Download Pulmonary Surfactant Protein A Activates a

The Journal of Immunology
Pulmonary Surfactant Protein A Activates a
Phosphatidylinositol 3-Kinase/Calcium Signal Transduction
Pathway in Human Macrophages: Participation in the
Up-Regulation of Mannose Receptor Activity1
Alison A. Beharka,* Joy E. Crowther,*† Francis X. McCormack,‡ Gerene M. Denning,*
Jason Lees,* Eric Tibesar,* and Larry S. Schlesinger2†
Surfactant protein A (SP-A), a major component of lung surfactant, binds to macrophages and has been shown to alter several
macrophage biological functions, including up-regulation of macrophage mannose receptor (MR) activity. In the present study,
we show that SP-A induces signal transduction pathway(s) that impact on MR expression. The addition of human, rat, or
recombinant rat SP-A to human monocyte-derived macrophages significantly raised the level of cytosolic Ca2ⴙ above baseline
within 10 s of SP-A addition, as measured by spectrofluorometric analysis. SP-A induced a refractory state specific for SP-A
consistent with homologous desensitization of a receptor(s) linked to calcium mobilization because a second application of SP-A
did not induce a rise in cytosolic Ca2ⴙ whereas the addition of platelet-activating factor did. Using site-directed mutations in
SP-A, we determined that both the attached sugars and the collagen-like domain of SP-A are necessary to optimize Ca2ⴙ mobilization. SP-A triggered the increase in cytosolic Ca2ⴙ by inducing activation of phospholipase C, which leads to the hydrolysis of
membrane phospholipids, yielding inositol 1,4,5-trisphosphate and mobilizing intracellularly stored Ca2ⴙ by inositol triphosphatesensitive channels. Finally, inhibition of PI3Ks, which appear to act upstream of phospholipase C in Ca2ⴙ mobilization, decreased
the SP-A-induced rise in MR expression, providing evidence that SP-A induction of MR activity involves the activation of a
pathway in which PI3K is a component. These studies provide further evidence that SP-A produced in the lung plays a role in
modulating macrophage biology, thereby contributing to the alternative activation state of the alveolar macrophage. The Journal
of Immunology, 2005, 175: 2227–2236.
I
nhaled particles and infectious agents represent a constant
threat to the respiratory system. To defend itself, the respiratory tract has a complex system consisting of mechanical
reflexes, physical barriers, and a diverse set of biochemical and
cellular defenses. Acquired humoral and cellular immunity are of
obvious importance in lung defense; however, it is becoming increasingly clear that components of innate immunity also play important roles in determining the response to and fate of inhaled
particles. Pulmonary surfactant is a multimolecular complex, including proteins and lipids, that serves to reduce the surface tension of the alveoli, allowing expansion of the lung during inspiration. Recently, components of surfactant, specifically surfactant
protein A (SP-A)3 and surfactant protein D (SP-D), have been
*Interdisciplinary Program in Immunology and Department of Internal Medicine and
Department of Microbiology, University of Iowa, and Department of Veterans Affairs, Iowa City, IA 52240; †Department of Medicine, Department of Molecular Virology, Department of Immunology, Department of Medical Genetics, and Center for
Microbial Interface Biology, Ohio State University, Columbus, OH 43210; and ‡Department of Internal Medicine, University of Cincinnati, Cincinnati, OH 45267
Received for publication September 15, 2004. Accepted for publication June 2, 2005.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by National Institutes of Health Grants AI 33004 (to
L.S.S.), HL-51990, and HL-61612 (to F.X.M.) and the Department of Veterans
Affairs.
2
Address correspondence and reprint requests to Dr. Larry Schlesinger, Department
of Internal Medicine, Ohio State University, 420 West 12th Avenue, 200 MRF, Columbus, OH 43210. E-mail address: [email protected]
3
Abbreviations used in this paper: SP-A, surfactant protein A; SP-D, surfactant protein D; CRD, carbohydrate recognition domain; MR, mannose receptor; SP-R210,
Copyright © 2005 by The American Association of Immunologists, Inc.
shown to play important roles in the lung innate immune
response (1, 2).
SP-A, a member of the collectin family, is the most abundant
protein component (by weight) of human pulmonary surfactant (2).
SP-A is produced as a 28- to 36-kDa (rat) or 35-kDa (human)
polypeptide that contains a hydroxyproline-rich collagen-like domain, a carbohydrate recognition domain (CRD), and one (human)
or two (rat) N-linked oligosaccharide attachment sites at Asn1 in
the N terminus (rat) and Asn187 (human and rat) in the CRD (3).
In the lung microenvironment, SP-A monomers assemble into an
octadecamer, a structure that provides high valency-binding sites
for a diverse set of molecules, including cell surface receptors and
surface components of microorganisms, and is believed to be necessary for optimal activity of the protein (4, 5).
The interaction of SP-A with macrophages has multiple effects
on the macrophage functional state, including chemotaxis, increased phagocytosis, and modulation of cytokine and oxidant production (1, 2, 6, 7). We have determined that the increase in macrophage phagocytosis is related in part to SP-A-enhanced activity
of the mannose receptor (MR) (8), a pattern recognition receptor
(9). SP-A effects on macrophages are believed to be mediated by
interactions of this protein with its receptor(s) on macrophages (1).
Chroneos et al. (10) have reported a 210-kDa SP-AR on U937 cells
and rat macrophages called SP-R210. Other SP-A-binding proteins
have also been reported (reviewed in Ref. 11). Little is known
210-kDa SP-A receptor; InsP3, D-myo-inositol 1,4,5-triphosphate; ER, endoplasmic
reticulum; PLC, phospholipase C; MDM, monocyte-derived macrophage; HSA, human serum albumin; PMB, polymyxin B sulfate; PAF, platelet-activating factor; APP,
alveolar proteinosis protein; SP-Ahyp, recombinant rat SP-A; [Ca2⫹]cyt, cytosolic calcium; MFI, mean fluorescence intensity; PKC, protein kinase C.
0022-1767/05/$02.00
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SP-A ACTIVATES PI3K/Ca2⫹ SIGNALING IN HUMAN MACROPHAGES
about the signaling pathways activated by SP-A binding to its receptor(s). Ohmer-Schröck et al. (12) reported that human SP-A
activates a phosphoinositide/calcium-signaling pathway involved
in phagocytosis by rat alveolar macrophages. However, the signaling pathway(s) induced by SP-A in primary human macrophages is unknown.
Several mechanisms induce the release of small bursts of Ca2⫹
into the cytosol, which acts as a second messenger for signal transduction (13). In macrophages, the generation of D-myo-inositol
1,4,5-triphosphate (InsP3) is important in regulating Ca2⫹ release
from the endoplasmic reticulum (ER) (reviewed in Ref. 14). Signals initiated by either G protein-linked receptors or receptors
linked directly or indirectly to tyrosine kinases (14) activate phospholipase C (PLC), which hydrolyzes phosphatidylinositol (4,5)bisphosphate to form InsP3 and 1,2-diacylglycerol. In some cases,
PI3Ks may act upstream of PLC activation because PLC␥ can be
activated by lipid products of PI3Ks (15, 16). Although a variety
of agonists are capable of activating both PLC and PI3Ks in human
macrophages (17), it is not known whether SP-A is one of them.
We hypothesized that SP-A binding to human macrophages activates a signal transduction pathway that uses Ca2⫹ as a second
messenger. In this study, we examined Ca2⫹ mobilization in human monocyte-derived macrophages (MDM) in response to SP-A.
Using wild-type and variant SP-A proteins with site-directed mutations, we investigated whether the attached carbohydrates and/or
collagen-like domain of SP-A are necessary for activation. Using
inhibitors, we studied the possible link between components of the
Ca2⫹/InsP3 signal transduction pathway and SP-A up-regulation
of MR expression. Our data indicate that SP-A induces Ca2⫹
mobilization by activating PLC, which hydrolyzes membrane
phospholipids and yields InsP3, and that activation of PI3K(s),
potentially through a PLC-signaling pathway, plays a role in SP-A
up-regulation of MR.
Materials and Methods
Buffers, reagents, and media
RPMI 1640 medium with L-glutamine was purchased from Invitrogen Life
Technologies. RPMI 1640 medium was used alone or with 20 mM HEPES
(Sigma-Aldrich) and 1 mg/ml human serum albumin (HSA) (Calbiochem)
at pH 7.2. Fura 2-AM was purchased from Molecular Probes. BAPTAAM, wortmannin, LY294002, U73122, and U73343 were purchased from
Calbiochem. Thapsigargin, polymyxin B sulfate (PMB), and LPS from
Escherichia coli 055:B5 strain were purchased from Sigma-Aldrich. Platelet-activating factor (PAF) was a kind gift from Dr. L. Stoll (University of
Iowa, Iowa City, IA). PE-conjugated mouse anti-human MR mAb and
PE-conjugated anti-mouse IgG1␬ Ab were purchased from BD Pharmingen. Anti-SP-R210 Ab was a generous gift from Dr. Z. A. Chroneos (University of Texas, Tyler, TX).
SP-A proteins
The SP-A proteins used in this study were produced and purified as previously described (18) and shown schematically in Beharka et al. (8). In
brief, bronchoalveolar lavage was used to obtain SP-A from patients with
alveolar proteinosis protein ((APP) SP-A) and from healthy volunteers (native human SP-A) (19). Native rat SP-A was purified from silica-pretreated
Sprague-Dawley rat lungs. Recombinant rat SP-A (SP-Ahyp), which is deficient in hydroxyproline content and hence designated SP-Ahyp, was produced from SF-9 insect cells following infection with a recombinant baculovirus containing a 1.6-kb cDNA for rat SP-A (18). SP-Ahyp proteins
devoid of oligosaccharides at one or both of the consensus sequences for
N-linked glycosylation were generated by amino acid substitutions at the
Asn1 (SP-Athr1) or Asn187 site (SP-Aser187) or at both the Asn1 and Asn187
sites (SP-Athr1ser187) (18). Carbohydrate-deficient SP-A proteins retained
structural and biologic functions, including partial oligomerization, aggregation of phospholipid liposomes, binding to immobilized carbohydrate,
inhibition of lipid secretion from type II cells, and competition for receptor
occupancy on type II cells (18). The synthesis of the mutant SP-Ahyp proteins containing a nested deletion of the proximal collagen-like domain
(Gly8-Gly44) (TM2), a truncation of the protein at the neck region resulting
in a protein lacking the collagen-like and the N-terminal regions (TM1-23), and an amino acid substitution at the Asn187 site (TM1-2-3ser187) were
made as described previously (20, 21). SP-A proteins were suspended in
buffer containing 10 mM HEPES ⫹ 20 mM NaCl and kept at ⫺20°C until
use. Purity of the SP-A proteins was assessed by SDS-PAGE and silver
staining. Endotoxin levels in SP-A preparations were determined using an
Limulus amoebocyte lysate kit (BioWhittaker) and ranged from undetectable to 300 pg/␮g protein, with an average of 15 pg/␮g protein.
Human macrophages
Blood was obtained from healthy adult volunteers using an approved Institutional Review Board protocol for human subjects at the University of
Iowa and the Veterans Affairs Medical Center. PBMC from single donors
were isolated from heparinized blood on Ficoll-sodium diatrizoate (Pharmacia) and cultured in Teflon wells (Savillix) in the presence of 20%
autologous serum at 37°C for 5 days (22). On the day of each experiment,
PBMC containing MDM were removed from Teflon wells, washed extensively, and counted. For Ca2⫹ experiments, 3 ⫻ 105 MDM were adhered
to 25-mm acid-cleaned glass coverslips in single wells for 2 h in the presence of medium containing RPMI 1640 medium ⫹ 20 mM HEPES ⫹ 10%
autologous serum. For measurement of InsP3, 1 ⫻ 105 MDM were adhered
to 10-mm acid-cleaned glass coverslips for 2 h in single wells.
Cell line culture
U937 cells, obtained from the American Type Culture Collection, were
maintained at 37°C in a humidified 5% CO2 incubator in RPMI 1640
medium with heat-inactivated 10% FBS (HyClone), 1 mM glutamine, 20
mM HEPES, and penicillin/streptomycin (100 U/ml and 100 ␮g/ml, respectively). Cells were maintained in 175-cm2 flasks and passaged every
3– 4 days by scraping the flask with a cell scraper. Cells were not used after
passage 30.
Measurement of cytosolic calcium ([Ca2⫹]cyt)
Measurement of [Ca2⫹]cyt was performed as described previously (23, 24).
Briefly, MDM on 25-mm round glass coverslips were washed and loaded
with fura 2 by addition of the cell-permeate form, fura 2-AM, in RPMI
1640 medium for 30 min at 37°C in 5% CO2. MDM were then washed with
HEPES-buffered saline containing glucose and stimulated by the addition
of SP-A proteins, LPS or PAF, as indicated (⬍5% v/v). Measurements of
the apparent [Ca2⫹]cyt were done using two different spectrofluorometers.
In the first set of experiments, [Ca2⫹]cyt was measured using the Photoscan
II spectrofluorometer (Photon Technology International) with a Nikon microscope (Nikon) at the Cell Fluorescence Core Facility (Veterans Affairs
Medical Center). The second set of experiments used an ImageMaster Ratio Fluorescence imaging system (Photon Technology International) at the
Central Microscopy Research Facility (University of Iowa). In both cases,
the final Ca2⫹ concentrations were determined from the ratio of emission
intensities (emission wavelength (␭em) 510 nm) to excitation wavelengths
of (␭ex) 340 and 380 nm using a Photon Technology International software
package. Background fluorescence intensities for each ␭ex were obtained
using non-fura 2-loaded cells and were subtracted from the raw data. The
ratios of the corrected fluorescence intensities were then converted to
[Ca2⫹] values using the formula [Ca2⫹] ⫽ Kd ⫻ (R ⫺ Rmin)/(Rmax ⫺ R),
where the maximum and minimum ratios (Rmax and Rmin, respectively), as
well as the apparent dissociation constant (Kd), were empirically derived
from [Ca2⫹] curves generated using the instrument. Positive (PAF), vehicle
(Tris buffer; DMSO), and HSA controls were done for each experiment.
Experiments were repeated using MDM isolated from a minimum of three
different blood donors.
In the second method, MDM were adhered to 24-well tissue culture
plates in RPMI 1640 medium ⫹ 10% autologous serum, then washed and
repleted in Dulbecco’s PBS plus 10 mM HEPES plus 1 mg/ml human
serum albumin plus 0.1% glucose (DPBS-HHG) containing 10 ␮M fluo-4
AM (Molecular Probes) for 1 h at 37°C. Cells were then washed three
times to remove unhydrolyzed dye and repleted with DPBS-HHG. Calcium
measurements were made using a BMG FluoStar microplate fluorometer
(BMG Lab Technologies) with excitation and emission wavelengths of 485
and 520 nm, respectively. Readings were taken every 3 s for 2 min to
establish baseline, then 10 ␮g/ml SP-A were added and readings taken
every 3 s for 10 min. A second bolus of SP-A (10 ␮g/ml)- or PAF (100
nM)-positive control was added at 3– 60 min after the first addition of SP-A
and fluorescence changes monitored. The maximal increase in fluorescence
over baseline was determined for each SP-A or PAF addition. Responses
of SP-A-treated cells to a second treatment with SP-A were expressed as
percentage of initial response.
The Journal of Immunology
Competitive binding assay with inhibitors
SP-A was fluorescently labeled using an Alexa Fluor 647 protein labeling
kit (Molecular Probes).4 Cells were adhered to 4-well plates, washed, and
rested overnight in RPMI 1640 medium ⫹ 10% serum. Macrophages were
lifted using rubber policemen and resuspended in DPBS-HHG. Macrophages in suspension at 1E5/tube were incubated in triplicate tubes with 10
␮g/ml unlabeled SP-A, 10 –100 nM wortmannin, 3–9 ␮M Ly294002, 5
␮M U73122, 5 ␮M U73343, or medium controls for 30 min at 37°C, then
incubated with 1 ␮g of fluorescent-labeled SP-A at 4°C for 2 h. For conditions of SP-A preincubation, cells were pelleted once to remove medium
containing unbound SP-A before the addition of labeled SP-A. Cells were
washed, fixed in 2% paraformaldehyde, and fluorescence indicative of
SP-A binding was measured in triplicate tubes using a Beckman
FACSCalibur flow cytometer. Background fluorescence (macrophages
with no SP-A) was subtracted from all test conditions and values set relative to the positive control (macrophages ⫹ SP-A, no inhibitors).
Measurement of InsP3
Two different methods were used to determine whether SP-A induced
InsP3 formation. In the first method, adherent MDM were incubated with
20 ␮g/ml APP SP-A for 0, 15, 60 s, and 10 min (time dependence) or with
0, 2.5, 5, 10, 20, or 40 ␮g/ml APP SP-A for 15 s (concentration dependence). The reaction was stopped, and the cells were lysed by the addition
of 0.2⫻ volume of ice-cold 20% perchloric acid on ice for 20 min. After
centrifugation for 15 min at 2000 ⫻ g at 4°C, supernatants were removed
and neutralized with ice-cold 1.5 M KOH containing 60 mM HEPES.
KClO4 was sedimented by centrifugation at 2000 ⫻ g for 15 min. Determination of InsP3 in the supernatant was done using a competitive RIA
(Amersham Biosciences), according to the manufacturer’s instructions.
In the second method, adherent MDM were incubated for 48 h in RPMI
1640 medium containing 1 ␮Ci/ml myo-[3H]inositol (Amersham Biosciences), and turnover of inositol phosphates was measured as described
previously (23, 25). Briefly, cells were washed with HEPES-buffered saline
supplemented with 10 mM glucose (HBS-G) and incubated in HBS-G for
20 min at 37°C, followed by incubation for 20 min with HBS-G containing
10 mM lithium chloride. Finally, cells were treated with SP-A as in the first
assay. Inositol phosphates were extracted overnight at 4°C with 0.5 M
perchloric acid. The acid extract was neutralized with 2.5 M KOH and 0.5
M HEPES (pH 7.4) and centrifuged to remove the precipitate. The inositol
phosphate species were then collected using anion-exchange column chromatography (Dowex AG, 1– 8⫻, 100 –200 mesh, formate form; Bio-Rad)
as described previously (23, 25).
2229
using a Photoscan II spectrofluorometer. In these studies, the basal
[Ca2⫹]cyt values for MDM were ⬃75–150 nM and were similar
between experiments. A trace of a typical experiment using an
optimal concentration of APP SP-A (10 ␮g/ml) is shown in Fig.
1A. Exposure of MDM to SP-A resulted in an significant increase
in [Ca2⫹]cyt concentration over baseline ( p ⬍ 0.05). The effect
was rapid and transient. Cytosolic Ca2⫹ levels began to rise within
10 s of SP-A addition, and the maximal concentration was reached
in ⬃100 s with an amplitude of 125 ⫾ 21 nM over baseline. The
maximal level of [Ca2⫹]cyt was sustained for ⬍100 s, after which
the level of [Ca2⫹]cyt declined over a period of 5 ⫾ 1 min. However, even after 20 min, [Ca2⫹]cyt levels never returned completely
to baseline (data not shown). The kinetics of the rise in [Ca2⫹]cyt
observed after addition of SP-A were similar to those observed
with an optimal concentration (50 nM) of PAF (Fig. 1B), except
the magnitude of the response to SP-A was less. PAF, a known
activator of the InsP3/Ca2⫹ pathway in macrophages (27), was
used as the positive control in all experiments.
Flow cytometry
PBMC were incubated with inhibitors (thapsigargin, wortmannin, U73343,
Ly294002, or BAPTA), vehicle control (DMSO), or medium for 30 min
followed by SP-A proteins (10 ␮g/ml) or HSA protein control (10 ␮g/ml)
in Teflon wells for 2 h. As a positive control for up-regulation of MR,
MDM were incubated with IL-4 (Genzyme) for 20 h before staining (26).
PBMC (5 ⫻ 106) were incubated with PE-conjugated mouse anti-human
MR or PE-conjugated subtypic control mAb, mouse IgG1␬. Cells were
washed and fixed in paraformaldehyde and analyzed for mean fluorescence
intensity (MFI) and percentage of positive cells (95/5% cutoff) using a
FACScan Flow Cytometry System (BD Biosciences). Macrophages were
identified using side scatter vs forward scatter. MFI due to nonspecific
binding as determined using subtypic control Ab was subtracted from experimental data to provide a specific MFI. Each experiment was done in
duplicate a minimum of three times. Cell viability was unaffected by the
presence of inhibitors during the time course examined and was ⬎90% by
trypan blue exclusion.
Statistics
Data were subjected to an analysis of normality. Normally distributed data
were analyzed by ANOVA followed by Student’s t test using SPSS statistical program software. Statistical significance was defined as p ⬍ 0.05.
Results
SP-A induces Ca2⫹ mobilization in MDM
To determine the influence of SP-A on Ca2⫹ mobilization, we
measured the level of [Ca2⫹]cyt in adherent fura-2 loaded MDM
4
J. E. Crowther and L. S. Schlesinger. Defining the endocytic pathway for surfactant
protein A in human macrophages: binding, clathrin-dependent uptake, and trafficking
through the endolysosomal pathway. Submitted for publication.
FIGURE 1. SP-A increases cytosolic Ca2⫹ concentration. MDM were
loaded with fura-2 and stimulated with 10 ␮g/ml APP SP-A alone (A), LPS
(0.5 ␮g/ml) for 400 s followed by PAF (10 nM) (B), or APP SP-A (10
␮g/ml) and PMB (10 ␮g/ml) (C). The [Ca2⫹]cyt response was analyzed by
Photoscan II spectrofluorometer. APP SP-A increased [Ca2⫹]cyt after a
time lag of 5–10 s. Addition of PMB did not significantly reduce the APP
SP-A increase in [Ca2⫹]cyt. LPS stimulation did not result in Ca2⫹ mobilization at the concentrations used, and MDM were still capable of responding to the positive control, PAF. Representative of three to eight
independent experiments.
2230
SP-A ACTIVATES PI3K/Ca2⫹ SIGNALING IN HUMAN MACROPHAGES
LPS does not affect the SP-A-induced rise in [Ca2⫹]cyt
The preparations of SP-A used contained variable levels of LPS.
However, our data provide evidence that the increase in [Ca2⫹]cyt
is due solely to SP-A and not to contaminating LPS because neutralization of LPS through the addition of PMB (5 ␮g/ml) before
stimulation with APP SP-A did not significantly diminish the APP
SP-A-induced [Ca2⫹]cyt response (Fig. 1C). Furthermore, addition
of LPS alone, either at levels found in the SP-A preparations
(⬍300 pg/ml; data not shown) or higher levels used as controls
(0.5 ␮g/ml; Fig. 1B), did not mobilize [Ca2⫹] cyt. Failure of LPS
to mobilize Ca2⫹ was not due to an inability of the MDM to
respond because the addition of PAF after LPS treatment resulted
in a significant [Ca2⫹]cyt response (Fig. 1B).
SP-A induced Ca2⫹ mobilization is concentration dependent
The SP-A-induced elevation in [Ca2⫹]cyt was concentration dependent (Fig. 2). APP SP-A induced a rise in [Ca2⫹]cyt when
added at 0.5 ␮g/ml (threshold concentration), and the increase over
baseline was significant at 1 ␮g/ml (Fig. 2). The dose response was
linear at concentrations of up to 5 ␮g/ml and then plateaued, with
no additional effect at higher concentrations.
SP-R210 has been reported as a receptor for SP-A that is present
on rat alveolar and bone marrow-derived macrophages and the
human myeloid cell line U937 (10). To determine whether signaling through SP-R210 was responsible for the SP-A-induced rise in
[Ca2⫹]cyt, we blocked binding of SP-A to SP-R210 through the
addition of 10 ␮g/ml rabbit anti-SP-R210 (10) or control rabbit
IgG to MDM for 20 min before stimulation with 5 ␮g/ml APP
SP-A. SP-R210 did not affect the subsequent rise in [Ca2⫹]cyt by
APP SP-A (174 ⫾ 35 vs 167 ⫾ 21 nM for MDM treated with
anti-SP-R210 Ab or isotype control, respectively, n ⫽ 3). Expression of SP-R210 and C1qR, but not the putative SP-AR identified
by Strayer et al. (28) and Stevens et al. (29), has been demonstrated
on U937 cells (10). Addition of APP SP-A did induce a rise in
[Ca2⫹]cyt in U937; however, a 10-fold greater concentration of
SP-A was required for an optimal response in these cells compared
FIGURE 2. SP-A-dependent increase in cytosolic Ca2⫹ is concentration
dependent. The [Ca2⫹]cyt response of MDM was analyzed by Photoscan II
spectrofluorometer for 10 min after addition of the indicated concentrations
of APP SP-A (as described in the legend to Fig. 1). The data show the
maximum increase in [Ca2⫹]cyt (nM) reached over baseline. The amount of
Ca2⫹ mobilized was concentration dependent. Results are expressed as
mean ⫾ SEM of three to eight independent experiments. ⴱ, Significantly
higher levels of [Ca2⫹]cyt compared with unstimulated controls; p ⬍ 0.05
by Student’s t test.
with human MDM (50 ␮g/ml APP SP-A for U937 vs 5 ␮g/ml for
MDM, n ⫽ 3).
The rise in [Ca2⫹]cyt is refractory to a second treatment of SP-A
The SP-A-induced rise in [Ca2⫹]cyt was not seen after a second
stimulation with SP-A at the same concentration as the first (10
␮g/ml), even after 3 min (Fig. 3A). When 20 ␮g/ml APP SP-A
(Fig. 3B) or 40 ␮g/ml APP SP-A (Fig. 3C) were used as the secondary stimulus, [Ca2⫹]cyt increased within 20 s of APP SP-A
addition but never reached levels obtained by the first SP-A stimulus. However, the refractory state was specific to SP-A, as MDM
exposed to APP SP-A retained their ability to respond to a second
unrelated agonist, PAF (Fig. 3D), and MDM exposed to PAF were
capable of responding to SP-A (Fig. 3E) to the same extent as
MDM treated with SP-A alone.
We examined the length of this refractory period in MDM and
found full recovery of the calcium response to a second stimulation
with SP-A by 60 min (Fig. 4). The reduced rise in [Ca2⫹]cyt seen
upon a second stimulation with SP-A at time points of ⬍60 min
was not due to a reduction in the ability of SP-A to bind to MDM
because preincubation of MDM with 10 ␮g/ml SP-A for 15 or 30
min at 37°C did not inhibit binding of subsequently added SP-A
(Fig. 4). These data suggest that SP-A induces a refractory state in
FIGURE 3. SP-A-treated MDM are refractory to a second stimulation
with SP-A. MDM exposed to 10 ␮g/ml APP SP-A became refractory to a
second stimulation with the same concentration of APP SP-A (10 ␮g/ml)
(A). Secondary exposure to two times (20 ␮g/ml) (B) or four times (40
␮g/ml) (C) the concentrations of APP SP-A resulted in a rise in [Ca2⫹]cyt,
but the rise was significantly lower than that achieved with the first exposure. MDM exposed to APP SP-A retained their ability to respond to a
second unrelated agonist, PAF (D), and MDM stimulated with PAF retained their ability to respond to APP SP-A (E). Results are expressed as
ratiometric calcium concentration in relative units (see Materials and
Methods for equation) over time. Representative of three independent
experiments.
The Journal of Immunology
2231
induced similar ( p ⬎ 0.05) levels of [Ca2⫹]cyt that were significantly higher ( p ⬍ 0.005) than that induced by the wild-type SPAhyp. Additionally, the amount of SP-A required to achieve a
threshold [Ca2⫹]cyt response varied with SP-A type (0.5, 1, 1, and
2 ␮g/ml for APP, human native, native rat, and SP-Ahyp, respectively). These results indicate that the source of SP-A (APP, and
native, recombinant) affects the degree of Ca2⫹ mobilization.
The attached carbohydrates and collagen-like domain of SP-A
are both involved in SP-A-induced Ca2⫹ mobilization
FIGURE 4. The MDM refractory period to SP-A is ⬍60 min and is not
a result of decreased SP-A binding to MDM. For measurements of
[Ca2⫹]cyt, MDM were loaded with fluo-4 and stimulated with 10 ␮g/ml
APP SP-A for 3– 60 min, followed by a second stimulation with the same
concentration of APP SP-A (10 ␮g/ml). The [Ca2⫹]cyt response was analyzed by Flurostar microplate fluorometer. [Ca2⫹]cyt results (F) are expressed as mean percent of initial response ⫾ SEM of two independent
experiments. To determine SP-A binding, MDM were incubated with 10
␮g/ml APP SP-A or medium controls for 15–30 min at 37°C, then incubated with 1 ␮g of fluorescent-labeled SP-A ⫾ at 4°C for 2 h, washed, and
bound protein determined by flow cytometry. Binding data (f) are expressed as percentage of binding to MDM incubated with medium alone ⫾
SD and are representative of two independent experiments.
macrophages that is specific for SP-A and thus resembles homologous receptor desensitization with regard to mobilization of intracellular Ca2⫹.
We next explored which of the domains or structural elements of
SP-A mediated the Ca2⫹ response. To study the potential influence
of the carbohydrate moieties of SP-A in mediating its effect on
Ca2⫹mobilization, we used SP-Ahyp proteins devoid of oligosaccharides at one (Asn1 site (SP-Athr1) or Asn187 site (SP-Aser187)) or
both (Asn1 and Asn187 (SP-Athr1ser187)) of the consensus sequence
sites for glycosylation. The absence of carbohydrates at these sites
on the variant proteins used in these studies has been demonstrated
previously (7, 18). Stimulation of fura 2-labeled MDM with 10
␮g/ml SP-A devoid of one (SP-Aser187 or SP-Athr1) or both (SPAthr1ser187) N-linked carbohydrates did not elicit a significant rise
in [Ca2⫹]cyt over baseline (Fig. 6). When the amount of mutant
SP-A protein added to the cells was increased to 40 ␮g/ml, all
SP-A proteins were able to induce small, short duration (⬍100 s)
rises in [Ca2⫹]cyt; however, only the rise in [Ca2⫹]cyt induced by
SP-A devoid of carbohydrate at Asn1 (SP-Athr1) was significantly
higher than baseline (Fig. 6). Moreover, the rise induced by SPAthr1 was significantly lower than that induced by SP-A in which
both carbohydrates were present (SP-Ahyp). These results provide
evidence that the presence of carbohydrate at both glycosylation
sites is necessary for optimal mobilization of intracellular Ca2⫹.
In parallel experiments, we investigated the importance of the
collagen-like domain of SP-A for induction of Ca2⫹ mobilization
SP-A source affects the degree of Ca2⫹ mobilization
We next determined the influence of the source of SP-A protein on
Ca2⫹ mobilization. When added to MDM at 10 ␮g/ml, the increase
in [Ca2⫹]cyt by APP SP-A was significantly higher than from all
other sources (Fig. 5). Native human SP-A and native rat SP-A
FIGURE 5. The [Ca2⫹]cyt response varies with SP-A type. The
[Ca2⫹]cyt response of MDM was analyzed by Photoscan II spectrofluorometer after the addition of the indicated sources of SP-A at 10 ␮g/ml. Addition of APP SP-A gave the greatest response. Addition of native human
or native rat SP-A resulted in similar rises in [Ca2⫹]cyt. Addition of SPAhyp produced from SF-9 insect cells (hyp) resulted in a lower response
than the other SP-As tested. Results are expressed as mean ⫾ SEM of three
to eight independent experiments. ⴱ, Significantly different from baseline,
p ⬍ 0.05.
FIGURE 6. SP-A carbohydrate moieties are involved in SP-A-induced
mobilization of Ca2⫹. To determine which carbohydrate(s) of SP-A are
necessary for increased [Ca2⫹]cyt, MDM labeled with fura 2 were stimulated with the following SP-A proteins at 10 and 40 ␮g/ml: SP-Ahyp (hyp),
recombinant protein devoid of oligosaccharide at the Asn1 (thr1), Asn187
(ser187), or both the Asn1 and Asn187 sites (thr1ser187). The [Ca2⫹]cyt response was analyzed by ImageMaster Ratio Fluorescence imaging system.
When the proteins were added at 10 ␮g/ml, none was capable of inducing
a level of [Ca2⫹]cyt greater than baseline. At 40 ␮g/ml, the proteins all
induced small, short duration rises in [Ca2⫹]cyt. Only the rise in [Ca2⫹]cyt
induced by the SP-Athr1 protein was significantly greater than baseline.
Results are expressed as mean ⫾ SEM of three to eight independent experiments. ⴱ, Significant compared with baseline [Ca2⫹]cyt, p ⬍ 0.05.
2232
SP-A ACTIVATES PI3K/Ca2⫹ SIGNALING IN HUMAN MACROPHAGES
(Fig. 7). Truncated SP-A proteins lacking the first half of the collagen-like domain but still possessing both carbohydrate attachment sites (TM2), lacking the collagen-like domain and the Nterminal segment (Asn1 to Ala7) but still possessing the
carbohydrate attachment site at Asn187 (TM1-2-3), or lacking the
collagen-like domain, N-terminal segment, and both carbohydrate
attachment sites (TM1-2-3ser187) were added to fura 2-labeled
MDM. None of the truncated mutants was capable of inducing a
Ca2⫹ response similar in strength or duration to that achieved by
APP SP-A or SP-Ahyp. However, similar to the response seen using carbohydrate-deficient SP-A proteins, all of the collagen-deficient mutants induced a very low-level increase of Ca2⫹, which
was similar between the mutants. Taken together, these studies
demonstrate a critical role for both the carbohydrates and collagenlike domain in the Ca2⫹ response.
SP-A induces Ca2⫹ mobilization from intracellular stores
The rise in [Ca2⫹]cyt induced by agonists is accomplished through
two mechanisms: the release of Ca2⫹ from intracellular stores, of
which the ER is the largest contributor, and by influx across the
plasma membrane (30). We next evaluated the relative contribution of these two mechanisms to the SP-A-induced calcium response. Thapsigargin is an irreversible ER Ca2⫹-ATPase inhibitor
whose application leads to depletion of intracellular Ca2⫹ stores
(31). Fura 2-loaded MDM were pretreated with thapsigargin (0.1,
1, or 10 ␮M) for 60 min. Preliminary experiments confirmed that
this pretreatment did not adversely affect cell viability (data not
shown). Pretreatment with thapsigargin resulted in the dose-dependent loss of APP SP-A-induced Ca2⫹ mobilization (Table I).
Thapsigargin at 10 ␮M essentially prevented any increase in Ca2⫹
in response to APP SP-A. These results provide evidence for the
involvement of intracellular Ca2⫹ stores in the initial SP-A-induced rise in [Ca2⫹]cyt. However, the data do not exclude the
possibility that calcium influx plays a role later in the Ca2⫹
response.
FIGURE 7. The collagen-like domain of SP-A is involved in the SPA-induced mobilization of Ca2⫹. The [Ca2⫹]cyt response of MDM was
analyzed by Photoscan II spectrofluorometer after the addition of the indicated sources of SP-A at 10 ␮g/ml. Wild-type SP-Ahyp (hyp) induced a
rise in [Ca2⫹]cyt, which was significantly above baseline. Recombinant rat
proteins containing a deletion of the proximal collagen-like domain (TM2)
and protein lacking the collagen-like and the N-terminal regions (TM1-2-3)
and devoid of oligosaccharide at the Asn187 site (TM1-2-3ser187) induced
small, short duration rises in [Ca2⫹]cyt; however, the level of [Ca2⫹]cyt
achieved was not significantly different from baseline. Results are expressed as mean ⫾ SEM of three to eight independent experiments. ⴱ,
Significantly different from baseline, p ⬍ 0.05.
Table I. SP-A-induced [Ca2⫹]cyt is decreased by inhibition of PLC and
PI3K and reduction of intracellular calcium storesa
Pretreatment
Percentage of Inhibition of
SP-A-Induced [Ca2⫹]cyt
(Mean ⫾ SEM)b
n
Thapsigargin (0.1 ␮M)
Thapsigargin (1 ␮M)
Thapsigargin (10 ␮M)
U73122 (5 ␮g/ml)
U73343 (5 ␮g/ml)
Wortmannin (10 nM)
Wortmannin (100 nM)
62 ⫾ 15ⴱ
83 ⫾ 8ⴱ
97 ⫾ 3ⴱ
73 ⫾ 10ⴱ
12 ⫾ 22
45 ⫾ 19
60 ⫾ 11ⴱ
3
5
3
3
3
3
4
a
Fura 2-labeled MDM were preincubated with buffer, SP-A, or the indicated
treatments (thapsigargin, an ER Ca2⫹ ATPase inhibitor; wortmannin, an inhibitor of
PI3K; U73122, a PLC inhibitor; U73343, a structural analog of U73122 used as a
negative control) for 30 min before the addition of APP SP-A (10 ␮g/ml). The response of MDM was analyzed by Photoscan II.
b
% inhibition ⫽ (SP-A increase in [Ca2⫹]cyt over baseline ⫺ treatment increase
in [Ca2⫹]cyt over baseline) ⫼ SP-A increase in [Ca2⫹]cyt over baseline.
ⴱ, Significant compared with SP-A alone by Student’s t test, p ⬍ 0.05.
SP-A activates an InsP3-signaling pathway
To further explore the role of the InsP3/Ca2⫹ pathway in SP-A
signaling, we next investigated whether SP-A induced generation
of InsP3 in MDM. The level of InsP3 in MDM was measured using
a commercially available competitive RIA, which determines the
InsP3 content, as well as an assay that measures turnover of inositol phosphates (23, 32). Both assays gave similar results (data not
shown); therefore, only the results of the competitive RIA are
shown. APP SP-A induced production of InsP3 in a time- and
dose-dependent manner (Fig. 8). A typical time course of InsP3
formation in response to SP-A is shown in Fig. 8A. Within 15 s of
SP-A addition, InsP3 levels rose significantly above baseline. Maximal InsP3 levels were reached at 15 s post-SP-A addition. InsP3
formation returned to basal values by 10 min after stimulation.
These kinetics are similar to those observed for the Ca2⫹ response
to SP-A. Additionally, the level of InsP3 formed was dependent on
the concentration of APP SP-A (Fig. 8B). Although the effective
range of SP-A in these assays was similar to that of the Ca2⫹
response, slightly more APP SP-A was needed to induce InsP3
formation compared with Ca2⫹ mobilization, a result that may be
related to differences in assay sensitivity.
InsP3 is typically formed through PLC hydrolysis of membrane
phospholipids (14). Incubation of MDM with an aminosteroid inhibitor of PLC (U73122), but not its analog (U73343), inhibited
the SP-A-induced rise in [Ca2⫹]cyt (Table I), suggesting that SP-A
activation of PLC leads to Ca2⫹ mobilization. SP-A binding to
MDM was not decreased by treatment with U73122 compared
with its analog control U73343 (data not shown), indicating that
inhibitors of PLC had no effect on SP-AR expression. Thus, these
data provide support for SP-A activation of a PLC/InsP3 pathway
leading to the rise in [Ca2⫹]cyt.
Involvement of PI3K in SP-A-induced Ca2⫹ mobilization
PI3K, through its lipid products, has been reported to regulate
PLC␥-mediated calcium signaling under some conditions (16).
Therefore, we next determined whether inhibition of PI3K affected
the SP-A-induced rise in [Ca2⫹]cyt. We pretreated MDM with the
PI3K inhibitor wortmannin, a fungal metabolite, before the addition of APP SP-A (Table I). Pretreatment with wortmannin resulted in a significantly decreased Ca2⫹ response (Table I). However, wortmannin did not completely inhibit Ca2⫹ mobilization in
response to APP SP-A, suggesting that activation of PI3K plays a
role in but may not be essential for Ca2⫹ mobilization.
The Journal of Immunology
2233
Table II. Determination of signaling molecules involved in upregulation of human macrophage MR expression by SP-Aa
Treatmentb
Percentage of Increase in
MFI (Mean ⫾ SEM)c
n
APP SP-A
IL-4
Wortmannin (10 nM)
Wortmannin (100 nM)
Ly294002 (9 ␮M)
Ly294002 (3 ␮M)
Ly294002 (1.5 ␮M)
Ly294002 (0.75 ␮M)
Thapsigargin (10 ␮M)
U73122 (5 ␮M)
U73343 (5 ␮M)
BAPTA (0.5 ␮M)
BAPTA (1 ␮M)
BAPTA (5 ␮M)
102 ⫾ 21ⴱ
253 ⫾ 96ⴱ
75 ⫾ 19ⴱ
56 ⫾ 31
22 ⫾ 12
65 ⫾ 28
77 ⫾ 18ⴱ
83 ⫾ 28ⴱ
95 ⫾ 38ⴱ
88 ⫾ 30ⴱ
94 ⫾ 22ⴱ
61 ⫾ 11
23 ⫾ 20
⫺28 ⫾ 12
8
10
2
2
2
3
3
3
4
3
3
2
2
2
a
MR MFI measured using flow cytometry.
MDM were incubated with SP-A proteins (10 ␮g/ml) for 2 h in all but the IL-4
group. IL-4 added for 20 h was used as the positive control. Inhibitors were added 30
min before APP SP-A treatment.
c
Values shown represent the % increase in MFI ⫽ (treated MFI-control MFI) ⫼
control MFI ⫻ 100. Values are corrected for nonspecific binding by subtracting the
MFI from the appropriate nonspecific control (IgG1␬). Mean ⫾ SEM; n ⫽ 6.
ⴱ, Significant relative to MR expression on untreated MDM by Student’s t test,
p ⬍ 0.05.
b
FIGURE 8. InsP3 formation in response to SP-A is time and concentration dependent. MDM (1 ⫻ 105) were incubated with 20 ␮g/ml SP-A for
the indicated time periods (A) or the indicated concentrations of APP SP-A
for 15 s (B). Incubation was stopped, and cells were lifted and lysed by
adding ice-cold 20% perchloric acid for 20 min. After centrifugation at 4°C
for 15 min at 2000 ⫻ g, supernatants were removed and neutralized with
ice-cold 1.5 M KOH containing 60 mM HEPES. The resultant KClO4 was
precipitated and InsP3 in supernatant samples determined using a specific
protein binding assay. Results are expressed as mean ⫾ SEM of two independent experiments; ⴱ, p ⬍ 0.05.
PI3K plays a role in SP-A-induced up-regulation of MR
expression
The interaction of SP-A with the macrophage results in modification of macrophage functions, including, but not limited to, upregulation of MR expression (1, 7, 8). To determine whether SP-A
signaling through the InsP3/Ca2⫹ pathway results in up-regulation
of MR, we used inhibitors against the previously identified signaling molecules activated by SP-A and measured MR expression
using flow cytometry. As previously reported (8), APP SP-A or
IL-4 significantly increased MR expression on MDM (Table II).
The addition of the general PI3K inhibitors wortmannin or
Ly294002 before SP-A treatment significantly reduced the SP-Ainduced up-regulation of MR in a dose-dependent manner. Wortmannin did not reduce basal MR expression levels (data not
shown), nor did it significantly inhibit SP-A binding to MDM at
concentrations that inhibited MR up-regulation by SP-A (100 nM)
compared with concentrations that had no effect (10 nM; data not
shown). Additionally, Ly294002 treatment of MDM had no effect
on binding of SP-A to MDM (data not shown), indicating that
inhibition of PI3K(s) has no effect on SP-AR expression. There-
fore, a PI3K(s) plays an important role in the SP-A-induced signaling pathway that leads to MR up-regulation. The role of PLClinked Ca2⫹ mobilization in SP-A-induced MR up-regulation is
less clear. Inhibition of PLC by U73122 did not significantly affect
SP-A induction of MR. Furthermore, while preincubation with the
Ca2⫹ inhibitor BAPTA reduced SP-A-induced MR expression, inhibition of calcium mobilization by thapsigargin did not significantly affect SP-A induction of MR. However, it should be noted
that 5 ␮M BAPTA tended ( p ⫽ 0.07) to reduce the basal level of
MR surface expression on MDM, suggesting that Ca2⫹ is involved
in the normal trafficking of this receptor.
Discussion
SP-A is increasingly recognized as a key component of innate
immunity in the lung. SP-A plays a critical role in host defense
against inhaled pathogens, as demonstrated by the increased susceptibility of the SP-A⫺/⫺ mouse to infection with a variety of
extracellular bacteria (33–35). SP-A has been shown to have direct
antimicrobial activity against both bacterial (36) and fungal (37)
microorganisms. However, it is also evident that part of the role of
SP-A in pulmonary immunity is due to the ability of SP-A to
modify alveolar macrophage immunological function (2). Addition of SP-A to macrophages in vitro increases chemotaxis and
phagocytosis, decreases NO production, and alters the respiratory
burst (reviewed in Ref. 1). Recently, our laboratory has demonstrated that SP-A increases the surface expression and activity of
the macrophage MR (8) and that this up-regulation plays a role in
the observed increase in Mycobacterium tuberculosis phagocytosis
by SP-A-treated cells (7).
SP-A has been shown to associate with macrophages in a saturable and specific manner, consistent with receptor-mediated
binding (38). Although several SP-A binding proteins have been
identified on rat macrophages, myeloid cell lines, and in purified
protein systems (10), the receptor(s) responsible for the effects of
SP-A on MR expression and activity have not yet been determined.
Interactions between SP-A and its macrophage receptor(s) activate
signaling pathway(s) that ultimately effect the reported alterations
2234
SP-A ACTIVATES PI3K/Ca2⫹ SIGNALING IN HUMAN MACROPHAGES
in macrophage functions (12, 39 – 43). Our laboratory is particularly interested in the identification of signaling pathway(s) leading
to up-regulation of the MR. The results presented here indicate that
at least one signal transduction pathway activated in human macrophages by SP-A involves Ca2⫹ signaling and that PI3K contributes to the SP-A-induced increase in MR expression.
Ca2⫹ is an important second messenger in phagocytic cells and has
been linked to many cellular processes, including vesicle trafficking
(reviewed in Ref. 44). [Ca2⫹]cyt in resting cells is low; upon stimulation, [Ca2⫹]cyt increases 5- to 10-fold, activating Ca2⫹-sensitive
processes. Our data demonstrate that SP-A significantly increases
[Ca2⫹]cyt within seconds of SP-A addition to cultured MDM. The
immediate increase in [Ca2⫹]cyt in response to APP SP-A is similar to
that reported by Ohmer-Schröck et al. (12) using rat alveolar macrophages. However, there may be species differences in threshold responsiveness to SP-A: we found that 10 ␮g/ml APP SP-A induced
mobilization of calcium in 100% of MDM. In contrast, OhmerSchröck et al. (12) reported 30 ␮g/ml APP SP-A stimulated Ca2⫹
mobilization in 36% of rat alveolar macrophages, and 120 ␮g/ml APP
SP-A achieved mobilization in 69% of these cells (12).
The effects of SP-A on [Ca2⫹]cyt were observed in our system at
concentrations as low as 1 ␮g/ml. Although the precise SP-A concentration in pulmonary surfactant is not known, the concentration
of SP-A in rat lung hypophase has been estimated at 300 ␮g/ml to
1.8 mg/ml (reviewed in Ref. 1) based on SP-A levels in lavage
fluid. However, this does not account for the possibility that microenvironments could exist where the local concentrations of
SP-A are much higher or lower (1). It is important to note that
SP-A activity may also be affected by its association with lipid,
as SP-A has been shown to be concentrated in tubular myelin in
the human lung (45).
In our studies, after the maximum [Ca2⫹]cyt was reached, the
[Ca2⫹]cyt declined over the following 10 min until reaching a
steady-state level slightly above baseline. Interestingly, when these
macrophages were stimulated with a second dose of SP-A (lesser
or equal concentration) within 10 min of the first, no second rise in
[Ca2⫹]cyt was detected. When 2- or 4-fold greater APP SP-A was
used as the secondary stimulus following an initial SP-A treatment,
the [Ca2⫹]cyt was increased but never achieved the level reached in
response to the first stimulus. However, SP-A-treated MDM were
still able to fully respond to the second stimulus PAF. This suggests that the inhibition is not due to depletion of available Ca2⫹
but that SP-A facilitates an agonist-specific refractory period,
which is consistent with the hypothesis that SP-A induces homologous desensitization of its receptor(s) on the macrophage surface.
Desensitization facilitates decreased responsiveness of a cell to
successive exposure of the same extracellular stimulus over time.
We envision that the functional state of the SP-AR(s) is under
continuous regulation in response to changes in SP-A levels in the
lung, such as has been reported in bacterial pneumonia (46). Additionally, the degree of SP-AR desensitization is likely influenced
by the differentiation and/or activation state of the cell. In the alveoli, alveolar macrophages are in continuous contact with surfactant containing SP-A and ingest abundant amounts of this material
(47); therefore, it is possible that resident alveolar macrophages are
in a constant state of desensitization to SP-A. In other words,
maintenance of the alevolar macrophage phenotype may require
constant (tonic) ligation and desensitization of SP-AR(s).
In contrast, monocytes or interstitial macrophages that migrate
into the lung would be sensitive to the effects of SP-A upon first
encounter. In this model, SP-A would serve as a local tissue factor
that contributes to the induction and maintenance of the unique
biological properties of alveolar macrophages compared with macrophages in other tissue compartments. These include increased
surface expression of receptors of innate immunity such as the MR
(9), greater phagocytosis of both nonopsonized and opsonized particles (48), decreased production of inflammatory cytokines (48), a
reduced oxidative burst in response to stimuli (6, 49), and decreased expression of costimulatory molecules (50)—an antiinflammatory phenotype consistent with a state referred to as “alternative activation” (51). Homologous receptor desensitization is
dependent on agonist occupancy of the receptor; consequently, under conditions in which SP-A concentration is significantly decreased, the alveolar macrophages would become resensitized to
SP-A. This would explain why alveolar macrophages are capable
of responding to SP-A in vitro (7, 12).
In the current study, the threshold level of SP-A needed to induce a Ca2⫹ response and the magnitude of this response were
both dependent on the source of SP-A used. Although human APP,
native human and rat, and SP-Ahyp demonstrate shared functional
characteristics, differences in the magnitude of select biological
responses related to SP-A type have been observed previously (52,
53). Our laboratory has reported previously that APP SP-A was
more effective than either native human or SP-Ahyp in up-regulating MR receptor expression and enhancing the phagocytosis of M.
tuberculosis by MDM (7, 8).
One notable difference among the SP-As used is the degree of
higher-order molecular organization (54); our results suggest that
these differences are related to the ability of SP-A to induce Ca2⫹
mobilization. APP SP-A, which can form complexes larger than
the native SP-A octadecamer (52, 55), demonstrated the greatest
amount of Ca2⫹ mobilization. In contrast, SP-Ahyp, which oligomerizes to a lesser extent than native SP-A due to a lack of
proline hydroxylation, demonstrated the lowest amount of Ca2⫹
mobilization. Additionally, mutants that lacked the collagen-like
domain and/or the N-terminal region, both of which are believed to
be essential for forming higher order multimers (3), were capable
of stimulating only a slight Ca2⫹ response. These data suggest that
higher order oligomers are essential for maximally effective receptor engagement, which may involve recognition of a multiunit
epitope, and/or receptor clustering by ligation of multiple CRD. It
should also be noted that, in addition to differences in multimerization, human APP, native human and rat, and SP-Ahyp also demonstrate differences in glycosylation patterns (3), which may play
a role in the current results.
To further investigate the role of individual structural components of SP-A on Ca2⫹ mobilization, we used SP-Ahyp variants
lacking part or all of the collagen-like domain or one or both attached carbohydrates. Wild-type SP-Ahyp contains two sugars, one
at Asn1 in the N-terminal segment and one at Asn187 in the CRD
(3). Removal of one or both sugars significantly decreased the
SP-A-induced Ca2⫹ burst, which is consistent with studies showing that these carbohydrate moieties are important for some of its
biological effects (7, 56). When both sugars remained intact but the
collagen-like domain was partially or completely removed, the SPA-induced calcium response was also diminished. This is supported by work by Chroneos et al. (10), which showed that collagen V inhibits SP-A binding to macrophages, as well as work by
Vandivier et al. (57), which showed that C1q collagen tails inhibit
macrophage uptake of SP-A-coated erythrocytes. It is not yet clear
whether the collagen-like domain participates in direct proteinprotein interactions that result in a calcium flux or whether it acts
primarily as scaffolding that amplifies the binding activities of
other domains of the molecule. None of the mutations completely
eliminated the Ca2⫹ response, and inhibition of the Ca2⫹ response
by these mutations was not additive because the removal of both
sugars and the collagen-like domain or of the N-terminal and collagen-like domains did not further diminish the Ca2⫹ response. It
The Journal of Immunology
is unclear whether the small remaining Ca2⫹ response is physiologically relevant. Viewed as a whole, our studies demonstrate that
both the protein-associated carbohydrates and collagen-like domain of SP-A play a role in SP-A-induced Ca2⫹ mobilization.
The possibility exists that there is more than one receptor for SP-A
on macrophages, each one of which induces a different signal pathway
and biological effect. Several proteins have been identified as putative
receptors for SP-A, not all of which are present on human macrophages (38, 58). One SP-A-binding protein reported to be on U-937
cells and rat alveolar macrophages is the 210-kDa protein SP-R210
(10). Our assays failed to reveal a role for this receptor in the Ca2⫹
response or up-regulation of the MR because Abs against SP-R210
failed to inhibit SP-A-induced increases in [Ca2⫹]cyt or MR expression (data not shown). U937 cells also required 10 times more APP
SP-A for the induction of calcium than did human MDM, suggesting
that the SP-AR(s) on U937 cells that signals through Ca2⫹ is expressed at an extremely low level compared with that on primary
human macrophages, has a lower affinity for SP-A, or lacks expression of a coreceptor (39, 40, 59).
The calcium mobilized into the cytoplasm in response to stimulation originates from either the extracellular space or the ER
(14). One standard approach to examine whether extracellular
Ca2⫹ plays a role in induced rises in [Ca2⫹]cyt is to chelate extracellular Ca2⫹ using EGTA. However, this approach cannot be used
in our system to study the contribution of extracellular Ca2⫹ to
signaling by SP-A, as we have recently shown that binding of
SP-A to its receptor(s) on primary human macrophages is Ca2⫹
dependent.4 We have shown that depletion of ER Ca2⫹ stores by
preincubation with thapsigargin completely blocks the SP-A-induced [Ca2⫹]cyt rise, indicating that the initial calcium response is
mobilized from intracellular stores. We also found that SP-A induces a rise in InsP3, an important molecule involved in release of
Ca2⫹ from ER stores (14). Based on these observations, we have
concluded that SP-A-induced Ca2⫹ mobilization at least initially
originates from intracellular stores. The level of [Ca2⫹]cyt above
baseline seen as late as 20 min after SP-A addition may be due to
an influx of extracellular Ca2⫹ through capacitative calcium entry,
which is controlled by the efflux of calcium from the ER. Recent
evidence supports InsP3R involvement in the initiation of this entry process (60). Therefore, we propose that the SP-A-induced rise
in [Ca2⫹]cyt is composed of two phases: an initial spike in
[Ca2⫹]cyt through InsP3-induced release of Ca2⫹ from specific intracellular stores, followed by a plateau phase (essential for replenishment of the intracellular stores) that is sustained by Ca2⫹
influx from the extracellular medium.
Our data further demonstrate that SP-A induction of Ca2⫹ mobilization is mediated through activation of PLC. PLC not only
catalyzes the formation of InsP3 from phosphatidylinositol 4,5bisphosphate but also controls cellular phosphatidylinositol 4,5bisphosphate concentrations, thereby intersecting with other signal
transduction pathways such as that of PI3K. Pretreatment with
wortmannin before stimulation with SP-A resulted in a markedly
decreased calcium response, suggesting that PI3K is an upstream
signaling molecule involved in Ca2⫹ mobilization by SP-A.
Taken together, these studies demonstrate that SP-A activates a
Ca2⫹/PLC/InsP3 signal transduction pathway. We next investigated
whether signaling through this pathway by SP-A results in up-regulation of MR expression. This hypothesis is supported by inhibition of
MR up-regulation by the calcium chelator BAPTA. However,
BAPTA may also chelate local concentrations of calcium or other
cations needed at the membrane for vesicular fusion, thereby inhibiting processes in addition to calcium release that are necessary for
MR up-regulation. The role of SP-A signaling through PLC in MR
up-regulation is less clear because inhibitors of PLC did not signifi-
2235
cantly reduce the effect of SP-A on MR expression. However, inhibitors of PLC decreased but did not completely abolish the calcium
response to SP-A; the residual low-level calcium response may have
been sufficient to induce MR up-regulation in these cells. Alternatively, redundancy in signaling is common in immunological systems,
and inhibition of PLC may therefore cause a compensatory increase in
the activity of other contributing enzymes or pathways, which may
also account for our results.
Using the inhibitors wortmannin and Ly294002, we have shown
that PI3K are involved in both SP-A-induced mobilization of Ca2⫹
and in SP-A up-regulation of MR. PI3K, as with calcium, is involved in intracellular trafficking and acts as a regulator of multiple
aspects of membrane trafficking (61– 63). A PI3K may be the common molecule involved in two separate signaling cascades— one
resulting in MR up-regulation and the other involving the Ca2⫹/
PLC/InsP3 pathway; or, more likely, a different member of the
PI3K family is involved in each event. Interestingly, the lipid products of PI3K have been found to directly activate certain protein
kinase C (PKC) isoforms. If PKC plays a role in MR up-regulation, it would provide an additional explanation for our data using
BAPTA because treatment of cells with BAPTA-AM leads to a
translocation and inactivation of PKC (64).
In conclusion, SP-A binding to human macrophages activates a
signal transduction pathway(s) that results in increased levels of
[Ca2⫹]cyt. The magnitude of this increase is dependent on 1) the
type of SP-A used and 2) the sugars and collagen-like domain of
the protein. Our experiments make the novel observation that macrophages become refractory to SP-A after initial exposure, suggesting that the receptor(s) for SP-A undergoes homologous desensitization. This finding may be particularly important for setting
the anti-inflammatory “tone” of macrophages within the lung. Our
data indicate that SP-A triggers the increase in calcium by inducing PLC activation, leading to the hydrolysis of membrane phospholipids and yielding InsP3. We have shown that activation of
PI3K(s) plays a role in SP-A up-regulation of MR and may act as
a component of the PLC signal pathway. These studies provide
further insight into the signaling pathways activated in human
macrophages by SP-A which play direct roles in SP-A modulation
of macrophage biology.
Acknowledgments
We thank Thomas Kaufman, Jennifer Ufnar, Amanda Dawson, and
Thomas Moninger for their expert technical assistance and
Deb Nollen-Richter for help with the preparation of this manuscript.
Disclosures
The authors have no financial conflict of interest.
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