(Received for publication, October 24, 1996, and in revised form, January 17, 1997)
From the Cultured human umbilical vein endothelial cells
inhibited tumor necrosis factor- Tumor necrosis factor- The first objective of this study was to examine whether endothelial
cells can influence the production of TNF from monocytes. For
comparative purposes, elastase release was also measured as a marker of
neutrophil activation in the same whole blood system.
The second objective was to identify the mediators of such effects. In
this report we describe the purification of an active species from the
conditioned media of human umbilical vein endothelial cells (HUVEC) and
its unexpected identification as calmodulin. Furthermore, we provide
preliminary evidence that there are cell-surface receptors for
calmodulin on myeloid cells. The results imply that calmodulin released
from endothelial cells serves the function of an extracellular
signaling molecule, which regulates the activation of inflammatory
cells.
Monoclonal antibodies were raised
against recombinant human TNF- HUVEC were cultured in medium 199 (Mediatech) containing 15% v/v heat-inactivated bovine calf serum,
0.5% v/v endothelial cell growth supplement (prepared as described by
Maciag et al. (16)), 10 µg/ml heparin (Sigma), 2 mM L-glutamine, 50 IU/ml penicillin, and 50 µg/ml streptomycin (Mediatech) as described (17, 18) and used at
first through fourth passage.
For the production of conditioned media, 2 m2 of
endothelial cells were grown to confluence in cell culture factories
(Nunc, Denmark). After washing with Hanks' balanced salt solution
without phenol red (HBSS composition (in mM):
CaCl2, 1.26; KCl, 5.36; KH2PO4,
0.44; MgCl2, 0.49; NaCl, 136.9; NaHCO3, 4.17;
Na2HPO4, 3.38; glucose, 5.56) (Life
Technologies, Inc.), they were incubated in 1.5 liters of modified
Eagle's medium without phenol red (Mediatech) with the calcium
ionophore A23187 (Sigma) at 3 × 10 24-Well culture plates
(Costar) were coated with 2% w/v gelatin (Sigma). For experiments
involving endothelial cell monolayers, HUVEC were seeded onto
gelatin-coated wells at 3-5 × 105 cells per well
48 h prior to experimentation. All wells were washed with HBSS.
Blood from healthy volunteers was drawn within 5 min of use into
heparin (10 units/ml final; UpJohn, Kalamazoo, MI) or, where specified,
hirudin (10 units/ml; Sigma) and 200 µl added per well. To minimize
evaporation, only the center eight wells were used, and the outer wells
were filled with sterile HBSS. Test reagents dissolved in HBSS (8-10
µl) were added to the wells immediately after the blood; calmodulin
was diluted in HBSS supplemented with 0.1% sterile pyrogen-free
gelatin (Sigma), which served as its buffer control. In most
experiments, LPS (from Escherichia coli strain O55:B5,
Difco) was used to stimulate TNF release. Plates were incubated at
37 °C in room air, 5% CO2 on an orbital shaker at 150 rpm, for 4 h unless otherwise noted. Plasma was separated by
centrifugation at 500 × g for 10 min and assayed for
TNF and elastase.
Human
peripheral blood mononuclear cells (PBMC) and neutrophils were prepared
from freshly drawn heparinized blood by density gradient separation on
Mono-Poly Resolving Medium (Flow Laboratories, McLean, VA) (19). Cells
were washed three times with RPMI 1640 (Mediatech) and resuspended at a
concentration of 2.5-9 × 106 cells/ml in RPMI 1640 plus 10% autologous heparinized plasma or in 100% plasma. 200-µl
aliquots were added to 24-well tissue culture plates, incubated, and
assayed as described for whole blood.
The human monocytic and myelomonocytic cell lines, THP-1, HL-60,
MonoMac-6, and U937 (American Type Culture Collection, Rockville, MD)
were cultured in RPMI 1640 supplemented with 10% v/v fetal bovine
serum (Hyclone), 2 mM L-glutamine, 50 IU/ml
penicillin, and 50 µg/ml streptomycin (Mediatech). Cells were washed,
resuspended, and used as described for PBMC.
