Hydrolysis of surfactant-associated phosphatidylcholine by
mammalian secretory phospholipases
A2
R. Duncan
Hite1,
Michael C.
Seeds1,
Randy B.
Jacinto1,
R.
Balasubramanian1,
Moseley
Waite2, and
David
Bass1
1 Section on Pulmonary and
Critical Care, Department of Internal Medicine, and
2 Department of Biochemistry, Wake
Forest University School of Medicine, Winston-Salem, North Carolina
27157
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ABSTRACT |
Hydrolysis of
surfactant-associated phospholipids by secretory phospholipases
A2 is an important potential
mechanism for surfactant dysfunction in inflammatory lung diseases. In
these conditions, airway secretory phospholipase A2
(sPLA2) activity is increased, but
the type of sPLA2 and its impact
on surfactant function are not well understood. We examined in
vitro the effect of multiple secretory phospholipases
A2 on surfactant, including their
ability to 1) release free fatty
acids, 2) release lysophospholipids, and 3) increase the minimum surface
tension (
min) on a pulsating bubble surfactometer. Natural porcine surfactant and Survanta were
exposed to mammalian group I (recombinant porcine pancreatic) and group
II (recombinant human) secretory phospholipases
A2. Our results demonstrate that
mammalian group I sPLA2 hydrolyzes phosphatidylcholine (PC), producing free fatty acids and
lysophosphatidylcholine, and increases
min. In contrast, mammalian
group II sPLA2 demonstrates limited hydrolysis of PC and does not increase
min. Group I and group II
secretory phospholipases A2 from
snake venom hydrolyze PC and inhibit surfactant function. In summary,
mammalian secretory phospholipases
A2 from groups I and II differ
significantly from each other and from snake venom in their ability to
hydrolyze surfactant-associated PC.
lysophospholipid; lung injury; asthma; pulsating bubble
surfactometer
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INTRODUCTION |
SURFACTANT is a complex mixture of phospholipids
(80-90% wt/wt), neutral lipids (5-10%), and proteins
(5-10%) that lines the alveolar surface in a monomolecular film
and principally serves to reduce the work of breathing by lowering the
surface tension of the alveolus and distal conducting airways (12).
Synthesis, secretion, and reuptake of surfactant is controlled
primarily by alveolar type II epithelial cells. Phosphatidylcholine
(PC) is the most abundant phospholipid (80%), and the fatty acid acyl composition of PC is predominantly saturated palmitic acid (65%). The
surfactant-associated apoproteins [surfactant protein (SP) A-D] markedly enhance the adsorption, spreading, and
maintenance of the phospholipid monolayer (27, 44).
Disease states that result in a deficiency of surfactant are
characterized by severe dysfunction of the lungs, including reduced gas
exchange, decreased lung compliance, and increased airway resistance.
Premature infants with insufficient surfactant synthesis by type II
epithelial cells develop respiratory distress syndrome (RDS) (32).
Children and adults can also develop an acute RDS (ARDS) that is
similar to RDS but is the result of a severe systemic inflammatory
insult (16, 19). Similarly, in asthma, the function of surfactant to
maintain the patency of conducting airways is inhibited by inflammation
(21, 29). Several mechanisms for inflammation-mediated inhibition of
surfactant have been suggested, including
1) direct surfactant injury by
hydrolytic enzymes, proteases, and metabolic by-products,
2) disruption of the surfactant
monolayer by serum proteins that leak into the alveolus, and
3) reduction in surfactant synthesis
by direct injury to type II epithelial cells (28).
One specific mechanism for surfactant injury is hydrolysis of
surfactant-associated phospholipids by phospholipases
A2 (22, 25). Intratracheal
administration of phospholipases A2 to adult rats results
in severe lung injury and serves as an in vitro model of ARDS (13). Phospholipases A2
have been categorized into at least 10 groups (I-X) based on amino
acid sequence data (10, 11). Many of these enzymes are secreted
extracellularly and are commonly referred to as secretory
phospholipases A2. The secretory phospholipases A2 share many
important characteristics including 1) small size (13-18 kDa),
2) stability in acidic pH,
3) millimolar calcium requirement
for activity, and 4) minimal
preference for any specific fatty acid in the
sn-2 position of
the phospholipid. The secretory phospholipase
A2 (sPLA2) groups are
differentiated by their structural configuration, including amino acid
sequence and the number and location of disulfide bonds. However, the
biological activity of the enzymes between groups can differ
significantly as subtle differences in the sequences regulate
interfacial substrate binding and catalysis (41).
