Leukotriene B4 augments neutrophil phagocytosis of Klebsiella pneumoniae.
ABSTRACT Neutrophils play a critical role in the clearance of bacteria from the lung and other organs by their capacity for phagocytosis and killing. Previously, we identified an important role for the leukotrienes in rat alveolar macrophage phagocytosis of Klebsiella pneumoniae. In this report, we explored the possibility that the leukotrienes play an important role in phagocytosis by neutrophils as well. Inhibition of endogenous leukotriene synthesis by 5-lipoxygenase knockout in mice or by pharmacologic means in human peripheral blood neutrophils attenuated phagocytosis of opsonized K. pneumoniae. Reduced phagocytosis was also observed in human neutrophils pretreated with a leukotriene B4 receptor but not a cysteinyl-leukotriene receptor antagonist. While leukotriene B4 reconstituted defective phagocytosis in leukotriene-deficient neutrophils and enhanced phagocytosis in neutrophils capable of leukotriene synthesis, leukotriene C4, leukotriene D4, 5-hydroperoxyeicosatetraenoic acid, and 5-oxo-eicosatetraenoic acid were ineffective. To determine the opsonin dependence of the leukotriene B4 augmentation of phagocytosis, we assessed the ability of leukotriene B4 to modulate neutrophil phagocytosis and the adherence of sheep erythrocytes opsonized with immunoglobulin G or the complement fragment C3bi. While leukotriene B4 augmented both Fc receptor- and complement receptor-mediated phagocytosis, increased adherence to leukotriene B4-treated neutrophils was limited to complement opsonized targets. In conclusion, we have identified a novel role for leukotriene B4 in the augmentation of neutrophil phagocytosis mediated by either the Fc or complement receptor.
- SourceAvailable from: Nobukazu Araki[show abstract] [hide abstract]
ABSTRACT: Phosphoinositide 3-kinase (PI 3-kinase) has been implicated in growth factor signal transduction and vesicular membrane traffic. It is thought to mediate the earliest steps leading from ligation of cell surface receptors to increased cell surface ruffling. We show here that inhibitors of PI 3-kinase inhibit endocytosis in macrophages, not by interfering with the initiation of the process but rather by preventing its completion. Consistent with earlier studies, the inhibitors wortmannin and LY294002 inhibited fluid-phase pinocytosis and Fc receptor-mediated phagocytosis, but they had little effect on the receptor-mediated endocytosis of diI-labeled, acetylated, low density lipoprotein. Large solute probes of endocytosis reported greater inhibition by wortmannin than smaller probes did, indicating that macropinocytosis was affected more than micropinocytosis. Since macropinocytosis and phagocytosis are actin-mediated processes, we expected that their inhibition by wortmannin resulted from deficient signaling from macrophage colony-stimulating factor (M-CSF) receptors or Fc receptors to the actin cytoskeleton. However, video microscopy showed cell surface ruffling in wortmannin-treated cells, and increased ruffling after addition of M-CSF or phorbol myristate acetate. Quantitative measurements of video data reported slightly diminished ruffling in wortmannin-treated cells. Remarkably, the ruffles that formed in wortmannin-treated macrophages all receded into the cytoplasm without closing into macropinosomes. Similarly, wortmannin and LY294002 did not inhibit the extension of actin-rich pseudopodia along IgG-opsonized sheep erythrocytes, but instead prevented them from closing into phagosomes. These findings indicate that PI 3-kinase is not necessary for receptor-mediated stimulation of pseudopod extension, but rather functions in the closure of macropinosomes and phagosomes into intracellular organelles.The Journal of Cell Biology 01/1997; 135(5):1249-60. · 10.82 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Leukotrienes (LTs) are potent mediators of inflammation derived from the 5-lipoxygenase pathway of arachidonic acid metabolism. Although they are known to enhance leukocyte recruitment and function, their role in antimicrobial host defense has not been established. To determine the role of endogenous LTs in the host response to pulmonary infection, wild-type mice and mice rendered LT-deficient by targeted disruption of the 5-lipoxygenase gene (knockout mice) were studied following intratracheal challenge with Klebsiella pneumoniae. Wild-type mice demonstrated a marked increase in lung LT levels and neutrophil numbers following bacterial challenge. As compared with wild-type animals, knockout animals manifested a greater degree of lethality as well