To guide the purification of an active factor,
chromatographic fractions of HUVEC-conditioned media were assayed for
the ability to inhibit LPS-induced TNF release by their addition in a
0.1 volume to the whole blood just prior to stimulation with LPS. In
some cases, the fractions were first desalted into TBS on a PD-10 gel
filtration column (Pharmacia Biotech Inc.). Active fractions were
subjected to further isolation procedures until an essentially pure
protein (as judged by SDS-PAGE) was obtained. The purification will be
described in brief since the active fraction was shown to be
calmodulin. The irrelevant proteins were precipitated from the
concentrated conditioned media by acidification to pH 5 and addition of
12% polyethylene glycol followed by ultracentrifugation. The
supernatant was separated by anion exchange (Mono Q HR10/10, Pharmacia), gel filtration (1.5 × 100 cm Sephacryl S-100HR,
Pharmacia), and chromatofocusing (Mono P HR 5/20, Pharmacia). To
further concentrate the material purified as above, and to remove the
chromatofocusing buffer (Polybuffer, Pharmacia) to prepare the specimen
for sequencing, the active fractions were applied to a reverse-phase
microbore HPLC column (PLRP-S, 1 × 50 mm, Michrom BioResources,
Pleasanton, CA) and eluted with acetonitrile, yielding a single major
peak.
Samples to be evaluated for calcium-dependent binding to
phenyl groups were loaded onto a phenyl-Superose column (HR 5/5, Pharmacia) and washed with 0.1 mM CaCl2, 0.5 M NaCl, 0.02 M Tris-HCl, pH 7.5, and then with
0.1 mM CaCl2, 0.02 M Tris-HCl, pH
7.5. The column was eluted with 1 mM EDTA, 0.02 M Tris-HCl, pH 7.5.
To determine heat stability, the purified endothelial factor and
calmodulin samples were heated to 90 °C for 5 min, immediately chilled on ice, and then assayed for activity.
Peak fractions from the HPLC were partially
evaporated in a SpeedVac (Savant Instruments Inc., Farmingdale, NY) to
remove acetonitrile. Approximately 15 µg of material was subjected to enzymatic digestion with trypsin according to the method of Stone et al. (20). The tryptic fragments were separated by HPLC on a 1 × 50 mm microbore C18 column (Michrom BioResources) and
eluted using a linear gradient of acetonitrile to 50%. The eluate from the column was monitored by absorption at 215 nm and by in-line mass
spectrometry (AP III LC/MS/MS system, Sciex, Thornhill, Ontario, Canada).
Amino acid sequence analyses were performed using automated Edman
degradation with a model 470A gas-phase protein sequencer equipped with
a model 120A on-line phenylthiohydantoin amino acid analyzer (Applied
Biosystems, Inc., Foster City, CA) (21).
Protein samples and chromatography fractions were analyzed on 12%
SDS-PAGE gels according to the method of Laemmli (22). Gels were
stained with a Bio-Rad silver stain kit (Bio-Rad).
Calmodulin-dependent phosphodiesterase was
used to assay calmodulin function as described by Wallace et
al. (23). Calmodulin was also measured by a radioimmunoassay kit
(Calmodulin RIAgents, Amersham Corp.) according to the manufacturer's
instructions, using the supplied unheated calmodulin preparation for
the standard. Samples and supplied standard were heated at 90 °C for
1 h to enhance reactivity with the antibodies.
Bovine testis calmodulin was prepared according to the method of Dedman
and Kaetzel (24). Protein concentration of purified calmodulin samples
was estimated, using an extinction coefficient at 280 nm of 0.18 ml
mg Recombinant calmodulin was prepared and expressed in E. coli
by inserting the human calmodulin I gene into the
pIN-III-pelB-Neo vector, a modification of the
pIN-III-pelB plasmid (26) which includes a tandem
neor gene. The construct contains an N-terminal
extension, EFEDQVDPRLIDGKIEGR, corresponding to the epitope for
monoclonal antibody HPC-4 (27). Otherwise, the recombinant protein
differs from mammalian calmodulin only in that it is expected to lack
the post-translational trimethylation of lysine 115. The protein was
isolated from periplasmic extracts by calcium-dependent
affinity chromatography on phenyl-Sepharose followed by HPC-4 affinity
chromatography (26).
