS has been shown to result in a depression in contractility of skeletal (15), cardiac (3, 33), and smooth muscle, especially vascular smooth muscle (22, 32), in several animal models. In a previous study, we demonstrated that LPS administration to rats resulted in a decrease in contractility of ileal longitudinal smooth muscle (40). The reduction in contractility was associated with an upregulation of the inducible isoform of nitric oxide synthase (NOS II) and could be reversed by addition of a competitive inhibitor of NOS.
The current study was designed to test the hypothesis that the reduction in contractility and the upregulation of NOS activity induced by LPS are mediated by TNF and IL-1. We studied the roles of these two cytokines by pretreating the animals with their respective cytokine antagonists, TNF binding protein (TNFbp) and IL-1 receptor antagonist (IL-1ra), which specifically inhibit their action.
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METHODS |
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Cytokine antagonists. TNFbp and IL-1ra were gifts of Synergen (Boulder, CO). Production of the TNFbp has been described in detail by Su et al. (35). Soluble TNF receptor I was produced by the introduction of a synthetically constructed gene encoding the free extracellular domain of human TNF receptor I into an Escherichia coli expression vector. The recombinant protein contains an additional amino terminal methionine and an asparagine/cysteine substitution (amino acid 105) to allow the attachment of a polyethylene glycol (PEG) linker. After the soluble TNF receptor I monomer was extracted and purified, a dimeric TNFbp was synthesized by covalent attachment of two soluble TNF receptor I monomer molecules to a bifunctional 20-kDa PEG group via cysteine residue 105 of the protein. The resulting TNFbp dimer has a molecular mass of ~56 kDa. It was dissolved in neutral PBS, and before administration it was diluted to a concentration of 1.5 mg TNFbp/ml. Pharmacokinetic studies indicate that the efficacy (minimal effective dose) of TNFbp dimer in animal models occurs at doses ranging from 0.3 to 5 mg/kg body wt administered every other day (35).
A nonglycosylated form of human IL-1ra has been isolated from human monocytes in conditioned medium (16) and produced by recombinant DNA technology in E. coli (8). Recombinant human IL-1ra is identical to the naturally occurring nonglycosylated human form of IL-1ra with the exception of the addition of one N-terminal methionine (12). It was formulated at a concentration of 100 mg IL-1ra/ml in a salt solution (CSE vehicle) containing 10 mM citrate, 140 mM sodium chloride, and 0.5 mM EDTA, at pH 6.5 (31).
Protocols. Two experimental protocols
were followed: the first was designed to test the ability of the
combined administration of TNFbp and IL-1ra to attenuate the effects of
LPS administration; the second was designed to test the effects of each
cytokine antagonist separately. To complete the first protocol, male
Sprague-Dawley rats, 275-300 g, were divided into four groups of
six animals each and were fasted overnight. One group (TNFbp + IL-1ra + LPS) was injected with TNFbp (1.5 mg/kg iv) plus IL-1ra (100 mg/kg sc)
1 h before [time (t) = 1 h] an intraperitoneal injection (at
t = 0 h) of LPS from
E. coli (serotype 0111:B4; 20 mg/kg
body wt; Sigma Chemical, St. Louis, MO). The second group (TNFbp + IL-1ra + saline) was similarly injected with both cytokine
antagonists at t =
1 h but
received at t = 0 h an intraperitoneal
injection of an equal volume of normal saline, the vehicle for LPS. The third group (vehicles + LPS) was injected at
t =
1 h with the respective vehicles for the two cytokine antagonists followed with LPS
at t = 0 h. The vehicle for IL-1ra was
CSE buffer. The vehicle for TNFbp was neutral PBS. The fourth group
(vehicles + saline), which served as the control group, was injected
with cytokine antagonist vehicles at t =
1 h and with saline at t = 0 h. Because the effective half-lives of TNFbp and IL-1ra administered in
this manner are ~30 and 6 h, respectively (Synergen, personal communication), a second injection of either IL-1ra or its vehicle was
administered at t = +2 h. At
t = +5 h after LPS or saline injection, the animals were killed, and full-thickness sections of
ileum were taken from each animal. These sections were taken such that
paired determinations of contractility and NOS II activity could be
determined for each animal.
