|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 11 (December 1), 1999:
pp. 3897-3905
Granulocyte-Macrophage Colony-Stimulating Factor Upregulates Reduced
5-Lipoxygenase Metabolism in Peripheral Blood Monocytes and
Neutrophils in Acquired Immunodeficiency Syndrome
By
Michael J. Coffey,
Susan M. Phare,
Sandro Cinti,
Marc Peters-Golden, and
Powel H. Kazanjian
From the Divisions of Pulmonary and Critical Care Medicine and
Infectious Diseases, University of Michigan Medical Center, Ann Arbor,
MI.
 |
ABSTRACT |
Leukotrienes (LT) are mediators derived from the 5-lipoxygenase
(5-LO) pathway, which play a role in host defense, and are synthesized
by both monocytes (peripheral blood monocyte [PBM]) and neutrophils
(PMN). Because 5-LO metabolism is reduced in alveolar macrophages and
PMN from acquired immunodeficiency syndrome (AIDS) subjects, we
investigated the synthesis of LT by PBM and PMN from these subjects.
There was a reduction (74.2% ± 8.8% of control) in LT synthesis in
PBM from human immunodeficiency virus (HIV)-infected compared with
normal subjects. Expression of 5-LO (51.2% ± 8.8% of control), and
5-LO activating protein (FLAP) (48.5% ± 8.0% of control) was
reduced in parallel. We hypothesized that this reduction in LT
synthetic capacity in PBM and PMN was due to reduced cytokine
production by CD4 T cells, such as granulocyte-macrophage colony-stimulating factor (GM-CSF). We treated 10 AIDS subjects with
GM-CSF for 5 days. PBM 5-LO metabolism ex vivo was selectively increased after GM-CSF therapy and was associated with increased 5-LO
and FLAP expression. PMN leukotriene B4
(LTB4) synthesis was also augmented and
associated with increased 5-LO, FLAP, and cytosolic phospholipase
A2 expression. In conclusion, as previously demonstrated for PMN, PBM from AIDS subjects also demonstrate reduced
5-LO metabolism. GM-CSF therapy reversed this defect in both PBM and
PMN. In view of the role of LT in antimicrobial function, cytokine
administration in AIDS may play a role as adjunct therapy for infections.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
INFECTION WITH human immunodeficiency
virus (HIV) results in the development of acquired immunodeficiency
syndrome (AIDS) by causing depletion and dysfunction of CD4 T
lymphocytes.1 However, the dysfunction of other cell types
including macrophages2,3 and neutrophils4 also
occurs in AIDS. There is evidence that chemotaxis, phagocytosis, and
killing of microorganisms by macrophages, monocytes (peripheral blood
monocyte [PBM]),5 and neutrophils (PMN)6 is
reduced with the progression of HIV disease. Furthermore, there is
evidence of reduced synthesis of specific mediators associated with the
reduction in CD4 count. 5-lipoxygenase (5-LO) is the enzyme, which
catalyzes the formation of leukotriene (LT) from the fatty acid
arachidonic acid (AA) bound to membrane phospholipids.7,8 These proinflammatory mediators are involved in the pathogenesis of a
number of disease states including asthma.9 Our laboratory has previously shown that LT synthesis is reduced in alveolar macrophages (AM) from HIV-infected subjects compared with healthy controls.10 Furthermore, in a recent study, we demonstrated that PMN from AIDS patients demonstrated reduced LT synthetic capacity
compared with controls.11 However, there is no information about the effects of HIV on the 5-LO pathway in PBM, which are precursor cells for AM.
LT also play an important role in host defense mechanisms against
microorganisms.12,13 5-LO knockout mice are more
susceptible to pneumonia from Klebsiella pneumoniae
than are wild-type mice.14 LT deficiency is associated
with impaired phagocytosis and killing of microorganisms.15
The addition of exogenous LTs in vitro to LT-deficient cells boosts
phagocytosis.14 Likewise, in vivo treatment of AIDS
patients with granulocyte colony-stimulating factor (G-CSF) boosted PMN
5-LO metabolism, and this resulted in increased anticryptococcal
activity.11
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine
produced predominantly by activated T lymphocytes.16-18 With the depletion and dysfunction of CD4 T lymphocytes in AIDS, levels
of GM-CSF decline.19-21 GM-CSF has been shown to be an
important constituent of lymphocyte-derived conditioned medium, which
is capable of increasing 5-LO metabolism by mononuclear
phagocytes.18,22 In addition, treatment of PMN with GM-CSF
in vitro has resulted in increased 5-LO metabolism via increased
expression of 5-LO enzyme23 and its helper protein 5-LO
activating protein, termed FLAP.24 Furthermore, treatment
of patients with GM-CSF results in increased urinary LTE4
levels.25,26 In view of these observations, we examined
5-LO metabolism in PBM from AIDS subjects and investigated the effect
of GM-CSF therapy on PBM and PMN 5-LO metabolism after treatment of
these patients, as GM-CSF was expected to affect both mononuclear and
granulocytic cells.
| |
MATERIALS AND METHODS |
Clinical study protocol.
We performed a prospective trial of GM-CSF therapy in 10 consecutively
enrolled nonsmoking HIV-infected subjects with low (<100
cm2) CD4 counts without evidence of opportunistic infection
and cytokine therapy as part of their medical regimen. The experimental
protocol was approved by the University of Michigan Institutional
Review Board for Approval of Research Involving Human Subjects. Full informed consent was obtained from all subjects before the study. All
subjects were on their regular antiretroviral therapy. Subjects were
treated with GM-CSF at a dose of 250 µg/m2 subcutaneously
on each of days 1 to 5. PMN and PBM were isolated on day 1 before
GM-CSF therapy, day 4 during therapy, and on day 8, 3 days after
stopping GM-CSF. Total white cell and absolute PMN
(ANC) and absolute PBM counts (AMC)
were performed on day 1, day 4, and day 8. In addition, HIV load, as
measured by bDNA,27 was determined before and after the
drug study. Subjects were carefully monitored for adverse events during
the study. PMN and PBM 5-LO metabolism in vitro were also determined
(see below). HIV-negative healthy control subjects without evidence of
systemic infection were also studied. These subjects were volunteers,
both male and female, nonsmokers on no medication, who were recruited from the employees at University Hospital.