as bacteremia following challenge. Interestingly, they displayed no defect in neutrophil recruitment to the lung. However, alveolar macrophages from knockout animals exhibited impairments in bacterial phagocytosis and killing, and these defects were overcome by in vitro addition of exogenous LTB4. We conclude that endogenous LTs play a critical role in the defense against bacterial pneumonia in this murine model.The Journal of Immunology 01/1997; 157(12):5221-4. · 5.52 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Leukotriene (LT) synthesis is initiated by the enzyme 5-lipoxygenase (5-LO). Prolonged cell stimulation causes the translocation of 5-LO to the nuclear envelope and the synthesis of LT, with subsequent inactivation and persistent membrane association of 5-LO. In this study, we examined whether persistent membrane association of 5-LO, as well as the inactivation of 5-LO, could be prevented by shortening the length of cell stimulation or by blocking LT synthesis. As expected, stimulation of rat basophilic leukaemia (RBL) cells, a mast cell model, or alveolar macrophages (AMs) with calcium ionophore for 15 min caused 5-LO translocation, LT generation and the inactivation and persistent membrane association of 5-LO. When RBL cells or AMs instead were stimulated for 0.5-5 min, translocation of 5-LO and synthesis of LT still occurred. However, after washing and resting, the 5-LO enzyme returned to its original intracellular distribution. Furthermore these cells showed a retained capacity for LT synthesis on subsequent re-stimulation. Similar results were obtained when cells were stimulated with either formyl peptide or zymosan, instead of ionophore. In contrast, blockade of LT synthesis during the initial stimulation, with the selective inhibitors zileuton or MK-886, did not inhibit 5-LO translocation, inactivation or persistent membrane association resulting from prolonged cell stimulation. We conclude that, in long-lived immune cells, 5-LO translocation is reversible when cell stimulation is short, but persistent after prolonged stimulation. In addition 5-LO remains active and LT synthetic capacity is retained after transient stimulation, whereas significant inactivation of 5-LO occurs after prolonged stimulation. Finally, results with LT synthesis inhibitors indicate that inactivation and persistent membrane association of 5-LO can result independently of 5-LO activation.Biochemical Journal 03/1998; 329 ( Pt 3):519-25. · 4.65 Impact Factor
INFECTION AND IMMUNITY,
Copyright © 2001, American Society for Microbiology. All Rights Reserved.
Apr. 2001, p. 2011–2016Vol. 69, No. 4
Leukotriene B4Augments Neutrophil Phagocytosis of
PETER MANCUSO, PATRICK NANA-SINKAM, AND MARC PETERS-GOLDEN*
Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine,
University of Michigan Medical Center, Ann Arbor, Michigan 48109-0642
Received 11 September 2000/Returned for modification 11 October 2000/Accepted 26 December 2000
Neutrophils play a critical role in the clearance of bacteria from the lung and other organs by their capacity
for phagocytosis and killing. Previously, we identified an important role for the leukotrienes in rat alveolar
macrophage phagocytosis of Klebsiella pneumoniae. In this report, we explored the possibility that the leuko-
trienes play an important role in phagocytosis by neutrophils as well. Inhibition of endogenous leukotriene
synthesis by 5-lipoxygenase knockout in mice or by pharmacologic means in human peripheral blood neutro-
phils attenuated phagocytosis of opsonized K. pneumoniae. Reduced phagocytosis was also observed in human
neutrophils pretreated with a leukotriene B4receptor but not a cysteinyl-leukotriene receptor antagonist. While
leukotriene B4reconstituted defective phagocytosis in leukotriene-deficient neutrophils and enhanced phago-
cytosis in neutrophils capable of leukotriene synthesis, leukotriene C4, leukotriene D4, 5-hydroperoxyeicosa-
tetraenoic acid, and 5-oxo-eicosatetraenoic acid were ineffective. To determine the opsonin dependence of the
leukotriene B4augmentation of phagocytosis, we assessed the ability of leukotriene B4to modulate neutrophil
phagocytosis and the adherence of sheep erythrocytes opsonized with immunoglobulin G or the complement
fragment C3bi. While leukotriene B4augmented both Fc receptor- and complement receptor-mediated phago-
cytosis, increased adherence to leukotriene B4-treated neutrophils was limited to complement opsonized
targets. In conclusion, we have identified a novel role for leukotriene B4in the augmentation of neutrophil
phagocytosis mediated by either the Fc or complement receptor.
