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Expression of functional cell surface C1 -inactivator by U937 cells
CLINICAL
IMMUNOLOGY
AND
Expression
IMMUNOPATHOLOGY
49,
463-477 (1988)
of Functional Cell Surface CT-Inactivator U937 Cells’*2
by
BRUCE P. RANDAZZO,*~ HOWARD B. FLEIT,~ ALLEN P. KAPLAN,* AND BERHANE GHEBREHIWET*+~ *The Department fThe Department
of Medicine, Division of Allergy, of Pathology, State University
Rheumatology, of New York,
and Clinical Stony Brook,
Immunology, and New York 11794
-We have previously shown that th_e human monocyte-like cell line U937 synthesizes Cl-INA and expresses cell surface Cl-INA. In this report we provide evidence that this surface-expressed Cl-INA is functionally active. Intact U937 cells demonstrated functional Cl-INA activity in a hemolytic assay. Th& activity was blocked when the cells were incubated with monospecific antibody to Cl-INA, and was not detectable in cellfree supematants of U937 cells. SDS-PAGE analysis of radiolabeled U937 cell surface proteins purilied by anti-Cl-INA affinity chromatography revealed two distinct bands. One protein had a M, of 105 kDa identical to plasma Cl-INA, and the second had a M, of 200 kDa. We werEunable to determine the identity of the 200 kDa protein by Western blotting with anti-Cl-INA. However, the possibility _exists that this 200 kDa molecule may represent a Cl-INA regeptor, a dimeric form of Cl-INA, or an unrelated cell surface protein with affinity for Cl-INA. Furthermore, we show that treatment of U937 cells w$h phorbol ester resulted in an increase in the percentage of cells expressing surface Cl-INA. These results suggest that U937 cells express functional cell surface Cl-INA, which could function in vivo to protect these human tumor cells from lysis by host COIIlphllent.
0 1988 Academic
Press, Inc.
INTRODUCTION Human CT-inactivator (CT-INA) or a,-neuraminoglycoprotein is a plasma protease inhibitor with activity against a number of serine proteases belonging to both the complement system and the intrinsic coagulation pathway. While it is the only known plasma inhibitor of activated Cl? and ClS (l-3), Ci-INA also plays a major role in inhibiting the activities of several coagulation enzymes including factors XIIa and XIIf (HFa and HFfJ4 (4-6) and plasma kallikrein (7, 8). Inactivation of plasmin by Ci-INA in a purified system has been demonstrated, but the relative contribution of Ci-INA to the inactivation of plasmin in plasma is probably not ’ This investigation was supported by Grants AI 16337-05 and CA 41047 from the National Institute of Health, and 860742 from the American Heart Association (National Center). ’ This publication represents part of B.P.R.‘s work, performed in partial fulfillment of the Ph.D. degree in experimental pathology. 3 To whom correspondence should be addressed at the Division of Allergy, SUNY at Stony Brook, HSC T-16 Room 040, Stony Brook, NY, 11794-8161. 4 Abbreviations used: HFa, activated Hageman factor (or factor XIIa); HR, Hageman factor fragment (or factor XIIf); DMSO, dimethyl sulfoxide; EDTA, ethylenediaminetetraacetic acid; EACA, epsilon-amino-caproic acid; PMSF, phenylmethylsulfonyl fluoride; C4D GPS, C4 deficient guinea pig serum; EA, antibody sensitized sheep red blood cell; BSA, bovine serum albumin; ELISA, enzymelinked immunosorbent assay; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 463 0090-1229/88 $1.50 Copyright All rights
Q 19% by Academic Press, Inc. of reproduction in any form reserved.