Bovine testis calmodulin was radiolabeled with iodine-125
using Enzymobeads (Bio-Rad) according to the manufacturer's
instructions. Free iodine and any denatured calmodulin were removed by
calcium-dependent affinity purification on a 1-ml stop-flow
phenyl-Sepharose column equilibrated in 0.1 M NaCl, 0.02 M MOPS, 0.1 mM CaCl2, pH 7.5. After
washing with 5 ml of the same buffer, the 125I-calmodulin
was eluted with 0.1 M NaCl, 0.02 M MOPS, 1 mM EDTA.
Unless otherwise specified, cell binding studies were done with all
steps, including washing of the cells, performed at 4 °C. Cells were
washed once in HBSS without Ca2+ or Mg2+
containing 20 mM HEPES, pH 7.5, supplemented with 1% human
serum albumin and 1 mM EDTA (HHA/EDTA), and twice in HBSS
with Ca2+ and Mg2+ containing 20 mM
HEPES, pH 7.5, and 1% albumin (HHA/Ca2+). The cells were
then resuspended in HHA/Ca2+ at a concentration of
1.25 × 108 cells/ml. 80 µl (1 × 107 cells) were aliquoted into 1.5 ml of siliconized
Eppendorf tubes (PGC Scientific, Gaithersburg, MD) and 10 µl of
HHA/Ca2+ with varying concentrations of unlabeled
calmodulin (from 10 To show whether bound calmodulin was internalized, binding studies were
performed at 4 and 37 °C, as described above except that after the
incubation period was complete, the cells were washed in HHA/EDTA and
resuspended in HHA/EDTA before layering over dibutyl phthalate/apiezon
oil.
To determine if calmodulin-binding sites were sensitive to proteolytic
digestion, HL-60 cells were washed once in HBSS with 20 mM
HEPES, pH 7.5, and 1 mM EDTA, without albumin (HH/EDTA) and
twice with HH/Ca2+ and resuspended in HH/Ca2+at
107 cells per ml. L-1-Tosylamido-2-phenylethyl
chloromethyl ketone-treated trypsin (Sigma) was then added to a final
41 units/ml and allowed to incubate at 37 °C for 30 min. The trypsin
was then neutralized by the addition of diisopropyl fluorophosphate to
2 mM final. The cells were then washed once in
HH/Ca2+ and resuspended in HHA/Ca2+and binding
studies performed as described.
Cells were washed once in HH/EDTA, twice in
HH/Ca2+, and then resuspended in HH/Ca2+ at a
concentration of 1.25 × 107 cells/ml. 800 µl were
aliquoted into 1.5-ml siliconized Eppendorf tubes (PGC Scientific) and
100 µl of HH/Ca2+ or HH/EDTA with 20 mM EDTA
(2 mM final) added. Radiolabeled calmodulin in 100 µl of
HH/Ca2+ was then added to a final concentration of
10 Statistical comparisons were by paired
t test. Data are presented as mean ± S.E.
Incubation of whole blood with LPS for 4 h evoked a
dose-dependent release of TNF (Fig. 1). In
control wells not treated with LPS, TNF levels were usually below the
limit of detection by our ELISA. When blood treated with LPS was
incubated over an endothelial monolayer for 4 h, TNF release was
reduced by about half at all concentrations of LPS (Fig. 1). Donors
varied in the extent of inhibition by endothelial cells, and in 8 of 70 total experiments, no inhibition of TNF release was observed.
In contrast to the effect on TNF, blood incubated over a HUVEC
monolayer exhibited increased elastase release at all concentrations of
LPS (Fig. 2). In some subjects (8 of 35 total
experiments), elastase release was not augmented by endothelium; five
subjects appeared to show inhibition.
One contribution to these cellular responses might be the release of
active peptides from the endothelium. To test this possibility, conditioned media from endothelial cells were assayed for TNF inhibiting activity. After concentrating 66-fold by ultrafiltration, conditioned media (collected without A23187 stimulation) were able to
inhibit TNF release by 43% (n = 2). Empirically, it
was observed that stimulation of the endothelial cells with the calcium ionophore A23187 yielded increased inhibitory activity in the conditioned media, and therefore, purification was attempted from ionophore conditioned media. The purification was performed as detailed
under "Materials and Methods." At each chromatographic step a
single peak of activity was identified. The activity eluted from a Mono
Q anion exchange column at 0.35 M NaCl at pH 7.5. The
isoelectric point predicted by chromatofocusing was pH 3.4. After
chromatofocusing, the sample was essentially pure as judged by 12%
SDS-PAGE. Silver staining demonstrated a single negative staining
(ghost) band with an apparent mass of ~21 kDa (Fig.
