Production of superoxide and TNF-
from alveolar macrophages
is blunted by glycine
Michael D.
Wheeler and
Ronald G.
Thurman
Laboratory of Hepatobiology and Toxicology, Department of
Pharmacology, University of North Carolina at Chapel Hill, Chapel
Hill, North Carolina 27599
 |
ABSTRACT |
Glycine blunts lipopolysaccharide (LPS)-induced
increases in intracellular calcium concentration
([Ca2+]i)
and tumor necrosis factor-
(TNF-
) production by
Kupffer cells through a glycine-gated chloride channel. Alveolar
macrophages, which have a similar origin as Kupffer cells, play a
significant role in the pathogenesis of several lung diseases including
asthma, endotoxemia, and acute inflammation due to inhaled bacterial
particles and dusts. Therefore, studies were designed here to test the
hypothesis that alveolar macrophages could be inactivated by glycine
via a glycine-gated chloride channel. The ability of glycine to prevent endotoxin [lipopolysaccharide (LPS)]-induced increases in
[Ca2+]i
and subsequent production of superoxide and TNF-
in alveolar macrophages was examined. LPS caused a transient increase in
intracellular calcium to nearly 200 nM, with
EC50 values slightly greater than 25 ng/ml. Glycine, in a dose-dependent manner, blunted the increase in
[Ca2+]i,
with an IC50 less than 100 µM.
Like the glycine-gated chloride channel in the central nervous system,
the effects of glycine on
[Ca2+]i
were both strychnine sensitive and chloride dependent. Glycine also
caused a dose-dependent influx of radiolabeled chloride with EC50 values near 10 µM, a
phenomenon which was also inhibited by strychnine (1 µM). LPS-induced
superoxide production was also blunted in a dose-dependent manner by
glycine and was reduced ~50% with 10 µM glycine. Moreover, TNF-
production was also inhibited by glycine and also required nearly 10 µM glycine for half-inhibition. These data provide strong
pharmacological evidence that alveolar macrophages contain
glycine-gated chloride channels and that their activation is protective
against the LPS-induced increase in
[Ca2+]i
and subsequent production of toxic radicals and cytokines.
glycine-gated chloride channel; intracellular calcium; tumor
necrosis factor-
 |
INTRODUCTION |
ALVEOLAR MACROPHAGES ARE the lung's central defense
against inhaled particles and pathogens. In the phagocytosis of foreign particles, the macrophage becomes activated and releases many toxic
mediators such as tumor necrosis factor-
(TNF-
), superoxide (O
2·), nitric oxide,
granulocyte-macrophage colony-stimulating factor, and other
inflammatory cytokines (2, 3). In particular, the inhalation of organic
or cotton dusts, which are highly contaminated with bacterial endotoxin
[lipopolysaccharide (LPS)], results in the overproduction and release
of radicals and TNF-
largely from alveolar macrophages (33).
Moreover, LPS in septic patients stimulates alveolar macrophages to
release cytokines, particularly TNF-
(11), which lead to adult
respiratory distress syndrome (ARDS), a disease associated with a high
rate of mortality (26, 29, 30). Accumulating evidence also indicates a
role for macrophages in the pathogenesis of both hypersensitivity reactions and bronchial asthma (8, 18). The macrophage releases cytokines that induce histamine release from basophils and enhance the
inflammatory potential of eosinophils (12, 17).
LPS-induced macrophage activation is typically characterized by an
initial increase in intracellular calcium concentration ([Ca2+]i),
which is necessary for the production of superoxide and TNF-
and the
induction of several inflammatory responses (6). LPS activates
macrophages, in part, via the cell surface receptor CD14, which leads
to the release of calcium from the endoplasmic reticulum through
inositol trisphosphate-dependent channels (6, 31). Furthermore,
[Ca2+]i
is increased by calcium influx through voltage-dependent calcium channels, which are activated by membrane depolarization (13). The
increase in
[Ca2+]i
is involved in the signaling of macrophage production of TNF-
and
free radicals, most likely by activating calcium-dependent kinases (10,
14).
