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Blood, Vol. 95 No. 3 (February 1), 2000:
pp. 1086-1092
PHAGOCYTES
Polytrauma induces increased expression of pyruvate kinase in
neutrophils
Rudolf Oehler,
Gertrude Weingartmann,
Nicole Manhart,
Ulrich Salzer,
Michael Meissner,
Werner Schlegel,
Andreas Spittler,
Michael Bergmann,
Daniela Kandioler,
Christiane Oismüller,
Heidi M. Struse, and
Erich Roth
From the Surgical Research Laboratories, Institute of Biochemistry,
Institute of Tumor Biology-Cancer Research, and Department of
Anesthesiology, University of Vienna, Vienna, Austria, and the
Department of Human Biological Chemistry and Genetics, University of
Texas, Galveston.
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Abstract |
Polytrauma (PT) leads to systemic activation of polymorphonuclear
neutrophils (PMNs). Organ damage commonly found in these patients is
ascribed to respiratory bursts of activated PMNs. With the use of
sodium dodecyl sulfate-polyacrylamide gel electrophoresis, PMN
extracts from PT patients were found to contain a clear protein band
not seen in control PMNs from healthy volunteers. This band was
identified by amino acid sequencing and Western blotting as pyruvate
kinase (PK). Enzymatic assays revealed a 600-fold increase in PK
activity in PMNs of PT patients, with the highest levels occurring
between the fifth and seventh posttraumatic day. In lymphocytes, no
such increase was detectable. As PK is a major regulatory enzyme in
glycolysis, glucose-dependent lactate production in PMNs from PT
patients was assayed. These cells showed a higher glycolytic lactate
production than controls. It was additionally demonstrated that acute
activation of respiratory burst activity depends mainly on breakdown of
glucose to lactate via the pentose-phosphate pathway and glycolysis. In
PMNs from PT patients, this glucose-dependent respiratory burst
activity was more than twofold higher than in controls. The increase in
expression and activity of PK in PMNs from PT patients may contribute
to the high glucose-dependent respiratory burst activity seen in these cells.
(Blood. 2000;95:1086-1092)
© 2000 by The American Society of Hematology.
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Introduction |
Severe polytrauma (PT) remains a major cause of
morbidity and mortality despite modern techniques of resuscitation and
intensive care and an ever-increasing number of powerful and effective
antibiotics.1 In PT patients, a local inflammatory process
spills over and causes an exaggerated systemic response, with
inflammatory damage to otherwise healthy cells and organs distant to
the primary site of injury. Severe traumatic injury leads to the
systemic activation of various humoral systems and cellular
systems2; it also induces metabolic changes in protein
catabolism3 and reduces the endogenous storage of
glutamine,4 an important immunonutrient.5
Polymorphonuclear neutrophils (PMNs) play an important role in this
inflammatory process. Mediators released by activated PMNs, such as
oxygen radicals, proteinases, and phospholipid-derived products, cause
organ damage that often results in multiple failure of vital
organs.6 Additionally, these mediators affect the hydration
state of cells, leading to abnormalities in protein catabolism in these
patients.7 The first step in organ damage, injury to
vascular endothelial cells, is mediated by the toxic oxygen metabolites
of activated PMNs.8
When exposed to appropriate stimuli, PMNs change their pattern of
oxygen metabolism, increasing their oxygen uptake sharply while
releasing large amounts of superoxide anion into the environment. The
key reaction in this respiratory burst is the 1-electron reduction of
oxygen to superoxide anion using the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH), catalyzed by the
membrane-bound NADPH oxidase.9 NADP+ is then
reconverted to NADPH by the pentose phosphate pathway (PPP) by
breakdown of glucose. Thus, respiratory burst activity of PMNs depends
on glucose metabolism. In the present study, it is described, for the
first time: an increase in expression of pyruvate kinase (PK) in PMNs
of PT patients, compared with healthy volunteers. PK catalyzes a major
regulatory step in the glycolytic pathway, the conversion of
phosphoenolpyruvate (PEP) to pyruvate. PMNs of PT patients show an
enhanced breakdown of PEP to pyruvate, which correlates with increased
glycolytic lactate production in these cells. It is suggested that the
increased expression of PK in PMNs of PT patients results in a higher
PPP rate for higher NADPH production and is therefore a requirement for
the increased respiratory burst activity found in these cells.
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Materials and methods |
If not otherwise indicated, all chemicals used were
obtained from Sigma (St. Louis, MO).
