EUGENE A. SPRAGUE, PhD, JIM L. KELLEY, PhD,. C. ALAN SUENRAM, PhD, and. COLIN J. SCHWARTZ, MD. A mononuclear cell chemoattractant ofhigh ...
Initial Characterization
of a Peripheral
Blood
Chemoattractant Derived From Cultured Arterial Smooth Muscle Cells
Mononuclear Cell
ANTHONY J. VALENTE, PhD, STEPHEN R. FOWLER, PhD, EUGENE A. SPRAGUE, PhD, JIM L. KELLEY, PhD, C. ALAN SUENRAM, PhD, and COLIN J. SCHWARTZ, MD
From the Department of. Pathology,; The LUni.versity of Texas Health S&ience Center San A ntonifc Texas
A mononuclear cell chemoattractant of high specific activity produced by baboon (Papio cynocephalus) aortic
tactic and chemokinetic activity for peripheral blood mononuclear cells but not for polymorphonuclear leukocytes. Production of SMC-CF by the cells was significantly inhibited in the presence of cycloheximide, and its activity was abolished after incubation with the bacterial protease subtilisin. Chromatofocusing experiments indicate that SMC-CF is a cationic protein with a pI of >10.5. The role SMC-CF may play as an inflammatory mediator in monocyte recruitment to the arterial intima in atherogenesis is discussed. (Am J Pathol 1984, 117: 409-417)
THERE IS an increasing interest in the role of the peripheral blood monocyte in the pathogenesis of atherosclerosis. Monocytes as progenitors of tissue macrophages play an important role in inflammation, serving as scavengers, secretory cells"2 and regulators of lymphocyte function.3'4 They undoubtedly serve as precursors of foam cells in both vascular-'6 and extravascular sites,7 although the question of the relative contributions of the monocyte-macrophage, on the one hand, and the smooth-muscle cell (SMC), on the other, to the plaque foam cell population remains uncertain. Recruitment of blood monocytes to the arterial intima has been observed in many species, including rat,'-9 pig,6 '0 'l pigeon,'2 rabbit,5"3"4 nonhuman primate, and man,5 '.1'1 and is enhanced during atherogenesis and experimental hyperlipidemia. Mechanisms responsible for the margination and subsequent emigration of peripheral blood monocytes to the arterial intima are not fully understood; but, clearly, processes of cell contact, adhesion, locomotion, and finally directed migration into and retention within the subendothelial space must
be involved. Chemotactic gradients originating within the arterial wall could regulate the directed migration of blood monocytes into the arterial intima, thus accounting for the inflammatory monocytic infiltrate which is a hallmark of the atherogenic process. Recently we described the presence of mononuclear cell chemoattractant activity in the conditioned medium of cultured baboon aortic medial SMCs.'8"9 Because of its potential relevance to a better understanding of the phenomenon of blood monocyte recruitment to the arterial intima occurring during atherogenesis, we have further explored the nature of this chemoattractant activity. In this paper we report on some of its physical and chemical properties.
medial smooth-muscle cells (SMCs) in culture has been partially characterized. Smooth-muscle cells, between the third and eighth passage, were grown to confluence in Medium 199 containing 10% fetal calf serum and then incubated for 24 hours in either serumless medium (Neuman and Tytell) or Medium 199 containing 0.2% bovine serum albumin. The 24-hour SMC-conditioned medium was fractionated on Sephadex G100-Superfine and potent chemoattractant activity (SMC-CF) eluted in the 10,000-12,000 dalton region. SMC-CF displayed chemo-
409
Supported by NHLBI Grants 1-POI-HL-26890 and HL-07446. Accepted for publication July 5, 1984. Address reprint requests to Anthony J. Valente, PhD, Department of Pathology, The University of Texas Health Science Center, 7703 Floyd Curl Dr., San Antonio, TX 78284.
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Materials and Methods Serumless medium (Neuman and Tytell),20 M199, and Hanks' balanced salt solution (HBSS) were obtained from GIBCO Laboratories (Grand Island, NY). Fetal calf serum (FCS) was obtained from K.C. Biologicals (Lenexa, Kans). Media were supplemented with 10 mM HEPES, 2 mM L-glutamine, 100 U/ml penicillin, and 100 lg/ml streptomycin sulfate before use. Bovine serum albumin (BSA, #A-8022) and cycloheximide (#C6255) were obtained from Sigma Chemical Co., (St. Louis, Mo). Disposable tissue-culture flasks and tubes were obtained from Corning (Corning Glass Works, Corning, NY). Chemical reagents were of analytical grade.