The alveolar epithelial surface represents the body’s largest
interface with the external environment, and it is continually
challenged with bacteria aspirated from the oropharynx that
evade the mechanical clearance mechanisms of the upper re-
spiratory tract. The alveolar macrophage (AM) is the primary
means of innate host defense against bacterial pathogens that
attempt to reach the alveolar space. When the bacterial burden
in the alveolar space is great enough to activate a sufficient
number of AMs to secrete chemotactic substances such as
chemokines, leukotriene B4(LTB4), and complement frag-
ments, polymorphonuclear leukocytes (PMNs) are recruited
from a marginated pool in the pulmonary vasculature to the
alveolar epithelial surface (19). PMNs play a critical role in
clearing encapsulated bacteria, such as Klebsiella pneumoniae,
from the lung and in preventing bacterial dissemination to the
systemic circulation (26). The ability of these cells to perform
this function can be enhanced by inflammatory mediators that
activate the PMN for increased phagocytosis and killing of
The LTs are potent lipid mediators of inflammation derived
from the 5-lipoxygenase (5-LO)-catalyzed oxygenation of ara-
chidonic acid (AA). The enzyme 5-LO in concert with the
5-LO activating protein can oxygenate AA to form the inter-
mediate metabolite, 5-hydroperoxyeicosatetraenoic acid. This
5-LO product can either be dehydrated to LTA4or reduced to
5-hydroxyeicosatetraenoic acid (5-HETE). LTA4can be hy-
drolyzed to form the potent PMN chemoattractant LTB4or
conjugated with glutathione to form the cysteinyl-LTs (LTC4,
LTD4, and LTE4), well known to elicit smooth muscle contrac-
tion and microvascular permeability (15).
LTs are readily acknowledged as important mediators of
pathologic states of inflammation such as asthma. To ascertain
whether LTs participate in the homeostatic form of inflamma-
tion, i.e., antimicrobial host defense, we subjected 5-LO knock-
out (KO) mice to an intrapulmonary challenge with K. pneu-
moniae (2). Compared to their wild-type (WT) counterparts,
the LT-deficient mice exhibited enhanced lethality and re-
duced bacterial clearance from the lung in this model. This
defect in 5-LO KO mice in vivo was associated with reduced
AM phagocytosis and killing of K. pneumoniae in vitro. Further
in vitro studies indicated that the inhibition of LT synthesis by
pharmacologic means also reduced AM phagocytosis of bac-
teria. Moreover, exogenous LTs restored phagocytosis when
added to LT-deficient AMs and also augmented phagocytosis
above baseline capacity when added to LT-competent AMs
(17) (2). Finally, mechanistic studies revealed that the LT aug-
mentation of phagocytosis in AMs was largely Fc receptor
(FcR) mediated and protein kinase C dependent in AMs (16).
Since PMNs are professional phagocytes known to synthe-
size LTs and express plasma membrane receptors for LTs, we
explored the possibility that these mediators modulate PMN
phagocytosis of K. pneumoniae as they do in AMs. In this study,
we demonstrate that PMN phagocytosis of K. pneumoniae is
indeed augmented by LTB4and that the enhancement in PMN
phagocytosis can occur through FcR- or complement receptor
MATERIALS AND METHODS
Cell isolation and culture. Human PMNs, isolated from venous blood from
healthy volunteers, were purified by centrifugation through Polymorphprep (Ny-
comed Pharma, Oslo, Norway), followed by hypotonic lysis of erythrocytes (3).