We studied 16 patients with PT (11 male, 5 female), with a mean age of
37.7 years (range, 21-73). The trauma involved the head in 12 patients,
the thorax in 9 patients, the spine in 3 patients, the abdomen in 4 patients, the pelvis in 7 patients, and the extremities in 13 patients.
All patients had trauma in at least 2 of the above-mentioned areas. The
disease was graded according to the Injury Severity
Score.10 Patients had an average Injury Severity Score of
44 (range, 25-58). All patients needed mechanical
ventilation and showed the signs of a trauma-induced systemic
inflammatory response syndrome upon arrival in the intensive care unit.
Throughout the study, blood loss frequently had to be substituted with
erythrocyte transfusions, a mean 19.4 red packed cell units (range,
0-101). At the time of investigation, no patient was classified as
septic according to standard clinical and laboratory parameters. Blood
cultures were negative in all patients. The control group comprised 22 healthy volunteers (10 male, 12 female), aged 31 ± 8 years. The
protocol for obtaining human blood was approved by the Ethics Committee
of the University of Vienna.
Leukocytes of 14 mL anticoagulated
(ethylenediaminetetraacetic acid [EDTA]-treated) fresh venous blood
were purified, as described,11 within 20 minutes after
blood samples were collected. Briefly, PMNs were separated from
mononuclear cells by centrifugation of blood cells loaded on
Ficoll-Paque (Pharmacia, Uppsala, Sweden). PMNs were collected from the
pellet of the Ficoll gradient, washed in 0.9% NaCl, and separated from
the erythrocytes by dextran sedimentation. Remaining red blood cells
were removed by hypotonic lysis. PMN recovery was greater than 92%,
and red blood cell contamination was less than 3%. To isolate
lymphocytes, cells were collected from the interface region of the
Ficoll gradient, washed twice in phosphate buffered saline (PBS) (10 mmol/L Na2HPO4/NaH2PO4, pH = 7.4; 135 mmol/L NaCl; 2.7 mmol/L KCl; 5 mmol/L EDTA; 0.5% bovine serum albumin), and resuspended in PBS containing
magnetically labeled anti-CD14 antibodies. Monocytes were separated
from lymphocytes by magnetic cell sorting (MACS, Miltenyi Biotec,
Bergisch Gladbach, Germany) according to the manufacturer's
instructions. The purity of the lymphocyte fraction was usually 90%,
as determined by flow cytometry. The remaining 10% were mostly
thrombocytes and monocytes.
Cells were lysed by hypotonic treatment. The lysis buffer consisted of
10 mmol/L Tris-HCl, pH = 7.8; 1 mmol/L
EDTA; 10 mmol/L KCl; 0.3% Triton X-100; and 1 mmol/L phenylmethylsulfonyl fluoride (PMSF). After
homogenization, the cell lysate was collected and clarified from the
nuclei by centrifugation at 800g for 3 minutes. The cell lysate
was centrifuged a second time (17 000g for 3 minutes), and the
supernatant was stored in aliquots at  70°C. The protein content was quantified as described by Bradford.12
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
was performed according to Laemmli.13 Briefly, 10 µg of
cell lysate was loaded per lane on a 10% SDS-polyacrylamide gel and run at 160 V for 50 minutes. Protein bands were
visualized by Coomassie staining. Two-dimensional PAGE was performed as
described by O'Farrell.14 Protein spots were visualized by
silver staining according to Merril.15
For Western blot analysis, proteins from a 10 µg cell lysate sample
were separated by SDS-PAGE and transferred to a nitrocellulose membrane
by electroblotting. PK expression was revealed with goat antibodies
against rabbit PK-M (Rockland, Gilbertsville, PA), which shows strong
cross-reactivity with human PK-M. Bound antibodies were detected with a
peroxidase-conjugated anti-goat immunoglobulin (Ig) G antibody (Sigma,
St. Louis, MO) in the presence of Supersignal substrate (Pierce,
Rockford, IL).