464
RANDAZZO
ET
AL.
significant (9-l 1). Sodium dodecyl sulfate-polyacrylamide gel electrophoretic (SDS-PAGE) analysis of Ci-INA-enzyme complexes has shown that CT-INA forms nondissociable 1: 1 stoichiometric bimolecular complexes with the enzymes it inhibits (1, 5, 12-14). In addition to its ability to inactivate serine proteases through complex formation, Ci-INA also serves as a major homeostatic component of the classical pathway of complement, since it prevents the spontaneous autocatalytic activation of the first component of complement (Cl) (15, 16). Thus, Ci-INA plays several critical roles in complement activation including stabilization of macromolecular Cl, the inactivation of Ci subcomponents, and serves as a major inhibitor of the initiation phase of the contact coagulation-kinin-forming pathways. While the majority of plasma CT-INA is probably synthesized in the liver f 17, IQ, it is becoming increasingly clear that cells of the monocyte-macrophage series represent another potential source of Ci-INA (19,20). The U937 cel1 line is believed to represent a promonocyte cell type, and manifests several features of normal human monocytes (21-23). Using the technique of metabolic labeling, we have previously shown that U937 cells synthesize Ci-INA, and demonstrated that these cells express cell surface CT-INA as assessed by fluorescence-activated cell sorter analysis (20). It has been shown recently that U937 cells express cell surface factor H (24) and the complement C3b receptor CR1 (25), both of which function to markedly enhance the proteolytic inactivation of C3b (26, 27). Thus, U937 cells express a variety of cell surface proteins, which normally serve to control complement activation and could perhaps serve to protect these cells from complement-mediated lysis. While the functional activity of U937 surfaceexpressed CR1 and factor H has been established (24), no information on the functional state of cell surface CT-INA has been reported. In this study we present evidence that CT-INA expressed on the surface of U937 cells is functionally active, and present data which show that two molecular species are affinity purified (anti-Ci-INA Sepharose 4B) from detergentsolubilized U937 membranes. The first has a molecular weight identical to serum Ci-INA and is identified in Western blots by antibody to Ci-INA. The second has a molecular weight of 200 kDa. We will discuss the possible biologic significance of this cell surface-associated multispecific enzyme inhibitor. MATERIALS
AND METHODS
Buffers and reagents. The following buffers were used: HBSS. Hanks’ balanced salt solution, pH 7.4, containing calcium (1.4 g/liter), magnesium (1.0 gi liter), and phenol red (GIBCO, Grand Island, NY); PBS, phosphate-buffered saline, consisting of 100 mM phosphate and 0.9% NaCl, pH 7.2; GVB, isotonic veronal-buffered saline, containing 0.15 mM CaC12. 0.5 m&4 MgCl?, 0.02% NaN,, and 0.1% gelatin, pH 7.2; and 4B-phorbol If-myristate 13-acetate (PMA) purchased from Sigma Chemical Co. (St. Louis, MO), dissolved in DMSO at a stock concentration of 0.3 mg/ml, and stored frozen at - 80°C. Purifiedproteins and antibodies. CT-INA was purified according to a procedure described by Reboul et al. (28), ClS was purified and activated according to the method of Gigli et al. (29). Production of monospecific polyclonal rabbit anti-
U937 CELLS EXPRESS FUNCTIONAL
Ci-INA
465
human CT-INA IgG and F(ab’), fragment has been described elsewhere (20). The antibody is monospecific by immunoelectrophoresis against normal human serum, and does not cross-react with fetal calf serum as assessed by Ouchterlony or ELISA. The cell line producing monoclonal antibody OKMl was obtained from the American Type Culture Collection (Rockville, MD) and was grown in Iscove’s modified Dulbecco’s medium supplemented with 20% FCS. OKMl has been shown to bind to the complement C3bi receptor (CR3) at a site distinct from the ligand-binding site (30). Spent culture supematants were used as the source of OKMl . Fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit Ig and FITC-conjugated goat anti-mouse Ig were purchased from Cappel Worthington Biochemicals (Malvern, PA). Guinea pig C4 and C4 deficient guinea pig serum (C4D GPS) used