Glycine, a nonessential amino acid, has been shown to be protective
against hypoxia, ischemia, and various cytotoxic substances in
renal proximal tubules via glycine-gated chloride channels (23, 35).
Recently, it was reported that dietary glycine prevents liver and lung
injury due to lethal doses of LPS in the rat (15). It has since been
shown that glycine blunts the transient increases in
[Ca2+]i
and production of TNF-
in response to LPS in the resident hepatic
macrophage, the Kupffer cell (16). It was concluded that Kupffer cells
contain a glycine-gated chloride channel that hyperpolarizes the plasma
membrane, making voltage-dependent calcium influx more difficult, thus
preventing activation of the macrophage (16).
Because alveolar macrophages are critically involved in the
pathogenesis of many pulmonary diseases caused by inhaled particles and
endotoxins, these studies were designed to test the hypothesis that
alveolar macrophages could be inactivated by glycine via a
glycine-gated chloride channel. Alveolar macrophages are important because of their diverse role of phagocytosis of exogenous particles such as cotton dust. The release of inflammatory cytokines and toxic
free radicals by alveolar macrophages stimulated by LPS is dependent on
increases in
[Ca2+]i
(10, 14). Therefore, the ability of glycine to prevent LPS-induced
increases in
[Ca2+]i
and to modulate the subsequent production of
O
2· and TNF-
was also examined.
Preliminary accounts of this work have appeared elsewhere (36).
 |
METHODS |
Isolation of alveolar macrophages.
Alveolar macrophages were isolated from Sprague-Dawley rats
(300-350 g) by bronchoalveolar lavage. Briefly, rats were
anesthetized by intraperitoneal injection of pentobarbital sodium (100 mg/kg body wt). The lungs were lavaged eight times with 8-ml aliquots of PBS (145 mM NaCl, 1.9 mM
NaH2PO4,
and 9.35 mM
Na2HPO4,
pH 7.4) via a cannula inserted into the trachea. Lavage suspensions
were centrifuged at 500 g for 7 min at
4°C. Red blood cells were lysed with 0.15 M
NH4Cl, and cells were suspended in
HEPES-buffered medium (in mM: 145 NaCl, 5 KCl, 10 HEPES, 5.5 glucose,
and 1 CaCl2, pH 7.4). Cell
viability determined by trypan blue exclusion was >94%.
Cells were resuspended in DMEM (4,500 mg/l glucose) supplemented with
10% FBS and antibiotics (100 U/ml penicillin G and 100 µg/ml
streptomycin sulfate) and were plated at the desired density and
incubated for 1 h at 37°C. Nonadherent cells were removed by
replacing medium with fresh DMEM. Adherent cells were cultured at
37°C with 5% CO2 for
24-48 h before experiments.
Measurement of
[Ca2+]i.
Changes in
[Ca2+]i
of single cells were measured fluorometrically using the calcium
indicator fura 2 (16). Briefly, cells were plated on glass coverslips
at a density of 3.0 × 105
cells/coverslip and were incubated in 2 ml modified HBSS
(m-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-AM (Molecular Probes, Eugene, OR) at room
temperature for 30 min. After loading, cells were rinsed and placed in
a measurement chamber with m-HBSS buffer at room temperature. A
microspectrofluorometer (Photon Technology International, South
Brunswick, NJ) attached to an inverted microscope (Diaphot, Nikon) was
used to monitor changes in
[Ca2+]i.
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 alveolar macrophages. The ratio of emission at 340 to 380 nm
was determined, and the corresponding value of
[Ca2+]i
was calculated using the relationship
|
|
where
F0/Fs
is the ratio of fluorescent intensities in buffers containing 3 mM EGTA
and 1 µM ionomycin
([Ca2+]min)
or 10 mM Ca2+ and 1 µM ionomycin
([Ca2+]max).