3).
No N-terminal sequence was generated from the purified protein,
suggesting that the N terminus was blocked. To obtain internal sequence
information, a trypsin digest was performed and the resulting fragments
separated by HPLC. The eluted fragments were analyzed in-line with
electro-spray mass spectrometry. Sequence was obtained from two
fractions; one (eluate peak 3) contained clearly distinguishable major (VFDKDGNGYISAAELR) and minor (MKDTDSEEEIREAFR) sequences, and the
other (eluate peak 4) contained a faint sequence
(XXMTNLXEXLTDXXXDXXXX). In total 40 amino acid residues were identified in the three
fragments. This sequence information was compared with known sequences
in the Swiss-Prot data base using the GCG software package (Version 7;
Genetics Computer Group, Madison, WI), which revealed complete identity
with three contiguous sequences of calmodulin, corresponding to amino
acids 76-126. The sequences of the three fragments and their measured
masses (as well as the masses of the other HPLC eluate peaks, which
were not sequenced) corresponded to known tryptic fragments of
calmodulin (28-30) whose masses were predicted by the GCG software
program (Table I).
Table I.
Tryptic fragments of endothelial factor in comparison with
calmodulin
Department of Internal Medicine and Manitoba
Institute of Cell Biology, University of Manitoba, Winnipeg,
Manitoba R3E 0V9, Canada and the ¶ Cardiovascular Biology
Program,
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
release from whole blood or
isolated mononuclear cells exposed to endotoxin. In contrast, the
endothelial cells augmented neutrophil elastase release in the same
blood. A protein with these functional properties was isolated from
endothelial cell-conditioned media and, surprisingly, was identified as
calmodulin. Authentic calmodulin mimicked the effect of endothelium.
125I-Calmodulin bound to a high affinity site on
monocytic cell lines (Kd ~30 nM, in
agreement with its functional activity). Cross-linking of
125I-calmodulin to monocytic cells identified a candidate
calmodulin receptor. We conclude that calmodulin possesses an
extracellular signaling role in addition to its intracellular
regulatory functions. Calmodulin released at sites of tissue injury or
possibly by specific mechanisms in the endothelium can bind to
receptors, modulating the activities of inflammatory cells.
(TNF),1 a
product chiefly of monocytes and their descendants, the tissue
macrophages, is a multifunctional cytokine that enhances host immune
responses but that is also implicated in diverse pathological processes
such as septic shock, rheumatoid arthritis, tumor cachexia, multiple
sclerosis, and graft-versus-host disease (1). A number of
inflammatory stimuli can evoke its release from monocytes and
macrophages, such as bacterial endotoxin (lipopolysaccharide; LPS),
interferon-
, and IL-1. Although several substances have been shown
to inhibit the production of TNF in vitro, including
adenosine (2), epinephrine (3), PGE2 (4), PGI2
(5), transforming growth factor-
(6, 7), IL-4 (8), IL-6 (9), IL-10
(10-12), and IL-13 (13, 14), the physiological circumstances under
which monocytes would be exposed to these mediators remain undefined.
The possibility that the endothelium, through these or other mediators,
may control the activation of TNF-producing cells in blood has not been
addressed directly.
TNF and Elastase ELISAs
(a generous gift of Genentech, South
San Francisco, CA) and a complex of human neutrophil elastase and human
1-antitrypsin (Athens Research and Technology, Athens,
GA) by standard techniques. (15) Polyclonal antibody to TNF was raised
in a goat. TNF was assayed in plasma diluted 1:10 using mAb TNF1286 for
capture and the goat polyclonal for detection or with mAb TNF1311 for
capture and TNF1289 for detection. The lower limit of sensitivity for this assay was ~0.5 ng/ml. Elastase-
1-antitrypsin
complexes were assayed in plasma diluted 1:50 using anti-elastase mAb
HEL1076 for capture and anti-antitrypsin mAb HAT1099 for detection.
