Laboratory of Hepatobiology and Toxicology, Department of Pharmacology, University of North Carolina, Chapel Hill, North Carolina 27599-7365
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ABSTRACT |
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Mortality associated with endotoxin shock is likely
mediated by Kupffer cells, alveolar macrophages, and circulating
neutrophils. Acute dietary glycine prevents mortality and blunts
increases in serum tumor necrosis factor- (TNF-
) following
endotoxin in rats. Furthermore, acute glycine blunts activation of
Kupffer cells, alveolar macrophages, and neutrophils by activating a
glycine-gated chloride channel. However, in neuronal tissue, glycine
rapidly downregulates chloride channel function. Therefore, the
long-term effects of a glycine-containing diet on survival following
endotoxin shock were investigated. Dietary glycine for 4 wk improved
survival after endotoxin but did not improve liver pathology, decrease serum alanine transaminase, or effect TNF-
levels compared with animals fed control diet. Interestingly, dietary glycine largely prevented inflammation and injury in the lung following endotoxin. Surprisingly, Kupffer cells from animals fed glycine for 4 wk were no
longer inactivated by glycine in vitro; however, isolated alveolar
macrophages and neutrophils from the same animals were sensitive to
glycine. These data are consistent with the hypothesis that glycine
downregulates chloride channels on Kupffer cells but not on alveolar
macrophages or neutrophils. Importantly, glycine diet for 4 wk
protected against lung inflammation due to endotoxin. Chronic glycine
improves survival by unknown mechanisms, but reduction of lung
inflammation is likely involved.
endotoxin shock; Kupffer cells; alveolar macrophages; neutrophils; tumor necrosis factor-
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INTRODUCTION |
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INFLAMMATORY CELLS SUCH
AS alveolar macrophages and Kupffer cells, the resident hepatic
macrophages, are thought to play critical roles in organ failure due to
endotoxin (3, 31). Both cell types produce
tumor necrosis factor- (TNF-
), interleukin-1 (IL-1), and other
toxic mediators that lead to tissue injury (11). Serum TNF-
is an important mediator because it is involved in signaling between several cell types and may potentiate macrophage activation and
tissue injury following endotoxin (4, 21).
The precise role of TNF-
due to endotoxin shock and the primary
source of serum TNF-
are still largely unknown; however, injection
of recombinant TNF-
mimics most of the effects of endotoxin
(22).
Importantly, it was reported recently that administration of dietary
glycine for 3 days blunted the increase in serum TNF- after
endotoxin injection, which markedly improved survival and minimized
liver and lung injury due to endotoxin in rats (6). Furthermore, glycine directly inactivated Kupffer cells and alveolar macrophages via activation of a glycine-gated chloride channel (7, 29). Therefore, it was concluded that
glycine protects the liver and the lung from injury due to endotoxin by
inactivating Kupffer cells and alveolar macrophages by blunting the
production of TNF-
(7). The pharmacological properties
of the glycine-gated chloride channel in the Kupffer cell parallel
those described for the spinal cord glycine-gated chloride channel
(7, 15). Glycine activates the channel, and
chloride enters the cells and hyperpolarizes the plasma membrane. This
prevents lipopolysaccharide (LPS)-induced increases in intracellular
calcium via voltage-dependent calcium channels. Furthermore, recent
molecular evidence showed that the glycine-gated chloride channel
exists in Kupffer cells with molecular properties similar to the
channel in spinal cord (27). Other white blood cells such
as neutrophils and lymphocytes recently have also been shown to possess
glycine-gated chloride channels (20, 28,
30).
It was hypothesized that the receptor in the Kupffer cell and alveolar macrophage would also be downregulated due to chronic exposure to glycine, like the glycine-gated chloride channel in the central nervous system (15). Therefore, the purpose of this study was to evaluate the effects of long-term feeding of glycine in an endotoxin shock model on Kupffer cell, alveolar macrophage, and neutrophil activation. Herein, it is reported that 4 wk of dietary glycine improved survival following endotoxin shock and indeed caused downregulation of the glycine receptor on Kupffer cells. However, long-term dietary glycine treatment had no effect on the glycine receptor in alveolar macrophages or neutrophils. Importantly, glycine blocked the accumulation of neutrophils in the lung.
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MATERIALS AND METHODS |
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Animals and diet. Male Sprague-Dawley rats weighing 200-250 g were given free access to a AIN-76 synthetic powdered diet containing 5% glycine or casein as a nitrogen-balanced control diet for up to 32 wk (17). Diets were a generous gift from Novartis Nutrition. Animals were individually caged under identical housing conditions, given humane care, and maintained in compliance with institutional guidelines.