in the hemolytic assays were purchased from Cordis Laboratories Inc. (Miami, FL). Bovine serum albumin (fraction V powder) was purchased from Sigma Chemical Co. Cell line. The U937 cell line was propagated in suspension culture in RPM1 1640 containing 10% fetal calf serum, 100 units/ml penicillin, and 100 @/ml of streptomycin (GIBCO). It was maintained in a humidified atmosphere consisting of 5% CO2 and 95% air. Viability was assessed by trypan blue exclusion before all experiments, and was always greater than 95%. Radioiodination of proteins. Purified human serum Ci-INA was labeled with 1251by the method of Bolton and Hunter (31). Briefly, CT-INA was suspended in 0.1 A4 sodium borate buffer, pH 8.5, and 250 &i of Bolton Hunter reagent (New England Nuclear, Boston MA) was added, and the reaction proceeded for 3 hr on ice. Unbound ‘25I was removed by extensive dialysis of the protein against PBS, pH 7.4. Radioiodination of cell surface proteins. Surface radiolabeling of U937 cell membrane proteins was carried out by the lactoperoxidase-catalyzed reaction method (32). Viable U937 cells (generally, 5 x 10*-l x 109) were removed from log phase cultures, and were washed three times in HBSS. The cells were then resuspended in 1 ml of HBSS containing 65 kg of lactoperoxidase (Sigma Chemical Co.) and 1 mCi of carrier-free 1251(New England Nuclear), and the reaction mixture was kept on ice. Next, a 15+1 aliquot of a l:lO,OOO dilution of 30% H202 (Sigma Chemical Co.) in HBSS was added every 15 set until a total of five additions had been made. The radiolabeled cells were then pelleted by centrifugation, the supernatant was removed, and the cells were washed five times with HBSS. Isolation of U937 cell surface Ci-ZNA. Radiolabeled U937 cells were resuspended in 5 ml of buffer containing 10 mJ4 Na,HPO,, 20 mM NaC 1,2 m&Z EDTA, 10 mM EACA, 20 mA4 iodoacetamide, 0.5 rr&! PMSF, and 0.02% NaN,, pH 7.4 (lysis buffer). The cells were then lysed by three cycles of freezing and thawing. The cell membranes were pelleted by centrifugation for 1 hr at 30,OOOg at 4”C, and were then washed and pelleted three times in 40 ml of lysis buffer. The pelleted membranes were next solubilized in 50 ml of lysis buffer containing 1% NP-40 (v/v) and stirred for 20 hr at 4°C. The solubilized membrane proteins were separated from insoluble material by centrifugation at 30,OOOg (60 min at 4”C), and were then dialyzed against lysis buffer containing 0.1% NP-40. This material was
466
RANDAZZO
ET AL.
applied to an anti-CT-INA aftinity column, which was constructed as previously described (20, 33), and equilibrated with the same buffer used for dialysis (starting buffer). The column was washed with starting buffer until the radioactivity in the effluent was negligible. The specifically bound material was then eluted with a linear NaCl concentration gradient consisting of 50 ml of starting buffer and 50 ml of the same buffer containing 1 M NaCl. The eluted material was pooled, dialyzed against starting buffer, and concentrated using an Amicon ultrafiltration cell fitted with a UM-10 membrane (Amicon Corp., Danvers, MA). In some experiments the radiolabeled detergent-solubilized U937 membrane proteins, prepared as described above, were applied onto an anti-albumin Sepharose 4B column in an attempt to remove nonspecifically adsorbing proteins. The membrane proteins were loaded onto the column, and the column was washed with 10 bed volumes of starting buffer. Material that passed through this column unbound was then applied onto the anti-CT-INA column as described above. SDS-PAGE analysis of U937 cell surface Ci-INA. The material eluted from the affinity column was subjected to 8% SDS-PAGE using the method described by Laemmli (34). Samples to be reduced were boiled for 3 min in sample buffer containing P-mercaptoethanol at a final concentration of 1%. Each gel was run with prestained high molecular weight markers (Bethesda Research Laboratories, Gaithersburg, MD) and radiolabeled human serum CT-INA. After electrophoresis, the gel was fixed in a mixture of 7% acetic acid, 40% methanol in H,G, vacuum dried, and autoradiographed at -80°C using Kodak XAR film (Kodak, Rochester, NY). Western blot analysis affinity chromatography.