Cell isolation.
PMN were isolated from venous blood drawn from HIV-infected subjects
with low CD4 counts and healthy controls. A volume of 5 mL of
heparinized whole blood was layered over 3.5 mL of a mixture of sodium
metrizoate and Ficoll (1-Step Polymorphs; Accurate Chemical & Scientific Corp, Westbury, NY) in a 15-mL centrifuge tube. The sample
was centrifuged at 450g for 30 minutes in a swing-out rotor at
22°C. After centrifugation, the distinct lower PMN-containing band
was aspirated and diluted in 0.45% NaCl solution to restore normal
osmolality. The cells were washed twice in 0.5 N saline and resuspended
in Iscove's medium. This resulted in less than 5% contamination with
erythrocytes and greater than 95% PMN, as determined by Diff-Quik
staining. Human PBM were isolated from HIV-infected subjects with low
CD4 counts and healthy controls. They were purified by centrifugation
through Ficoll-Paque (Pharmacia LKB, Uppsala, Sweden), followed by
adherence of the mononuclear layer on plastic for 1 hour at 37°C at
5% CO2. After vigorously washing 3 times, the remaining
cells were greater than 90% pure as determined by nonspecific esterase
testing. There was no difference in the purity of PMN and PBM obtained
from healthy control subjects and HIV-infected subjects. Viability of
PMN and PBM was greater than 90% as determined by trypan blue exclusion.
Quantitation of maximal LT synthetic capacity.
Freshly isolated PMN were suspended in Iscove's medium at 1 × 106 /mL in eppendorf tubes, and incubated for 30 minutes at
37°C with 10 µmol/L calcium ionophore A23187 (Calbiochem, La
Jolla, CA) or vehicle (0.5% dimethyl sulfoxide [DMSO]) alone. The
cells were then centrifuged and the supernatant was frozen at
 70°C for subsequent LTB4 analysis by
enzyme-linked immunoassay (EIA) (Cayman Chemical, Ann Arbor, MI).
Isolated PBM were adhered for 1 hour, washed, and then stimulated with
A23187 (10 µmol/L) and exogenous AA (50 µmol/L) for 30 minutes.
There was no difference in adherence properties between PBM from
healthy controls and HIV-infected subjects. We have shown that the
combination of A23187 and high dose exogenous AA is the maximal
stimulus for LT synthesis in PBM (data not shown). To directly compare
EIA results from controls and HIV-infected subjects, the sample from
each HIV-infected subject was analyzed in parallel with that of a
healthy control subject studied on the same day. For each sample, the
average of duplicate determinations was calculated. Data are expressed as pg product per 1 × 106 cells.
Antiretroviral agent preparation in vitro.
The nucleoside inhibitor azidothymidine (AZT)
(Retrovir) was prepared by dissolving a 100-mg capsule in 40 mL water
and plated onto cells at a final concentration (Cmax clinical dose) of
0.63 µg/mL. 3TC (Epivir), also a nucleoside inhibitor, was prepared by dissolving a 150-mg tablet in 40 mL water, and
plated onto cells at a final concentration (Cmax clinical dose) of 1.5 µg/mL. The protease inhibitor Indinovir (Crixivan) was prepared by
dissolving a 400-mg capsule in 40 mL water and plated onto cells at a
final concentration (Cmax clinical dose) of 8.9 mg/L. The GM-CSF dose used in vitro was 250 U/mL.
Analysis of [3H]AA release and metabolism.
Isolated PMN from HIV-infected subjects were incubated overnight.
Release and metabolism of AA was assessed in PMN whose lipids were
prelabeled by incubation with [3H]AA. This was
accomplished by including 0.5 µCi [3H]AA (specific
activity, 60 to 100 Ci/mmol) (Dupont-New England Nuclear, Boston, MA)
in the medium during culture for 1.5 hours. Cells were then
centrifuged, washed, and AA metabolism was determined by incubation in
Iscoves medium for 30 minutes with 10 µmol/L A23187. To assess total
AA release, cells were stimulated with A23187 in the presence of 0.1%
bovine serum albumin (BSA) and radioactivity in the medium determined.
PBM were prelabeled for 16 hours with 0.5 µCi
[3H]AA in the medium. Cells were then washed and
stimulated with A23187. Cell supernatants were extracted using
C18 Sep-Paks, and radiolabeled eicosanoids were separated
by reverse-phase high-performance liquid chromatography
(HPLC),28 identified by coelution with authentic standards
(Cayman Chemicals), and quantitated using an on-line radioactivity
detector (Radiomatic Model 515; Packard, Downers Grove, IL).
Radiolabeled products released were expressed as a percentage of
radioactivity incorporated into cellular membrane phospholipids.
Immunoblot analysis of total cellular 5-LO, FLAP, and cytosolic
phospholipase A2.
Steady-state quantities of 5-LO, FLAP, and cytosolic phospholipase
A2 (cPLA2) proteins were assessed by immunoblot
analysis using a modification of methods described
previously.29 Briefly, equal amounts of protein (5 to 20 µg) were separated on 10% sodium dodecyl sulfate
(SDS)-polyacrylamide gels by the method of Laemmli.30 High-
and low-molecular-weight rainbow markers (Amersham, Arlington Heights,
IL) were also loaded on each gel. After overnight transfer to
nitrocellulose membranes (Bio-Rad Laboratories, Richmond, CA), blots
were blocked by incubating for 1 hour with 10% nonfat dried milk in
Tris-buffered saline (TBS), washed in TBS containing 0.1% Tween
(TBS-T), and incubated at room temperature for 1 hour with rabbit
polyclonal antisera raised against either human leukocyte 5-LO (1:3,000
dilution), amino acid residues 41-52 of the human FLAP sequence
(1:5,000 dilution), or recombinant human cPLA2 (1:1,000 dilution). Antisera against 5-LO and FLAP were provided by Dr J. Evans
(Merck Frosst, Pointe Claire-Dorval, Quebec, Canada); anti-cPLA2 antiserum was provided by Dr J. Clark (Genetics
Institute, Cambridge, MA). After washing, blots were incubated for 1 hour with horseradish peroxidase-conjugated goat anti-rabbit IgG
(Amersham) at a dilution of 1:5,000 in TBS-T. Membranes were then
washed and developed using the ECL chemiluminescent Western blotting system (Amersham). Multiple exposures of each blot were obtained. Densities of luminescent bands were quantitated in appropriately exposed autoradiographs by video densitometry using NIH Image software
(Scion Corp, Frederick, MD). Samples obtained from
healthy controls and HIV-infected subjects were loaded on the same gel and underwent immunoblot analysis in parallel.