The first mammalian sPLA2 (group
I) has been isolated and purified from porcine, bovine, equine, and
human pancreas, with 75-80% homology of the amino acid sequences
between the species (39). Although the principal role of the pancreatic
sPLA2 is in digestion, the protein
and mRNA for human group I sPLA2
are also present in human lung (35). The mammalian group I
sPLA2 preferentially hydrolyzes PC
and can hydrolyze surfactants in vitro (22, 37).
Mammalian group II sPLA2 was
initially isolated and purified from inflamed peritoneal and synovial
fluids (6, 20). Expression of group II
sPLA2 has been demonstrated in
several inflamed and noninflamed tissues, including the lung (25). The principal roles of the group II
sPLA2 are believed to be in host defense through potent antibacterial effects and in inflammation through signal transduction and generation of arachidonic acid metabolites (36, 42). The group II secretory phospholipases A2 prefer
phosphatidylethanolamine and phosphatidylserine and hydrolyze PC less efficiently than group I
sPLA2 (6, 20). New mammalian
low-molecular-weight phospholipases
A2 have been identified, groups V
and X, but their principal roles are not fully understood (1, 10).
The bronchoalveolar lavage (BAL) fluid (BALF) from humans with ARDS
contains less total phospholipid (including PC and
phosphatidylglycerol), higher amounts of lysophosphatidylcholine
(lysoPC), and increased sPLA2 activity (16). Similarly,
BALF from asthmatic patients who are challenged with endobronchial
antigen instillation demonstrates increased amounts of
sPLA2 activity and
1-palmitoyl-lysoPC (5, 7). In combination, the BALF
characteristics of patients with ARDS and asthma strongly support the
presence of secretory phospholipases A2. The group and cellular origins
of the secretory phospholipases A2
responsible for the changes in phospholipid composition are unknown.
Examination of BALF from ARDS patients with antibodies to group II and
heparin affinity confirms the presence of a group II
sPLA2 and a second, non-group II
sPLA2 (25). Furthermore, the
proenzyme of group I sPLA2 is
present in the serum of patients with ARDS (30).
In this study, we examined the capacity of the mammalian group I
(pancreatic) and group II (inflammation) enzymes to
1) release free fatty acids,
2) increase the formation of
lysophospholipids, and 3) cause
surfactant dysfunction. Our results identify significant differences in
the hydrolysis of surfactant-associated PC by these enzymes and provide
important information in understanding the role of
sPLA2-mediated hydrolysis of
surfactant in inflammatory lung diseases.
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MATERIALS AND METHODS |
Phospholipid and lysophospholipid standards were purchased from Avanti
Polar Lipids (Alabaster, AL). Arachidonic acid was purchased from
Cayman Chemical (Ann Arbor, MI). The 17:0 fatty acid standard was
purchased from NuPrep (Elysian, MN). Radiolabeled [2-palmitoyl-9,10-3H(N)]dipalmitoyl-L-
-phosphatidylcholine
([3H]DPPC) was
purchased from DuPont NEN (Boston, MA). COS-1 cells were obtained from
the American Type Culture Collection (Manassas, VA). All solvents were
purchased from Fisher Scientific (Pittsburgh, PA). Diethylaminoethyl
dextran was purchased from Pharmacia (Piscataway, NJ). All additional
chemicals were purchased from Sigma (St. Louis, MO), including the
following phospholipases: recombinant porcine pancreas (group I),
Naja naja (snake venom group I), and
Crotalus atrox (snake venom group II).
Surfactants. Natural porcine
surfactant (NPS) was isolated from juvenile pigs (10-15 kg) that
were euthanized with intravenous Pentothal Sodium. Repetitive saline
lavage (60-80 ml/kg) was performed via an endotracheal tube.