Endotoxin treatment. After dietary treatment, various amounts of LPS (1-40 mg/kg; Escherichia coli serotype 0111:B4; Sigma, St. Louis, MO) in saline were injected intravenously via the tail vein, and survival was assessed over 24 h. At the end of experiments, serum and tissue were collected under pentobarbital sodium (75 mg/kg; Nembutal; Abbott Laboratories, North Chicago, IL) anesthesia. All treatments with LPS were performed simultaneously in both glycine- and casein-fed animals. Liver and lung specimens were fixed in phosphate-buffered Formalin and embedded in paraffin, and sections were stained with hematoxylin and eosin for histological evaluation.
Kupffer cell preparation and culture. Kupffer cells were isolated by collagenase digestion and differential centrifugation with Percoll (Pharmacia, Uppsala, Sweden) as described elsewhere with slight modifications (19). Briefly, the liver was perfused through the portal vein with Ca2+- and Mg2+-free Hanks' balanced salt solution (HBSS) at 37°C for 5 min at a flow rate of 40 ml/min. Subsequently, perfusion was with HBSS containing 0.025% collagenase IV (Sigma) at 37°C for 5 min at a flow rate of 26 ml/min. After the liver was digested, it was excised and minced in collagenase buffer. The suspension was filtered through nylon gauze, and the filtrate was centrifuged at 450 g for 10 min at 4°C. Cells were resuspended in buffer, parenchymal cells were removed by centrifugation at 50 g for 3 min, and the nonparenchymal cell fraction was washed twice with buffer. Cells were centrifuged on a density cushion of 50:25% Percoll (Amersham Pharmacia Biotech, Uppsala, Sweden) in HBSS at 1,000 g for 15 min. The Kupffer cell fraction was collected and washed with buffer. Cells were seeded onto 25-mm glass coverslips and cultured in Dulbecco's modified Eagle's medium (DMEM, GIBCO Laboratories Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum and antibiotics (100 U/ml of penicillin and 100 µg/ml of streptomycin sulfate) at 37°C in a 5% CO2 atmosphere. After 1 h of incubation, the medium was exchanged with fresh medium to remove nonadherent cells (i.e., stellate and endothelial cells). Cells were cultured for 24-48 h before experiments. Purity was verified by uptake of FITC-labeled latex beads.
Alveolar macrophage preparation and culture. Alveolar macrophages were isolated by bronchoalveolar lavage as previously described (2). Briefly, the trachea was cannulated, and the lungs were lavaged four times with 10 ml of phosphate-buffered saline warmed to 37°C. Cells were centrifuged at 500 g, and the pellet was resuspended in DMEM. Yield of alveolar macrophages following lavage was greater than 8 × 106 cells and was determine to be greater than 95% viable by trypan blue exclusion. Alveolar macrophages were seeded onto 25-mm glass coverslips and cultured as described above for Kupffer cells.
Neutrophil preparation and culture. Saline (35 ml) containing 1% oyster glycogen type II (Sigma) was administered intraperitoneally to male Sprague-Dawley rats (300-350 g) under light anesthesia with methoxyflurane. Four hours later, animals were reanesthetized and exsanguinated, and cells in the peritoneum were removed by lavage with saline (35 ml) containing heparin (1,000 U/l). The suspension was centrifuged at 500 g for 7 min, and the pellet was resuspended in 0.15 M NH4Cl to lyse erythrocytes. Cells were pelleted again by centrifugation at 500 g for 7 min. The cell pellet was suspended in RPMI 1640 containing L-glutamine (1 mM), 10% heat-inactivated fetal bovine serum, penicillin (10 IU/ml), and streptomycin (10 µg/ml). Cells (0.5 × 106 cells) were seeded onto 25-mm glass coverslips and incubated for 1 h at 37°C. The medium was then replaced with fresh medium, and cells were cultured for 24 h at 37°C. Neutrophils were identified microscopically by the lobular shape of their polymorphic nuclei and were >90% in all cell preparations.
Measurement of intracellular calcium concentration.
Changes in intracellular calcium concentration
([Ca2+]i) of single cells were measured
fluorometrically using the calcium indicator fura 2 (7).