Proteins were separated by SDS-PAGE and blotted onto a polyvinylidene
difluoride protein sequencing membrane (PVDF) (Immobilon-P, Millipore,
Bedford, MA) with the use of a BioRad electroblotting unit. Protein
bands were visualized by Coomassie staining, and the band of interest
was excised. The piece of membrane was destained in 50% methanol,
blocked for 30 minutes at 37°C with 0.5%
polyvinylpyrrolidone-40 in 0.1 mol/L acetic acid, and
then extensively washed with water. The membrane was incubated
overnight with 1µg trypsin (sequencing grade, Boehringer-Mannheim,
Mannheim, Germany) in 200 µL 100 mmol/L Tris/Cl buffer, pH = 8.0,
10% acetonitrile. The membrane was stripped twice with 200 µL 50% acetonitrile. The supernatants were pooled, acidified with 6 µL 1 mol/L HCl and
subjected to reversed phase high performance liquid chromatography
(Nucleosil 300, 5 µm, C18, 4 × 250 mm). The peptides were
eluted at 1 mL/min with the use of a 0 to 50%
acetonitrile gradient in 0.1% trifluoroacetic acid (1% per minute),
and 0.5 mL fractions were collected. The absorbance was
measured at 214 nm. Sequence analysis of peak fractions was carried out
by automated sequencing (model 477A sequenator, Applied Biosystems,
Foster City, CA).
PK activity was assayed by the widely used standard method described by
Fujii and Miwa.16 Briefly, cell lysate was diluted in
tri-potassium-magnesium-EDTA (TKME)-buffer (100 mmol/L
Tris-Cl, pH = 8.0; 100 mmol/L KCl; 10 mmol/L MgCl2; 0.5 mmol/L EDTA)
containing 0.2 mmol/L nicotinamide adenine dinucleotide
(reduced form) (NADH), 1.5 mmol/L adenosine
diphosphate, 5 mmol/L PEP, and 60 milliunits(mU)/mL lactate dehydrogenase
(LDH) and incubated at 37°C for up to 60 minutes. In this assay,
the conversion of PEP to pyruvate (catalyzed by PK) is combined with
the reduction of pyruvate to lactate (catalyzed by LDH). The latter
reaction is accompanied by an oxidation of NADH. LDH activity added to
the assay mixture was in excess and does not interfere with the results
of this combined assay. The conversion of 1 µmol PEP to pyruvate per
minute, as determined by the decrease in absorbance at 340 nm due to
oxidation of NADH, was defined as 1 unit. Triton X-100 contained in the
cell lysate reduces the sensitivity of the PK assay but also leads to
higher reproducibility.17
To determine lactate production, we incubated cells at 37°C for up
to 4 hours and measured the lactate content of the medium photometrically by the lactate oxidase-peroxidase assay (Sigma Diagnostics, St. Louis, MO). To exclude any negative effects of the
long incubation time without any metabolic substrate on the viability
of PMNs, we analyzed cell viability and function before and after
incubation. After 4 hours of incubation without
substrate, more than 90% of cells were still viable. To
determine cell function, we stimulated cells with phorbol myristate
acetate (PMA) and measured the superoxide anion production as described
below. Neither PMNs from PT patients nor those from healthy volunteers
showed a significant reduction in superoxide anion formation by the
4-hour incubation.
Superoxide anion production by isolated PMNs was determined by
measuring the superoxide dismutase (SOD)-inhibitable reduction of
ferricytochrome c to the ferrous form, as described
previously.18 Briefly, 106 cells were incubated
in 1 mL PBS (138 mmol/L NaCl, 2.7 mmol/L KCl, 0.6 mmol/L CaCl2, 1.0 mmol/L
MgCl2, 10 mmol/L
NaH2PO4/Na2HPO4, pH = 7.4) containing 40 µmol/L cytochrome c with or
without 20 µg/mL SOD. Samples were equilibrated for 5 minutes at 37°C, and the reaction was initiated by adding 100 ng/mL PMA. Absorbance was measured at 550 nm every 2 minutes for up to 1 hour. After this incubation, still more than 91%
of cells were viable as assayed by trypan blue exclusion. The reference
sample containing SOD allowed estimation of the superoxide specific
cytochrome c reduction. The absorbance determined in the reference
was subtracted from that in the sample; the resulting value
was the rate of SOD-inhibitable cytochrome c reduction. The
maximum rate was calculated from the maximum slope (usually
between the sixth and the 25th minute after addition of PMA)
by the use of the specific millimolar extinction coefficient
of reduced cytochrome c of 21.1 [(mmol/L 1·cm1)].