* Corresponding author. Mailing address: Division of Pulmonary
and Critical Care Medicine, 6301 MSRB III, University of Michigan
Medical Center, Ann Arbor, MI 48109-0642. Phone: (734) 763-9077.
Fax: (734) 764-4556. E-mail: email@example.com.
Elicited PMNs were obtained from 129/SvEv WT and 5-LO KO mice by peri-
toneal lavage 4 h after an intraperitoneal injection of 1% glycogen solution in
saline. Ninety percent of the cells obtained by peritoneal lavage were identified
as PMNs by a modified Wright-Giemsa stain (Diff-Quik; American Scientific
Products, McGaw Park, Ill.). Following PMN isolation, the cells were enumer-
ated using a hemocytometer and suspended in RPMI 1640 (Gibco, Grand Island,
N.Y.) to a final concentration of 5 ? 105cells/ml. For phagocytosis experiments,
105PMNs were adhered to glass eight-well Falcon culture slides (Becton Dick-
inson, Franklin Lakes, N.J.) for 1 h in RPMI 1640.
K. pneumoniae preparation. K. pneumoniae strain 43816 (serotype 2; American
Type Culture Collection) was grown in tryptic soy broth (Difco, Detroit, Mich.)
for 18 h at 37°C. The concentration of bacteria in culture was determined
spectrophotometrically (A600) (10).
Pharmacologic modulation of PMNs. In some experiments, PMNs were pre-
treated for 15 min with the 5-LO enzyme inhibitor zileuton (10 ?M; Abbott
Laboratories, Chicago, Ill.), the 5-LO activating protein inhibitor MK-886 (1
?M; Merck-Frosst, Montreal, Quebec, Canada), the cysteinyl-LT receptor an-
tagonist LY171883 (1 ?M), or the LTB4receptor antagonist LY292476 (1 ?M)
(the latter two from Eli Lilly, Indianapolis, Ind.), each diluted in Hanks balanced
salt solution. The use of these pharmacologic agents at these doses was previ-
ously shown to inhibit LT synthesis and block the LT receptors (4, 7) and reduce
phagocytosis of K. pneumoniae in AMs (17).
Phagocytosis of K. pneumoniae. Following the addition of 106K. pneumoniae
opsonized with 1% immune serum (17), PMN cultures were incubated for 30 min
at 37°C. Following the incubation period, the extracellular bacteria were re-
moved by three washes with Hanks balanced salt solution. The monolayers
containing bacteria were stained with Diff-Quik and enumerated. For each slide,
a standard pattern of high-powered fields was examined by light microscopy
(1,000 ? objective) to enumerate 100 cells. By comparing the phagocytic index
in the presence and absence of cytochalasin D (20), we determined that 90% of
the cell-associated bacteria using this method were actually internalized.
Preparation of E opsonized with IgG or C3bi. Sheep erythrocytes (E; ICN
Biomedicals, Costa Mesa, Calif.) were washed and opsonized with either rabbit
anti-E immunoglobulin G (IgG) (ICN Biomedicals, Inc.) or IgM (ICN Biomedi-
cals) and C3bi (Sigma) as described previously (1, 29). Opsonized E were enu-
merated using a hemocytometer and diluted to a final concentration of 20 ? 107
Phagocytosis of opsonized E. E-IgG or E-C3bi (107) was added to the PMN
monolayers following pretreatment with medium alone (control), LTs, or LT
modulators for 5 to 10 min; 15 min later, internalization of E-C3bi was initiated
by the addition of 15 nM phorbol myristate acetate (PMA). After a 30-min
incubation at 37°C, water was added to each well for 10 s followed by 3? saline
to lyse the surface-bound E and to restore isotonic conditions. A phagocytic
index was calculated as described previously (17).