of U937 membrane
proteins
isolated
by anti-Cl-INA
U937 cells (5 x lo*) were surface radiolabeled using the lactoperoxidase method. The membrane proteins were solubilized in NP-40 and applied onto an anti-Ci-INA aflinity column as described above. The radiolabeled material eluted from the affinity column with a NaCl gradient was pooled, concentrated by ultrafiltration, and subjected to 6% SDS-PAGE. One lane containing prestained rainbow molecular weight markers (Amersham, Arlington Heights, IL) was included in all gels to estimate molecular weights and to evaluate the efficiency of transfer. Following electrophoresis, the proteins were passively transferred to nitrocellulose paper (pore size 0.2 pm, Schleicher and Schuell Inc., Keene, NH) using a method described by Southern (35). Briefly, the gel was placed on a piece of blotter paper on a flat piece of glass. The blotter paper was made continuous with a reservoir containing a buffer consisting of 8 mM Tris base (Sigma Chemical Co.) and 58 nuV glycine. The nitrocellulose paper was placed in contact with the gel with a piece of filter paper placed over it. An approximately 3-in. layer of paper towel was placed over the filter paper, and a heavy book was placed on top. The transfer was carried out for 4 days at room temperature. After transfer, the nitrocellulose paper was incubated for 60 min at 37°C in 0.1% BSA (w/v) in PBS with 0.05% Tween 20 (v/v). Next, the paper was incubated for 2 hr at 37°C in PBS-Tween with polyclonal rabbit IgG anti-CT-INA at approximately 1 kg/ml. The paper was washed for 90 min with PBS-Tween, and was then incubated for 1 hr at 37°C with alkaline phosphatase-conjugated affmity-purified
U937 CELLS EXPRESS FUNCTIONAL
Ci-INA
467
goat anti-rabbit IgG (Cappel Corp.) diluted 1: 1000 in PBS-Tween. After a 90-min wash in PBS-Tween and a brief wash in HzO, a 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium-based substrate in Tris buffer (Kirkegaard and Perry Labs. Inc., Gaithersburg, MD) was added and incubated at 37°C until purple bands appeared on the paper. After the nitrocellulose paper had air dried, it was autoradiographed at - 80°C using Kodak XAR film (Kodak). Determination of Ci-ZNA functional activity. CT-INA functional activity was assayed using a C4 dependent hemolytic assay essentially as described by Minta and Aziz (36). Briefly, we first established that 10 CHsO units of C4 would reconstitute the maximal hemolytic activity of 200 ~1 of a 1:40 dilution of C4 deficient guinea pig serum (C4D-GPS) in GVB when incubated with 100 l.~l of antibody sensitized sheep erythrocytes (EA) at 1 x 109/ml. Next, we determined that preincubation of 20 ng of ClS with 10 CHS, units of C4 for 45 min at 37°C prior to addition of the diluted C4D-GPS caused a reduction in C4 dependent lysis to approximately 10% of maximal. Finally, we established that preincubation of varying doses of purified human serum Ci-INA with the 20 ng of Ci could cause a dose-dependent inhibition of Cl<-induced C4 destruction. To assay the functional activity of U937 cell surface CT-INA, various numbers of U937 cells were suspended in GVB and 20 ng of ClS was added. After adjusting the volume of each sample to 0.2 ml with GVB, the suspensions were incubated for 2.5 hr at 4°C with gentle rocking. The cells were then pelleted by centrifugation at 8OOg, and 0.175 ml of the supernatant was removed from each sample and transferred to a separate tube. Next, 10 CHso units of C4 were added to each supernatant, and the mixture was further incubated for 45 min at 37°C. Finally, 200 ~1 of a 1:40 dilution of C4D-GPS and 100 l.~l of EAs at 1 x 109/ml were added to each tube. After a l-hr incubation at 37”C, each tube was centrifuged at 8OOg and the percentage of lysis of each sample was determined by assaying for free hemoglobin in the supernatant spectrophotometrically at 412 nm. Fluorescence-activated cell sorter analysis of 11937 cells with and without PMA-induced differentiation. U937 cells were cultured in plastic bacteriological culture dishes at 5 x lo5 cells/ml in a total volume of 4 ml of RPM1 1640 with 10% FCS, with or without PMA at a final concentration of 160 ti. After 2 days in culture, cells were gently scraped from the plastic dishes with a Teflon policeman, washed three times in HBSS, counted in the presence of trypan blue, and resuspended in HBSS containing 1% BSA and 0.02% NaN3 (HBSS-BSA). Then, 1 x lo6 U937 cells in HBSS-BSA were incubated either with rabbit F(ab’), antiCi-INA (100 kg/ml) or OKM 1 monoclonal antibody (2 pglrni) for 30 min at 4°C. After incubation, the cells were washed three times with HBSS-BSA. Cells stained for CT-INA were incubated with FITC-conjugated goat anti-rabbit IgG at 40 pg/ml while cells stained with OKMl were incubated with FITC-conjugated goat anti-mouse Ig at 50 p&ml in HBSS-BSA for 30 min at 4°C. After incubation, the cells were washed three times in HBSS-BSA and were resuspended at 1 x LO6 cells/ml in HBSS-BSA with 1% formalin. Flow microfluorometry was performed using an EPICS V fluorescence-activated cell sorter (FACS, Coulter, Hialeah, Fla). Data are shown as the log of integrated green fluorescence gated on forward
468
RANDAZZO
ET AL.