Antibodies used for detection were biotinylated using NHS-LC-biotin
(Pierce), and streptavidin-alkaline phosphatase and an amplified
substrate system (Life Technologies, Inc.) were used for readout.
6 M.
After 4 h the conditioned media were collected and the cells placed in an additional 2.5 liters of medium without ionophore overnight. The conditioned media were pooled and concentrated to 150 ml
by ultrafiltration with a 3,000 molecular weight cut-off membrane
cartridge (S1Y3, Amicon, Beverly, MA).
1 cm
1 (25). Calmodulin derived from human
erythrocytes (Sigma) or hog brain (Boehringer Mannheim) was also
obtained and used in some experiments. It should be noted that the
peptide sequences of the calmodulins of these species are
identical.
9 to 2 × 10
5
M final) added. Immediately thereafter, 10 µl of
125I-calmodulin was added to a final concentration of
5 × 10
9 M and allowed to incubate for
40 min at 4 °C with intermittent mixing. After the incubation period
the cells were carefully layered over 9:1 dibutyl phthalate:apiezon oil
(J. T. Baker Inc. and Apiezon Products Ltd., London, UK) and
centrifuged at 200 × g for 2 min in siliconized
400-µl centrifuge tubes. The tip of the centrifuge tube containing
the cell pellet was amputated and counted in a gamma counter (Iso-Data,
Rolling Meadows, IL) for the determination of bound
125I-calmodulin. To determine nonspecific binding, 10 µl
HHA/EDTA containing 20 mM EDTA (2 mM final) was
added instead of unlabeled calmodulin. Nonspecific binding determined
in the presence of 2 × 10
5 M cold
calmodulin gave similar values. The Ligand computer software program
(Elsevier-BIOSOFT, Cambridge, UK) was used to estimate the
Kd and the number of binding sites per cell.
7 M and allowed to bind for 40 min. The
cross-linking reagent, bis(sulfosuccinimidyl) suberate (Pierce),
dissolved in Me2SO was then added in a final concentration
of 1 mM, and cross-linking was allowed to proceed for
1 h at 4 °C. The unbound and uncross-linked 125I-calmodulin was then removed by washing twice in
HH/EDTA, and the cell pellet was extracted in buffer consisting of 0.15 M NaCl, 0.02 M Tris-HCl, pH 7.5, 1% Triton
X-100, 0.1 mM phenylmethylsulfonyl fluoride, 0.1 mM pepstatin, 0.1 mM leupeptin, 1 mM EDTA, 5 mM benzamidine, 0.02% sodium azide
overnight at 4 °C with rocking. The next morning the
Triton-insoluble material was removed by centrifugation at 10,000 × g. The supernatant, containing the solubilized membrane
proteins, was added to Laemmli sample buffer, electrophoresed, dried,
and autoradiagraphed on X-OMAT AR film (Kodak).
Fig. 1.
Inhibition of LPS-induced TNF release in
whole blood by endothelium. Each point represents the mean
response of blood from 10 subjects, each subject's blood having been
incubated on endothelial cells from three different cords. Statistical
comparison shown is by t test on data for the 100 ng/ml dose
of LPS. Identical results are obtained if the analysis is performed on
the data for the 1- and 10-ng/ml doses.
[View Larger Version of this Image (15K GIF file)]
Fig. 2.
Augmentation of LPS-induced elastase release
in whole blood by endothelium. Elastase was measured as complexes
with 1-antitrypsin (
1-AT).
Mean responses from eight subjects are shown. The comparison between
groups with and groups without endothelium is by t test on
data for the 100 ng/ml dose of LPS.
[View Larger Version of this Image (17K GIF file)]
Fig. 3.
Silver-stained 12% SDS-PAGE gel of the
active fraction from the Mono P chromatofocusing column. The
purified protein appears as a negatively staining Ghost
band.
[View Larger Version of this Image (33K GIF file)]
Endothelial
factor
Calmodulin
Peak no.
Measured
mass
Residues
Expected mass
daltons
daltons
1
804.3
31 -37
805.4
2
1984.3
75
-90
1983.7
3
1754.9
91 -106
1755.9
4
1562.9
1 -13
1563.7
5
1844.1
14
-30
1844.9
6
2258.5
87 -106
2260.2
7
2400.9
107 -126
2402.2
9
3722
75
-106
3721.6
10
4930.9
?