Adherence of opsonized E. To assess adherence of E-IgG or E-C3bi to the
surface of PMNs, monolayers were incubated with cytochalasin D (5 ?g/ml) at
37°C in 5% CO2, which would allow surface binding of E’s, but not phagocytosis
(13). Thirty minutes after the addition of opsonin-coated E’s, the total number
of adherent targets was enumerated for 100 cells, using the microscopic proce-
dure described above for phagocytosis. The adherence index was calculated by
determining the total number of adhered E-IgG or E-C3bi per 100 PMNs.
Statistical analysis. A minimum of three replicate wells per condition was
studied in each experiment, and the number of individual experiments is indi-
cated in the figure legends. Data are expressed as the mean ? standard error
(SE). Where appropriate, mean values were compared using a paired t test, a
one-way analysis of variance, or a Kruskal-Wallis test on ranks for nonparametric
data. The Dunnett’s test or the Student-Newman-Keuls tests were used for mean
separation. In all cases, a P value of ?0.05 was considered significant.
Inhibition of endogenous LT synthesis reduces PMN phago-
cytosis of K. pneumoniae. To determine if endogenously pro-
duced LTs have a functional role in PMN phagocytosis, human
PMN monolayers were pretreated with either zileuton (10
?M) or MK-886 (1 ?M) at concentrations known to inhibit LT
synthesis. They were then incubated with K. pneumoniae op-
sonized with immune serum. Both agents, which block LT
synthesis by distinctly different mechanisms of action, reduced
phagocytosis of opsonized K. pneumoniae by approximately
35% (P ? 0.05) (Fig. 1). The results of this experiment indicate
that endogenously produced LTs are necessary for maximal
bacterial phagocytosis in human PMNs.
Decreased phagocytosis of opsonized K. pneumoniae by elic-
ited PMNs from 5-LO KO mice. During bacterial infection of
the lung and other organs, PMNs are recruited to tissue from
the peripheral circulation in response to a chemoattractant
gradient. During the recruitment process, the PMN encounters
a number of mediators which prime the cells for enhanced
antimicrobial defense. These include tumor necrosis factor
alpha, gamma interferon, interleukin-12, and LTs. To assess
the importance of endogenously generated LTs in phagocyto-
sis by PMNs recruited to a site of inflammation, we assessed
phagocytosis of opsonized K. pneumoniae in PMNs recruited in
response to glycogen instillation into the peritoneal cavity of
5-LO KO and WT mice. Phagocytosis by elicited peritoneal
PMNs obtained from 5-LO KO mice was reduced by approx-
imately 50% compared with cells from WT animals (P ? 0.05)
(Fig. 2). This reduction in phagocytosis underscores the im-
portance of LTs in the antimicrobial function of PMNs at a site
LTB4receptor antagonist attenuates phagocytosis. In a pre-
vious study (17), we determined that receptor antagonists to
both LTB4and cysteinyl-LTs in rat AMs reduced phagocytosis.
This indicated that endogenous production of both classes of
LTs augmented phagocytosis. We used this same approach to
determine the contribution of endogenously produced LTs of
each class to phagocytosis by human PMNs. The LTB4recep-
tor antagonist LY292476 reduced bacterial phagocytosis by
approximately 40% (P ? 0.05) (Fig. 3), indicating that endo-
genously produced LTB4was produced during phagocytosis,
exited the cell, and activated the plasma membrane receptor
for LTB4. In contrast, the cysteinyl-LT receptor antagonist
LY171886 had a minimal effect on PMN phagocytosis.
Exogenous LTB4restores phagocytosis in LT-deficient hu-
man and murine PMNs. To determine the specificity of the
relationship between various LTs and phagocytosis, we exam-
ined whether exogenous LTs could restore the impairment of
phagocytosis observed in human PMNs pretreated with zileu-
ton and elicited PMNs from 5-LO KO mice. Prior to the
addition of opsonized K. pneumoniae, exogenous LTB4or
LTC4(1 nM) was added to human PMNs pretreated for 15 min
with zileuton (10 ?M) or to murine elicited PMNs. The addi-
tion of exogenous LTB4completely restored phagocytosis in
human PMNs (Fig. 4A) and nearly restored phagocytosis in
murine elicited PMNs (Fig. 4B). In contrast, the addition of
exogenous LTC4did not enhance phagocytosis in either in-
stance (data not shown).