R is the measured ratio of fluorescent intensities at excitation wavelengths of 340 and 380 nm, and
Rmax and
Rmin are values of R at
[Ca2+]max
and
[Ca2+]min,
respectively. A dissociation constant
(Kd) of 135 nM
was used.
Measurement of radiolabeled chloride influx.
To determine if glycine could stimulate influx of extracellular
chloride into alveolar macrophages, a radiolabeled chloride flux assay
described by Morrow and Paul (25) was used. Briefly, 5 × 105 cells were plated on
coverslips in 60-cm2 culture
dishes in DMEM supplemented with 10% FBS and antibiotics (100 U/ml
penicillin G and 100 µg/ml streptomycin sulfate). Cells were
incubated at 37°C in a 5%
CO2-containing atmosphere for 24 h
and washed with HEPES buffer (in mM: 20 HEPES, pH 7.4, 118 NaCl, 4.7 KCl, 1.2 MgSO4, and 2.5 CaCl2) before the assay. Glycine
(1 µM to 1 mM) was diluted in HEPES buffer and was added to
60-cm2 petri dishes. Radiolabeled
chloride (36Cl final concentration
2 µCi/ml) was added to the glycine solution and kept at room
temperature. Coverslips with adherent cells were then incubated in
chloride solution at room temperature for 5 s, removed rapidly into
ice-cold wash buffer for 3 s, and then transferred into a second
ice-cold wash buffer for 7 s. Coverslips were broken, transferred to
scintillation vials containing 1.6 ml of 0.2 N NaOH, and incubated for
1 h. A 160-µl aliquot was taken for protein quantification by the
Lowry method (20). The sample was then diluted in 10 ml scintillation
fluid, and radioactivity was counted.
Measurement of O
2·
production.
Alveolar macrophage O
2· production
was measured by the superoxide dismutase (SOD)-inhibitable reduction of
ferricytochrome c (22). Cells were
plated in 24-well tissue culture plates at
106 cells/well and cultured at
37°C for 24 h in DMEM with 10% FBS. Supernatant was replaced with
m-HBSS and Ca2+ supplemented with
ferricytochrome c (0.8 mg/ml final
concentration). Glycine (0.1 µM to 1 mM) was added 5 min before LPS.
The reduction of ferricytochrome c was
measured both in the presence and absence of SOD (85 U/ml). The
difference in absorbance of ferricytochrome c, measured at 550 nm, was used to
calculate O
2· concentration using
a molar extinction coefficient of 17,500.
Measurement of TNF-
release in culture media.
Isolated alveolar macrophages were cultured in 24-well culture plates
at a density of 0.5 × 106
cells/well in glycine-free DMEM for 24 h, as described by Ikejima et
al. (16). Cells were then incubated with LPS (1-1,000 ng/ml) in
the presence or absence of glycine (1 µM to 1 mM) at 37°C for 4 h. TNF-
in the culture medium was determined using an ELISA kit
(Genzyme, Cambridge, MA).
 |
RESULTS |
Glycine blocks LPS-induced increases in
[Ca2+]i
in alveolar macrophages.
The
[Ca2+]i
in individual alveolar macrophages was determined fluorometrically with
the calcium indicator fura 2 as described in
METHODS. The addition of LPS plus 5%
rat serum caused a transient increase in
[Ca2+]i
that reached maximal levels within 60 s and rapidly returned to basal
levels (i.e., ~10-50 nM) within 3-5 min (Fig.
1). LPS (plus 5% rat serum) induced
increases in
[Ca2+]i
to a peak concentration of nearly 200 nM in a dose-dependent manner (half-maximal effect with 26 ng/ml LPS) (Fig.
2). The addition of 5% rat serum
provided the LPS-binding protein (LBP) that enhanced the response of
the alveolar macrophage, making the cell more sensitive to LPS by
nearly 100-fold, confirming work by others (data not shown) (21).

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Fig. 1.
Effects of lipopolysaccharide (LPS) and glycine on intracellular
calcium concentration
([Ca2+]i)
in isolated alveolar macrophages.