Data analysis.
Where indicated, data were expressed as the mean ± standard error of mean (SEM). Differences between the mean values of
HIV-infected and healthy control groups were analyzed by analysis of
variance (ANOVA), with statistical significance
assessed by the Scheffe test. A P value <.05 was considered significant.
| |
RESULTS |
AA metabolism in PBM from AIDS patients.
Figure 1 shows the maximal LT synthetic
capacity of PBM obtained from healthy control and AIDS patients,
assessed by quantitating immunoreactive LTB4 synthesis
after activation with A23187 and exogenous AA. LTB4
synthesis was significantly reduced in PBM from HIV-infected subjects
compared with healthy control subjects.
View larger version (28K):
[in this window]
[in a new window]
| Fig 1.
LTB4 synthesis in PBM from healthy controls
and AIDS subjects. PBM were isolated as described in Materials and
Methods and plated at 1 × 106/mL in Dulbecco's modified
Eagle's medium (DMEM). Maximal LTB4 release was determined
after stimulation with A23187 (10 µmol/L) and AA (50 µmol/L) for 30 minutes at 37°C. Medium was analyzed for LTB4 by EIA
and expressed as pg/106 cells, n = 10, *P < .001. Healthy control PBM LTB4 levels were
4,797.2 ± 884.5 pg/106 cells.
|
|
One possible explanation for the reduction in 5-LO metabolic capacity
of PBM from AIDS subjects could be a decrease in
PLA2-mediated availability of AA. Therefore, we
radiolabeled endogenous AA in PBM and stimulated the cells with the
agonist, A23187. We found that this decrease in LT synthesis not due to
a decrease in capacity for AA release (AIDS patients, 6.5% ± 2.7%
incorporated AA; healthy controls, 5.4% ± 1.5% incorporated AA, n = 4, P = .8). Further evidence that the LT synthetic defect was
not explained by reduced AA release is that other metabolites of AA,
the prostaglandins (PG), were increased in PBM from AIDS subjects
(153.9% ± 13.2% of control, n = 9, P < .05) compared
with cells from healthy control subjects. This is in keeping with
evidence of increased cyclooxygenase (COX) metabolism in PBM and AM
obtained from HIV-infected subjects.10,31,32
5-LO and FLAP expression in PBM from HIV-infected subjects.
Reduced 5-LO metabolic capacity in PBM from HIV-infected subjects
might reflect altered expression of 5-LO and/or FLAP proteins in cells.
Therefore, we performed Western blot analysis for 5-LO and FLAP on
crude lysates of PBM from healthy controls and HIV-infected subjects
(Fig 2). Eight subjects were studied
because of inadequate cellular protein from 2 of the enrolled subjects
to perform Western blot analysis. Cells from AIDS subjects exhibited a
reduction in both 5-LO and FLAP expression relative to healthy control
subjects (taken as 100%). The expression of cPLA2 protein
was unchanged in PBM from AIDS subjects compared with those obtained
from healthy control subjects (Fig 3). This
is in keeping with the similar magnitude of AA release in these 2 groups and emphasizes the selectivity of the defect involving the 5-LO
pathway proteins in AIDS.
View larger version (27K):
[in this window]
[in a new window]
| Fig 2.
5-LO and FLAP expression in PBM from control and AIDS
subjects. Equal amounts (20 µg of protein) of crude cellular lysate
from PBM were subjected to immunoblot analysis for 5-LO and FLAP as
described in Materials and Methods. Top panel, representative
autoradiograph of a Western blot demonstrating the amount of 5-LO
(left) and FLAP (right) in PBM from healthy control and HIV-infected
subjects. Lower panel, relative expression of 5-LO and FLAP in PBM from
healthy control and HIV-infected subjects, as assessed by densitometry
and expressed as a percent of values derived from cells from healthy
control subjects. Data represent the mean ± SEM from n = 8 subjects. *P < .001.
|
|
View larger version (30K):
[in this window]
[in a new window]
| Fig 3.
cPLA2 expression in PBM from healthy control
and AIDS subjects and after GM-CSF therapy of AIDS patients. Equal
amounts (20 µg of protein) of crude cellular lysate from PBM were
subjected to immunoblot analysis for 5-LO and FLAP as described in
Materials and Methods. Top panel, a representative Western blot and
lower panel, mean densitometric data. (A) cPLA2 expression
in PBM from healthy control and HIV-infected subjects expressed as a
percent of values obtained from healthy control subjects. (B)
cPLA2 expression in PBM from HIV-infected subjects treated
with GM-CSF therapy, as assessed by densitometry and expressed as a
percent of values derived from day 1 PBM. n = 9, P = not
significant (NS).
|
|
Effect of GM-CSF on AIDS patients.
The HIV group consisted of 8 males and 2 females, with a mean age of
44.9 ± 2.5 years. One subject withdrew from the study on day 4 because of side effects (syncope) of GM-CSF therapy. The mean CD4 count
was 59 ± 10.8 cm2. All patients were receiving
antiviral therapy before and during the study; 100% were receiving 2 nucleoside agents and 90% were receiving protease inhibitor therapy.