12
lead
3390.3
1 -30
3390.6
12 trail
4874.1
107
-148
4874.2
15
4071.3
38 -74
4069.9
Amino acid analysis was performed in our institution and by a reference laboratory (Harvard Micro Chem, Cambridge, MA). Similar results were obtained from both analyses (Table II), which compare well to the known amino acid composition of calmodulin (28).
|
To exclude the possibility that the endothelial factor was a modified form of calmodulin, both authentic calmodulin from hog brain (which shares amino acid sequence identity with human calmodulin) and the purified endothelial factor were subjected to ion-spray mass spectrometry for the determination of molecular mass. Both species of calmodulin had a similar spectral pattern. The value obtained for the endothelial factor (16,785.77 ± 1.75 daltons) agrees well with the predicted molecular mass of calmodulin of 16,790 daltons (16,706 as calculated by the GCG software for the amino acid sequence, plus 84 daltons for the known trimethylation of lysine 115 and the acetylation of the N terminus) and with the value obtained for hog brain calmodulin (16,788.65 ± 1.11 daltons).
Calmodulin was obtained from human erythrocytes, hog brain, and bovine
testes, and recombinant epitope-tagged calmodulin was expressed in
E. coli. Each of these calmodulins demonstrated inhibition of TNF production in the whole blood assay system (Fig.
4). Maximum inhibition was observed at 1 × 107 M and the half-maximal effect occurred at
approximately 3 × 10
8 M (Fig. 4 and
Fig. 5). Calmodulin and the material purified from endothelial cell-conditioned media, like HUVEC monolayers, also augmented elastase release from whole blood (Fig. 5). The concentration of calmodulin evoking half-maximal augmentation of elastase was similar
to that required for inhibition of TNF release.
The responses to calmodulin were more consistent than the responses to
the HUVEC monolayers, although individual variability was still
evident. Failure to inhibit TNF release with 107
M calmodulin was seen in only 3 of 62 total experiments,
and failure to augment elastase release was seen in 12 of 56 total experiments, with just one of the 56 demonstrating inhibition of
elastase release by >20%.
The amount of calmodulin present in the active fraction from the Mono P column was estimated by comparing the activity of authentic hog brain calmodulin with dilutions of the active chromatographic fraction. The resulting estimate, 134 µg/ml, agreed well with a functional phosphodiesterase assay, which yielded an estimate of 128 µg/ml.
On SDS-PAGE gels, the purified endothelial factor co-migrated with authentic calmodulin and demonstrated the same negative staining with silver. It also demonstrated the same shift in mobility when electrophoresis was performed in the presence of calcium as compared with EDTA (not shown), which occurs because calmodulin retains the ability to bind calcium and undergo conformational change in the presence of SDS (31, 32).
The binding of Ca2+ to calmodulin induces the exposure of a hydrophobic patch at either end of the molecule allowing calcium-dependent binding to media such as phenyl-Sepharose (33). TNF-inhibiting activity was selectively removed from the purified endothelial factor preparation by passage over a phenyl-Superose column in the presence of calcium, and TNF-inhibiting activity could subsequently be eluted from this column by EDTA (data not shown). Another unusual property of calmodulin is its resistance to thermal denaturation. Neither authentic calmodulin nor the purified endothelial cell factor was inactivated by heat treatment (data not shown).
To exclude the possibility that calmodulin may act by accelerating the
degradation of TNF in blood or by interfering with the ELISA for TNF,
rather than by inhibiting TNF release from monocytes, experiments were
performed in which blood samples were spiked with exogenous recombinant
human TNF- (9 or 36 ng/ml final) with or without authentic
calmodulin (10
7 M final), and the recovery,
as a percentage of the added concentration, was determined by the
ELISA. After 10 min incubation at 37 °C, the recovery averaged
87.1 ± 4.1% in control and 82.3 ± 4.0% in calmodulin-supplemented samples; after 4 h incubation, recovery was 72.2 ± 3.4 and 71.1 ± 2.8%, respectively
(p = not significant for difference between control and
calmodulin-supplemented samples at either time).