[Ca2+]i
was measured on an individual cell fluorometrically using fluorescent
calcium indicator fura 2 as described in
METHODS. LPS (1 µg/ml) plus 5% rat
serum was added in modified HBSS (m-HBSS), and glycine (1 mM) was added
3 min earlier. Data are representative of experiments performed on
cells isolated from 4-6 individual animals.
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Fig. 2.
Dose dependence of LPS on increase in
[Ca2+]i
in alveolar macrophages.
[Ca2+]i
was measured on an individual cell fluorometrically using fluorescent
calcium indicator fura 2 as described in
METHODS. LPS plus 5% rat serum in
m-HBSS was added to isolated alveolar macrophages. Rat serum alone
yielded no response and was used as control.
Inset: data plotted on a logarithmic
scale to demonstrate linearity. Data are represented as peak
[Ca2+]i
above basal concentrations and are expressed as means ± SD of
experiments performed on cells isolated from 4-6 individual
animals (linear regression: * P < 0.05, ANOVA with Tukey's post hoc analysis for comparison with
control).
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Glycine (1 mM) added 3 min before stimulation with LPS nearly
completely prevented the increase in
[Ca2+]i
due to LPS, with values only increasing to 15 nM (Fig. 3). Modulation of LPS-induced increases in
[Ca2+]i
by glycine was also shown to be dose dependent, with an
IC50 observed with nearly 10 µM
glycine (Fig. 3). Glycine alone had no
measurable effect on
[Ca2+]i
(data not shown).

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Fig. 3.
Dose-response curve for glycine on LPS-induced peak
[Ca2+]i.
Experimental conditions are as described in Fig. 1. Alveolar
macrophages were incubated in glycine-containing m-HBSS for 3 min
before addition of LPS (1 µg/ml). Data are represented as peak
[Ca2+]i
above basal
[Ca2+]i
for each individual cell. Inset: data
plotted on a logarithmic scale to demonstrate linearity. Data are
expressed as means ± SD of experiments from cells isolated from
3-6 individual animals (linear regression:
* P < 0.05, ANOVA with
Tukey's post hoc analysis for comparison with control).
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Strychnine antagonizes inhibition of LPS-induced increases in
[Ca2+]i
by glycine.
The glycine-gated chloride channel that has been characterized in the
spinal cord is known to be inhibited by low concentrations of
strychnine. Therefore, to test the hypothesis that alveolar macrophages
contain a strychnine-sensitive glycine-gated chloride channel,
strychnine was added 3 min before glycine. Strychnine (1 µM) largely
prevented the inhibitory effects of glycine (1 mM) on LPS-induced
increases in
[Ca2+]i
(Fig. 4). The peak
[Ca2+]i
values due to LPS in the presence of glycine (1 mM) were not significantly different from control levels in the presence of strychnine (1 µM). Strychnine (1 µM) alone also had no effect on
[Ca2+]i
levels (data not shown).

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Fig. 4.
Effects of strychnine on LPS-induced increases in
[Ca2+]i.
Experimental conditions are as described in Fig. 1. Strychnine (1 µM)
was added to buffer 3 min before glycine (1 mM). After incubation with
strychnine and glycine for 3 min, LPS (1 µg/ml plus 5% serum) was
added. In experiment using a high concentration of strychnine in
absence of glycine, strychnine (1 mM) was added to buffer 3 min before
LPS. Data are expressed as peak
[Ca2+]i
above basal
[Ca2+]i
for each individual cell. Data are expressed as means ± SD of
experiments performed on cells isolated from 3 or 4 individual animals
(a P < 0.05 compared with LPS-treated controls,
b P < 0.05 compared with LPS-treated cells in presence of glycine; 2-way
ANOVA with Tukey's post hoc analysis for comparison with LPS-treated
control).
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As a partial agonist to the glycine-gated chloride channel, high
concentrations of strychnine mimic the effects of glycine in several
models (24). High-dose strychnine (1 mM) added 3 min before the LPS
stimulation totally inhibited the increase in
[Ca2+]i
in alveolar macrophages due to LPS like glycine (Fig.