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Results |
Peripheral blood PMNs of patients with polytrauma (PT-PMNs) express
a set of proteins different from that of PMNs of healthy volunteers
(HV-PMNs). When these proteins are separated by molecular weight by means of SDS-PAGE, a sharp protein band that
does not appear in HV-PMN samples is found in PT-PMNs (Figure
1a). This additional band migrates at a
molecular weight of about 56 to 58 kd. It showed rather
strong staining in every PT-PMN extract analyzed (n = 10). However,
in most of the analyzed HV-PMN extracts, no band was seen at the
corresponding position. The PT-PMN-specific band was seen in all
samples taken from patients within 48 hours after trauma. Between the
fifth and the seventh day after trauma, the band became even stronger
in 4 out of 10 patients (data not shown).
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| Fig 1.
Protein expression in PMNs from PT patients and healthy
volunteers.
(A) Coomassie blue-stained SDS-polyacrylamide gel showing protein
pattern of lysates (10 µg per lane) from PMNs from a healthy
volunteer (lane 1) and 2 PT patients (lanes 2 and 3). The arrow
indicates the additional band in lanes 2 and 3. (B) Western blot
showing expression of PK in PMNs from a healthy volunteer (lane 1) and
a PT patient (lane 2).
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To investigate whether this protein band, which was found only in
PT-PMN extracts, corresponded to a single protein or consisted of
different proteins, 2-dimensional gel electrophoresis was performed. A
PT-PMN extract was separated by SDS-PAGE, the PT-PMN-specific band was
excised, and the protein was eluted from the gel. After a 2-dimensional
electrophoretical separation of the eluate (first dimension,
isoelectric focusing; second dimension, SDS-PAGE), only 1 spot could be
seen in the silver stained gel, which demonstrated that the
PT-PMN-specific band consisted of a single protein. This spot
corresponded to a protein with a molecular weight between 56 and 58 kd, with a pI between 7.4 and 7.6 (data not shown).
In order to identify this protein, we analyzed the amino acid sequence
of 2 trypsinized fragments as described in the "Materials and
methods" section. Both sequences were identical to the glycolytic enzyme PK-M (PK; EC 2.7.1.40; SWISSPROT Acc. No. P14 618): the sequence of trypsin-fragment 1 (A-P-I-I-A-V-T-R) corresponded to the PK
sequence at position 446-454, and the sequence of trypsin-fragment 2 (G-P-E-I-R-T-G-L-I-K-G-S) corresponded to the PK sequence at position
114-126. The theoretical molecular weight of PK (57 746 d) and the calculated pI (7.57) corresponds
to the values obtained in the 2-dimensional PAGE (MW = 56-58
kd; pI = 7.4-7.6).
For further confirmation of these data, we analyzed PK expression by
Western blotting using a specific antibody. In Figure 1B, a much
stronger staining is clearly observed in the PT-PMN lane than in the
HV-PMN lane. In sum, these results strikingly illustrate that PK
expression is much greater in PT-PMNs than in HV-PMNs.
PK catalyzes the essentially irreversible transfer of the phosphate
group from PEP to ADP, yielding adenosine triphosphate (ATP) and
pyruvate. To investigate whether the high expression of PK in PT-PMNs
causes an increased breakdown of PEP to pyruvate, we assayed the enzyme
activity in these cells. Peripheral blood from PT patients and from
healthy volunteers was collected, and PK activity in the lysate of
isolated PMNs was measured. In PT-PMNs (Figure
2A), the PK activity was much higher than
in HV-PMNs. The PK activities found in HV-PMNs were in the same range
as described previously.19
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| Fig 2.
PK activity in PMNs of PT patients.
Cells were isolated from venous blood, and PK activity was measured in
cell lysates. PK activity is expressed as milliunits (mU)
per mg protein. (A) PK activity in PMNs of healthy volunteers
(n = 19) and PT patients (n = 9) between the second and fifth
posttraumatic day. (B) PK activity in PMNs at different times after
trauma: within the first 48 hours (n = 5) after trauma, and between
120 and 170 hours after trauma (n = 9). (C) PK activity in PMNs and
lymphocytes of PT patients. PMNs and lymphocytes were isolated
simultaneously from venous blood of PT patients (n = 6) between the
second and fifth posttraumatic day. The data are shown in a box-chart
plot, with markings that indicate the 0, 25th 50th, 75th, and 100th
percentiles.