Within 30 min, cells were centrifuged (600 g for 30 min) from the lavage fluid. A
surfactant pellet was isolated from the cell-free supernatant by
ultracentrifugation (15,000 g for 60 min). The pellet was resuspended in saline and washed three times with
normal saline and repeat ultracentrifugation. After the surfactant
pellet was washed, it was resuspended in saline and the phosphorus
content was determined with the method of Bartlett (2). The surfactant
suspension was separated into aliquots, which were stored at
70°C. Survanta (Ross Laboratories, St. Louis,
MO) samples were stored at 0-5°C. Individual aliquots of the
surfactants were thawed on the day of each experiment.
Preparation of human group II
phospholipases. Recombinant human group II (rhGpII)
sPLA2 was obtained by transfecting
COS-1 cells with a pCMV-5 plasmid containing an
sPLA2 gene construct with the
method of Wong et al. (43). rhGpII
sPLA2 was collected after 3 days
of transfection and partially purified by overnight extraction in 0.18 M
H2SO4.
A second example of group II sPLA2 was obtained by partial purification of human synovial fluid (HuSF) from the inflamed joints of patients with rheumatoid arthritis by acid
extraction. All acid extracts were dialyzed against a buffer (pH = 7.40) containing 0.05 M Tris and 0.05 M NaCl and stored as aliquots at
70°C. Aliquots were thawed and used fresh daily for each
experiment. The protein content of the rhGpII and HuSF
sPLA2 was measured with
Bradford's Coomassie blue reagent (Pierce, Rockford, IL).
Distribution of [3H]DPPC in
surfactant.
A stock solution of labeled surfactant was prepared fresh daily.
Aliquots of [3H]DPPC
(10 µCi/ml in 1:1 toluene-ethanol, 0.1 µCi/incubation) were
transferred to microcentrifuge tubes and dried under
N2 gas. NPS or Survanta was added
and diluted to 1.0 mg phospholipid/ml in saline buffered (pH = 7.4)
with Tris (5.0 mM) and CaCl2 (5.0 mM) and mixed three times with a Branson sonicator (Heat
Systems-Ultrasonics, Farmingdale, NY) with a stepped microtip set at
~40 W for 15 s. Sonicated samples were loaded at the bottom of a
discontinuous sucrose gradient (in
H2O): 0 M (3 ml), 0.25 M (11 ml),
0.35 M (11 ml), and 0.60 M (11 ml including sample). The gradient was ultracentrifuged (64,000 g for 60 min
at 4°C), and fractions were removed and analyzed for radioactivity
and phosphorus. Data are expressed as the percentage of total
radioactivity and the absolute phosphorus (in nmol) in each fraction.
Hydrolysis of
[3H]oleate-labeled Escherichia
coli.
[3H]oleate-labeled
E.
coli was prepared with a modification
of the method of Kramer and Pepinsky (26). Labeled E. coli (400 pmol lipid/sample, 0.1 µCi/ml) was then
exposed to secretory phospholipases A2 for 1 h at 37°C.
The incubation was terminated by lipid extraction of the phospholipids
and fatty acids with the method of Bligh and Dyer (4). The
phospholipids and fatty acids were separated by thin-layer
chromatography (TLC) with Silica G plates (Analtech, Newark, DE) and a
mobile solvent phase of hexane, ethyl ether, and formic acid (90:60:6
by vol). Phospholipid and free fatty acid fractions were visualized on
the TLC plates with I2 vapor and
scraped and analyzed for radioactivity with a scintillation counter.
Before TLC, each sample was supplemented with unlabeled arachidonic
acid (40 µg) to enhance free fatty acid staining by the
I2 vapor. The data are expressed
as the percentage of the total radioactivity recovered in the free
fatty acid fraction.
Hydrolysis of
[3H]DPPC-labeled
surfactant.
Aliquots (200 µl) of the sonicated,
[3H]DPPC-labeled
surfactant stock were diluted to a final phospholipid concentration of 0.5 mg/ml with saline containing 5 mM Tris (pH 7.4) and 5 mM
CaCl2 and incubated in the
presence or absence of secretory phospholipases A2 for 2 h
at 37°C. Samples were then processed and analyzed as outlined in
Hydrolysis of
[3H]oleate-labeled Escherichia coli.