Exogenous LTB4dose dependently augments phagocytosis
of opsonized K. pneumoniae in PMNs capable of LT synthesis.
Having established the importance of endogenously produced
LTB4for full phagocytic capacity of PMNs, we considered the
possibility that exogenously added LTs may augment PMN
phagocytosis even in cells competent for LT synthesis. Human
PMNs were incubated with various doses (0.01 to 100 nM) of
exogenous LTB4, LTD4, LTC4, 5-oxo-eicosatetraenoic acid (5-
oxo-ETE), or 5-HETE for 5 min prior to the addition of
opsonized K. pneumoniae. As shown in Fig. 5, phagocytosis was
enhanced above the basal level with LTB4in a dose-dependent
fashion, reaching statistical significance at 1 to 100 nM. In
contrast, none of the other lipids were able to significantly
2012 MANCUSO ET AL.INFECT. IMMUN.
enhance phagocytosis at any of the doses tested (data not
LTB4increases human PMN phagocytosis of E-IgG. The
phagocytic target used in all of the previously described exper-
iments was K. pneumoniae opsonized with 1% immune serum.
Immune serum would be expected to contain two distinct op-
sonins, IgG and complement. To determine whether FcR-
dependent phagocytosis in human PMNs is modulated by
LTB4, cells were pretreated with increasing doses of LTB4for
10 min prior to a 30-min incubation with E-IgG. After incu-
bation, extracellular Es were removed by hypotonic lysis and a
phagocytic index was calculated to determine the number of
ingested E-IgG per 100 PMNs. In a dose-dependent fashion,
LTB4enhanced phagocytosis of E-IgG, with the maximum
effect (?3.5-fold the control level) observed at 10 to 100 nM
(Fig. 6). We next explored the possibility that LTB4enhanced
phagocytosis of E-IgG by increasing target adherence. Follow-
ing pretreatment with LTB4, PMN monolayers were cultured
at 37°C and cooled to 15°C prior to the addition of E-IgG, in
order to allow adherence but not phagocytosis (9). In contrast
to the effects on phagocytosis demonstrated in Fig. 6, E-IgG
adherence to human PMNs was not significantly affected by
LTB4pretreatment at doses up to 100 nM (control phagocytic
index of 100 versus phagocytic index with 100 nM LTB4of
99 ? 11). These results indicate that LTB4augments the ability
of the PMN to internalize, rather than to bind, E-IgG.
LTB4enhances human PMN adherence and PMA-stimu-
lated phagocytosis of E-C3bi. Since LTB4is known to cause
mobilization of CR to the cell surface (18), we next evaluated
whether LTB4increased the ability of the PMN to bind com-
plement-opsonized targets. PMNs were pretreated with in-
creasing doses of LTB4(0.01 to 100 nM) prior to the addition
of E-C3bi. LTB4at 0.1 to 100 nM significantly increased
E-C3bi adherence to human PMNs (Fig. 7). To determine if
LTB4-mediated enhancement of E-C3bi adherence is associ-
ated with increased phagocytosis, human PMNs were pre-
treated with various concentrations of LTB4prior to the addi-
tion of E-C3bi. Phagocytosis was initiated 15 min later with the
addition of 15 nM PMA, since phagocytic cells must be acti-
vated by an additional stimulus in order to phagocytose com-
plement-opsonized targets (9). LTB4dose dependently aug-
mented phagocytosis of E-C3bi following treatment with PMA
(Fig. 8). These results indicate that LTB4enhances CR-depen-
dent phagocytosis, and that this may occur, at least in part, via
an effect on increasing target adherence.
In this study, we used both genetic and pharmacologic ap-
proaches to demonstrate that endogenously produced LTs
promote PMN phagocytosis of bacteria opsonized with im-
mune serum. This conclusion was based on the observations
that PMNs elicited from the peritoneum of 5-LO KO mice and
human PMNs pretreated with zileuton and MK-886, two drugs
that block LT synthesis by distinctly different mechanisms (5,
23), exhibited reduced phagocytosis. It is very likely that LTB4
is the endogenous 5-LO product responsible for this effect on
phagocytosis, since (i) it is the principal 5-LO product in PMNs
and (ii) LTB4receptor antagonist, but not cysteinyl-LT recep-
tor antagonist, also reduced phagocytosis. Although they do
not synthesize LTC4, some PMN effector functions have been
shown to respond to cysteinyl-LTs (11, 14). Taken together,
the results of these experiments suggest that endogenously
produced LTB4acts as an autocrine stimulus for enhanced
PMN phagocytosis of opsonized K. pneumonia.
Experiments demonstrating the ability of exogenous LTB4
to restore phagocytosis in elicited PMNs from 5-LO KO mice
and human PMNs pretreated with the LT synthesis inhibitor
zileuton supported the conclusion that reduced phagocytosis of
opsonized bacteria was indeed due to the lack of LT synthesis
rather than other potential phenotypic abnormalities. Based
on our previous studies with AMs (17), we considered the
possibility that LTs might have pharmacologic effects on PMN
phagocytosis of bacteria. While LTB4, LTC4, 5-HETE, and
5-oxo-ETE all enhanced phagocytosis of opsonized K. pneu-
monia by normal AMs in our previous study (17), only LTB4
stimulated increased PMN phagocytosis in the present study. It
is also noteworthy that the concentration (1 to 100 nM) of
exogenously added LTB4that restored phagocytosis in LT-
deficient PMNs and enhanced phagocytosis in LT-competent
cells was within the physiologic range. Nanomolar quantities of
LTB4have been recovered from the bronchoalveolar lavage
fluid of pneumonia patients (12). Moreover, concentrations of
LTB4at which binding to the two types of PMN LTB4recep-
FIG. 1. Effect of endogenous LT synthesis inhibition on PMN
phagocytosis of opsonized K. pneumoniae. Human PMNs were pre-
treated in the absence (control) or presence of zileuton (10 ?M) or
MK-886 (1 ?M) for 15 min prior to the addition of K. pneumoniae
opsonized with 1% immune serum. The phagocytic index of the con-
trol was 93 ? 23. Data are presented as the mean ? SE (n ? 5). ?, P ?
0.05 with respect to the control, using a paired t test.
FIG. 2. Effect of 5-LO gene knockout on phagocytosis of opsonized
K. pneumonia by elicited peritoneal PMNs. Data are presented as the
mean ? SE (n ? 3). ?, P ? 0.05 with respect to the control, using a
paired t test.
VOL. 69, 2001LTB4AUGMENTS NEUTROPHIL PHAGOCYTOSIS2013
tors (BLT) are half-maximal are 1.1 nM (BLT1) and 20 nM
(BLT2) (30). These results indicate that only LTB4, the prin-
cipal 5-LO product of PMNs (8), is capable of enhancing
phagocytosis in these cells at physiologic levels.
The positive effect of LTB4on PMN phagocytosis is not
surprising since there is ample evidence demonstrating that
LTB4activates many PMN functions, including upregulation
of the CR (18), superoxide generation (28), and increased
calcium mobilization (22). PMNs recruited to a site of inflam-
mation have been exposed to various inflammatory mediators
and are representative of the activated cells called upon for
antimicrobial effector functions (24). The fact that the phago-
cytic capacity of elicited PMNs from 5-LO KO mice was com-
promised suggests that LTs play an important role in PMN
activation for enhanced bacterial phagocytosis.