To determine whether the effects observed with calmodulin were exerted
directly on monocytes and neutrophils, or mediated by an intermediary
cell type, PBMCs and neutrophils were isolated from whole blood. Plasma
was added back to the washed cells as a source of LPS-binding protein
(to restore responsiveness to LPS) and of 1-antitrypsin
(to permit detection of complexes with elastase in our ELISA). In
response to 100 ng/ml LPS, PBMCs in 10% plasma produced quantities of
TNF at the threshold of detection by our ELISA, but secreted markedly
more when suspended in 100% plasma. Neutrophils released similar
amounts of elastase under either condition. The release of TNF by PBMCs
was significantly and dose-dependently inhibited by
calmodulin, whereas the release of elastase by neutrophils was
augmented (Fig. 6).
Heparin, used as an anticoagulant in most of these experiments, may have nonspecific effects; for example, it can bind TNF directly and induce the release of TNF-binding proteins in vivo (34). Therefore, experiments were done using the specific thrombin antagonist, hirudin. The inhibitory effect of calmodulin was similar in blood anticoagulated with hirudin as with heparin (27 ± 3 and 40 ± 2% inhibition, respectively; quadruplicate determinations in one subject). Likewise, the augmentation of elastase release was similar (52 ± 7 versus 46 ± 5%).
The inhibition of monocyte TNF production by extracellular calmodulin would most likely involve a membrane receptor. To investigate this possibility, binding studies were performed using 125I-calmodulin. A time course showed that binding was maximal by 40 min. Two classes of binding sites were identified on several, but not on all, monocytic cell lines investigated (Table III). A high affinity site on THP-1 cells was identified with a Kd of approximately 30 nM and 11,000 sites per cell (Fig. 7). This affinity matches the half-maximal biological activity for both TNF and elastase release, suggesting that this binding site may represent the calmodulin receptor that mediates these effects. A second, low affinity site of Kd = 1.8 µM and 420,000 sites per cell was also identified.
|
A binding study was performed at 37 °C to determine whether calmodulin is internalized by THP-1 cells. Although nonspecific binding was increased at 37 °C compared with 4 °C, specific binding after 40 min incubation was the same. Furthermore, if the cells were washed with EDTA at the end of the period of incubation at either temperature, essentially all of the counts were removed. Only 5.8 ± 1.1% of specifically bound counts remained after washing with cells incubated at 4 °C, compared with 4.9 ± 0.4% with cells incubated at 37 °C, indicating that active internalization of the ligand had not occurred.
Trypsin treatment of HL-60 cells prior to binding caused a 90% reduction in high affinity binding sites and 75% reduction in low affinity sites, suggesting that the receptor is a trypsin-sensitive protein.
To further characterize the cell-surface calmodulin-binding proteins,
we performed cross-linking studies. Autoradiograms of SDS-PAGE gels of
125I-calmodulin cross-linked to monocytic cell membrane
proteins showed two bands, one with an apparent molecular mass of 110 kDa and a second, less distinct complex of 44 kDa. No complexes were observed when cross-linking was done in the presence of EDTA, confirming that the interaction of calmodulin with its membrane receptor is calcium-dependent (Fig. 8). The
fact that an excess concentration of unlabeled calmodulin blocked the
appearance of the complexes indicates that they form as a result of
specific binding. The calmodulin antagonist trifluoperazine also
blocked binding, suggesting that the interaction of calmodulin with its receptor involves the hydrophobic patch characteristically involved in
calmodulin binding to its intracellular targets.
These studies demonstrate that calmodulin derived from endothelial cells can interact with receptors on leukocytes, dampening the elaboration of TNF from monocytes while enhancing neutrophil elastase release.
Based on the chromatographic results, the major protein obtained from endothelial cultures that is able to inhibit LPS-stimulated TNF production appears to be calmodulin. The inhibitor was identified as calmodulin based on chromatographic properties, electrophoretic characteristics, thermal stability, peptide sequence, and mass identities with calmodulin. Futhermore, calmodulin purified from diverse tissue sources, and recombinant calmodulin expressed in E. coli, possessed the same potency and functions as the protein preparation from the endothelial cell supernatants. Thus, it is extremely unlikely that the activities observed are due to a co-purifying contaminant in these preparations.