4).

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Fig. 5.
Effects of glycine on LPS-induced increases in
[Ca2+]i
in chloride-free buffer. Experimental conditions are as described in
Fig. 1. Alveolar macrophages were incubated in chloride-free buffer by
substitution of sodium chloride with sodium gluconate for 3 min before
addition of glycine (1 mM). After macrophages were incubated in
chloride-free buffer containing glycine for 3 min, LPS (0.1 µg/ml
plus 5% serum) was added. Data are represented as peak
[Ca2+]i
above basal
[Ca2+]i
for each individual cell and expressed as means ± SD of experiments
performed on cells isolated from 3 or 4 individual animals
(a P < 0.01 compared with LPS-treated control, ANOVA with Tukey's post
hoc comparison).
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Inhibition of LPS-induced increases in
[Ca2+]i
by glycine is dependent on extracellular chloride.
Activation of the glycine-gated chloride channel allows the influx of
chloride ions that hyperpolarize the cell membrane, thus preventing
increases in
[Ca2+]i
via voltage-dependent channels (32). Therefore, replacing sodium
chloride with sodium gluconate in the extracellular assay buffer should
prevent glycine from blunting LPS-induced increases in
[Ca2+]i.
Indeed, glycine (1 mM) in normal chloride-containing buffer nearly
completely inhibited the increase of
[Ca2+]i
due to LPS; however, in chloride-free buffer, glycine had no effect
(Fig. 5). LPS could still increase
[Ca2+]i
in the absence of extracellular chloride to peak levels similar to
those observed in the presence of chloride, suggesting that extracellular chloride depletion does not directly influence the LPS-induced increase in
[Ca2+]i.
Therefore, it is concluded that the inhibitory effects observed by
glycine are dependent on the presence of chloride, consistent with the
hypothesis that glycine acts via a glycine-gated chloride channel.
Glycine stimulates influx of radiolabeled chloride.
The glycine-gated chloride channel promotes the influx of chloride into
the cell that is hypothesized to hyperpolarize the plasma membrane,
preventing LPS-induced increases in
[Ca2+]i.
Although the pharmacological data above support the hypothesis that
glycine acts through a glycine-gated chloride channel, influx of
radiolabeled chloride into the cells stimulated by glycine would
provide hard physical evidence that chloride movement across the
membrane is stimulated by glycine and would strongly support the
presence of a glycine-gated chloride channel in alveolar macrophages. Indeed, radiolabeled chloride influx was stimulated with glycine in a
dose-dependent manner, with an
EC50 of ~10 µM. Glycine (0.1 mM) caused a significant 2.5-fold influx of radiolabeled chloride (Fig.
6A).
Furthermore, the effect of glycine was blocked by 1 µM strychnine
(Fig. 6B). These data provide strong
evidence for the presence of a glycine-sensitive chloride channel in
alveolar macrophages.

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Fig. 6.
Glycine promotes influx of radiolabeled chloride. Cells (5.0 × 105) were plated onto
25-mm2 coverslips and incubated
for 24 h at 37°C in DMEM supplemented with 10% FBS and
antibiotics. Cells were washed with HEPES buffer before experiments
described in METHODS.
A: coverslips were added to buffer
containing radiolabeled chloride and increasing concentrations of
glycine for 5 s and washed twice in cold buffer.
B: cells were added to buffer
containing radiolabeled chloride alone, buffer containing radiolabeled
chloride and 0.1 mM glycine, or buffer containing radiolabeled
chloride, 0.1 mM glycine, and 1 µM strychnine for 5 s, then washed
twice in cold buffer. Counts were normalized to amount of protein on
each coverslip. Data are representative of at least 4 experiments done
in triplicate and are expressed as percent of control in each
experiment (linear regression:
* P < 0.05, ANOVA with
Tukey's post hoc comparison).
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Glycine blocks the LPS-induced production of
O
2·.
