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

1997; Elsevier BV; Volume: 272; Issue: 18 Linguagem: Inglês

10.1074/jbc.272.18.11778

1083-351X

Donald S. Houston, C W Carson, Charles T. Esmon,

Neutrophil, Myeloperoxidase and Oxidative Mechanisms

Cultured human umbilical vein endothelial cells inhibited tumor necrosis factor-α 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 (K d ∼30 nm, in agreement with its functional activity). Cross-linking of125I-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. Cultured human umbilical vein endothelial cells inhibited tumor necrosis factor-α 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 (K d ∼30 nm, in agreement with its functional activity). Cross-linking of125I-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. Tumor necrosis factor-α (TNF), 1The abbreviations used are: TNF, tumor necrosis factor-α; 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. 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 (1Fiers W. FEBS Lett. 1991; 285: 199-212Google Scholar). A number of inflammatory stimuli can evoke its release from monocytes and macrophages, such as bacterial endotoxin (lipopolysaccharide; LPS), interferon-γ, and IL-1. Although several substances have been shown to inhibit the production of TNF in vitro, including adenosine (2Le Vraux V. Chen Y.L. Masson I. De Sousa M. Giroud J.P. Florentin I. Chauvelot-Moachon L. Life Sci. 1993; 52: 1917-1924Google Scholar), epinephrine (3Severn A. Rapson N.T. Hunter C.A. Liew F.Y. J. Immunol. 1992; 148: 3441-3445Google Scholar), PGE2 (4Kunkel S.L. Spengler M. May M.A. Spengler R. Larrick J. Remick D. J. Biol. Chem. 1988; 263: 5380-5384Google Scholar), PGI2(5Crutchley D.J. Conanan L.B. Que B.G. J. Pharmacol. Exp. Ther. 1984; 271: 446-451Google Scholar), transforming growth factor-β (6Espevik T. Figari I.S. Shalaby M.R. Lackides G.A. Lewis G.D. Shepard H.M. Palladino Jr., J.A. J. Exp. Med. 1987; 166: 571-576Google Scholar, 7Tsunawaki S. Sporn M. Ding A. Nathan C. 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Immunol. 1993; 151: 6370-6381Google Scholar), 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. Monoclonal antibodies were raised against recombinant human TNF-α (a generous gift of Genentech, South San Francisco, CA) and a complex of human neutrophil elastase and human α1-antitrypsin (Athens Research and Technology, Athens, GA) by standard techniques. (15Esmon C.T. Esmon N.L. LeBonniec B.F. Johnson A.E. Methods Enzymol. 1993; 222: 359-385Google Scholar) 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-α1-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. 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. (16Maciag T. Cerundolo J. Ilsley S. Kelley P.R. Forand R. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 5674-5678Google Scholar)), 10 μg/ml heparin (Sigma), 2 mml-glutamine, 50 IU/ml penicillin, and 50 μg/ml streptomycin (Mediatech) as described (17Moore K.L. Andreoli S.P. Esmon N.L. Esmon C.T. Bang N.U. J. Clin. Invest. 1987; 79: 124-130Google Scholar, 18Carson C.W. Hunder G.G. Kaplan K.L. Johnson C.M. Am. J. Pathol. 1991; 139: 199-200Google Scholar) 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 × 10−6m. 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). 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) (19Furmaniak-Kazmierczak E. Hu C.Y. Esmon C.T. Blood. 1993; 81: 405-411Google Scholar). 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 mml-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, 0.5m 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, 0.02m 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 Stoneet al. (20Stone K.L. Lo Presti M.B. Crawford J.M. De Angelis R. Williams K.R. Matsudaira P.T. A Practical Guide to Protein and Peptide Purification for Microsequencing. 2nd Ed. Academic Press, San Diego1993Google Scholar). 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) (21Hewick R.M. Hunkapiller M.W. Hood L.E. Dreyer W.J. J. Biol. Chem. 1981; 256: 7990-7997Google Scholar). Protein samples and chromatography fractions were analyzed on 12% SDS-PAGE gels according to the method of Laemmli (22Laemmli U.K. Nature. 1970; 227: 680-685Google Scholar). 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. (23Wallace R.W. Tallant E.A. Cheung W.Y. Methods Enzymol. 1983; 102: 39-47Google Scholar). 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 (24Dedman J.R. Kaetzel M.A. Methods Enzymol. 