angle light scatter and log of 90” light scatter. In each case, control histograms were obtained for cells stained in the absence of primary antibody, and were subtracted from appropriate experimental samples to account for nonspecific staining. RESULTS Isolation of surface-expressed CT-INA. Elution of the radiolabeled detergentsolubilized U937 membrane proteins from the Sepharose 6B-anti-Ci-INA affinity column was accomplished with a linear NaCl gradient. As the elution profile in Fig. 1 shows, one single peak of radioactive material was eluted under these conditions. The first, unadsorbed peak contained a total of approximately 3.3 1 x IO6 cpm of radiolabeled U937 membrane proteins, while a total of 76,370 cpm were contained in the second peak, which was eluted with the NaCl gradient. Thus, the specifically bound and eluted material represents approximately 2.3% of the total detergent-solubilized surface-labeled U937 membrane proteins. SDS-PAGE analysis of surface-expressed CT-INA. The material eluted from the anti-Ci-INA affinity column was concentrated, and was then analyzed by 8% SDS-PAGE and autoradiography. As shown in Fig. 2, the U937-derived material contains two distinct proteins with different molecular weights. One migrates with an apparent M, that is similar to that of the radiolabeled human serum CT-INA, which is 105 kDa, and the other has an apparent molecular weight of approximately 200 kDa. No shift in mobility was observed for either the 105 kDa or 200
600 i 500.
l\+
I\
.Y.400+ / * - 200c” 3 100- i1 z I 30 20 5 8 roi! + 0 * 0
.“.
+\+ \*\ \+ \
16o . .-I50 t - -{40 +\+ \ +\ . i 130 I /20 +. 14: ~ +-•. ‘* j ‘0 +-+, 1. .. . . . .C’*-+.+-+.~ /. ‘* *llfllll ; .~~ ~ : .~.~ .2?2d 0 50 6” 70 80 90 400 FRACTIONS
-FIG. I. Affinity chromatography of solubilized U937 cell membrane proteins on an antiCl-INA-Sepharose 6B affinity column. U937 cells (5 x 10’) were surface labeled with 12’1 by lactoperoxidase catalyzed iodination, and lysed by freeze thawing in 5 ml of buffer consisting of 10 mM Na2HP0,, 20 m&f NaCl, 2 mM EDTA, 10 mM EACA, 20 mM iodoacetamide, 0.5 mM PMSF, and 0.02% NaN,, pH 7.4 (lysis buffer). The membrane fraction was isolated by centrifugation at 30,OOOg and was solubilized in lysis buffer made 1% with NP-40. The membrane proteins were then apphed onto an anti-Cl-INA-Sepharose 6B affinity column equilibrated with the above buffer made 0.1% with NP-40. After washing, the specifically bound protein was eluted with a linear NaCl gradient and 3.5-m] fractions were collected. This figure shows a typical elution profile from a series of four independent experiments.
U937
CELLS
A
EXPRESS
B
Ci-INA
FUNCTIONAL
C
469
D
200
(kD)
66
43
FIG:~. SDS-PAGE analysis of U937 cell surface-expressed CT-INA. The material eluted from the anti-Cl-INA-Sepharose 6B afftnity column was dialyzed against starting buffer, concentrated by ultrafiltration, and run on an 8% SDS-PAGE gel. The gel wa_s fixed, dried, and autoradiographed. Lanes A and B contain purified, radiolabeled human serum Cl-INA run under nonreducing (A) and reducing (B) conditions. The band in each lane toward the bottom of the figure is at the level of the dye front. Lanes C and D contain the isolated U937 membrane proteins run under nonreducing (C) and reducing (D) conditions.