Briefly, cells were plated on glass coverslips at a density of 3.0 × 105 cells/coverslip and were incubated in 2 ml of HBSS
[(in mM) 15 HEPES, 110 NaCl, 5 KCl, 0.3 Na2HPO4, 0.4 KH2PO4,
0.8 MgSO4 · 7H2O, 1.25 CaCl2 · 2H2O, 4 NaHCO3, and 5.6 glucose] containing 5 µM fura 2 acetoxymethyl ester (Molecular Probes, Eugene, OR) at room temperature
for 30 min. After being loaded, cells were rinsed and placed in a
measurement chamber with HBSS buffer at room temperature. A
microspectrofluorometer (Photon Technology, South Brunswick, NJ)
attached to an inverted microscope (Diaphot, Nikon, Japan) was used to
monitor changes in intracellular calcium. Changes in fluorescent
intensity of fura 2 at excitation wavelengths of 340 and 380 nm and
emission at 510 nm were recorded continuously in individual cells. The
ratio of emission at 340 and 380 nm was determined, and the
corresponding value of [Ca2+]i was calculated
using the relationship
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Clinical chemistry. Serum was collected 8 h after injection of saline or endotoxin (10 mg/kg). Serum alanine transaminase and alkaline phosphatase levels were measured by standard enzymatic methods (Sigma). The peak in serum transaminases following LPS occurred 6-12 h after injection (data not shown).
Measurement of serum TNF- levels.
Rats were anesthetized with methoxyflurane (Metofane; Pittman-Moore,
Mundelein, IL), and an intravenous catheter was inserted into the
femoral vein for serial blood sampling. Whole blood (200 µl) was
collected at each time point coincident with the injection of 200 µl
of lactated Ringer solution. Aprotinin (75 µl; Sigma) was added to
the serum immediately after collection, and samples were stored at
80°C until TNF-
measurement. Serum TNF-
was measured using an
enzyme-linked immunosorbent assay (ELISA) kit (Genzyme, Cambridge, MA).
Glycine measurement. Rat serum and lavage fluid were collected, and the glycine concentration was determined (12, 13). For the collection of lung fluid, the lungs were lavaged with 10 ml/kg body wt of neutral-buffered saline. In brief, glycine was extracted and benzoylated, and the resulting hippuric acid was extracted and dried in a nitrogen stream. The concentration of a colored conjugate of hippuric acid with dimethylaminobenzaldehyde was determined spectrophotometrically at 458 nm.
Tissue preparation and histological analysis. Liver and lung specimens were harvested when the rats were killed, fixed in 10% phosphate-buffered Formalin for 24 h, and mounted in paraffin. Sections (8 µm) were cut and counterstained with eosin and hematoxylin. The number of neutrophils per high-power field (×400) was counted based on nuclear shape in 10 random fields.
Statistical analysis. All results are expressed as means ± SE. Statistical differences between means were determined using analysis of variance followed by Tukey's post hoc analysis or by Student's t-test as appropriate. P < 0.05 was selected before analysis to reflect statistical significance.
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RESULTS |
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Routine parameters.
Sprague-Dawley rats were given free access to AIN-76 synthetic powdered
diet containing 5% glycine or casein as a nitrogen-balanced control
diet. The body weight and food consumption were measured daily (Table
1). Body weights of animals and food
consumption in both groups over 4 wk were not significantly different.
Importantly, glycine-fed rats had blood glycine concentrations nearly
eight times higher than those in the control group (Table 1).
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Effect of long-term glycine diet on survival after LPS injection.
To evaluate the efficacy of long-term dietary glycine, animals received
either powdered diet containing 5% glycine or nitrogen-balanced casein
control diet for 4 wk. After dietary treatment, an intravenous injection of LPS (5-40 mg/kg) was given via the tail vein, and survival after 24 h was assessed. All animals survived after the injection of 5 mg/kg of LPS. However, mortality rates of 20, 80, 100, and 100% were observed with 10, 20, 30, and 40 mg/kg of LPS, respectively, in animals that received control diet for 4 wk (Fig. 1). In animals that received
glycine diet, mortality after 10 and 20 mg/kg of LPS was completely
prevented and slightly attenuated in animals given 30 mg/kg of LPS
compared with casein-fed control animals. These data demonstrate that
glycine is protective against of LPS-induced mortality even after
chronic (>4 wk) consumption of glycine.
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Long-term glycine diet minimizes lung but not liver injury due to
LPS.
