Endothelial Cells and Extracellular Calmodulin Inhibit Monocyte Tumor Necrosis Factor Release and Augment Neutrophil Elastase Release*

(Received for publication, October 24, 1996, and in revised form, January 17, 1997)

Donald S. Houston Dagger §, Craig W. Carson par and Charles T. Esmon **

From the Dagger  Department of Internal Medicine and Manitoba Institute of Cell Biology, University of Manitoba, Winnipeg, Manitoba R3E 0V9, Canada and the  Cardiovascular Biology Program, Oklahoma Medical Research Foundation and ** Howard Hughes Medical Institute, Oklahoma City, Oklahoma 73104

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Cultured human umbilical vein endothelial cells inhibited tumor necrosis factor-alpha 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.


INTRODUCTION

Tumor necrosis factor-alpha (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-gamma , 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-beta (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.

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.


MATERIALS AND METHODS

TNF and Elastase ELISAs

Monoclonal antibodies were raised against recombinant human TNF-alpha (a generous gift of Genentech, South San Francisco, CA) and a complex of human neutrophil elastase and human alpha 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-alpha 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.

Endothelial Cell Culture

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 × 10-6 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).

Ex Vivo Culture of Whole Blood

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.

Mononuclear Cell and Neutrophil Preparations

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.

Purification and Characterization of Endothelial TNF-suppressing Activity

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.

Trypsin Digestion, Sequence Analysis, and Mass Spectrometry

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).

Purification and Assay of Calmodulin

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-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.

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).

125I-Calmodulin Binding to Monocytic Cell Lines

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 10-9 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.

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.

Cross-linking of 125I-Calmodulin to Surface Receptors

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 10-7 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).

Statistics

Statistical comparisons were by paired t test. Data are presented as mean ± S.E.


RESULTS

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.


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.
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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.


Fig. 2. Augmentation of LPS-induced elastase release in whole blood by endothelium. Elastase was measured as complexes with alpha 1-antitrypsin (alpha 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.
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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).


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.
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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


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).

Table II. Amino acid composition of purified endothelial factor, and comparison with calmodulin


Amino acid Measured (our lab) Reference lab Predicted number (calmodulin)

Asx 24 23 23
17 ASP + 6 ASN
Glx 27 27 27
21 GLU + 6 GLN
Ser 4 3 4
Gly 11 11 11
His 1 1 1
Arg 6 9 6
Thr 12 12 12
Ala 11 11 11
Pro 2 3 2
Tyr 2 2 2
Val 7 7 7
Met 7 9 9
Ile 8 7 8
Leu 10 9 9
Phe 9 8 8
Lys 7 7 8
Cys 0
Trp 0

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 × 10-7 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.


Fig. 4. TNF release by whole blood in response to 100 ng/ml LPS and varying concentrations of authentic calmodulin. Top, responses of two donors are shown, with calmodulin derived from human erythrocytes and hog brain. Bottom, responses of one donor with calmodulin derived from bovine testis and recombinant calmodulin expressed in E. coli. Error bars are range of duplicate blood samples. Axis scales in the panels differ but attention is drawn to the fact that with each calmodulin the half-maximal inhibition occurred at ~3 × 10-8 M.
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Fig. 5. TNF and elastase release by whole blood in response to 100 ng/ml LPS and varying concentrations of authentic calmodulin (top) or the purified active material from HUVEC-conditioned media (peak fraction from the Mono P chromatofocusing column, bottom). Responses of two donors are shown; error bars are range of duplicate blood samples. The abscissa of each plot represents the final concentration in the incubation mixture. Buffer indicates the response to LPS with the addition only of sterile Tris-buffered saline with 0.1% pyrogen-free gelatin (the dilution vehicle for calmodulin). Polybuffer indicates the response to LPS with the addition of the column elution buffer (Polybuffer 74, pH 2.9) that had first been neutralized by the addition of 0.1 volume of 200 mM HEPES, pH 7.5. The Mono P fraction was likewise neutralized with 0.1 volume of 200 mM HEPES, pH 7.5, and serial dilutions made in Tris-buffered saline with 0.1% gelatin. By reference to the authentic calmodulin curves, the concentration of calmodulin in the undiluted Mono P fraction was estimated to be 8 × 10-6 M or 134 µg/ml. alpha 1-AT, alpha 1-antitrypsin.
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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 10-7 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-alpha (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 alpha 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).


Fig. 6. Release of TNF from isolated PBMCs (top) and elastase from isolated neutrophils (bottom) in response to 100 ng/ml LPS and varying concentrations of calmodulin. Responses are the mean of cells from six subjects.
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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.

Table III. Binding of 125I-calmodulin to monocytic cell lines


Kd Sites/cell

THP-1 cells
  Site 1 30.2  ± 5.9 nM 11,000  ± 2,000
  Site 2 1.78  ± 0.11 µM 420,000  ± 10,000
HL-60 cells
  Site 1 29.8  ± 9.7 nM 7,900  ± 2,900
  Site 2 2.36  ± 0.20 µM 353,000  ± 13,000
U937 cells
  Site 1 20.2  ± 10.4 nM 3,000  ± 1,150
  Site 2 1.84  ± 0.58 µM 56,600  ± 7,600
MonoMac-6 cells
  Site ? 12.0  ± 1.0 µM 2,400,000  ± 180,000


Fig. 7. Binding of 125I-calmodulin to THP-1 cells. Nonspecific binding was determined by binding in the presence of EDTA. Inset, Scatchard plot.
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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.


Fig. 8. Autoradiogram of 125I-calmodulin cross-linked to THP-1 cell-surface proteins. Membrane extracts were run on a 6-12% gradient SDS-PAGE gel. Ca2+ indicates that the cross-linking was done in the presence of calcium; EDTA indicates cross-linking in the presence of EDTA; Cold indicates cross-linking in the presence of calcium and a large excess (2 × 10-5 M) of unlabeled calmodulin; TFP indicates cross-linking in the presence of calcium and the calmodulin antagonist trifluoperazine (300 µM). R and NR indicate that the gel was run under reducing and nonreducing conditions, respectively.
[View Larger Version of this Image (85K GIF file)]


DISCUSSION

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.


FOOTNOTES

*   This work was presented in preliminary form at the meeting of the American Society of Hematology, Seattle, WA, December 1-5, 1995 (Houston, D. S., Carson, C. W., and Esmon, C. T. (1995) Blood 86, Suppl. 1, 426a).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§   Supported by a Clinician-Scientist Award from the Medical Research Council of Canada. To whom correspondence should be addressed: Manitoba Institute of Cell Biology, 100 Olivia St., Winnipeg, Manitoba R3E 0V9 Canada. Tel.: 204-787-2184; Fax: 204-787-2190; E-mail: houston{at}umanitoba.ca.
par    Supported by National Institutes of Health NRSA Training Grant 5 T32 HL07207-17 and by the Oklahoma Chapter of the Arthritis Foundation.
1   The abbreviations used are: TNF, tumor necrosis factor-alpha ; LPS, lipopolysaccharide (endotoxin); IL, interleukin; HUVEC, human umbilical vein endothelial cell; ELISA, enzyme-linked immunosorbent assay; mAb, monoclonal antibody; HBSS, Hanks' balanced salt solution; PBMC, peripheral blood mononuclear cells; PAGE, polyacrylamide gel electrophoresis; PG, prostaglandin; HPLC, high performance liquid chromatography; HHA, Hanks/Hepes/albumin buffer; MOPS, 4-morpholinepropanesulfonic acid.

ACKNOWLEDGEMENTS

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.


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