kDa band when the U937-derived material was electrophoresed under reducing conditions. In experiments in which the radiolabeled detergent-solubilized U937 membranes were first passed over anti-albumin Sepharose, subsequent antiCi-INA affinity chromatography still isolated a 105 kDa and 200 kDa band (data not shown). Western blotting of radiolabeled detergent-solubilized U937 membrane proteins with anti-CT-ZNA. Western blotting was performed on radiolabeled deter-
gent-solubilized U937 membrane proteins post anti-Ci-INA affinity chromatography to determine if anti-CT-INA would identify the same bands seen by SDSPAGE in the experiment described for Fig. 2. After SDS-PAGE, proteins were transferred to nitrocellulose and blotted using the same polyclonal rabbit antiCi-INA IgG antibody used in the affinity column described above. In Fig. 3 lanes A to D on the left are the nitrocellulose paper of the Western blot. Lanes A and B contain purified serum CT-INA run under nonreducing (A) and reducing (B) conditions. A single band with a M, of 105 kDa is seen in each lane. Lanes C and D contain U937 membrane proteins run unreduced (C) and reduced (D). Under these conditions, only a single band with a M, of 105 kDa identical to that of native human serum Ci-INA is seen in each lane, Lanes E and F on the right side of the figure are an autoradiogram of the Western blot shown on the left side of the figure. The bands seen in lanes E and F correspond to the position of lanes C and D in the blot, which contain the radiolabeled U937 membrane proteins. Thus, a 200 kDa band is present on the nitrocellulose, in addition to the 105 kDa band, yet
470
RANDAZZO
A
ET AL.
6
FIG,~. Western blot of surface radiolabeled detergent-solubilized U937 membrane proteins with anti-Cl-INA. U937 cells were radiolabeled using the lactoperoxidase method. Membrane proteins were solubilized in lysis buffer (see Materials and Methods) containing the detergent NP-40. The membrane proteins were then subjected to affinity chromatography on anti-CT-INA-Sepharose 4B. Specifically bound proteins were eluted from the affinity column with a linear NaCl gradient, The eluted material was subjected to 6% SDS-PAGE and transfemed passively to nitrocellulose paper. The nitrocellulose paper was then blotted with rabbit anti-CT-INA, followed by alkaline phosphataseconjugated goat anti-rabbit lg. Finally. an appropriate chromogenic substrate was added. After air drying, the blot was autoradiographed. Lanes A through D are the nitrocellulose paper of the Western blot. Lanes A and B contain purified human serum Ci-INA run under nonreducing (A) and reducing (B) conditions. Lanes C and D contain lJ937 membrane proteins run under nonreducing (C) and reducing (I)) conditions. Lanes E and F are an autoradiogram of the Western blot. The bands that are seen correspond in position to lanes C and D in the Western blot containing the U937 membrane proteins.
this 200 kDa protein does not appear to be identified by anti-CT-INA in the Western blot, while the 105 kDa protein is. Demonstration of the functional activity of U937 cell surface CT-INA. The Ci-INA functional activity of intact U937 cells was assessed by a hemolytic assay, which utilizes the ability of Ci-INA to inactivate Cl? and thus prevent CIS-induced destruction of C4. The results of a representative experiment are shown in Fig. 4. Maximal or 100% C4 dependent hemolysis for these experiments is defined as the amount of EA lysis caused by C4D GPS reconstituted with an optimal dose of C4 as described under Materials and Methods. It can be seen that preincubation of this C4 dose with ClS causes destruction of C4 and reduced C4 dependent hemolysis to 8% of maximal in this experiment. Incubation of increasing amounts of intact U937 cells with the ClJ prior to the addition of the C4 caused a dose-dependent inhibition of C Is-induced destruction of C4, as is apparent from the increase in C4 dependent hemolysis. Neutralization of U937 cell surface CT-INA activity by anti-CT-INA. To ascertain that the CT-INA activity observed with intact U937 cells was in fact due to a protein antigenically related to plasma Ci-INA, we performed experiments in which U937 cells were incubated with exogenous ClS in the presence or absence of monospecific, polyclonal IgG anti-Ci-INA at 100 p&ml during the entire 2.5-h]
U937 CELLS EXPRESS FUNCTIONAL
: ,L 0
5
10
U937
CELL
NUMBER
471
Ci-INA
15
20
(x~O-~)
FIG. 4. Inactivation of exogenous ClS by intact U937 cells. The CT-INA functional activity-of intact U937 cells was assessed by a C6dependent hemolytic assay that utilizes the ability of Cl-INA to inactivate Cl?., and thus prevent ClGinduced destruction of C4. Intact U937 cells ranging from 0 to 20 X lo6 were incubated _with 20 ng of ClS for 2.5 hr at 4°C in GVB. The cells were pelleted by centrifugation and the Cl-INA activity of each supematant was assayed by adding 10 CH, units of C4 and incubating for 45 min at 37°C. Finally, C4D GPS diluted 1:40 and EA were added to each sample and the residual CCdependent hemolytic activity of each sample was determined by measuring the amount of free hemoglobin released into the supematant. These data represent a typical experiment and each point is the mean 2 SD of triplicate determinations.