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PT triggers a complex cascade of posttraumatic events that leads to a
multifocal pathophysiologic process. The sequence of events following
trauma has been divided into an acute early phase of traumatic shock
and a later phase of systemic inflammation.2 To determine
whether the increase in PK activity is an early or a late event in the
pathogenesis of polytrauma, we measured the enzyme activity in PMNs
within the first 2 posttraumatic days and between the fifth and the
seventh day after trauma (Figure 2B). In the early stage after injury,
the PK activity was higher in some patients than in healthy controls,
but when we considered the whole group, we found no significant
elevation. In the later phase, however, a strong and highly significant
increase in PK activity was detected. In 4 patients, we investigated PK
activity in PMNs at both times and found a similar increase between the early and the late phases. Since later posttrauma periods were not
investigated, it is not clear when the values return to normal.
To investigate whether the increase of PK in PMN is a cell
type-specific event or a general phenomenon of leukocytes, we measured the enzyme activity in extracts of PMNs and lymphocytes within the same
PT patients. Unfortunately, the low number of monocytes isolated from
the 14 mL blood available from each patient do not allow
PK activity assays. In lymphocytes, a lower PK activity was observed
than in PMNs (Figure 2C). In comparison with healthy volunteers, the PK
activity in lymphocytes of PT patients is not significantly elevated
(data not shown).
The catalysis of PEP to pyruvate by PK is 1 of the 2 major regulatory
steps in glycolysis. The other locus of control is at the level of
phosphofructokinase. In PMNs, PK has been described to correlate
closely to the lactate production and is assumed to be rate-limiting
for glycolysis.20 To delineate the consequences of an
increased PK activity on the metabolism of PMNs, we measured glycolysis
rate. PMNs have been described as converting glucose almost completely
to lactate21 (Figure 3).
Therefore, we analyzed lactate production of PT-PMNs between the second
and fifth posttraumatic day and lactate production of HV-PMNs. Cells
were incubated in PBS in the presence or absence of glucose, and
lactate production was measured for up to 4 hours. PT-PMNs showed a
glucose-dependent lactate production rate that was higher than that of
HV-PMNs (Figure 4). This indicates that the
higher expression of PK in PT-PMNs is associated with an elevated
glycolysis rate. An increase of lactate production by about 50%
appears to be modest in comparison with the 600-fold increase in PK
activity. However, the assay systems used to measure both parameters
are completely different and do not allow direct stoichiometric
comparisons (PK activity was measured in vitro in cell lysates, whereas
the lactate production was analyzed in vivo in intact cells).
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| Fig 3.
Schematic representation of the relationship between
glycolysis, pentose phosphate pathway, and formation of oxygen radicals
by NADPH oxidase.
The form of arrows indicates the specific enzyme activities in PMNs as
determined previously by Fauth et al17: thick arrows
correspond to an activity of 400 to 2000 mU/mg; dashed
arrows correspond to an activity lower than 100 mU/mg.
Abbreviations: PPP, pentose phosphate pathway; G6P, glucose
6-phosphate; G6PDH, glucose 6-phosphate dehydrogenase; PFK,
phosphofructokinase; FPA, fructose-1,6-bis-phosphate aldolase; GA3P,
glyceraldehyde 3-phosphate; PEP, phosphoenolpyruvate; PK,
pyruvate kinase; LDH, lactate dehydrogenase; PDH, pyruvate
dehydrogenase.
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| Fig 4.
Lactate production of PMNs from PT patients and healthy
volunteers.
PMNs were isolated from venous blood of healthy volunteers (n = 5)
and PT patients (n = 5) between the second and fifth posttraumatic
day. Cells were cultured in PBS with and without 11 mmol/L glucose. Lactate in the medium was measured every
hour for up to 4 hours. Glucose-dependent lactate production is
determined by the difference between lactate production in the presence
and the absence of glucose. Data shown represent the median ± standard deviation.
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PK catalyzes the final step in glycolysis (Figure 3). Glucose breakdown
via phosphofructokinase, as well as by the PPP, results in the
conversion of PEP to pyruvate via PK. In PK-deficient red blood cells,
the PPP was shown to be inhibited.22 The PPP provides cells
with NADPH, which serves as the substrate of respiratory burst activity
in PMNs. To investigate whether glucose breakdown via the PPP or
glycolysis directly affects superoxide anion production in PMNs, we
examined respiratory burst activity rates in the presence and absence
of glucose. HV-PMNs were isolated and treated with PMA in a medium
consisting of PBS with and without glucose. Superoxide anion production
was determined as SOD-inhibitable cytochrome c reduction. Superoxide
anion production was observed to be higher in the presence than in the
absence of glucose (Figure 5). In addition,
PMNs in the glucose-containing medium exhibited a lactate production
rate of 4.13 ± 0.82 µg/(106
PMN·hour), whereas in the absence of glucose, less
lactate was produced (0.76 ± 0.67 µg/(106
PMN·hour), P = .0001, n = 5). These data clearly show
that inclusion of glucose in the activation medium further increases
respiratory burst activity and that this increase is associated with a
higher level of glucose breakdown to lactate.