The hematologic characteristics of these subjects are shown in
Table 1. There was a significant increase
in white cell count and ANC after GM-CSF treatment. The AMC was also
increased during GM-CSF therapy: (day 1, 0.4 ± 0.06 × 103/mL; day 4, 0.6 ± 0.08 × 103/mL;
and day 8, 0.5 ± 007 × 103/mL, P = .035 comparing day 1 and day 4, n = 9). This increase in PBM
counts peaked at day 4 and returned to control levels on day 8. The
plasma HIV RNA was not significantly changed after GM-CSF therapy
(pretherapy, 3.04 ± 1.7 (range, <0.4 to 5.33)
104 particles bDNA/mL, n = 10; posttherapy 3.88 ± 1.83 (range, <0.4 to 5.54) 104 particles bDNA/mL, P = ns comparing pre and posttherapy bDNA, n = 9). The side effect profile
of GM-CSF therapy included syncope (n = 1), myalgia/bone pain (n = 2),
headache (n = 2), and vomiting (n = 1).
Effect of GM-CSF treatment on AA metabolism of PBM from HIV-infected
subjects.
Because PBM from AIDS subjects also demonstrate a defect in 5-LO
metabolism, we next examined the effect of GM-CSF therapy on PBM AA
metabolism. Figure 4 shows that GM-CSF
treatment increases LT synthetic capacity at day 4, which returns to
baseline levels by day 8. This increase in 5-LO metabolism could not be
accounted for by an increase in AA release, as shown in the
representative HPLC profile (Fig 5). In
additional support of the selective increase in 5-LO metabolism, there
was no increase in PBM COX metabolites after GM-CSF therapy (data not
shown).
View larger version (26K):
[in this window]
[in a new window]
| Fig 4.
Effect of in vivo GM-CSF therapy on PBM LT synthesis in
AIDS subjects. PBM were isolated on day 1, day 4, and day 8 of the
study protocol as described in Materials and Methods and plated at
1 × 106/mL in DMEM. Maximal LTB4
release was determined after stimulation with A23187 (10 µmol/L) and
exogenous AA (50 µmol/L) for 30 minutes at 37°C. Medium was
analyzed for LTB4 by EIA and the value obtained for
HIV-infected subjects was expressed as a percent of the
LTB4 values observed for AIDS subjects on day 1 (2,851.9 ± 448.7 pg/106 cells). *P = .05, n = 8.
|
|
View larger version (12K):
[in this window]
[in a new window]
| Fig 5.
[3H]Eicosanoid profile of stimulated PBM
from AIDS subjects before and during therapy with GM-CSF. Cells
prelabeled overnight were incubated with 1 µmol/L A23187 for 30 minutes. HPLC elution profiles are displayed for [3H]AA
metabolites synthesized by A23187-stimulated PBM from a representative
HIV-infected subject on day 1 (A) and on day 4 (B). Peaks were
identified by coelution with authentic standards, and the products were
expressed as a percentage of incorporated radioactivity. This HPLC
profile is representative of 6 subjects whose cells were
radiolabeled.
|
|
Effect of GM-CSF treatment on 5-LO, FLAP, and cPLA2
expression in PBM from HIV-infected subjects.
We next examined the effect of GM-CSF on 5-LO and FLAP expression.
There was an increase in the expression of both 5-LO and FLAP in PBM
after treatment of AIDS subjects with GM-CSF
(Fig 6). This increase in protein
expression peaked at day 4 and had returned to control levels by day 8. In contrast to the increase in expression of 5-LO and FLAP in PBM and
increase in cPLA2 expression in PMN after GM-CSF therapy,
there was no change in cPLA2 expression in PBM (Fig 3).
View larger version (75K):
[in this window]
[in a new window]
| Fig 6.
Effect of in vivo GM-CSF therapy on PBM 5-LO and FLAP
expression in AIDS subjects. PBM were isolated on day 1, day 4, and day
8 of the study protocol, as described in Materials and Methods. Equal
amounts (20 µg of protein) of crude cellular lysate from PBM were
subjected to immunoblot analysis for 5-LO and FLAP. Top panel,
representative autoradiograph of a Western blot demonstrating the
amount of 5-LO (A) and FLAP (B) in PBM from HIV-infected subjects on
protocol day 1, 4, and 8. Lower panel, relative expression of 5-LO (A)
and FLAP (B) in PBM from HIV-infected subjects treated with GM-CSF, as
assessed by densitometry and expressed as a percent of values derived
from day 1 PBM. n = 9, *P = .05.
|
|
Effect of GM-CSF treatment on AA metabolism of PMN from HIV-infected
subjects.
In view of the reduced CD4 count and the associated reduced capacity to
elaborate GM-CSF, which could account for their reduced LT synthetic
capacity, we hypothesized that if we treated these patients with
GM-CSF, we could reverse the defect in 5-LO metabolism. We studied PMN
before, during, and after 5 days of treatment with 250 µg/m2 GM-CSF administered subcutaneously daily. On day 4 of treatment with GM-CSF in vivo, there was a dramatic increase in LT
synthesis detected in vitro (Fig 7), as
compared with the markedly reduced baseline levels. This increase in
5-LO metabolism had waned by day 8 and returned to control levels.
Another mechanism by which LT synthesis can be increased by GM-CSF is
by augmenting AA release. This was indeed the case, as day 4 PMN
prelabeled with [3H]AA demonstrated increased total
release of radioactivity. Figure 8 shows a
HPLC profile, which exhibits increased quantities of radiolabeled free
AA and 5-LO products synthesized by PMN after in vivo GM-CSF treatment.
There was also a trend towards an increase (221% ± 21.5% of
control PMN, n = 8, P = .06) in
PGE2 synthesis by PMN after GM-CSF therapy.
View larger version (18K):
[in this window]
[in a new window]
| Fig 7.
Effect of in vivo GM-CSF therapy on PMN LT synthesis in
AIDS subjects. PMN were isolated on day 1, day 4, and day 8 of the
study protocol as described in Materials and Methods and plated at
1 × 106/mL in DMEM. Maximal LTB4
release was determined after stimulation with A23187 (10 µmol/L) for
30 minutes at 37°C. Medium was analyzed for LTB4 by
EIA, and the value obtained for HIV-infected subjects was expressed as
a percent of the value observed for AIDS subjects (LTB4
90.6 ± 14.4, pg/106 cells) on day 1. n = 9, *P
= .05.
|
|
View larger version (13K):
[in this window]
[in a new window]
| Fig 8.