Smooth-Muscle Cells Baboon (Papio cynocephalus) aortic medial SMCs were isolated, cultured, and characterized as previously described.2' Briefly, small pieces of the aortic arch and thoracic aorta were stripped free of the adventitial, endothelial cell, and outer medial layers; and the remaining inner media was diced and incubated in M199-10% FCS in 25 sq cm flasks. SMCs growing out of the explants were passaged by trypsinization with medium changes every 3 days. With an initial plating density of 1 x 104 cells/sq cm, confluence was achieved in 7-10 days. Mononuclear Cells and Polymorphonuclear Leukocytes Human mononuclear cells were isolated from venous blood of healthy volunteers by the method of Boyum.22 Blood was drawn into acid-citrate-dextrose (5 volumes blood + 1 volume ACD) and centrifuged at room temperature at 550g for 20 minutes. The platelet-rich plasma was aspirated, and the remaining cells were resuspended to twice the original blood volume in HBSS (Ca2+- and Mg2+-free) containing 0.27/o dextrose and 0.3707o sodium citrate. Two volumes of the diluted blood were layered over 1 volume of Histopaque-1077 (Sigma Chemical Co., St. Louis, Mo) in sterile 50-ml centrifuge tubes and centrifuged at 400g for 40 minutes. The mononuclear cell layers were pooled, washed once in HBSS, and resuspended in M199-0.2Wo BSA to 5.0 x 106 cells/ml for the chemotaxis assay. Polymorphonuclear leukocytes (PMNs) were isolated from the cell pellet remaining after the gradient centrifugation step. The cells were washed once in HBSS and resuspended in an equal volume of cold erythrocytelysing buffer (155 mM NH4C1, 10 mM KHCO3, 1 mM EDTA, pH 7.4) at 4 C for 15 minutes. The remaining
AJP * December 1984
cells were centrifuged; and, if necessary, the erythrocytelysing step was repeated. The cells (>960o PMNs) were then washed in HBSS and resuspended to 2-4.0 x 106 cells/ml in M199-0.2% BSA. Both the mononuclear cell and PMN preparations were greater than 95% viable as assessed by trypan blue dye exclusion.
Chemotaxis Assays Mononuclear cell chemotaxis was quantitated on 5 micropore polycarbonate filters in blind-well cham,p bers (Bio-Rad Laboratories, Richmond, CA) essentially as described by Snyderman et al.23 Solutions of chemoattractants to be assayed were diluted with at least an equal volume of M199-0.2% BSA, and 200,u were placed in the lower compartment of the chambers. Mononuclear cell preparations (500,ul) were added to the compartments above the filters. The chambers were incubated at 37 C for 90 minutes in a humidified 5% C02-air atmosphere. The filters were removed and stained in Giemsa, and chemotaxis was assayed by counting the number of cells on the attractant side of the filter in 10 oil-immersion fields (x 1000). Sequential fractions eluted from the gels in the gel filtration and chromatofocusing experiments were assayed singly; otherwise, the experiments were performed in
duplicate. The PMN chemotaxis assay was performed in duplicate in a similar manner, except that 3-,u micropore polycarbonate filters were used and the incubation time was 30 minutes.
SMC Chemoattractant Activity Baboon aortic SMCs, between the third and eighth passage, were grown in M199-1007o FCS in 75 sq cm flasks until confluent. The cell layer was rinsed once in M199, then further incubated for 24 hours in 15-ml volumes of either M199-0.2% BSA or serumless medium.20 Conditioned media containing the chemoattractant activity were collected and either stored at - 20 C until used or concentrated by ultrafiltration for immediate fractionation. Gel Filtration Conditioned media containing SMC chemoattractant activity were concentrated 10-fold by positive pressure ultrafiltration under N2 (Diaflo YM5 43 mm membrane, cutoff 5000, Amicon Corp., Lexington, Mass). Up to 4 ml of the concentrate was fractionated at 4 C on a 2.5 x 90-cm column of Sephadex G100 Superfine (Pharmacia Fine Chemicals, AB, Uppsala, Sweden) equilibrated in phosphate-buffered saline (PBS). The
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SMC-DERIVED MONONUCLEAR CHEMOATTRACTANT
eluted fractions were assayed for both absorbance at 280 nm and mononuclear cell chemoattractant activity.