Surfactant function. Aliquots (40 µl) of the surfactant-sPLA2
reaction mixtures were transferred into sample chambers designed for
the pulsating bubble surfactometer (14). The biophysical ability to
lower the surface tension of each sample (40 µl) was analyzed at
37°C with the pulsating bubble surfactometer. A bubble was formed
in the aqueous sample chamber, and pressure at the air-liquid interface
was continuously transduced during repeated expansion and contraction
(20 cycles/min) of the bubble between a minimum radius
(r) of 0.40 mm and a maximum
r of 0.55 mm. Surface tension (
)
was continuously calculated with the LaPlace equation (P = 2
/r). Pulsations were continued
for a maximum of 10 min or until
1.0 mN/m for at least 3 min.
The minimum surface tension
(
min) is defined as the
average of the lowest surface tension measurement from each minute over
three consecutive minutes during the entire 10-min analysis.
Phospholipid and free fatty acid
composition. Surfactant samples were prepared and
incubated in the presence or absence of sPLA2 as described. After lipid
extraction, specific phospholipid fractions were isolated by the TLC
method of Fine and Sprecher (15) by comparison to phospholipid
standards. Fractions were identified with the use of
I2 and scraped and analyzed for
phosphorus content. The data are expressed as the percentage of total
phospholipid recovered. Free fatty acid fractions were scraped and
esterified with the method of Rogozinski (33). The esterified samples
were separated with gas chromatography (Hewlett-Packard 5890) with a
bonded silica DB225 column (Alltech, Avondale, PA) and quantified with
flame ionization detection and comparison to a 17:0 internal standard.
Statistics. Data are the means ± SD of at least three experiments. Statistical significance was
determined by Student's t-test.
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RESULTS |
A model was established for measuring hydrolysis of surfactant
phospholipids by secretory phospholipases A2. A trace
amount of [3H]DPPC was
mixed with surfactant by sonication as described in MATERIALS AND METHODS such
that after exposure of the mixture to an
sPLA2, the release of the
[3H]palmitate from the
sn-2 position of the surfactant
phospholipid could be quantitated by TLC analysis of the free fatty
acids. To confirm that the
[3H]DPPC was
homogeneously mixed into the surfactant, the mixture was fractionated
over sucrose density gradients, and the colocalization of lipid
phosphorus and
[3H]DPPC was
established (Fig. 1). Free
[3H]DPPC in the
absence of surfactant migrated in the early gradient fractions
(fractions 1-3).
However, [3H]DPPC
comigrated with the major surfactant phospholipid peak (fractions 5-10) when
the two were mixed by sonication before gradient separation. Thus
trace-labeled surfactant appears to be a uniformly mixed substrate.

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Fig. 1.
Incorporation of
[2-palmitoyl-9,10-3H(N)]dipalmitoyl-L- -phosphatidylcholine
([3H]DPPC) in natural
porcine surfactant (NPS). NPS was mixed and sonicated
with 10 µCi/ml of
[3H]DPPC. Samples were
loaded at bottom of a discontinuous sucrose gradient. Data are means ± SD.
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Labeled surfactant was then subjected to hydrolysis by
sPLA2. Porcine pancreatic
sPLA2, a group I enzyme, readily
hydrolyzed the labeled E. coli and
surfactant in a dose-dependent manner (Fig.
2). Hydrolysis increased over the range of
10-1,000 U/ml of enzyme. To compare the ability of the various
secretory phospholipases A2 to
hydrolyze surfactant-associated phospholipids, we performed E. coli hydrolysis experiments. The
hydrolysis of E. coli serves as the
standard for comparison. In all experiments, we defined one unit of
enzyme activity as the amount required to result in 50% of the maximum
E. coli hydrolysis. The group I
sPLA2 also demonstrated
significant hydrolysis of the labeled E. coli.

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Fig. 2.