Because survival following LPS administration was improved after 4 wk
of glycine, it was hypothesized that glycine protected the liver and
lungs from injury due to endotoxin by inactivating Kupffer cells and
alveolar macrophages, respectively. Accordingly, animals fed 5%
glycine diet or casein diet for 4 wk were given 10 mg/kg of LPS, and
liver and lung specimens were collected 24 h later. This dose of
LPS was chosen because it was shown above to cause mortality only in
20% of control animals. LPS caused significant influx of lymphocytes
and neutrophils in the livers of animals fed casein- and
glycine-containing diets for 4 wk (Fig. 2). Mild liver necrosis was also observed
in both the casein- and glycine-fed animals following injection of LPS.
Serum alanine transaminase and alkaline phosphatase levels were
measured in both casein- and glycine-fed animals 8 after LPS (Table
2). Values increased four- to sixfold in
both groups. Furthermore, no significant differences in the increase in
serum enzymes were observed between the groups.
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Effect of long-term dietary glycine on LPS-induced serum
TNF- levels.
TNF-
has been hypothesized to mediate many symptoms observed
in endotoxin shock (22, 23), and it was shown
previously that 3 days of dietary glycine blunted the increase in serum
TNF-
levels after LPS injection (6). To test the
hypothesis that chronic dietary glycine would also blunt LPS-induced
increases in TNF-
, animals fed 5% glycine or casein for 4 wk were
injected with LPS (1 mg/kg), and TNF-
was measured in the serum
(Fig. 3). TNF-
levels in animals fed
casein rose to around 1,100 pg/ml within 60 min after injection of
endotoxin and diminished slowly over the course of 4 h. TNF-
levels in animals fed glycine peaked near 950 pg/ml, values not
significantly different from the casein group. These data are
consistent with the hypothesis that the protective effect of long-term
dietary glycine is not due to an effect of glycine on systemic TNF-
production.
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Effect of long-term dietary glycine on Kupffer cell activation.
To test the hypothesis that long-term glycine feeding would prevent the
activation of Kupffer cells due to LPS as in earlier short-term feeding
studies (6), animals received either 5% glycine or casein
control diet for 4 wk. Kupffer cells were isolated, and intracellular
calcium was measured after addition of LPS (10 µg/ml) (Fig.
4). Kupffer cells from animals fed casein
for 4 wk responded to LPS with a transient increase in intracellular
calcium within 30 s after stimulation that reached nearly 350 nM
and returned to basal levels within 2-3 min. When glycine (1 mM)
was added acutely to Kupffer cells from casein-fed animals 3 min before LPS, the increase in [Ca2+]i was blunted by
nearly 70%, confirming earlier work with cells from naïve rats
(7). In Kupffer cells from animals fed 5% glycine for 4 wk, LPS also caused a transient increase in calcium similar to the
increase observed in cells from the casein controls. However, unlike
Kupffer cells from the casein-fed controls, glycine added 3 min before
LPS had no effect on the increase in intracellular calcium in cells
from glycine-treated rats. Thus after long-term (>4 wk) dietary
glycine treatment, glycine is unable to inactivate the Kupffer cell.
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Effect of long-term dietary glycine on alveolar macrophages.
Acute respiratory failure and adult respiratory distress syndrome are
specific manifestations of endotoxin shock. Alveolar macrophages may
contribute to the development of lung injury due to endotoxin shock by
releasing toxic mediators such as TNF-, IL-1, and various free
radical species. It has been reported previously that alveolar
macrophages can be inactivated in vitro by glycine, most likely through
activation of a glycine-gated chloride channel (29).
Therefore, it was hypothesized that long-term dietary glycine would
prevent LPS-induced mortality by inactivating alveolar macrophages. To
test this hypothesis, alveolar macrophages were isolated from animals
fed either 5% glycine or casein for 4 wk, and the increase in
intracellular calcium due to LPS was assessed. Alveolar macrophages
from animals fed casein for 4 wk responded to LPS (10 µg/ml) with a
transient increase in intracellular calcium reaching nearly 200 nM and
returning to basal levels within 2-3 min (Fig.
6). When glycine (1 mM) was added 3 min
before LPS, the increase in [Ca2+]i was
blunted by nearly 90%, confirming earlier work (29). When
glycine (1 mM) was added to alveolar macrophages from glycine-fed animals, the LPS-induced increase in [Ca2+]i
was also blunted by about 90%, even after 4 wk of feeding (Fig. 7). These data indicate that the
glycine-gated chloride channel on alveolar macrophages, unlike the
receptor on Kupffer cells, is not downregulated by increased blood
levels of glycine.
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Effect of long-term dietary glycine on neutrophil activation.
Neutrophils are also involved in lung injury due to endotoxin by
infiltrating and adhering in the lung and releasing toxic mediators
such as hypochlorous acid, free radicals, and proteases (24). Because the increase in inflammatory cells (i.e.,
neutrophils) in the lung was largely blocked by glycine after LPS
challenge and because it has been reported that neutrophils are
inactivated by glycine (28), the possibility that glycine
acted directly on the neutrophil was investigated. Neutrophils were
isolated from animals fed either control diet or diet containing 5%
glycine diet for 4 wk, and the increase in intracellular calcium due to LPS was measured (Fig. 8). Neutrophils
from animals fed casein responded to LPS (100 mg/ml) with a transient
increase in intracellular calcium that peaked at 220 ± 20 nM, as
expected. Glycine added 3 min before LPS blunted this increase by
nearly 80%. Interestingly, the LPS-induced increase in intracellular
calcium in neutrophils from animals fed glycine diet was blunted by
greater than 60% with 1 mM glycine in vitro. Although the increase in
intracellular calcium was significantly blunted in vitro by glycine
even after chronic glycine feeding, it was not completely inhibited as
in casein-fed controls. Importantly, however, these data indicate that
the glycine-gated chloride channel may be minimally downregulated on
neutrophils and that neutrophil activation is blunted by glycine even
after long-term dietary glycine.
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DISCUSSION |
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Chronic dietary glycine improves survival against endotoxin.
Consumption of a glycine-containing diet has been shown to increase
survival in an endotoxin model (7). In pharmacological studies with isolated Kupffer cells, glycine blunted LPS-induced increases in intracellular calcium and TNF- production in a
chloride-dependent and strychnine-sensitive manner (7).
These data indicate that Kupffer cells are inactivated through
glycine-gated chloride channels similar to the inhibitory glycine-gated
chloride channels in the nervous system. Moreover, molecular evidence
supports the hypothesis that Kupffer cells contain glycine-gated
chloride channels similar to the receptor identified in the spinal cord
(27). These findings prompted the identification of
several other inflammatory cell types that possess glycine-gated
chloride channels, including alveolar macrophages (29),
neutrophils (28), T cells (20), and splenic
macrophages (30). Moreover, short-term dietary glycine has
been shown to be protective in several models involving inflammatory cells, such as liver ischemia-reperfusion, primary nonfunction after
organ transplantion, and several animal models of cancer (16-18, 34). However, the glycine-gated
chloride channel in the spinal cord becomes desensitized following
exposure to a high concentration of glycine (1).
Accordingly, it was hypothesized that survival following endotoxin
shock in animals fed glycine diet for long periods would not be
improved if the glycine-gated chloride channel was downregulated. In
these studies, the blood glycine concentrations in animals fed 5%
glycine were elevated to >1 mM from basal levels of 0.2 mM by feeding
(Table 1). In contrast to the hypothesis, animals fed glycine remained
tolerant to endotoxin (Fig. 1). Surprisingly, in animals that survived for 24 h after a sublethal dose of LPS, liver injury, measured by
serum alanine transaminase and alkaline phosphatase levels, and
inflammatory cell influx in glycine-fed animals were not significantly different from animals fed casein for 4 wk (Table 2 and Fig. 2).
However, lungs from the glycine-fed animals remained well preserved
compared with lungs from control animals, which had increased
cellularity of the alveolar wall, lung wet-to-dry weight ratio (Table
2) and inflammatory cell influx (Fig. 2). Most strikingly, the number
of infiltrating leukocytes in the lung of glycine-fed animals was
reduced by nearly 50% compared with that in the casein-fed control
animals. Lymphocyte infiltration and acute lung injury due to
endotoxemia have been described (25). In fact,
endotoxemia, acute pancreatitis, hemorrhagic shock, and many other
disorders have been associated with severe lung inflammation and injury (14, 33). Endotoxin-induced injury is
dependent on several inflammatory factors, and specific causes of death
have not been established. Thus glycine improves survival by unknown
mechanisms, but a reduction of lung inflammation is likely involved.
TNF- is likely an early mediator of LPS-induced lethality.