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| Fig 5.
Influence of glucose breakdown on superoxide anion
production in PMNs.
Superoxide anion production was determined as SOD-inhibitable
cytochrome c reduction. PMNs were isolated from venous blood of healthy
volunteers (n = 7) incubated in PBS with or without 11 mmol/L glucose (left 2 bars) and 500 µmol/L DHEA (right 2 bars). Cells were stimulated with
100 ng/mL PMA in the presence and absence of SOD.
Cytochrome c reduction was measured photometrically (550 nm) every 2 minutes for up to 1 hour, and maximum slope was used to calculate
superoxide anion production per minute. Data shown represent the median ± standard deviation.
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To determine whether the glucose-mediated increase in superoxide anion
production depends on the breakdown by means of PPP, we examined the
effect of the specific inhibitor of glucose 6-phosphate dehydrogenase:
dehydroepiandrosterone (DHEA). As is well established, glucose
6-phosphate dehydrogenase is the rate-limiting step in the PPP (Figure
3). The addition of DHEA to PMNs incubated in PBS with glucose reduced
superoxide anion production to levels seen without glucose (Figure 5).
In PMNs incubated in PBS without glucose, however, DHEA had no effect
on superoxide anion production. Thus, the glucose-dependent increase in
respiratory burst activity can be inhibited by blocking the PPP.
Blockage of the PPP is likely to result in a decreased level of
available NADPH. These data show that respiratory burst activity of
PMNs depends, at least in part, on the rate of glucose breakdown via
the PPP/glycolysis pathway where PK plays a regulatory role.
To investigate whether the increase of PK activity and glycolytic
lactate formation found in PT-PMNs is accompanied by higher glucose-dependent respiratory burst activity, we examined superoxide anion formation in the presence and absence of glucose. Blood samples
were taken from patients 48 to 120 hours after trauma and from healthy
volunteers. PMNs were isolated and treated with PMA in a medium
consisting of PBS with and without glucose. Superoxide anion production
was determined as SOD-inhibitable cytochrome c reduction. In the
absence of glucose, both groups showed similar respiratory burst rates
(PT-PMNs = 0.495 ± 0.190 nmol/(106
PMN·minute), n = 7; HV-PMNs = 0.549 ± 0.217 nmol/(106 PMN·minute), n = 9). The glucose-dependent
superoxide anion production, however, was more than twofold higher in
PT-PMNs than in HV-PMNs (Figure 6).
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| Fig 6.
Glucose-dependent respiratory burst activity in PMNs from
PT patients and healthy volunteers.
Superoxide anion production was determined as SOD-inhibitable
cytochrome c reduction. PMNs were isolated from venous blood of healthy
volunteers (n = 7) and polytrauma patients between the second and the
fifth posttraumatic day (n = 6) and incubated in PBS with or without
11 mmol/L glucose. Cells were stimulated with 100 ng/mL PMA in the presence and absence of SOD. Cytochrome
c reduction was measured photometrically (550 nm) every 2 minutes for
up to 1 hour, and maximum slope was used to calculate superoxide anion
production per minute. The superoxide anion production in the presence
of glucose minus that in its absence was defined as glucose dependent.
Data shown represent the median ± standard deviation.
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Discussion |
This study demonstrates that after severe trauma, a dramatic
increase in expression of PK is seen to occur in PMNs. PK, a key enzyme
in the glycolytic pathway, is found as at least 3 tissue-specific isozymes in mammals.23 By amino acid sequencing, it was
shown that the form expressed in PMNs of PT patients is the isozyme PK-M. This is in accordance with data from Tsutsumi et al, which showed
that in leukocyte preparations from healthy volunteers the isozyme PK-M
is predominantly expressed.24
PK-M has been further found to exist as 2 different isotypes: M1 and
M2, produced by alternative splicing of the same primary transcript
from the M gene.25 The amino acid sequences of these isotypes are almost identical and differ by only 45 residues coded by
exons specific for the isotype. Sequencing data, however, did not
enable us to definitively distinguish whether the PK found in PMNs was
of the M1 or the M2 type. However, since PK-M1 is expressed
predominantly in skeletal muscle, heart, and brain and the M2 isotype
is widely distributed in such adult tissues as leukocytes (as well as
in the fetus and in undifferentiated or proliferating
tissues26,27), the PK-isotype found in PMNs is most likely
the M2 type.