Porcine pancreatic group I secretory phospholipase
A2
(sPLA2) hydrolysis of
[3H]DPPC-labeled
surfactant and
[3H]oleate-labeled
Escherichia coli. Porcine pancreatic
sPLA2 was incubated at 37°C
with either
[3H]DPPC-labeled
surfactant (0.5 mg of phospholipid/ml) for 2 h or
[3H]oleate-labeled
E. coli (10 µg of phospholipid/ml)
for 1 h. Concentration of porcine pancreatic
sPLA2 ([Porcine
sPLA2]) is expressed in U/ml
where 1 unit is equivalent to amount of enzyme required to achieve 50%
of maximum E. coli hydrolysis.
Phospholipids and fatty acids were separated with thin-layer
chromatography (TLC). Data are means ± SD of %total radioactivity
in each free fatty acid fraction.
* P 0.02 compared with
control without group I sPLA2
(data not shown).
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rhGpII sPLA2 was examined for its
ability to hydrolyze surfactant phospholipids. The recombinant protein
expressed in COS-1 cells demonstrated significant hydrolysis of
[3H]oleate-labeled
E. coli (Fig.
3) but demonstrated only minimal hydrolysis
of [3H]DPPC-labeled
surfactant despite use of concentrations of up to 10,000 U/ml. In these
experiments, we again defined one unit of enzyme activity as the amount
required to result in 50% of the maximum E. coli hydrolysis. These results suggest that the mammalian group II sPLA2 is
significantly less able to hydrolyze [3H]DPPC than the
group I sPLA2. We considered the
possibility that the rhGpII sPLA2
expressed by the COS-1 cells might be an incomplete or altered product
and therefore might artificially demonstrate lower rates of surfactant
hydrolysis than the native enzyme. To investigate that possibility, we
performed identical experiments with the use of HuSF
sPLA2 obtained from inflamed
synovial fluid, and the rates of hydrolysis by HuSF for both
substrates,
[3H]DPPC-labeled
surfactant and E. coli, were identical
to the rates of hydrolysis with the rhGpII
sPLA2 (data not shown). Despite the use of higher concentrations than were used for the group I porcine
pancreas sPLA2, the hydrolysis of
surfactant by both group II enzymes was minimal. To examine the effects
of inhibitors (i.e., Clara cell protein) (23), which might potentially
be present in our NPS preparations, we studied the activity of both group II enzymes against
[3H]DPPC-labeled
Survanta. The rates of hydrolysis in the Survanta preparations were
identical to those in the labeled NPS preparations (data not shown).

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Fig. 3.
Recombinant human group II (rhGpII)
sPLA2 hydrolysis of
[3H]DPPC-labeled
surfactant and
[3H]oleate-labeled
E. coli. rhGpII
sPLA2 was incubated at 37°C
with either
[3H]DPPC-labeled
surfactant (0.5 mg phospholipid/ml) for 2 h or
[3H]oleate-labeled
E. coli (10 µg phospholipid/ml) for
1 h. Concentration of rhGpII sPLA2
([rhGpII sPLA2]) is
expressed in U/ml where 1 unit is equivalent to amount of enzyme
required to achieve 50% of maximum E. coli hydrolysis. Phospholipids and fatty acids were
separated with TLC. Data are means ± SD of %total radioactivity in
each free fatty acid fraction. Significant difference compared with
control without rhGpII sPLA2 (data
not shown): * P < 0.01;
P < 0.05.
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The hydrolysis of surfactant phospholipids by secretory phospholipases
A2 results in the production of a
lysophospholipid and a free fatty acid. Consequently, the group I
porcine pancreatic sPLA2
resulted in an increase in the lysoPC content and a
decrease in the PC content (Fig. 4) over
the same range as our
[3H]DPPC-labeled
surfactant experiments. The formation of lysoPC after exposure of
surfactant to the maximum concentration of rhGpII sPLA2 (10,000 U/ml) was low and
comparable to the levels of
[3H]palmitate released
as demonstrated in Fig. 3 (data not shown).

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Fig. 4.
Alterations in surfactant phospholipids by porcine pancreatic group I
sPLA2. NPS (0.5 mg of
phospholipid/ml) was incubated in presence and absence of porcine
pancreatic sPLA2 (0-1,000
U/ml) at 37°C for 2 h. Phospholipids were extracted and isolated
with TLC, and each fraction was analyzed for phosphorus content. Data
are means ± SD of %total phosphorus in phosphatidylcholine (PC)
and lysophosphatidylcholine (LPC) fractions.