Because LPS-induced Kupffer cell activation and systemic TNF-
levels
are blunted by short-term glycine dietary treatment (6),
it was initially hypothesized that Kupffer cells play a large role in
the inflammatory response to LPS. However, Kupffer cells are no longer
inactivated by glycine after 4 wk of 5% glycine diet, findings
inconsistent with the hypothesis that inactivation of Kupffer cells is
solely responsible for protection against LPS-induced injury due to
glycine (Figs. 4 and 5). Moreover, LPS-induced TNF-
production was
not different in glycine-fed animals from casein-fed animals after 4 wk
of feeding (Fig. 3), yet rats fed glycine long term had improved
survival after endotoxin shock (Fig. 1). Although these data do not
exclude TNF-
involvement in lethality, it is unlikely that systemic
elevation of TNF-
is solely responsible for mortality due to
endotoxin. Most likely, TNF-
plays a role in a complex chain of
inflammatory events upsteam from the point where glycine protects.
Also, these data are consistent with the hypothesis that glycine may
work through other unknown mechanisms, such as inhibiting TNF-
receptor signaling. It is also possible that glycine inhibits TNF-
production from some cells but that other cell types remain insensitive
to glycine and produce TNF-
locally in this model. Moreover, it is
reasonable to conclude that cell types other than Kupffer cells are
responsible for endotoxin-induced mortality.
Alveolar macrophages and circulating neutrophils are inactivated by
glycine following long-term dietary glycine.
Because LPS-induced Kupffer cell activation and TNF- levels after
LPS administration were not different in glycine- and casein-fed animals after 4 wk of feeding (Fig. 3), the possibility that glycine acts on other cell types (e.g., alveolar macrophages or neutrophils) was considered. The alveolar macrophage is inactivated by glycine and
remained sensitive to glycine after long-term glycine feeding (Figs. 6
and 7), indicating that the glycine-gated chloride channel in alveolar
macrophages is not downregulated, unlike the receptor in Kupffer cells.
A possible explanation for this phenomenon is that the glycine
concentration in the alveolar space does not increase with dietary
glycine to sufficient levels to downregulate the receptor. Indeed, the
glycine concentration in the tracheoalveolar lavage is elevated twofold
less than in blood after glycine feeding (Table 1). Moreover, it has
been previously reported that neutrophils are inactivated by glycine
(28). Here, neutrophils are nearly completely inactivated
by glycine even after chronic glycine feeding (Fig. 8), consistent with
the hypothesis that glycine-gated chloride channels present in
neutrophils are only partially downregulated by glycine. This
phenomenon is most likely due to the rapid turnover of circulating
neutrophils, which is much more rapid than Kupffer cells.
Role of neutrophil inactivation by glycine in survival against
endotoxin.
Neutrophils release reactive oxygen species, hypochlorous acid, and
many proteolytic enzymes that cause vascular thrombosis, tissue edema,
and necrosis (26). In adult respiratory distress syndrome
from severe sepsis or pancreatitis, patients with high concentrations
of IL-8 and a greater number of neutrophils in the lung had higher
rates of mortality (9, 10). In fact, it is
argued that infiltrating neutrophils are more critical in lung
pathology than tissue macrophages (14, 24).
TNF- is most likely not directly involved in LPS mortality based on
the finding that TNF-
levels were not different in casein- and
glycine-fed animals after LPS (Fig. 1). However, TNF-
may trigger
the recruitment of leukocytes by expression of IL-8 and intracellular
adhesion molecules (5, 8). The findings that
neutrophils are inactivated by glycine (Fig. 8) in response to
endotoxin and neutrophil infiltration in the lung is decreased after
endotoxin after 4 wk of consuming a glycine-containing diet are
consistent with the hypothesis that neutrophils play a critical role in
lung injury due to endotoxin. Whereas neutrophils are stimulated by LPS
and glycine blunts activation of neutrophils by LPS and other stimuli
(28), neutrophils are more likely activated by a variety
of endogenous inflammatory signals (i.e., TNF-
and adhesion
molecules). Thus it is reasonable to hypothesize that glycine
inactivates neutrophils to a variety of signals that involve
intracellular calcium signaling. Here, glycine improves survival by
unknown mechanisms, but inactivation of neutrophils and reduction of
lung inflammation are likely involved.
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ACKNOWLEDGEMENTS |
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This work was supported by the National Institute on Alcohol Abuse and Alcoholism and Novartis Nutrition.
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FOOTNOTES |
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Address for reprint requests and other correspondence: M. D. Wheeler, Laboratory of Hepatobiology and Toxicology, CB 7365 Mary Ellen Jones Bldg., Univ. of North Carolina, Chapel Hill, NC 27599-7365 (E-mail: wheelmi{at}med.unc.edu).
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.
Received 2 November 1999; accepted in final form 28 March 2000.
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