[3H]Eicosanoid profile of stimulated PMN
from AIDS subjects before and during therapy with GM-CSF. Prelabeled
cells were incubated with 1 µmol/L A23187 for 15 minutes. HPLC
elution profiles are displayed for [3H]AA metabolites
synthesized by A23187-stimulated PMN from a representative HIV-infected
subject on day 1 (A) and on day 4 (B). Peaks were identified by
coelution with authentic standards, and the products were expressed as
a percentage of incorporated radioactivity. This HPLC profile is
representative of 6 subjects whose cells were radiolabeled.
|
|
Effect of GM-CSF treatment on 5-LO, FLAP, and cPLA2
expression in PMN from HIV-infected subjects.
We next examined the effect of GM-CSF treatment on PMN expression of
the 2 proteins, 5-LO and FLAP, essential for LT synthesis. Both 5-LO
and FLAP expression were increased on day 4 compared with day 1 (Fig 9). The increase in protein expression
returned to control levels 3 days after GM-CSF therapy was discontinued (day 8). In addition, we examined the expression of cPLA2.
GM-CSF also increased cPLA2 expression (day 4: 343.8% ± 86.1% of day 1, P = .02 n = 8; day 8: 138.9% ± 45.6% of day 1). This increase in cPLA2 expression
correlated with the increase in AA release. These observations suggest
that increased expression of 5-LO, FLAP, and cPLA2 likely
account for the increase in LT synthetic capacity after GM-CSF therapy.
View larger version (70K):
[in this window]
[in a new window]
| Fig 9.
Effect of in vivo GM-CSF therapy on PMN 5-LO and FLAP
expression in AIDS subjects. PMN were isolated on day 1, day 4, and day
8 of the study protocol, as described in Materials and Methods. Equal
amounts (20 µg of protein) of crude cellular lysate from PMN were
subjected to immunoblot analysis for 5-LO and FLAP. Top panel,
representative autoradiograph of a Western blot demonstrating the
amount of 5-LO (A) and FLAP (B) in PMN from HIV-infected subjects on
protocol day 1, 4, and 8. Lower panel, relative expression of 5-LO (A)
and FLAP (B) in PMN from HIV-infected subjects treated with GM-CSF, as
assessed by densitometry and expressed as a percent of values derived
from day 1 PMN. n = 9, * P = .05.
|
|
Effect of antiretroviral agents and GM-CSF on PBM and PMN 5-LO
metabolism in vitro.
All of the AIDS patients were on antiretroviral therapy, including
nucleoside and protease inhibitors. Because there is an alteration in
lipid metabolism in patients receiving protease inhibitors, we
considered that this defect in 5-LO metabolism could be a reflection of
AIDS therapy. Furthermore, GM-CSF therapy is known to alter drug
metabolism within myeloid cells, particularly nucleosides. However,
incubation of PBM and PMN from healthy controls with the nucleoside
inhibitors, AZT and 3TC, and the protease inhibitor, Indinavir, singly
and in combination, had no effect on LTB4 synthetic
capacity (Table 2). This was also true for cells cotreated with GM-CSF and antiretroviral therapy in vitro. Therefore, the data suggest that the defect in 5-LO metabolism is a
reflection of the underlying disease and not its therapy.
View this table:
[in this window]
[in a new window]
|
Table 2.
Effect of Antiretroviral Agents on LTB4
Synthesis in PBM and PMN Treated With and Without GM-CSF In Vitro
|
|
| |
DISCUSSION |
With the progression of HIV disease, CD4 T-lymphocyte depletion results
in dysfunction of the normal cytokine profile. This results in the
reduction in levels of 1 such mediator, GM-CSF,19-21 which
is known to upregulate 5-LO metabolism.23 Such an
abnormality might contribute to the reduced LT synthetic capacity,
which occurs in AM, PBM, and PMN. We hypothesized that replacement
therapy with GM-CSF would restore cellular 5-LO metabolism. The major findings of this study show that (1) PBM from AIDS patients display a
selective reduction in 5-LO metabolism, which is associated with
reduced 5-LO and FLAP expression. (2) PBM LT synthetic capacity was
selectively increased after GM-CSF therapy and was associated with an
increase in 5-LO and FLAP, but not cPLA2 expression. (3) Treatment of AIDS subjects with GM-CSF resulted in increased PMN 5-LO
metabolism and AA release. This was associated with an increase in
5-LO, FLAP, and cPLA2 expression.
In view of the previously published data from our laboratory that 5-LO
metabolism was reduced in AM from AIDS subjects, we anticipated that LT
synthetic capacity would be also reduced in PBM. However, the defect in
5-LO metabolism was less dramatic in PBM, which may be explained in
part by the environment in which the two cell types reside. AM are
resident in the lung where they are exposed to a higher percentage of
activated memory CD4 T lymphocytes than are present in the peripheral
circulation. Cytokines like GM-CSF are released by activated
lymphocytes and may contribute to the upregulation of LT synthesis,
which occurs as PBM move into the lung and differentiate into
AM.18,22 This hypothesis is supported by the evidence that
reduced 5-LO metabolism and 5-LO and FLAP expression is observed in AM
from a CD4 T-cell-depleted murine
model.33 Therefore, a reduction in CD4
T-lymphocyte count would be expected to have a greater impact on AM
than PBM 5-LO metabolism.