The second protocol was similar to the first except that it tested the effects of single rather than combined cytokine antagonist treatment. Four additional groups of six male Sprague-Dawley rats, 275-300 g, were used. After an overnight fast, one group (IL-1ra + LPS) was treated with IL-1ra followed in 1 h with LPS, a second group (TNFbp + LPS) was treated with TNFbp followed in 1 h with LPS, a third group (vehicles + LPS) was treated with vehicle followed in 1 h with LPS, and a fourth group (vehicles + saline) was treated with vehicle followed in 1 h with saline. As above, the groups that received IL-1ra or its vehicle received a second injection of the same at t = +2 h. At t = +5 h after LPS or saline injection, full-thickness sections of ileum were taken from each animal.
For the contractility studies, strips of longitudinal muscle ~1 cm long were peeled from segments of ileum that were removed 1 cm orad from the ileocecal junction as we have previously described (40). Samples of full-thickness segments of ileum that were to be analyzed for NOS activity were frozen immediately after recovery from the animals.
Ileal muscle contractility. Strips,
two from each segment, were mounted in 10-ml organ baths filled with
Krebs-Ringer solution (in mM: 103 NaCl, 4.7 KCl, 2.5 CaCl2, 25 NaHCO3, 1.1 NaH2PO4, and 15 glucose). The solution was gassed with 5%
CO2-95%
O2. Isometric force was monitored
by an external force displacement transducer (Grass FT.03; Grass
Instrument, Quincy, MA) connected to a Beckman R411 recorder (Beckman
Instruments, Electronic Instrument Division, Schiller Park, IL; see
Ref. 40). Each strip was allowed to equilibrate for at least 20 min,
and then 105 M
carbamylcholine chloride [carbachol (CCh); Sigma Chemical] was added. After peak active force was reached, the bath was washed with fresh solution and allowed to stand for 20 min. The force of
contraction produced by CCh was recorded at increasing muscle lengths.
After the optimal length for active force development was found, each
strip was washed with fresh solution and allowed to equilibrate for 20 min at the optimal length. Next, L-arginine (L-Arg; 10
3 M;
Sigma Chemical) was added to the bath (40). Ten minutes later, CCh was
added stepwise to the organ bath to final concentrations of
10
8 to
10
4 M, and active force
development was recorded at each concentration to determine the
dose-response relationship. After recording contractile activity, the
length of each strip was measured, and each strip was removed, blotted
lightly, and weighed. The cross-sectional area of each strip was
calculated from length and weight data by assuming that the density of
smooth muscle was 1.05 g/cm3. All
force development was normalized to tissue cross-sectional area and is
expressed as stress. EC50 for CCh
was determined by probit analysis (14). One-way ANOVA and, where
appropriate, Duncan's multiple-range test were used to compare values
of EC50 and maximum stress among
the four groups (13). P < 0.05 was considered statistically significant.
NOS activity. NOS activity was assayed
by monitoring the conversion of
L-[3H]Arg
to
L-[3H]citrulline
using a modification of a procedure previously described (4). Tissue
was homogenized (250 mg tissue/ml buffer) utilizing three successive
20-s bursts in an Ultra-Turrax homogenizer set at maximum speed. The
homogenization buffer contained 0.05 M Tris (pH 7.4), 0.1 mM EDTA, 1 mM
dithiothreitol, 100 mU/ml aprotinin, 25 µg/ml phosphoramidon, 10 µg/ml leupeptin, 10 µg/ml soybean trypsin inhibitor, 10 µg/ml
phenylmethylsulfonyl fluoride, and 1% Nonidet P-40. The Bio-Rad
protein assay (Bio-Rad, Hercules, CA) was used to determine protein
concentrations in column effluents. NOS activity assays were conducted
by diluting samples containing 100 µg protein to a final volume of
100 µl, which contained 0.05 M Tris (pH 7.4), 1.0 mM dithiothreitol,
50 µM L-Arg containing 106 dpm
L-[3H]Arg,
1.0 mM NADPH, 100 U/ml calmodulin, and 10 µM tetrahydrobiopterin. To
assay the Ca2+-independent NOS
(NOS II) activity, Ca2+ was
chelated with EDTA (1 mM). Reactions were conducted in the presence and
absence of 0.3 mM
NG-nitro-L-arginine
methyl ester (L-NAME) and were stopped after 1 h with 1 ml
of ice-cold Tris buffer (pH 5.5) containing 2 mM EDTA. Samples were
applied to chromatography columns containing 2 ml of AG 50W-X8 resin
that then were washed two times with 1 ml of Tris buffer (pH 5.5). The
radioactivity present in 2 ml of column effluent was quantified by
liquid scintillation counting. Product formation that was inhibited by
incubation with L-NAME was taken as an index of NOS
activity and was calculated from the total of added substrate, as the
formation of citrulline
(pmol · h1 · mg
protein
1). NOS II
activities in tissues from the various groups were compared using the
same statistical approach as that outlined above for contractiltity.