The production of superoxide by alveolar macrophages was measured from
the SOD-inhibitable reduction of ferricytochrome
c to its ferrous form. LPS (1 µg/ml
plus 5% serum) increased O
2· production to 12.4 nmol · 106
cells
1 · 30 min
1 from basal values of
2.2 nmol · 106
cells
1 · 30 min
1 (Fig.
7). LPS-induced
O
2· production was inhibited by
glycine in a dose-dependent manner with an
IC50 of 1 µM and was blocked
completely by 1 mM glycine.

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Fig. 7.
Effect of glycine on LPS-induced superoxide production. Superoxide
production was measured by superoxide dismutase-inhibitable reduction
of ferricytochrome c as described in
METHODS. Cells were incubated in
either presence or absence of glycine 3 min before addition of LPS (1 µg/ml plus 5% serum). Inset: data
are plotted on a logarithmic scale to demonstrate linearity. Data are
expressed as means ± SD and are representative of 3 individual
experiments (linear regression:
* P < 0.05, ANOVA with
Tukey's post hoc analysis for comparison with control).
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Glycine blocks the LPS-induced release of TNF-
by
alveolar macrophages.
To evaluate the effects of glycine on cytokine release from alveolar
macrophages, LPS-induced TNF-
release was measured using an ELISA.
As expected, LPS (1 ng/ml to 1 µg/ml) increased TNF-
production,
with an EC50 of 10-30 ng/ml
(Fig.
8A),
consistent with the EC50 for
LPS-induced increases in
[Ca2+]i
(Fig. 1). Moreover, glycine blunted the LPS-induced release of TNF-
significantly in a dose-dependent manner, with an
IC50 value near 10 µM (Fig.
8B). This concentration of glycine
also caused a 50% decrease in the LPS-induced peak increase in
[Ca2+]i
(Fig. 3).

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Fig. 8.
Effect of glycine on LPS-induced tumor necrosis factor- (TNF- )
release. TNF- from isolated alveolar macrophages was measured in
culture medium by ELISA (Genzyme). A:
alveolar macrophages were cultured in glycine-free DMEM plus LPS
(1-1,000 ng/ml) plus 5% rat serum for 4 h.
B: alveolar macrophages were cultured
in glycine-free DMEM (controls) and DMEM supplemented with glycine (1 µM to 1 mM) and exposed to LPS (10 ng/ml) for 4 h. Data are
representative of 3-5 individual experiments and are expressed as
percent of control (linear regression:
* P < 0.05, ANOVA with
Tukey's post hoc analysis for comparison with glycine-free
controls).
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 |
DISCUSSION |
Glycine blunts increases in
[Ca2+]i
in alveolar macrophages.
There are many disease states in which the response of macrophages to
LPS leads to the overproduction of toxic mediators such as cytokines
and free radicals. In particular, alveolar macrophages respond to LPS
to produce inflammatory mediators such as TNF-
and superoxide that
are implicated in respiratory conditions in septic shock and ARDS.
Although the exact mechanisms that lead to mortality are uncertain, it
is thought that these mediators are involved (26, 29, 30).