incubation at 4°C. In Fig. 5, the CT-INA activity of 1 x IO7 U937 cells as determined by C4 hemolytic assay is shown in the bar second from the left labeled “Cl5 + U937,” and is taken as maximal activity. All data in the figure are calculated as a relative percentage of the maximal C4 dependent hemolysis. Thus,
FIG. 5. Neutralization of U937 cell surface CT-INA activity by anti-Ci-INA. CT-INA activity as assessed by inactivation of exogenous ClS was determined for 1 X 10’ U937 cells using the C4dependent hemolytic assay, and this activity is designated as 100% (bar labeled “Cl: + U937 cells”). The bar labeled “ClS + U937 cells + anti-Cl-INA” shows th_e effect of incubating the cells with exogenous ClS in the prese_nce of monospecitic polyclonal anti-Cl-INA at 100 &ml. The bars labeled “Cl? and “ClS + anti-Cl-INA” show the activity of the exogenous ClS in the absence of U937 cells in the presence and absence of antibody, respectively. Data are expressed as the mean 2 SD of triplicate determinations.
472
RANDAZZO
ET AL.
in the absence of U937 cells, exogenous ClS reduces the C4 dependent lysis to 18% of maximal as shown in the figure by the bar on the far left. Addition of anti-Ci-INA causes a significant (P < 0.005) decrease in the Ci-INA activity of U937 cells. The bar on the far right represents a control, in which the effect of the antibody on ClS destruction of C4 was examined in the absence of U937 cells. Assay of U937 supernatants for Ci-INA, and C4 functional activity. These experiments were performed in an effort to establish that the CT-INA activity demonstrated by intact U937 cells in the previous experiments was in fact due to cell surface CT-INA, and not due to secretion of Ci-INA by the cells. Thus, we incubated cells in GVB under conditions indentical to those used in the previous experiments, and then assayed the cell-free supernatants for various functional activities. The first line in Table 1 is a buffer control and shows that the exogenous ClS has reduced C4 dependent lysis to 23.4% of the amount obtained in the absence of exogenous CIS. The second line shows that iqcubation of U937 supernatant with the CIS does not cause an increase in C4 dependent lysis compared to that observed for the buffer control. Thus, there is no detectable CT-INA functional activity in the cell-free supernatant under these experimental conditions. Since any addition of C4 to the assay system by the U937 cells would give results indistinguishable from CT-INA activity, it was important to assay U937 supernatants for C4 functional activity. The last two lines of the table show that the U937 supernatant has no significant C4 activity. Effect of PMA treatment on expression of surfuce Ci-INA by U937 cells. The effect of a 2-day treatment of U937 cells with PMA on the expression of Ci-INA was studied by immunofluorescent analysis. As seen in Table 2 treatment of U937 cells with PMA (160 nM) caused an increase in the percentage of cells expressing surface CT-INA. There was also a shift in the mean peak channel of positively TABLE 1 ASSAY OF U937 CELL-FREE SUPERNATANT FOR CT-INA, AND C4 ACTIVITY ___-U937 supematant
Reaction mixture ClS
C4D-GPS
+ + -
+
EA f
t t
t t
$6 Lybis
23.4 t 0.85 18.9 2 1.4 0 L 0.4 -+ + 2.4 t 1.6 --___ -.-. ~~~~~-..- --.. ~-~~ -~ ~~. -~~~~~~ ~~~ Note. U937 cells were incubated in 0.2 ml of GVB for 2.5 hr at 4°C. Next, the cells were pelleted by centrifugation and 175 p,l of supematant were removed from each sample. To assay for CT-INA functional activity (data shown by the first pair of lines in the table), 175 ~1 of the supematant or buffer as control, was incubated with 20 ng of Clsfor 45 min at 37°C. Following the incubation, 10 CH,, units of C4 were added and the mixture was incubated an additional 45 min at 37°C. Next, 200 )LI of a 1:40 dilution of C4D GPS was added to the mixture along with 100 ~1 of EA (1 x lo9 cells/ml). The mixture was incubated (3X, 60 min), centrifuged, and the percentage of lysis was determined by assaying the supematant for free hemoglobin spectrophotometrically. To assay for C4 functional activity, 175 ~1 of U937 supematant or buffer was added to C4D GPS and EAs as above, and incubated (60 min, 37°C). after which time the mixture was centrifuged and the percentage of hemoIysis was determined. Data are expressed as the mean * SD of triplicate determinations. + +