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RESULTS |
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Ileal muscle contractility. Strips of
longitudinal muscle from the ileum of all animals studied under both
protocols contracted in response to CCh in a concentration-dependent
manner. Figure 1 shows the stress developed
by ileal muscle strips in response to increasing concentrations of CCh
for the four groups in the first protocol. LPS (vehicles + LPS) caused
a decrease in contractility below control levels (vehicles + saline; P < 0.05). Cytokine
antagonist treatment before LPS (TNFbp + IL-1ra + LPS) prevented the
LPS-induced decrease in contractility, but cytokine antagonist
treatment itself (TNFbp + IL-1ra + saline) had no effect
[P = not significant (NS)] on the contractility of strips from healthy rats.
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Figure 2 shows the maximum stress developed
by the strips of rat ileal longitudinal smooth muscle in response to
CCh (104 M) for the four
groups in the first protocol. The control group (vehicles + saline) had
a maximum stress of 508 ± 55 g/cm2 (mean ± SE), which was
not affected by cytokine antagonist treatment itself (TNFbp + IL-1ra + saline; 498 ± 23 g/cm2;
P = NS). LPS treatment (vehicles + LPS) significantly decreased the maximum stress to 355 ± 33 g/cm2
(P < 0.05). Cytokine antagonist
treatment before LPS (TNFbp + IL-1ra + LPS) prevented the
LPS-induced decrease in maximum stress (486 ± 48 vs. 355 ± 33 g/cm2;
P < 0.05), yielding a maximum stress
value not different from control (P = NS).
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Figure 3 shows the
EC50 values, derived from the
contractility curves in Fig. 1, for the four groups in the first
protocol. For the control group (vehicles + saline),
EC50 was 3.46 × 108 M
(log10 units,
7.46 ± 0.11, mean ± SE). EC50 was not
significantly affected by LPS or cytokine antagonist treatment.
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Figure 4 shows the maximum stress developed
by the strips of rat ileal longitudinal smooth muscle in response to
CCh (104 M) for the four
groups in the second protocol. These values were derived from full
dose-response curves (10
8
to 10
4 M CCh) similar to
those of Fig. 1. The control group (vehicles + saline) had a maximum
stress of 498 ± 50 g/cm2. As
in the first protocol, LPS (vehicles + LPS) significantly reduced the
maximum stress (386 ± 16 g/cm2;
P < 0.05). This LPS-induced
reduction in maximum stress was prevented by pretreatment with TNFbp
alone (TNFbp + LPS; 491 ± 24 g/cm2) and ameliorated by
pretreatment with IL-1ra alone (IL-1ra + LPS; 431 ± 24 g/cm2). The maximum stress for
pretreatment with TNFbp alone was significantly greater than that for
LPS (P < 0.05) and not different
from that of control (P = NS). Maximum
stress for IL-1ra pretreatment was intermediate between the control and
LPS values, but these differences did not reach statistical
significance. As observed in the first protocol, the
EC50 for CCh did not differ among
the four groups in the second protocol.
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NOS II activity. The NOS activity that
was calcium independent (NOS II activity) and inhibited by
L-NAME is presented in Fig. 5.
Activity was detected in ilea from control animals (vehicles + saline).
Cytokine antagonist treatment by itself (TNFbp + IL-1ra + saline)
tended to decrease this basal activity, but the change did not reach
significance. LPS (vehicles + LPS) increased ileal NOS II activity by
>10-fold (P < 0.01). This marked
increase in NOS II activity by LPS was largely prevented by cytokine
antagonist pretreatment (TNFbp + IL-1ra + LPS), which resulted in NOS
II activity that was significantly less than that for LPS treatment (P < 0.01) and not significantly
different from control (P = NS).
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Correlation between NOS II activity and maximum
stress. In the first protocol, both NOS II activity and
maximum stress were determined in the same animals. A plot of maximum
stress as a function of NOS II activity for these animals is shown in
Fig. 6. There was a statistically
significant (P < 0.03) inverse
relationship between NOS II activity and maximum stress.