PMN dysfunction occurs in AIDS despite the fact that there is no
evidence of integration of HIV into the cellular genome. Our laboratory
has previously described a dramatic decrease in PMN 5-LO metabolism in
AIDS subjects, which was associated with a decrease in FLAP
expression.11 These observations strongly suggest that the
primary cause for a defect in LT synthesis in AIDS is the result of an
alteration of the environmental milieu, most likely from cytokine
dysfunction. Furthermore, our in vitro treatments indicate that
antiretroviral therapy does not account for the decrease in 5-LO
metabolism. An explanation for the dramatic reduction in LT synthetic
capacity in PMN may be accounted for by the concomitant reduction in
G-CSF levels in AIDS,34 compounding the effect of the
reduction the GM-CSF levels on 5-LO metabolism. We did not measure
GM-CSF levels in healthy controls or in AIDS patients, but these
studies have been performed by other investigators.19 GM-CSF therapy partially restored PMN LT synthetic capacity to a
degree, which is similar to that of G-CSF therapy. This is consistent with previous reports demonstrating that GM-CSF in vitro upregulated both 5-LO and FLAP expression in PMN via transcriptional and
posttranscriptional mechanisms.23,24
PBM from AIDS patients responded to GM-CSF therapy by selective
upregulation of the 5-LO pathway, with no change in cPLA2 expression. This data is in contrast to the effect of GM-CSF treatment in vitro on PBM from control subjects.35 In these studies,
cPLA2 expression and associated AA release was selectively
increased, with a resultant increase in LT synthesis. However, there
was no effect of GM-CSF on 5-LO or FLAP expression in PBM from healthy controls. This could be explained by differences between PBM from healthy controls and HIV-infected subjects. Reduced in vivo exposure to
GM-CSF, with resultant reduction in 5-LO metabolism, may make PBM from
AIDS subjects more responsive to the cytokine's effects. Alternatively, it could be explained by a difference in cellular response to in vivo versus in vitro treatment with GM-CSF. Studies are
in progress to examine the effect of GM-CSF therapy on 5-LO metabolism
in healthy controls. Importantly, the effect of GM-CSF on PBM and PMN
5-LO metabolism is further augmented in vivo with the increase in both
the absolute PBM and PMN count. Notably, the effect of GM-CSF on
cellular 5-LO metabolism could be on all leukocytes in the circulation,
or alternatively GM-CSF could induce new PBM and PMN from the bone
marrow, which have normal levels of these enzymes.
What is the clinical significance of our findings? LT play an important
role in host defense mechanisms, eg, by upregulating phagocytosis,12 expression of cell surface CR3
molecules,13 secretion of O2 and
lysosomal hydrolases, and killing of microorganisms.36 Furthermore, 5-LO knockout mice, with defective production of LT,
display increased susceptibility to bacterial pneumonia.14 Therefore, a reduction in PBM, as well as AM and PMN 5-LO metabolism could predispose the host to infection. This would further compound the
immunosuppression seen in patients with progression of HIV disease to
AIDS. However, we have demonstrated that by treating these subjects
with GM-CSF we can reverse the reduction in 5-LO metabolism.
Furthermore, other investigators have demonstrated that GM-CSF therapy
can boost the host immune defense mechanisms and increase the clearance
of microorganisms.37-39 We speculate that the increase in
cellular 5-LO metabolism after GM-CSF therapy may account for the
mechanism by which the clearance of microorganisms is augmented. GM-CSF
may be more useful clinically because it increases both PMN and PBM
phagocytosis, whereas G-CSF only affects PMN function. Therapeutic
intervention with cytokine therapy may be a useful adjunct for
treatment of AIDS patients with opportunistic infections.
| |
FOOTNOTES |
Submitted December 23, 1998; accepted July 23, 1999.
Supported by a grant from the Immunex Pharmaceutical Company, the
American Lung Association of Michigan, and the General Clinical Research Center at University of Michigan (Grant No. M01-RR00042). M.J.C. was the recipient of National Institutes of Health Grant No.
R01-HL-02810. M.P-G. was supported by the National Institutes of Health
Grant No. R01-HL471.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Michael J. Coffey, MD, Associate Professor
of Internal Medicine, University of Michigan Medical Center, 6301 MSRB
III, 1150 W Medical Center Dr, Ann Arbor, MI 48109-0642; e-mail:
coffeym{at}umich.edu.
| |
REFERENCES |
1.
Centers for Disease Control:
Classification system for human T-lymphotropic virus type III/lymphadenopathy-associated virus infections.
Ann Intern Med
105:234, 1986
2.
Gartner S, Markovits P, Markovitz M, Kaplan M, Gallo R, Popovic M:
The role of mononuclear phagocytes in HTLV-III LAV infection.
Science
233:215, 1986[Abstract/Free Full Text]
3.
Ho D, Rota T, Hirsch M:
Infection of monocytes/macrophages by human T lymphotropic virus type III.
J Clin Invest
77:1712, 1986
4.
Roilides E, Mertins S, Eddy J, Walsh TJ, Pizzo PA, Rubin M:
Impairment of neutrophil chemotactic and bactericidal function in children infected with human immunodeficiency virus type 1 and partial reversal after in vitro exposure to granulocyte-macrophage colony-stimulating factor.
J Pediatr
117:531, 1990[Medline]
[Order article via Infotrieve]
5.
Lipscomb M, Russell S:
Lung macrophages and dendritic cells in health and disease, in
Twigg H
(ed):
Lung Macrophages in Human Immunodeficiency Viral Infection. New York, NY, Marcel Dekker, 1997, p 571
6.
Pitrak D, Bak P, De Marais P, Novak R, Andersen B:
Depressed neutrophil superoxide production in human immunodeficiency virus infection.
J Infect Dis
167:1406, 1993[Medline]
[Order article via Infotrieve]
7.
Needleman P, Turk J, Jakschik BA, Morrison AR, Lefkowith JB:
Arachidonic Acid Metabolism.
Annu Rev Biochem
55:69, 1986[Medline]
[Order article via Infotrieve]
8.
Samuelsson B, Funk CD:
Enzymes involved in the biosynthesis of leukotriene B4.
J Biol Chem
264:19469, 1989[Free Full Text]
9.
Israel E, Dermarkarian R, Rosenberg M, Sperling R, Taylor G, Rubin P, Drazen J:
The effects of a 5-lipoxygenase inhibitor on asthma induced by cold, dry air.
N Engl J Med
323:1740, 1990[Abstract]
10.
Coffey M, Phare S, Kazanjian P, Peters-Golden M:
Reduced 5-lipoxygenase metabolism in alveolar macrophages from subjects infected with the human immunodeficiency virus.