Chromatofocusing Chromatofocusing was carried out on Polybuffer Exchanger PBE 118 with a pH gradient formed by Pharmalyte pH 8-10.5 carrier ampholyte (Pharmacia Fine Chemicals, AB, Uppsala, Sweden). SMC chemoattractant-rich fractions isolated by gel filtration were concentrated 5-10 fold by ultrafiltration and dialyzed at 4 C in a Spectropor membrane (cutoff 3500, Spectrum Medical Industries, Inc., Los Angeles, Calif) against 25 mM triethylamine, pH 11.0. Approximately 3 ml of the dialyzed concentrate was then applied to a 0.9 x 30-cm column containing 10 ml of PBE 118 exchanger equilibrated in 25 mM triethylamine, pH 11.0. The column was eluted at 20 ml/hr with starting buffer until 20 ml of eluate had been collected. The pH gradient was then formed by further elution of the gel with 90 ml of Pharmalyte pH 8-10.5 carrier ampholyte diluted 1:45 in deionized water and adjusted to pH 7.5 with HCI. The pH and absorbance at 280 nm of the eluted fractions were measured before they were extensively dialyzed at 4 C against PBS and assayed for mononuclear cell chemoattractant activity.
Protease Sensitivity of SMC Chemoattractant Activity Sensitivity of SMC chemoattractant activity to the bacterial protease subtilisin, was tested as follows. Aliquots of agarose-conjugated Type VII-A subtilisin (Sigma Chemical Co., St. Louis, Mo) were washed five times in 30 volumes of PBS. To 100-jA aliquots of the packed gel were added either 200 pd volumes of PBS (control) or chemoattractant-rich fractions obtained from gel filtration of the conditioned media. The suspensions were incubated with gentle mixing at 37 C for 90 minutes. As an additional control, active fractions were incubated in a similar manner with Ultragel AcA 44 agarose-acrylamide beads (LKB Instruments Inc., Rockville, Md). The suspensions were centrifuged for 5 minutes on a microcentrifuge, and the supernatants were assayed for chemoattractant activity. Effect of Cycloheximide on Chemoattractant Production by SMCs SMCs were cultured in 25-sq cm flasks in M199-1007o FCS until confluent. The medium was discarded, and the cells were rinsed in M199, then further incubated in M199-0.2%7o BSA containing various concentrations (101-10-5 M) of cycloheximide. After 24 hours, the
medium was collected and stored at -20 C until assayed for chemoattractant activity. To determine the influence of cycloheximide alone on mononuclear cell migration, we used control assays employing the same concentrations of cycloheximide in nonconditioned medium (M199-0.207o BSA).