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DISCUSSION |
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Prolonged gastrointestinal stasis, or ileus, is a common complication of sepsis and, in fact, may perhaps be considered a single-organ example of the multiorgan dysfunction that characterizes severe sepsis. We and others have shown that a decrease in transit allows the overgrowth of bacteria within the lumen of the small intestine and eventual translocation of bacteria from the gut into the regional lymph nodes (23, 24, 30). These observations indicate that gut stasis may be not only a result of sepsis but also a cause of sepsis, particularly of persistent or recurrent sepsis, by virtue of such a "vicious cycle." The mechanisms of the impairment of gastrointestinal transit are not clearly defined but may be associated with impairment in the contractile function of smooth muscle.
We have previously demonstrated that LPS, given in vivo to healthy rats, causes a decrease in the contractility of ileal smooth muscle, measured in vitro (40). In addition, we showed that this decrease in contractility is associated with an LPS-induced upregulation of NOS II mRNA, immunoreactivity, and enzymatic activity in the mucosal and muscle layers of the small intestine (40). We also showed that the impaired contractility could be reversed by L-NAME, a competitive inhibitor of NOS. Similar results have been reported for vascular smooth muscle (32).
To investigate the mechanisms of LPS-induced impairment of gut smooth muscle contractile function, we took advantage of the large body of information on the mechanisms of cardiovascular impairment in sepsis. The cardiovascular derangement in severe sepsis is characterized by impairment of both cardiac and vascular smooth muscle function. When severe, these impairments result in vasorelaxation, hypotension, and hypoperfusion that are refractory to intravenous infusion of fluids and to vasoconstrictor and inotropic agents (25). If uncorrected, this cardiovascular compromise leads ultimately to multiorgan failure and death (26, 28). Extensive evidence now indicates that the cardiovascular derangement in sepsis is the result of a complex cascade of mediators. This cascade begins with a nidus of infection-releasing organisms or their products, such as LPS, which in turn cause the release by the host of numerous proinflammatory mediators. Principal among the proinflammatory mediators are TNF and IL-1. We (19-21) and others (2, 10, 18, 29, 37, 38) have shown that the cardiovascular derangement caused by LPS, TNF, or IL-1 is largely mediated by the induction of NOS II, resulting in excessive production of nitric oxide.
The discoveries of the natural occurrence of endogenous antagonists to TNF and IL-1 (6, 9, 27) provide a unique opportunity to study the roles of these cytokines in the LPS-induced derangement in gastrointestinal motility. The biological actions of TNF are mediated by two distinct cell surface receptors, a 55-kDa type I (p55) receptor and a 75-kDa type II (p75) receptor. Soluble forms of these receptors are shed by proteolytic cleavage (serine protease) of their extracellular domains. Soluble TNF receptors inhibit the action of TNF by the formation of high-affinity complexes, thereby preventing TNF from binding to target cell membrane receptors (6, 27). Soluble TNF receptors are found in the circulation of healthy individuals. Endotoxemia induces a severalfold increase in these receptors (34, 39), an effect mediated largely by the increase in TNF itself (17). In several animal models of sepsis and inflammation, administration of the soluble p55 TNF receptor has reduced inflammation and prolonged survival (6, 27). In other approaches, each of these soluble TNF receptors has been fused with the Fc portion of IgG1, forming chimeric "fusion proteins." In human patients with sepsis, a phase II trial of the p55-IgG1 construct had favorable results (the results of a phase III trial are pending; see Ref. 1). A phase III trial of the p75-IgG1 construct (11) was disappointing, but the difference has been attributed in part to the more stable kinetics (much slower exchange rate) of the p55 fusion protein (1).
IL-1ra was recently identified as the third member of the IL-1 gene
family, joining IL-1 and IL-1
(5). IL-1ra binds to the type I
cell-surface IL-1 receptor with an affinity similar to that of IL-1
and IL-1
, but it has no agonist effect, even at high concentrations.