J Immunol
157:393, 1996[Abstract]
11.
Coffey M, Phare S, George S, Peters-Golden M, Kazanjian P:
Granulocyte colony-stimulating factor administration to HIV-infected subjects augments reduced leukotriene synthesis and anticryptococcal activity in neutrophils.
J Clin Invest
102:663, 1998[Medline]
[Order article via Infotrieve]
12.
Demitsu T, Katayama H, Saito-Taki T, Yaoita H, Nakano M:
Phagocytosis and bactericidal action of mouse peritoneal macrophages treated with leukotriene B4.
Int J Immunopharmacol
11:801, 1989[Medline]
[Order article via Infotrieve]
13.
Marder P, Sawyer J, Froelich L, Mann L, Spaethe S:
Blockade of human neutrophil activation by 2-[2-propyl-3-[3-[2-ethyl-4-(4-fluorophenyl)-5-hydroxyphenoxy]propoxy]phenoxy]benzoic acid (LY29311), a novel leukotriene B4 receptor antagonist.
Biochem Pharmocol
49:1683, 1995[Medline]
[Order article via Infotrieve]
14.
Bailie M, Standiford J, Laichalk L, Coffey M, Strieter R, Peters-Golden M:
Leukotriene-deficient mice manifest enhanced lethality from klebsiella pneumonia in association with decreased alveolar macrophage phagocytic and bactericidal activities.
J Immunol
157:5221, 1996[Abstract]
15.
Skerrett S, Henderson W, Martin T:
Alveolar macrophage function in rats with severe protein calorie malnutrition: Arachidonic acid metabolism, cytokine release, and antimicrobial activity.
J Immunol
144:1052, 1990[Abstract]
16.
Burgess A, Metcalf D:
The nature and action of granulocyte-macrophage colony stimulating factors.
Blood
56:947, 1980[Abstract/Free Full Text]
17.
Kruger M, Van Gool S, Peng X, Coorevits L, Casteels-Van Daele M, Ceuppens J:
Production of granulocyte-macrophage colony-stimulating factor by T cells is regulated by B7 and IL-1 .
Immunology
88:49, 1996[Medline]
[Order article via Infotrieve]
18.
Ring W, Riddick C, Baker J, Munafo D, Bigby T:
Lymphocytes stimulate expression of 5-lipoxygenase and its activating protein in monocytes in vitro via granulocyte macrophage colony-stimulating factor and interleukin 3.
J Clin Invest
97:1293, 1996[Medline]
[Order article via Infotrieve]
19.
Hober D, Ajana F, Petit M, Sartiaux C, Boniface M, Caillaux M, Mouton Y, Wattre P:
Granulocyte-macrophage colony-stimulating factor and tumor necrosis factor alpha in patients with human immunodeficiency virus (HIV) type 1 infection.
Microbiol Immunol
37:785, 1993[Medline]
[Order article via Infotrieve]
20.
Herold M, Meise U, Gunther V, Rossler H, Zangerle R:
Serum concentrations of circulating endogenous granulocyte-macrophage colony-stimulating factor in HIV-1 seropositive injecting drug users.
Presse Med
23:1854, 1994
21.
Hittinger G, Poggi C, Delbeke E, Profizi N, Lafeuillade A:
Correlation between plasma levels of cytokines and HIV-1 RNA copy number in HIV-infected patients.
Infection
26:100, 1998[Medline]
[Order article via Infotrieve]
22.
Ring W, Riddick C, Baker J, Glass C, Bigby T:
Activated lymphocytes increase expression of 5-lipoxygenase and its activating protein in THP-1 cells.
Am J Physiol
273:C2057, 1997[Abstract/Free Full Text]
23.
Pouliot M, McDonald P, Khamzina L, Borgeat P, McColl S:
Granulocyte-macrophage colony-stimulating factor enhances 5-lipoxygenase levels in human polymorphonuclear leukocytes.
J Immunol
152:851, 1994[Abstract]
24.
Pouliot M, McDonald P, Borgeat P, McColl S:
Granulocyte/macrophage colony-stimulating factor stimulates the expression of the 5-lipoxygenase-activating protein (FLAP) in human neutrophils.
J Exp Med
179:1225, 1994[Abstract/Free Full Text]
25.
Denzlinger C, Kapp A, Grimberg M, Gerhartz H, Wilmanns W:
Enhanced endogenous leukotriene biosynthesis in patients treated with granulocyte-macrophage colony-stimulating factor.
Blood
76:1765, 1990[Abstract/Free Full Text]
26.
Denzlinger C, Tetzloff W, Gerhartz H, Pokorny R, Sagebiel S, Haberl C, Wilmanns W:
Differential activation of the endogenous leukotriene biosynthesis by two different preparations of granulocyte-macrophage colony-stimulating factor in healthy volunteers.
Blood
81:2007, 1993[Abstract/Free Full Text]
27.
Mellors J, Kingsley L, Rinaldo C, Todd J, Hoo B, Kokka R, Gupta P:
Quantitation of HIV-1 RNA in plasma predicts outcome after seroconversion.
Ann Intern Med
122:573, 1995[Abstract/Free Full Text]
28.
Peters-Golden M, McNish RW, Hyzy R, Shelly C, Toews GB:
Alterations in the pattern of arachidonate metabolism accompany rat macrophage differentiation in the lung.
J Immunol
144:263, 1990[Abstract]
29.
Coffey M, Peters-Golden M, Fantone J, Sporn P:
Membrane association of active 5-lipoxygenase in resting cells: Evidence for novel regulation of the enzyme in the rat alveolar macrophage.
J Biol Chem
267:570, 1992[Abstract/Free Full Text]
30.
Laemmli U:
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227:680, 1970[Medline]
[Order article via Infotrieve]
31.
Wahl L, Corcoran M, Pyle S, Arthur L, Harel-Bellan A, Farrar W:
Human immunodeficiency virus glycoprotein (gp120) induction of monocyte arachidonic acid metabolites and interleukin 1.
