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Blood, Vol. 94 No. 11 (December 1), 1999:
pp. 3897-3905
By
From the Divisions of Pulmonary and Critical Care Medicine and
Infectious Diseases, University of Michigan Medical Center, Ann Arbor,
MI.
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.
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.
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
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.
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.
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.
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).
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).
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.
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.
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.
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.
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.
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Effects of granulocyte and granulocyte-macrophage colony-stimulating factors in a neutropenic murine model of trichosporonosis.
Infect Immun
65:3422, 1997[Abstract]
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TOP
ABSTRACT
INTRODUCTION
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.
.
Immunology
88:49, 1996