PK-M2 was shown to be identical with the thyroid hormone binding
protein p58.28 Binding of 3.3',5-triiodo-L-thyronine
(T3) and its analogues results in the inhibition of the activity of PK-M2, preventing its conversion to an active tetrameric
form.29 The increased PK activity in PMNs of PT patients,
however, is associated with increased amounts of PK protein. Thus, the
up-regulation of PK activity after trauma is probably due to protein
expression rather than to increased tertramerization. Therefore, a
major involvement of T3 can be excluded.
The activity as well as the messenger RNA level of M2-type PK has been
reported to increase in rat thymocytes after induction of proliferation
by concanavalin A and interleukin 2.30 In contrast to
thymocytes, mature PMNs have lost proliferative ability. Normally, only
mature PMNs circulate in the blood, but in times of stress, progressively earlier neutrophilic forms can be seen in blood smears.31 This suggests that the increase in PK activity of PT-PMNs, as reported here, might be due to an increased pool of immature precursor cells. However, these PT-PMNs display an increased glucose-dependent respiratory burst activity. Thus, since respiratory burst is a late manifestation of PMN maturation,31 an
increased pool of PMN precursor cells in these preparations
is unlikely. The increase of PK expression found in the PMNs
of PT patients therefore seems not to be related to a
maturation effect.
An increase in PK activity in PMNs was also found in patients with
psoriasis vulgaris and psoriasis arthropathica.32 The activity of the enzyme was significantly increased during the active
stage of the disease and returned to normal during remission. In PT
patients, the PK activity was also shown to change during the course of
illness. PMNs of PT patients display an increase in PK activity within
the first 48 hours after trauma when compared with HV-PMNs. During the
course of illness, PK activity (Figure 2B) as well as PK expression
increases further. Even during remission, such changes are seen to
persist: a lower, but still elevated, PK activity level is detectable.
The reason for this increased PK expression remains unclear. Trauma
induces a complex cascade of pathophysiological events. Immediately
after trauma, various humoral and cellular systems become activated.
Platelets and PMNs are the cells chiefly affected. An increased number
of PMNs can be found in the liver, kidney, heart, spleen, and
brain.6 Mediators released by activated PMNs cause a
systemic inflammatory response syndrome. This commonly occurs within
the first 2 days after trauma.2 The results presented here
show that at this stage PK activity in PMNs is already elevated.
Within the first week after trauma, the exaggerated systemic response
leads to a severe impairment of organ function.2 This
accounts for the typical host response, which causes a further massive
activation of the monocyte-macrophage axis, endothelial cells, and
PMNs. Between the fifth and the seventh posttraumatic day, PK activity in PMNs is even higher than in the first 2 days after
trauma. In lymphocytes, in contrast, no increase in PK activity is ever
detected. These results indicate that PK activity is up-regulated in
peripheral blood PMNs and seems to be related to the activation state
of these cells during pathogenesis.
The circulating PMN pool reflects the balance between synthesis on the
one hand and margination and breakdown on the other. Biologically
activated PMNs marginate and leave the circulation. Therefore,
increased PK expression in PMNs may not be a direct result of increased
stimulation of these cells, but rather a response of the storage
compartment in the bone marrow to the pathological stimuli. The PMNs
produced after severe trauma may differ from those produced under
normal conditions. Additional corroboration for such a notion comes
from the observation that abnormalities are found in the functional
characteristics of circulating PMNs after serious injury. PMNs'
adherence increases proportionally in severely traumatized
patients.33 In addition, PMN chemotaxis is impaired
following major traumatic injury.34
It may be hypothesized that the increase in PK activity in PMNs after
trauma is closely related to the glucose metabolism of these cells.
Very little information is available on the glucose metabolism of PMNs.