* P 0.001 compared with
control without group I sPLA2.
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The profile of fatty acids released from surfactant was also directly
determined after hydrolysis of unlabeled surfactant with the porcine
pancreatic group I sPLA2 (1,000 U/ml). As expected, palmitic acid was the predominant free fatty acid
released, accounting for ~59% of the total (Fig.
5). In addition, a broad profile of released free fatty acids was seen, including myristic (14:0), palmitoleic (16:1), oleic (18:1), and linoleic (18:2).
This profile of fatty acids released after hydrolysis of our juvenile
NPS is comparable to the relative percentages estimated for the fatty acid compositions in the sn-2 position
reported for calf surfactant PC (24). However, small differences
between our NPS and calf PC do exist, including a smaller 16:1 fraction
and a larger 18:2 fraction. These differences likely reflect
differences in the composition of PC between the two species, and
sn-2 fatty acids are released as a
result of hydrolysis of other non-PC phospholipids (i.e.,
phosphatidylglycerol and phosphatidylethanolamine). A
similar but smaller release of palmitic acid
(P = 0.13) was seen in experiments in
which the maximum concentration of rhGpII
sPLA2 (10,000 U/ml) was used. This
result is consistent with the lack of specificity for individual fatty
acids in the sn-2 position typically
demonstrated by group I and group II sPLA2, which contrasts
with the preference of the high-molecular-weight cytosolic
PLA2 for arachidonic acid (8).

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Fig. 5.
Fatty acids released from surfactant phospholipids by
porcine pancreatic group I sPLA2.
NPS (0.5 mg of phospholipid/ml) was incubated in presence and absence
of porcine pancreatic sPLA2 at
37°C for 2 h. Free fatty acids were separated with TLC, HCl
methylated, extracted, and measured with gas chromatography with an
internal standard. Data are means ± SD of concentrations of each
fatty acid. * P 0.01 compared
with control without group I
sPLA2.
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The impact of sPLA2-mediated
hydrolysis of PC on surfactant function is demonstrated in Fig.
6. Increase in concentrations of the group
I porcine pancreatic enzyme (100-1,000 U/ml) results in an
increase in the
min, which
reflects a decline in surfactant function. Although the hydrolysis of
PC increases steadily over this dose range, the change in
does not.
Despite a further increase in hydrolysis between 500 and 1,000 U/ml,
min does not increase further
and appears to reach a plateau. This result suggests that the
relationship between hydrolysis of PC and surfactant function may
depend on a critical "threshold" of degradation that limits optimal packing of the phospholipid monolayer at the air-liquid interface of the bubble. In contrast, exposure of labeled NPS to the
maximum concentration of rhGpII
sPLA2 (10,000 U/ml) did not result
in an increase in
min (data not
shown).

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Fig. 6.
Comparison of porcine group I
sPLA2-mediated hydrolysis and
inhibition of surfactant function.
[3H]DPPC-labeled
surfactant (0.5 mg of phospholipid/ml) was incubated with porcine
pancreatic sPLA2 (0-1,000
U/ml) at 37°C for 2 h in buffered saline with
CaCl2 (5 mM). Data are means ± SD of minimum surface tensions recorded over 10 min on a pulsating
bubble surfactometer and of %total radioactivity in free fatty acid
fraction. Significant difference compared with control without group I
sPLA2:
* P < 0.001;
P < 0.02.
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To further compare the activity of secretory phospholipases
A2 within and between groups I and II, we studied the
ability of snake venom secretory phospholipases
A2 to cause surfactant dysfunction
(Fig. 7). In hydrolysis experiments, both
of the snake venom secretory phospholipases
A2, N. naja (group I) and C. atrox (group II), readily hydrolyze E. coli and labeled NPS, with activity for the
[3H]DPPC-labeled NPS
similar to that with the porcine pancreatic sPLA2 based on the comparable
hydrolytic rates of E. coli (data not
shown). Similarly, both snake venom secretory phospholipases A2, N. naja (100 U/ml) and C. atrox (100 U/ml), result in significant increases in
min. The ability of the
mammalian and snake venom group I enzymes to cause surfactant
dysfunction is remarkably similar. In contrast, the group II snake
venom causes surfactant dysfunction, whereas the rhGpII
sPLA2 does not.