1983; 102: 1-8Google Scholar). Protein concentration of purified calmodulin samples was estimated, using an extinction coefficient at 280 nm of 0.18 ml mg−1 cm−1 (25Watterson D.M. Harrelson Jr., W.G. Keller P.M. Sharief F. Vanaman T.C. J. Biol. Chem. 1976; 251: 4501-4513Google Scholar). 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. coliby inserting the human calmodulin I gene into the pIN-III-pelB-Neo vector, a modification of the pIN-III-pelB plasmid (26Rezaie A. Fiore M.M. Neuenschwander P.F. Esmon C.T. Morrissey J.H. Protein Expression Purif. 1992; 3: 453-460Google Scholar) which includes a tandemneo r gene. The construct contains an N-terminal extension, EFEDQVDPRLIDGKIEGR, corresponding to the epitope for monoclonal antibody HPC-4 (27Stearns D.J. Kurosawa S. Sims P.J. Esmon N.L. Esmon C.T. J. Biol. Chem. 1988; 263: 826-832Google Scholar). 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 (26Rezaie A. Fiore M.M. Neuenschwander P.F. Esmon C.T. Morrissey J.H. Protein Expression Purif. 1992; 3: 453-460Google Scholar). 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, 0.02m 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 mmHEPES, 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 × 10−5m final) added. Immediately thereafter, 10 μl of125I-calmodulin was added to a final concentration of 5 × 10−9m 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 bound125I-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 × 10−5m cold calmodulin gave similar values. The Ligand computer software program (Elsevier-BIOSOFT, Cambridge, UK) was used to estimate theK d 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 mmHEPES, 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 10−7m 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-linked125I-calmodulin was then removed by washing twice in HH/EDTA, and the cell pellet was extracted in buffer consisting of 0.15m 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). Statistical comparisons were by pairedt 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 (28Vanaman T.C. Methods Enzymol. 1983; 102: 296-310Google Scholar, 29Walsh M. Stevens F.C. Kuznicki J. Drabikowski W. J. Biol. Chem. 1977; 252: 7440-7443Google Scholar, 30Watterson D.M. Sharief F. Vanaman T.C. J. Biol. Chem. 1980; 255: 962-975Google Scholar) whose masses were predicted by the GCG software program (Table TableI).Table ITryptic fragments of endothelial factor in comparison with calmodulinEndothelial factorCalmodulinPeak no.Measured massResiduesExpected massdaltonsdaltons1804.331 –37805.421984.375 –901983.731754.991 –1061755.941562.91 –131563.751844.114 –301844.962258.587 –1062260.272400.9107 –1262402.29372275 –1063721.6104930.9?12 lead3390.31 –303390.612 trail4874.1107 –1484874.2154071.338 –744069.9 Open table in a new tab 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 TableII), which compare well to the known amino acid composition of calmodulin (28Vanaman T.C. Methods Enzymol. 1983; 102: 296-310Google Scholar).Table IIAmino acid composition of purified endothelial factor, and comparison with calmodulinAmino acidMeasured (our lab)Reference labPredicted number (calmodulin)Asx24232317 ASP + 6 ASNGlx27272721 GLU + 6 GLNSer434Gly111111His111Arg696Thr121212Ala111111Pro232Tyr222Val777Met799Ile878Leu1099Phe988Lys778Cys0Trp0 Open table in a new tab 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 inE. coli. Each of these calmodulins demonstrated inhibition of TNF production in the whole blood assay system (Fig.4). Maximum inhibition was observed at 1 × 10−7m and the half-maximal effect occurred at approximately 3 × 10−8m (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.Figure 5TNF 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 mmHEPES, 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 × 10−6m or 134 μg/ml. α1-AT, α1-antitrypsin.View Large Image Figure ViewerDownload (PPT) 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 10−7m 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 (31Wallace R.W. Tallant E.A. Cheung W.Y. Cheung W.Y. Calcium and Cell Function. Academic Press, New York1980: 13-40Google Scholar, 32Klee C.B. Crouch T.H. Krinks M.H. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 6270-6273Google Scholar). 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 (33Gopalakrishna R. Anderson W.B. Biochem. Biophys. Res. Commun. 1982; 104: 830-836Google Scholar). 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-α (9 or 36 ng/ml final) with or without authentic calmodulin (10−7m 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 calmodu