In whole leukocyte preparations, which are 60% to 70% PMNs, only a
slight conversion of glucose to CO2 (5%) is
described.21 Correspondingly, Ahmed and coworkers found
that only 2% of consumed glucose is converted to CO2 in
dimethyl sulfoxide-differentiated neutrophilic-like HL60
cells.35 Of the glucose consumed, 82% was
converted to lactate via glycolysis. The major rate-limiting step of
glycolysis is the conversion of PEP to pyruvate catalyzed by
PK.20 The increased PK activity in PT-PMNs coincides with increased glucose-specific lactate production, indicating that these
cells exhibit a higher glycolysis rate than HV-PMNs. Glycolysis supplies cells not only with ATP, but also with intermediates and
cofactors. In the course of glucose breakdown, glucose 6-phosphate can
be metabolized to glyceraldehyde-3-phosphate either via
phosphofructokinase and fructose-1,6-bis-phosphate aldolase or via the
PPP (Figure 3). Glyceraldehyde-3-phosphate is then metabolized to
pyruvate via PK. PMNs have been reported to show a low fructose-1,
6-bis-phosphate aldolase activity, whereas the activity of glucose
6-phosphate dehydrogenase in these cells is about 6 times
higher.17 Since glucose 6-phosphate dehydrogenase is the
rate-limiting step in the PPP,21 PMNs seem to metabolize
glucose preferentially via the PPP and PK to lactate. The PPP provides
cells with NADPH, which is the substrate of respiratory burst activity
in PMNs. It was demonstrated in this study that the glucose-dependent
respiratory burst can be almost completely suppressed by inhibiting
glucose 6-phosphate dehydrogenase. This indicates that in PMNs a direct connection exists between the glucose breakdown via the
pentose-phosphate-PK pathway and respiratory burst
activity. Additional corroboration for this comes from Tan et
al,36 who have recently reported that
PMA-stimulated superoxide anion production of PMNs was seen to increase
with the concentration of exogenous glucose and to plateau at 5 to 10 mmol/L glucose. In addition, PMN activation involved
increased glucose transport and intrinsic activation of glucose
transporter molecules. The superoxide anion production rate of PMNs
observed by Tan and coworkers, however, was remarkably higher than in
our experiments. This could be due to the different PMN isolation
procedures used. Tan et al isolated PMNs by density gradient
centrifugation as opposed to hypotonic lysis, which was used in the
present study. They observed a fivefold decrease in superoxide anion
production after hypertonic lysis, which correlates with the level of
superoxide anion production measured in the present study.
An increase in PK activity would permit higher glucose consumption,
which in turn would provide an additional substrate for respiratory
burst activity. Indeed, in PT-PMNs, elevated PK activity correlates
with a twofold increase of glucose-dependent superoxide anion
production after stimulation with PMA. To our knowledge, there are no
publications selectively describing the glucose-dependent respiratory
burst activity in PMNs from PT patients. However, several studies were
performed analyzing the overall superoxide anion production in the
presence of glucose, which measures the sum of glucose-dependent and
glucose-independent superoxide anion production. All groups found an
activation of PMNs and an increased superoxide anion production after
severe injury. However, their results differ with respect to the time
window when activation occurs. Botha and coworkers showed that PMNs
from patients with severe torso trauma are primed and activated in the
first 24 hours postinjury, but become unresponsive to PMA activation
after 48 hours.37 In contrast, other groups found an
elevated superoxide anion production in trauma patients up to 72 hours
after injury.38,39 Similar results were published recently
by Ogura and coworkers.40 They found an enhanced response
to fMLP from days 2 to 21 after injury. In the present study, the
elevated glucose-dependent superoxide anion production was measured
between the second and the fifth posttraumatic day, which is in
accordance with the latter publications.
Our data demonstrate that after trauma, an increase in expression and
activity of the glycolytic enzyme PK is seen to occur in PMNs. This
elevation is associated with an increased glucose-dependent superoxide
anion production. Since PK is the rate-limiting step in glycolysis in
PMNs, it might be hypothesized that the increased activity of this
enzyme in PT patients contributes to the higher glucose-dependent respiratory burst activity of these cells.
| |
Acknowledgments |
We are grateful to Susanne Oehler for helpful discussions and
proofreading the manuscript. We also thank Joachim Seipelt for providing the PK-specific antibodies.
| |
Footnotes |
Submitted December 2, 1998; accepted October 6, 1999.
Reprints: Rudolf Oehler, Surgical Research Laboratories,
Allgemeines Krankenhaus 8G9.05, Waehringer-Guertel 18-20, A-1090 Vienna, Austria (EU); e-mail: rudolf.oehler{at}akh-wien.ac.at.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction