| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Division of Infectious and Immunological Diseases, Department of Pediatrics, University of British Columbia, Vancouver, Canada
Correspondence: John-Paul Heale, University of British Columbia, Department of Pediatrics, Faculty of Medicine, The Research Institute, Room 381, 950 West 28th Avenue, Vancouver, BC, Canada, V5Z 4H4. E-mail: jpheale{at}interchange.ubc.ca
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONREFERENCES |
|---|
s) are incompetent to phagocytose
unopsonized Pseudomonas aeruginosa, but ingestion by other
macrophage phenotypes (i.e., peritoneal macrophages) occurs
efficiently. The purpose of this study was to explore factors that
might control such phenotypic differences. Our laboratory has
demonstrated that AMs exposed to sodium azide display enhanced
phagocytosis of P. aeruginosa. Here we report that the
phagocytic-enhancing effect of sodium azide was abrogated by inhibitors
of protein kinase C (PKC). Furthermore, the addition of PKC agonists,
such as phorbol myristate acetate (PMA), and tumor necrosis factor 
(TNF-), mimicked the phagocytic enhancing effect of sodium azide. We
conclude that AMs are normally incompetent to phagocytose P.
aeruginosa. Factors that up-regulate AM function (azide, PMA,
TNF-) can reverse the phagocytic incompetence in vitro.
Although these compounds are not appropriate candidate therapeutic
agents, their effects provide insights for understanding of the
pathways responsible for regulation of P. aeruginosa
phagocytosis.
Key Words: phorbol myristate acetate • tumor necrosis factor • azide
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONREFERENCES |
|---|
s) to phagocytose the bacteria
[2
]. Our experiments were designed to better understand
the control of P. aeruginosa phagocytosis and to determine
why freshly explanted AMs are unable to phagocytose unopsonized
P. aeruginosa. Our ultimate goal is to understand the
factors that regulate phagocytosis of P. aeruginosa in order
to enhance this primary host defense mechanism within the lungs of CF
patients.
We have previously demonstrated that freshly explanted murine AMs
are able to increase their capacity to ingest P. aeruginosa
when briefly exposed to sodium azide [3
]. Sodium azide
poisons oxidative respiration, specifically inhibiting cytochrome
oxidase [4
], the main metabolic pathway by which AMs
derive energy in the high O2 tension of the lung
[5
]. In response to sodium azide-induced stress, AM
kinase pathways are probably altered. We observed that staurosporine,
an inhibitor of protein kinase C (PKC) [6
], abrogates
the increase in phagocytosis brought about by sodium azide (Table 1
).
|
s were recovered from the lungs of 6-week-old, female BALB/c mice
by bronchoalveolar lavage (BAL); 3.0 x 105 AMs
were adhered to glass coverslips in 24-well Falcon tissue culture
plates in RPMI 1640 medium supplemented with 5% fetal calf serum
(GIBCO-BRL). AMs were incubated for 90 min in tissue culture medium
alone (control), with 1 mM sodium azide or with 1 mM sodium azide and
500 nM staurosporine. After the 90-min incubation, the medium was
replaced with fresh tissue culture medium without azide or
staurosporine for a further 90-min incubation to allow the AMs to
recover. The medium was then removed and replaced with 450 µL
phagocytosis buffer (PB: 100 mM Tris, pH 7.4, 150 mM NaCl, 0.6 mM
CaCl2, 1.0 mM MgCl2, 10 mM
D-glucose) and 50 µL of P. aeruginosa strain
P1 culture [7
] (grown overnight and adjusted to an
absorbance at A600 of 1.0 = 300 bacteria per AM).
The AMs and bacteria were incubated at 37°C for 60 min, after
which AMs were washed with fresh PB. Uningested bacteria were lysed
by exposure to 5 mg/mL lysozyme and subsequent osmotic shock with
dH2O [2
]. AMs were fixed with 4%
formaldehyde, stained with 2% Giemsa, and ingested bacteria enumerated
by visual inspection.
AMs incubated in the presence of sodium azide phagocytosed over
twice as many P. aeruginosa as controls (5.7±0.8 vs.
2.5±0.3 bacteria per AM, P<0.05, Table 1
). However,
AM incubated with sodium azide and staurosporine ingested 1.8 ± 0.2, significantly below control levels (P<0.05). Based
on these observations, PKC agonists such as phorbol myristate acetate
(PMA [6
]) and tumor necrosis factor (TNF-;
inflammatory cytokine [6
]) were chosen in addition to
sodium azide to investigate their capacity for up-regulation of
phagocytosis by AMs.