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Fig. 7.
Comparison of mammalian and snake venom
sPLA2. NPS (0.5 mg
phospholipid/ml) was incubated at 37°C for 2 h in buffered saline
with CaCl2 (5 mM) with the
following sPLA2: group I
[porcine pancreatic (100 U/ml) and Naja
naja (N. naja; 100 U/ml)] and group II [rhGpII (10,000 U/ml) and
Crotalus atrox (C. atrox; 100 U/ml)]. Samples were then analyzed for
function with pulsating bubble surfactometer. Data are means ± SD.
* P < 0.001 compared with
control without sPLA2.
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 |
DISCUSSION |
Our data support the hypothesis that secretory phospholipases
A2 may play a critical role in inflammatory lung diseases
like ARDS and asthma. The most abundant surfactant-associated
phospholipid, PC, is hydrolyzed by the mammalian group I
sPLA2 (Fig. 2). This interaction
results in the formation of lysoPC (Fig. 4) and free fatty acids (Fig.
5) and results in decreasing overall surfactant function (Fig. 6). The
mammalian group II sPLA2
hydrolyzes surfactant-associated PC but with significantly less
activity than the group I sPLA2 and does not increase
min. This
difference in the mammalian enzymes is not solely explained by the
class differences between group I and group II enzymes
because the snake venom group II C. atrox resulted in significant PC hydrolysis and caused
surfactant dysfunction.
Surprisingly, the group II sPLA2
rhGpII did not demonstrate significant hydrolysis of PC. The literature
suggests that the group II enzyme would be the most likely candidate
enzyme because of its release by phagocytes, including
neutrophils and alveolar macrophages, in inflammatory states (3, 40).
The increased levels of sPLA2
activity in patients with septic shock have been attributed to the
release of group II enzymes (17). In addition, a BAL study (25) of
patients with ARDS demonstrates an increase in the activity of
sPLA2, which
coelutes with purified group II standards on heparin
column fractionation (25). However, this same BAL study
also demonstrated an additional heparin column fraction, with
significant sPLA2 activity from an
unclear group or source. From our data, the second
sPLA2 could be group I because its
affinity for PC is substantially greater than that of the group II
sPLA2 based on the relative
activity for each enzyme toward E. coli as a substrate. The role for group I enzymes is supported by the propensity for ARDS in patients with pancreatitis and
by models of pancreatitis where high levels of serum
sPLA2 activity have been
demonstrated (34). Although pancreatitis is an uncommon cause of ARDS
and the pancreas is an unlikely source for increased
sPLA2 activity in patients with
asthma or in the majority of ARDS patients, there are additional
sources of group I sPLA2.
Expression of group I sPLA2 mRNA
has been reported from nonpancreatic tissues including the human lung,
and activated human granulocytes release the group I proenzyme (31,
35). Our data favor the group I
sPLA2 as a more likely candidate
enzyme than the group II sPLA2 for
causing surfactant damage in ARDS and asthma.
In addition, recently identified secretory phospholipases
A2 might also contribute to
surfactant damage. A group V enzyme has been identified from a human
cDNA library; it may be present in lung and appears to have a greater
affinity for hydrolysis of PC than the mammalian group II enzyme (11).
In addition, mRNA for a group X enzyme has been isolated from human
lung tissue, but activity of this enzyme against PC or other
surfactant-associated phospholipids is unknown (10). Studies similar to
those reported here are needed to define the role for those secretory
phospholipases A2 in surfactant
dysfunction.