It is well known that optimal recognition of phagocytic tar-
gets is mediated by surface receptors for specific opsonins (e.g.,
complement and IgG) (13). Initial experiments showing LTB4
enhancement of phagocytosis utilized bacteria opsonized with
complete immune serum that would contain complement and
IgG. Whether both or either of these opsonins was required for
the LTB4augmentation of phagocytosis was addressed by coat-
ing inert targets (E) with only a single opsonin, either IgG or
FIG. 3. Effect of LT receptor antagonists on phagocytosis of opso-
nized K. pneumoniae by human PMNs. The phagocytic index of the
control was 93 ? 23. Data are presented as the mean ? SE (n ? 4).
?, P ? 0.05 with respect to the control, using a paired t test.
FIG. 4. Ability of exogenous LTB4to restore phagocytosis in hu-
man PMNs pretreated with zileuton (10 ?M) for 15 min (A) or in
elicited murine PMNs from 5-LO KO mice (B). The phagocytic index
of the control (A) was 50 ? 2. Data are expressed as the mean ? SE
(n ? 3). ?, P ? 0.05 with respect to the control, using a paired t test.
FIG. 5. Effect of exogenous LTB4dose on human PMN phagocy-
tosis of K. pneumoniae opsonized with 1% immune serum. Human
PMNs were incubated with LTB4in the dose indicated for 5 to 10 min
prior to the addition of opsonized K. pneumoniae. The phagocytic
index of the control was 40 ? 3. Data are expressed as the mean ? SE
(n ? 3). ?, P ? 0.05 with respect to the control by analysis of variance.
FIG. 6. Effect of exogenous LTB4dose on human PMN phagocy-
tosis of E-IgG. Human PMNs were incubated with LTB4for 5 to 10
min prior to the addition of E-IgG. The phagocytic index of the control
was 20 ? 5. Data are expressed as the mean ? SE (n ? 3). ?, P ? 0.05
with respect to the control by Kruskal-Wallis test on ranks, using
Dunnett’s test for means separation.
2014 MANCUSO ET AL.INFECT. IMMUN.
C3bi. These experiments also served to exclude the possibilities
that other opsonins that may be present in the rat serum or
nonopsonic recognition of a moiety that may be present on the
surface of K. pneumoniae were responsible for the LTB4mod-
ulation of phagocytosis. They revealed that the effects of LTB4
on phagocytosis could be mediated through either the FcR or
the CR. This result was in contrast to our previous study that
concluded that the effects of exogenous LTs on rat AM phago-
cytosis were limited to IgG-opsonized targets. The fact that rat
AMs express low levels of the CR would explain this disparity
Subsequent experiments addressed the possibility that LTs
modulate FcR- and CR-dependent phagocytosis by increasing
adherence of targets to PMNs. This could result from increases
in receptor number or enhanced affinity of the receptor for its
ligand. Indeed, LTB4is recognized to enhance CR expression
in PMNs (18). While exogenous LTB4had no effect on the
adherence of E-IgG, it induced an increase in the internaliza-
tion of this target. In addition, LTB4enhanced E-C3bi adher-
ence in a dose-dependent fashion, indicating that its ability to
augment PMN phagocytosis may involve enhanced adherence
of complement-opsonized targets.
In summary, we have revealed an important role for endo-
genously produced LTB4in the augmentation of PMN phago-
cytosis of opsonized bacteria. This enhancement of phagocy-
tosis can be mediated through either the FcR or CR. In view of
the role of endogenous LTB4in promoting phagocytosis by
PMNs, increased susceptibility to infection observed in condi-
tions such as human immunodeficiciency virus infection (6)
and malnutrition (25) may relate to their recognized LT defi-
This work was supported by National Heart, Lung, and Blood In-
stitute grant RO1 HL58897. Support for P.M. was provided by an
American Lung Association of Michigan Research Fellowship Train-
We thank Joel A. Swanson, Thomas G. Brock, Michael Coffey, and
Theodore J. Standiford for helpful advice and discussion, and we thank
Maria Diakonova for technical assistance.
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FIG. 8. Effect of exogenous LTB4dose on human PMN phagocy-
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Editor: T. R. Kozel
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