AMs were incubated for 15 min with 1 mM sodium azide, 5 nM TNF-,
or 60 ng/mL PMA before the addition of P. aeruginosa (50
µL of A600 0.5=150 bacteria per AM) and the 1-h
phagocytosis assay in PB. PKC agonists enhanced AM phagocytosis
2.5-fold over untreated controls (4.4±0.2, sodium azide; 4.2±0.9,
TNF-; 4.1±0.9, PM; vs. 1.7±0.2, control, Fig. 1A
). Such a rapid up-regulation of phagocytosis suggests activation
of a preformed receptor on the surface of AMs. Cells that had been
washed of agonists after a 90-min exposure, and allowed to recover in
tissue culture medium alone, showed progressively lower levels of
phagocytosis (Fig. 1B)
. For those AMs incubated in each of the
agonists for 90 min, followed by a 270-min recovery in tissue culture
medium alone (350-min time point, Fig. 1B
), the phagocytosis levels
were not statistically different from control levels. A second
cytokine, interferon-
(IFN-), has previously been shown to
suppress phagocytosis of either opsonized or unopsonized P.
aeruginosa [8
]. When 100 U/mL IFN- was included
in the tissue culture medium, in a manner identical to that described
above, phagocytosis levels were suppressed 
50% below control levels
(Fig. 1) .
|
s
after prolonged incubation with the PKC agonists. Sodium azide was not
included in these experiments because of the toxicity observed.
Prolonged exposure of freshly explanted AMs to TNF- increased
phagocytosis from 2.6 ± 0.3 (control) to 8.4 ± 0.5 at
12 h (Fig. 2
). For TNF--exposed AMs, the phagocytic enhancement was
short-lived; at 24 h TNF- treatment, phagocytosis was 3.2 ± 0.4, comparable to control levels (2.7±0.5). TNF- effects at 36
and 48 h were indistinguishable from 24 h (3.2±1.4 and 3.2±0.3,
respectively). Prolonged PMA exposure also yielded enhanced
phagocytosis at 12 h (5.5±0.9), which persisted at 24 h (5.6±1.0) and
then fell to levels comparable with the control at 36–48 h (3.6±0.5
and 3.7±0.3, respectively). Phagocytosis by control AMs gradually
increased over time from 1.8 ± 0.3 at time 1.5 h, to 3.8 ± 0.3
at 48 h. IFN- treatment inhibited phagocytosis below control values
(Fig. 2)
. These results suggest that PKC agonists are able to activate
AMs to phagocytose P. aeruginosa to a level that is
statistically greater than control values. However, continued exposure
to PKC agonists does not result in phagocytic enhancement. Similar
effects have been documented for PKC-dependent activation of complement
receptor 3 [6
]. We are currently attempting to identify
the receptor that is pivotal in mediating ingestion of P.
aeruginosa by AMs.
|
s exposed to TNF-.
Murine resistance to P. aeruginosa lung infection is
directly related to the level of secreted TNF- within the animal’s
lung [9
]. Similarly, intratracheal inoculation of
recombinant TNF- in mice at the time of P. aeruginosa
lung infection improves bacterial clearance [10
].
Because gram-negative bacterial (i.e., P. aeruginosa)
lipopolysaccharide induces TNF- release from macrophages
[11
], our results may be relevant to the early
inflammatory response within the lung. When AMs detect LPS, they
release TNF-, which in turn promotes neutrophil emigration to the
site of infection [12
]. Given the in vitro
results described, we hypothesize that AMs may be rendered
phagocytically competent during the initial stages of P.
aeruginosa infection, until such time as neutrophils are
recruited. The dynamics of phagocytic cell recruitment and function,
combined with the modulatory activity of inflammatory cytokines, are
currently being investigated in a murine model of P.
aeruginosa lung infection.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  C. H. C. Serezani, D. M. Aronoff, S. Jancar, P. Mancuso, and M. Peters-Golden Leukotrienes enhance the bactericidal activity of alveolar macrophages against Klebsiella pneumoniae through the activation of NADPH oxidase Blood, August 1, 2005; 106(3): 1067 - 1075. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. C. Todt, B. Hu, A. Punturieri, J. Sonstein, T. Polak, and J. L. Curtis Activation of Protein Kinase C beta II by the Stereo-specific Phosphatidylserine Receptor Is Required for Phagocytosis of Apoptotic Thymocytes by Resident Murine Tissue Macrophages J. Biol. Chem., September 20, 2002; 277(39): 35906 - 35914. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