Recent reports have suggested that alveolar macrophages may be much
more sensitive to LPS than other macrophage/monocytes such as the
peritoneal macrophage and the Kupffer cell, the resident hepatic
macrophage (27). In this study, LPS caused a transient increase in
calcium and a rapid return to basal levels (~15-60 nM) within
minutes. Moreover, the EC50 for
LPS on the increase in
[Ca2+]i
was 26 ng/ml, a concentration which is consistent with the literature
(21). However, peak increases in
[Ca2+]i
due to LPS in alveolar macrophages occurred in response to 1.0 µg/ml
LPS in the presence of 5% rat serum (Fig. 2) as opposed to higher
values needed in other cell types (16). The differences in
responsiveness observed in these cells compared with other macrophages
may be related to their exposure to LPS. Kupffer cells are continuously
exposed to low circulating levels of LPS and may become desensitized
over time. Alveolar macrophages, present in surfactant lining of the
lung, are limited in their exposure to bloodborne LPS and thus may
remain highly sensitive to airborne pathogens. Because LPS binds the
LBP and interacts with the cell surface receptor CD14 expressed on a
variety of cell types, differences in sensitivity may also be related
to differences in the expression levels of receptor (21). This idea is
supported by two lines of work. First, exposure to acute ethanol in
vivo decreased responsiveness to LPS-induced production of TNF-
in
isolated hepatic macrophages (7). Second, this effect was inhibited by
reducing gut endotoxin with antibiotic treatment, suggesting that acute
ethanol increases gut-derived LPS, which desensitizes macrophages
against subsequent LPS stimulation (1). Other receptors are also
expressed on macrophages that bind LPS and elicit inflammatory
responses as well. Some of these include the scavenger receptors (SR-A
and SR-B) and
-integrins, and the participation of these receptors in the LPS response cannot be excluded in this model (9). In fact, it
is highly likely that all of these signaling pathways are to some
extent involved.
Not only were alveolar macrophages more sensitive to LPS, but they were
also more responsive to low concentrations of glycine. Glycine
inhibited transient increases in
[Ca2+]i
due to LPS in isolated alveolar macrophages in this study (Figs. 2 and
3) in a dose-dependent manner but with an
IC50 value >10-fold less than
values reported for Kupffer cells (16). This difference may also be
explained by differences in receptor density or by a downregulation
event due to levels of exposure of each cell type to glycine. Perhaps
glycine-sensitive chloride channels are expressed more abundantly in
alveolar macrophages as opposed to Kupffer cells. Alternatively,
preliminary long-term studies suggest that the effect of glycine on
LPS-induced increases in
[Ca2+]i
in Kupffer cells is lost after chronic exposure to dietary glycine
(Wheeler, unpublished data). This suggests that chronic elevated
glycine concentrations possibly lead to glycine receptor downregulation
or desensitization in Kupffer cells, causing the effect of glycine to
be lost or reduced. This possibility is likely, since Kupffer cells are
continuously exposed to blood concentrations of glycine of 100 µM or
higher, and alveolar macrophages exist in the alveolar space where the
glycine concentration is presumably lower than in blood. In fact,
glycine concentrations in alveolar fluid (50-60 µM) were
significantly lower than in the blood (90-160 µM). Thus
differences in local glycine concentrations may explain differences in
sensitivity of various macrophage populations to glycine.
Alveolar macrophages contain a glycine-gated chloride channel.
The data above suggest that alveolar macrophages possess a
glycine-gated chloride channel. The hypothesis is strongly supported by
the fact that the effects of glycine are both sensitive to strychnine
and are dependent on the presence of extracellular chloride (Figs. 4
and 6). First, glycine blunted the activation of the alveolar
macrophage with an IC50 slightly
greater than 10 µM, which is near the binding affinity of glycine for
the glycine-gated chloride channel in neuronal tissue (37). Strychnine
at low concentrations is a known antagonist of the glycine-gated
chloride channel in nervous tissue and has been useful in
pharmacological and biochemical studies of the channel in various
tissues and expression systems. In the spinal cord, strychnine binds
and inhibits the activity of the channel with nanomolar affinity;
however, strychnine mimics glycine by activating chloride movement
through the channel at higher concentrations (23, 24). The effects of
strychnine in alveolar macrophages (Fig. 4) are consistent with this
hypothesis, providing pharmacological evidence for the existence of the
glycine receptor with similar characteristics to the channel found in
both neuronal and renal tissue. All of the pharmacological data such as
strychnine sensitivity, extracellular chloride dependence, and
radiolabeled chloride influx are consistent with the hypothesis that
glycine activates a glycine-gated chloride channel similar to the
channel in neuronal tissue. Moreover, molecular evidence for a
glycine-gated chloride channel in alveolar macrophages using reverse
transcription-polymerase chain reaction has recently been obtained
(data not shown).
Glycine modulates the production of free radicals and cytokines in
alveolar macrophages.