Proc Natl Acad Sci USA
86:621, 1989[Abstract/Free Full Text]
32.
Foley P, Kazazi F, Biti R, Sorrell T, Cunningham A:
HIV infection of monocytes inhibits the T-lymphocyte proliferative response to recall antigens, via production of eicosanoids.
Immunology
75:391, 1992[Medline]
[Order article via Infotrieve]
33. Coffey M, Phare S, Peters-Golden M, Huffnagle G: Regulation of
5-lipoxygenase metabolism in mononuclear phagocytes in vivo by
pulmonary lymphocytes. Exp Lung Res (in press)
34.
Mauss S, Steinmetz H, Willers T, Manegold V, Kochanek M, Haussinger D, Jablonowski H:
Induction of granulocyte colony-stimulating factor by acute febrile infection but not by neutropenia in HIV-seropositive individuals.
J AIDS Hum Retrovirol
14:430, 1997[Medline]
[Order article via Infotrieve]
35.
Brock T, McNish R, Coffey M, Clark Ojo T, Phare S, Peters-Golden M:
Effect of granulocyte-macrophage colony-stimulating factor on eicosanoid production by mononuclear phagocytes.
J Immunol
156:2522, 1996[Abstract]
36.
Serhan C, Lundberg U, Weissmann G, Samuelsson B:
Formation of leukotrienes and hydroxy acids from human neutrophils and platelets exposed to monosodium urate.
Prostaglandins
27:563, 1984[Medline]
[Order article via Infotrieve]
37.
Denis M, Ghadirian E:
Granulocyte-macrophage colony-stimulating factor restricts growth of tubercle bacilli in human macrophages.
Microbiol Lett
24:203, 1990
38.
Mandujano J, D'Souza N, Nelson S, Summer W, Beckerman R, Shellito J:
Granulocyte-macrophage colony stimulating factor and pneumocystis carinii pneumonia in mice.
Am J Respir Crit Care Med
151:1233, 1995[Abstract]
39.
Muranaka H, Suga M, Nakagawa K, Sato K, Gushima Y, Ando M:
Effects of granulocyte and granulocyte-macrophage colony-stimulating factors in a neutropenic murine model of trichosporonosis.
Infect Immun
65:3422, 1997[Abstract]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
C. A. Sorgi, A. Secatto, C. Fontanari, W. M. Turato, C. Belanger, A. I. de Medeiros, S. Kashima, S. Marleau, D. T. Covas, P. T. Bozza, et al.
Histoplasma capsulatum Cell Wall {beta}-Glucan Induces Lipid Body Formation through CD18, TLR2, and Dectin-1 Receptors: Correlation with Leukotriene B4 Generation and Role in HIV-1 Infection
J. Immunol.,
April 1, 2009;
182(7):
4025 - 4035.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. N. Ballinger, L. L. N. Hubbard, T. R. McMillan, G. B. Toews, M. Peters-Golden, R. Paine III, and B. B. Moore
Paradoxical role of alveolar macrophage-derived granulocyte-macrophage colony-stimulating factor in pulmonary host defense post-bone marrow transplantation
Am J Physiol Lung Cell Mol Physiol,
July 1, 2008;
295(1):
L114 - L122.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. J. Coffey, C. H. Serezani, S. M. Phare, N. Flamand, and M. Peters-Golden
NADPH oxidase deficiency results in reduced alveolar macrophage 5-lipoxygenase expression and decreased leukotriene synthesis
J. Leukoc. Biol.,
December 1, 2007;
82(6):
1585 - 1591.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. Flamand, M. J. Tremblay, and P. Borgeat
Leukotriene B4 Triggers the In Vitro and In Vivo Release of Potent Antimicrobial Agents
J. Immunol.,
June 15, 2007;
178(12):
8036 - 8045.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. H. Serezani, J. H. Perrela, M. Russo, M. Peters-Golden, and S. Jancar
Leukotrienes Are Essential for the Control of Leishmania amazonensis Infection and Contribute to Strain Variation in Susceptibility.
J. Immunol.,
September 1, 2006;
177(5):
3201 - 3208.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. J. Serio, K. V. Reddy, and T. D. Bigby
Lipopolysaccharide induces 5-lipoxygenase-activating protein gene expression in THP-1 cells via a NF-{kappa}B and C/EBP-mediated mechanism
Am J Physiol Cell Physiol,
May 1, 2005;
288(5):
C1125 - C1133.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Peters-Golden, C. Canetti, P. Mancuso, and M. J. Coffey
Leukotrienes: Underappreciated Mediators of Innate Immune Responses
J. Immunol.,
January 15, 2005;
174(2):
589 - 594.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. V. Reddy, K. J. Serio, C. R. Hodulik, and T. D. Bigby
5-Lipoxygenase-activating Protein Gene Expression. KEY ROLE OF CCAAT/ENHANCER-BINDING PROTEINS (C/EBP) IN CONSTITUTIVE AND TUMOR NECROSIS FACTOR (TNF) alpha -INDUCED EXPRESSION IN THP-1 CELLS
J. Biol. Chem.,
April 11, 2003;
278(16):
13810 - 13818.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. M. Hayes, B. R. Lane, S. R. King, D. M. Markovitz, and M. J. Coffey
Peroxisome Proliferator-activated Receptor gamma Agonists Inhibit HIV-1 Replication in Macrophages by Transcriptional and Post-transcriptional Effects
J. Biol. Chem.,
May 3, 2002;
277(19):
16913 - 16919.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. THURNHER, C. ZELLE-RIESER, R. RAMONER, G. BARTSCH, and L. HOLTL
The disabled dendritic cell
FASEB J,
April 1, 2001;
15(6):
1054 - 1061.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. L. Baltch, R. P. Smith, M. A. Franke, W. J. Ritz, P. B. Michelsen, and L. H. Bopp
Effects of Cytokines and Fluconazole on the Activity of Human Monocytes against Candida albicans
Antimicrob. Agents Chemother.,
January 1, 2001;
45(1):
96 - 104.
[Abstract]
[Full Text]
|
|
|
|
|
TOP
ABSTRACT
INTRODUCTION