Thus IL-1ra functions as a naturally occurring, specific antagonist of
both IL-1
and IL-1
by blocking the binding and the biological
activities of these two cytokines (5). Blood levels of IL-1ra are
substantially increased by endotoxin, IL-1 itself, or TNF (5). In a
number of animal models of sepsis, early administration of IL-1ra
(before significant IL-1 synthesis and release) has been shown to
decrease both mortality and morbidity (5, 7) and to attenuate the
LPS-mediated induction of NOS II (36). Although the results of an
initial phase III trial of IL-1ra in human patients were encouraging
(12), a subsequent phase III trial was disappointing (6), perhaps due
in part to the relatively late administration of IL-1ra (up to 3 days after the onset of the infection causing the sepsis).
In the present study, we used specific formulations of the two cytokine antagonists, TNFbp and IL-1ra, to study the roles of their corresponding proinflammatory cytokines in mediating the LPS-induced decrease in ileal smooth muscle contractility and increase in NOS II activity.
The present study confirms our previous findings (40) that LPS causes a decrease in the contractility of ileal smooth muscle and an increase in ileal NOS II activity. As previously, we also showed that the reduction in contractility was characterized by a decrease in maximum response but with no significant change in the concentration of CCh that elicited 50% of that maximum response, that is, LPS did not result in a rightward shift in the dose-response curves.
In our earlier study (40), we found that 20 mg/kg LPS induced an increase in NOS II mRNA and immunoreactivity (protein) that could be detected both in full-thickness samples of ileum and samples of longitudinal muscle plus serosa. Furthermore, for each individual animal, the magnitude of the increases in the muscle layer appeared to correlate with that in full-thickness samples even though absolute levels in the muscle were lower. Only qualitative changes were assessed because the methods used to determine the increases in mRNA and immunoreactivity are not quantitative. To be quantitative, NOS II enzymatic activity was assayed in the present study. Although we successfully determined NOS II activity in full-thickness samples of ileum, our attempts to determine NOS II activity in samples of longitudinal muscle plus serosa were not successful, presumably due to the lower abundance of the enzyme in the muscle layer. In the present study, our goal of correlating muscle contractility with NOS II activity (see Fig. 6) required quantitative data. Thus we determined NOS II activity quantitatively in full-thickness samples. It is possible that the cytokine antagonists selectively inhibited the upregulation of NOS II only in the nonmuscle layers of the intestine. However, we are aware of no studies showing a selective action based on anatomical location. Thus it is more likely that the changes in NOS II activity monitored in the combined layers of the intestine reflected the changes in NOS II activity in the longitudinal muscle layer.
The principal new findings in the present study were that both the decrease in ileal muscle contractility and the increase in NOS II activity were largely prevented by pretreatment with cytokine antagonists that inhibit the action of TNF and IL-1. At the doses and time intervals we employed, TNFbp alone was more effective than was IL-1ra alone in preventing the decrease in contractility, indicating that TNF may be the more dominant mediator. However, full dose-response and time-response relationships need to be elucidated before any primacy can be assigned. An additional new finding was that the level of contractility was negatively correlated with the level of NOS II activity. Overall, these results suggest that LPS impairs ileal smooth muscle contractility predominantly by stimulating the production of TNF and IL-1, which in turn act by induction of ileal NOS II, which produces increased levels of the potent smooth muscle relaxant nitric oxide. This suggested cascade of mediators is consistent with that proposed for the pathogenesis of sepsis in general (26, 28).
The present study provides evidence that the LPS-induced derangement in gut smooth muscle function, similar to the LPS-induced derangements in cardiac and vascular smooth muscle functions, is the result of the proinflammatory cascade that includes TNF, IL-1, and ultimately nitric oxide. Further exploration of this proinflammatory cascade may provide new insight into the pathogenesis of the prolonged gastrointestinal stasis that often complicates the course of patients with sepsis and may suggest novel therapeutic approaches.
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ACKNOWLEDGEMENTS |
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We thank Dr. D. Mailman for advice on the design of the experiments and for discussion of the results, Synergen, Inc., for the generous gifts of TNFbp and IL-1ra, and J. L. Bastin for expertise in helping to prepare this manuscript.
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FOOTNOTES |
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This study was supported in part by National Institute of General Medicine Grant GM-38529.
Portions of this work have been published in abstract form (FASEB J. 9: A86, 1995).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: R. F. Lodato, Div. of Pulmonary & Critical Care Medicine, Dept. of Internal Medicine, The Univ. of Texas Health Science Center, 6431 Fannin St., Suite 1-274, Houston, TX 77030 (E-mail: rlodato{at}heart.med.uth.tmc.edu).
Received 27 May 1998; accepted in final form 22 February 1999.
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