There are variations and uncertainties in the functional activity of
native and recombinant group II enzymes that make it impossible to
conclude that all group II enzymes cannot contribute to the hydrolysis
of surfactant-associated PC. Both group II secretory phospholipases
A2 (rhGpII and HuSF) demonstrated
excellent activity on the bacterial substrate E. coli, which suggests that the intact protein with
proper folding was present. Various compounds can serve to modify
activation of secretory phospholipases
A2, including the inhibitory
low-molecular-weight Clara cell protein (23), and could therefore
impact our results if present. The similarity in our results for the
hydrolysis of NPS and Survanta, which is commercially purified and
prepared, suggests that contaminating inhibitors like the Clara cell
protein were not a factor in our experiments. In addition, our studies
do not exclude that higher rates of hydrolysis for the
labeled surfactant might not be seen if higher concentrations of rhGpII
sPLA2 (>10,000 U/ml) were used. Our comparisons of group I and II activities are based on the relative
activity of each toward E. coli as a
substrate and not the absolute levels of enzyme in surfactant. If
levels of group II sPLA2 within
the inflamed alveolar microenvironment in ARDS or asthma increased
beyond those used in this study, significant hydrolysis could occur.
There are also important variables in the physical state of the
substrate of sPLA2-mediated
hydrolysis that may have contributed to our results. The group I
secretory phospholipases A2 have a higher affinity for PC than group II secretory phospholipases A2 and might be expected to
demonstrate the results we have shown. Group II secretory
phospholipases A2 preferentially
hydrolyze phosphatidylethanolamine and phosphatidylglycerol as
substrates (20, 38). We did not examine the ability of the group I or II enzymes to hydrolyze surfactant-associated phospholipids other than
PC. In our experimental incubations, the air-liquid interface at the
top of the microcentrifuge tube was small. As a result, the greatest
percentage of the phospholipid was not in a monomolecular film but more
likely in vesicles. We cannot exclude the possibility that the group II
sPLA2 could more readily hydrolyze
surfactant-associated PC in a monomolecular film as found in the
alveolus. Another potential variable within the surfactant mixture that
could lead to altered presentation of the substrate to the enzyme is
the SPs. The similar activity of group I and II secretory
phospholipases A2 on NPS and
Survanta suggests that SP-A and -D, which are not present in Survanta,
play little or no role in determining enzymatic activity. Low levels of
SP-B and -C are present in Survanta, and, therefore, we cannot evaluate
their role in regulation of
sPLA2-mediated hydrolysis.
The mechanism of phospholipase-induced dysfunction of surfactant is
likely to be multifactorial. Because the enzymatic reaction results in
a reduction in the native phospholipids and an increase in
lysophospholipids and free fatty acids, both have been shown to inhibit
in vitro surface tension lowering of surfactant
activity (9, 18). Any one of these changes in the
surrounding milieu of the alveolar monolayer and subphase could
potentially interfere with the ability of the surfactant layer to pack
tightly, associate with the SPs, and subsequently lower
. In all
likelihood, it is a combination of these events that explains the
subsequent dysfunction, and clarification of this intricate
relationship warrants further investigation. From the data in Fig. 6,
it is intriguing to note the sharp contrast in function shown between 100 and 500 U/ml of the group I pancreatic enzyme despite hydrolysis steadily increasing within the same concentration range.
These data suggest that there may be a critical concentration or
relationship between the ratio of intact phospholipids,
lysophospholipids, and fatty acids that determines the function of the
surfactant film.
In summary, our data demonstrate that there are significant differences
between the mammalian group I and group II secretory phospholipases
A2 and their ability to hydrolyze
surfactant-associated PC and lead to surfactant dysfunction. These
findings have important implications in the role of
sPLA2 in inflammatory lung
conditions like ARDS and asthma.
 |
ACKNOWLEDGEMENTS |
We gratefully acknowledge the generosity of Dr. Robert Dillard
(Department of Pediatrics/Neonatology, Wake Forest University School of
Medicine, Winston-Salem, NC) for providing Survanta samples and Dr.
Lisa Marshall (Smith Kline Beecham, King of Prussia, PA) for providing
the cDNA that was utilized for our recombinant human group II
sPLA2 gene construct.
 |
FOOTNOTES |
Fatty acid analyses were performed by the Analytic Chemistry Laboratory
of the Comprehensive Cancer Center of Wake Forest University, supported
in part by National Cancer Institute Grant CA-12107. This work was
supported in part by National Heart, Lung, and Blood Institute Grant
P01-HL-50395.
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: R. D. Hite, Section on Pulmonary and
Critical Care Medicine, Wake Forest Univ. School of Medicine,
Winston-Salem, NC 27157.
Received 29 January 1998; accepted in final form 29 May 1998.
 |
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