Two classes of binding sites for calmodulin were detected on monocytic cell lines. The affinity of the first site matches the biological activity of calmodulin, suggesting that this may be the transducing receptor. Cross-linking studies also demonstrate two species. The absence of an increase in cell-associated 125I-calmodulin at 37 °C compared with 4 °C also suggests that calmodulin signals by binding to a receptor, rather than by being internalized and supplementing the cytoplasmic calmodulin pool.
It is possible that the inhibitory effect of calmodulin is not exerted directly upon the monocytes but mediated instead by an indirect mechanism involving other blood cells. For example, it has been reported that elastase and cathepsin-G released by neutrophils can degrade TNF (35). This possibility was examined by experiments using isolated peripheral blood mononuclear cells. These cells seemed, in general, to be less sensitive to inhibitory stimuli, as demonstrated by their decreased responsiveness to PGE2, perhaps due to partial activation during the process of isolation. Nonetheless, they also demonstrated inhibition of TNF release when exposed to calmodulin, supporting a direct mechanism of inhibition. Likewise, elastase release from isolated neutrophils was enhanced by calmodulin. The possibility that an interaction with plasma proteins is required to support these effects is not excluded by these experiments.
Although the identification of calmodulin as an extracellular signaling molecule was unexpected (after all, calmodulin is best known as an intracellular protein important in transducing cytoplasmic calcium signals (36, 37)), it is not implausible. Calmodulin is an abundant protein found in almost all cells and may be up to 0.4% of the total cell protein content of endothelial cells (38). Calmodulin has been found to accumulate in the conditioned media of endothelial cells over 24 h (39). It is not known whether calmodulin is released constitutively or through regulated secretion in response to inflammatory stimuli. Calmodulin release might also occur nonspecifically in response to cell injury or death. In this regard, calmodulin may be analogous to adenosine, which is released with cell injury and inhibits TNF release by binding to adenosine receptors on monocytes (2). Release of calmodulin from activated or injured cells may constitute a feedback mechanism modulating self-directed inflammation, preventing further cytotoxicity by inhibiting TNF release, and enhancing cleanup of debris by augmenting elastase release. By having calmodulin, a ubiquitous and abundant intracellular protein, as a modulator of the inflammatory response ensures rapid dampening of the local response whenever cell death or injury ensues and bypasses the need for a delayed biosynthetic response to cell injury. In this regard, it is interesting that calmodulin is found in all eukaryotic cells but is absent from prokaryotes (40) and therefore could constitute a primitive self-injury recognition system.
Other activities of extracellular calmodulin have been reported intermittently. It may stimulate the proliferation of cultured hepatocytes, keratinocytes, melanoma cells, K562 leukemia cells, HUVECs, and fibroblasts (39, 41-45). However, effects of calmodulin on inflammatory responses have not previously been described, and the effects of calmodulin on other monocyte and neutrophil functions, and on other cell types, are not known. The apparent contrasting activity of calmodulin on monocyte TNF release and on neutrophil elastase release suggests that calmodulin may have effects on multiple inflammatory cell functions.
Although the original observations were made with endothelium, it is unclear from these studies whether the calmodulin was released in a specific fashion or emerged as a result of limited cell disruption. Questions regarding the specificity of the effect for endothelial cells, the influence of other endothelial cell products on the TNF and elastase release, and other signals that might work synergistically with calmodulin are being examined.
In summary, these experiments have shown that calmodulin can modulate inflammatory responses of two different leukocyte populations. These observations argue for the likely physiological importance of this new inflammation-modulating mechanism. They further suggest that some forms of cell injury or dysfunction may lead to altered calmodulin signaling, consequently changing TNF- and elastase-mediated inflammatory activity, which could play a role in vascular and inflammatory diseases.
We thank the staff of the New Life Center, Presbyterian Hospital, Oklahoma City, OK for providing umbilical cords. Protein sequence determinations, amino acid analyses, and mass spectroscopy were performed with the help of Dr. Ken Jackson at the molecular biology resource facility of the W. K. Warren Medical Research Institute, Oklahoma City, OK. We acknowledge Monica Brase and Mei Cheng for their technical help and senses of humor, Gary Ferrell for producing the monoclonal antibodies, and Dr. Alireza Rezaie, Oklahoma Medical Research Foundation, for help in producing the recombinant calmodulin.