Results
Molecular Size and Charge Properties of
SMC-Chemoattractant Activity Fractionation of the concentrated SMC-conditioned medium (MI99-0.2%to BSA) on Sephadex G100 Superfine is shown in Figure 1. The major peak of chemoattractant activity eluted in the region corresponding to a protein with a molecular weight (MW) of 10,000-12,000 daltons. No absorbance was discerned at 280 nm in those fractions containing this low MW activity, indicating a high specific activity. Two minor peaks of activity of higher MW were also observed, one near the void volume (>100,000 daltons) and one coeluting with the main protein (BSA) peak. To eliminate the possibility that the SMC chemoattractant activity was derived by interaction of the SMC with the added BSA (Fraction V) proteins, and to determine the source of the minor high MW activities, we incubated SMCs in serumless medium for 24 hours and then concentrated and separated the medium on the same column of Sephadex GI0O (Figure 2a). Chemoattractant activ2.6
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measured at 280 nm. Chemotactic activity (cells/high-power field) measured on Fractions 17-60 is shown as vertical bars. The gel column was calibrated with bovine thyroglobulin (670 kilodaltons. kd: Vo), BSA (66 kd), ovalbumin (44 kd), horse myoglobin (17 kd). and cyanocobalamin (1.35 kd). The elution volume of phenol red was taken as the total included volume Vi. The major peak of chemoattractant activity (SMC-CF) eluted in the region corresponding to a protein with a MW of 10-12 kd (arrow). Two minor peaks of activity can also be seen. one eluting near the void volume (>100 kd) and the other coeluting with BSA. was
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VALENTE ET AL
AJP * December 1984
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abundance and potency, further characterization studies were confined to the low MW (10,000-12,000) SMC chemoattractant (SMC-CF). Initial experiments carried out to characterize the net charge of SMC-CF by flat-bed isoelectric focusing failed to locate a peak of activity over the pH range 3.0-9.5. Chromatofocusing on PBE 118 with a pH gradient for 8.0-11.0 was therefore undertaken (Figure 3). The bulk of the recovered SMC-CF was unretained by the gel at pH 10.5-11.0, which indicated that it was highly cationic with a pl of >10.5. Further experiments using an increased volume of the exchanger gel did not modify the result, indicating that the capacity of the gel had not been exceeded, and that SMC-CF was not retained under these conditions.
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nm and mononuclear cell chemoattractant activity are displayed as in Figure 1. The major peak of chemoattractant activity (SMO-OF) again eluted in the 10-12-kd region. No high MW activity (>100 kd) was observed, but a minor peak eluted in the 75 kd region, corresponding to the major cell-derived protein present in the medium. Additional chemotactic activity eluted over a broad low MW region bwhere lactal bum in hydrolysate peptides are present. Separation of concentrated nonconditioned serumless medium on Sephadex G100 Superfine. Absorbance at 280 nm and mononuclear cell chemoattractant activity are shown. Discrete peaks of chemoattractant activity were not discerned. Specifically, it can be seen that the nonconditioned serumless medium does not exhibit the 10-12-kd activity present in the SMO-conditioned serumless medium (a). Further, no high MW proteins were identified.
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ity and absorbance at 280 nm were compared with those obtained from an identical fractionation of concentrated nonconditioned serumless medium (Figure 2b). As in the previous experiments, the major peak of activity eluted in the 10,000-12,000-dalton region and did not correspond to any peak of absorbance at 280 nm. Of the two minor high MW activities, the one of >100,000 daltons was not observed, whereas the other appeared to co-elute with a protein of 75,000 daltons, the major protein peak found in the SMC-conditioned medium. Additionally, significant chemoattractant activity was found in the small peptide region co-eluting with components of the lactalbumin hydrolysate present in the serumless medium. Because of its relative
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Figure 3-Chromatofocusing of the SMC-CF (10-12 kd) fraction on PBE 118. The upper panel shows the pH gradient formed by elution of the gel with Pharmalyte, pH 8.0-10.5, carrier ampholyte. In the lower panel, the absorbance at 280 nm of the eluted fractions is illustrated. The vertical bars in the central panel indicate the chemoattractant activity (cells/high-power field) present in the eluted fractions. The majority of the recovered SMC-CF was unretained by the gel at pH 10.5-11.0, indicating that the SMC-CF was highly cationic (pi > 10.5).
SMC-DERIVED MONONUCLEAR CHEMOATTRACTANT
\;ol. 11. * No. i1
CHECKERBOARD ANALYSIS OF SMC-CF (Migrated Cells/High Power Field. Mean±SD)
trafiltration, and doubling dilutions were tested for chemoattractant activitv. Mononuclear cells responded
Relative Concentration Above The Filter 25 50 75 100
in a dose-dependent manner to the increasing concentration of SMC-CF, whereas little or no PMN migration was observed except marginally at the highest concentrations of SMC-CF tested. In control experiments both mononuclear cells and PMNs responded with significant migration to the synthetic peptide f-Met-LeuPhe (10-8 M), (Sigma Chemical Co., St. Louis, Mo). Thus, SMC-CF appears to be a mononuclear-cellspecific chemoattractant. As the migrating mononuclear cells in these and other experiments were predominantly monocytes morphologically, it is further concluded that SMC-CF is a blood-monocyte-
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