The increase in
[Ca2+]i
due to LPS serves as a second messenger in many signaling pathways and
is important in the production of free radicals and some inflammatory
cytokines that are released by macrophages (14, 28). These studies
demonstrate that glycine significantly reduces the LPS-induced
production of O
2· and TNF-
in
alveolar macrophages, most likely by blunting the increase in
[Ca2+]i
necessary for their production.
O
2· from macrophages is largely
generated from molecular O2
through NADPH oxidase, an enzyme complex activated by phosphorylation by calcium-dependent protein kinases (4, 38). By blunting the increase
in
[Ca2+]i
with glycine, the production of
O
2· is reduced most likely by
inhibiting calcium-dependent signaling required to activate NADPH
oxidase (Fig. 7).
TNF-
production is stimulated by LPS and is dependent on an increase
in
[Ca2+]i,
which is involved in signaling and protein synthesis. LPS is known to
activate macrophages through CD14,
2-integrins, and scavenger
receptors (9). In long-term in vitro assays, such as the measurement of
TNF-
, both CD14 and scavenger receptor pathways most likely become
activated and initiate cytokine production. Even though there is a
significant effect of glycine on LPS-induced TNF-
production,
glycine is not able to completely blunt LPS-induced TNF-
production
like
[Ca2+]i
and O
2· production. The reason for
these differences is not understood yet but may be due to the
involvement of scavenger receptors or other receptors and pathways that
may signal via receptors independent of CD14. In fact, it is reported that TNF-
production in response to long-term exposure to LPS can
increase independent of the transient increase in
[Ca2+]i
(19). Together, these data suggest that it is likely that several
receptors and pathways are involved in LPS-induced TNF-
production.
In this case, glycine would be expected to have minimal effects. On the
other hand, the transient LPS-induced increase in
[Ca2+]i
and O
2· production is most likely
mediated entirely through CD14, since it occurs within minutes. In this case, glycine is most effective.
The inhibition of free radical and TNF-
production by glycine is an
important phenomenon, since others have shown that mediators produced
by alveolar macrophages play a large role in the pathological changes
in sepsis, ARDS, and possibly asthma (5, 34). Moreover, these data may
provide an additional explanation for the protective effect of dietary
glycine on endotoxin-induced mortality and lung injury (15), since
alveolar macrophages play a large role in lung pathogenesis in that model.
Moreover, alveolar macrophages play a significant role in the
inflammatory process after exposure to inhaled particles such as cotton
dust, a rich source of endotoxin, or other organic dusts (33). TNF-
production from alveolar macrophages mediates a cascade of events
including histamine release, neutrophil and lymphocyte inflammation,
and potential chronic lung injury. Because glycine inactivated alveolar
macrophages and blunted the production of TNF-
, glycine may
potentially be useful in the treatment or prevention of lung
inflammation due to inhaled particles.
In conclusion, it was shown that glycine-gated chloride channels are
present in alveolar macrophages and can prevent activation by allowing
the protective influx of extracellular chloride, which opposes
increases in
[Ca2+]i.
Because increases in
[Ca2+]i
are required for free radical production as well as production of many
cytokines in alveolar macrophages, glycine may be useful in preventing
tissue injury due to inflammatory agents in response to LPS or other
antigens. Because many diseases such as sepsis, ARDS, bronchial
allergies, and acute inflammatory responses are mediated, in part, by
alveolar macrophage activation, glycine may be especially useful in
their treatment if shown to be effective in clinical trials.
 |
ACKNOWLEDGEMENTS |
This work was supported by grants from the National Institutes of Health.
 |
FOOTNOTES |
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. G. Thurman,
Laboratory of Hepatobiology and Toxicology, Dept. of Pharmacology, CB
no. 7365, Mary Ellen Jones Bldg., Univ. of North Carolina at Chapel
Hill, Chapel Hill, NC 27599-7365 (E-mail:
thurman{at}med.unc.edu).
Received 25 February 1999; accepted in final form 14 July 1999.
 |
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