+ + -
c4
U937
CELLS
EXPRESS
FUNCTIONAL
473
Ci-INA
TABLE2 EFFECT
--__ Antibody
anti-Ci-INA OKMl -~
OF
PMA
TREATMENT
OF
U937 CELLS ON SURFACE
PMA concentration 0 160 0 160
(nM)
EXPRESSION
OF
CT-INA % Positive cells 15.3 36.4 8.3 19.4
f 2 ” 2
1.2* 0.9* 1.5** 1.2**
Note. U937 cells were examined for cell surface CT-INA and CR3 using immunofluorescence and FACS analysis. U937 cells were cultured for 48 hr in the absence or presence of PMA (160 nM) prior to immunofluorescent staining. These data represent the mean 2 SD of three separate experiments. *P
cells following PMA treatment. An experiment examining the effect of PMA on expression of U937 CR3 (C3bi receptor) as detected by the OKMI monoclonal antibody was included as a control to show that PMA-induced differentiation had occurred. PMA treatment has been shown by others to increase the number of U937 cells staining with OKMl (37). The results shown in Table 2 confirm this observation. staining
DISCUSSION The data presented here indicate that U937, a human monocyte-like cell line, expresses functional Ci-INA as a cell surface protein. We have shown previously (20) that U937 cells synthesize CT-INA, and since there is not antigenic crossreactivity between our anti-human CT-INA antibody and fetal calf serum, in which the U937 cells are grown, it is clear that the cell surface-expressed CT-INA is being synthesized by the cells and is not absorbed from the culture media. However, it is important to state at this point that all of our studies have been done with U937 cells cultured in the presence of unheated FCS, since we find that culture of the cells in heat-inactivated FCS causes a significant decrease in U937 CT-INA synthesis, as well as expression of cell surface Ci-INA (B. P. Randazzo, H. B. Fleit, A. P. Kaplan, and B. Ghebrehiwet, unpublished data). Thus, FCS, and specifically some as yet undetermined heat labile factor(s) in FCS, seems to be important in the regulation of Ci-INA synthesis and surface expression in this experimental system. Isolation of Ci-INA antigen from detergent-solubilized U937 membranes by affinity chromatography yields two species: one migrates identically with native serum Ci-INA in SDS-PAGE, while the second has a M, of approximately 200 kDa (Fig. 2). Neither of these two proteins were adsorbed to an irrelevant antibody affinity column (data not shown), thus lending support to a specific interaction of the 105 kDa and 200 kDa protein with the anti-Ci-INA affinity column. While both of these species were isolated by affinity column with anti-CT-INA antibody raised to human serum Ci-INA, the 200 kDa protein was not recognized by the same antibody in a Western blot (Fig. 3). This may indicate that the 200 kDa molecule is not antigenically related to Ci-INA. However, the intensity of the 200 kDa band in the autoradiogram (Fig. 3) is approximately one order of
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magnitude less than that of the 105 kDa band. Assuming that the two molecules radiolabel equally well (which may not be the case), then the concentration of the 200 kDa species is significantly lower than that of the 105 kDa in this experiment. Since the intensity of the 105 kDa band is only moderate in the Western blot (Fig. 3) it is quite possible that the Western blot is not sensitive enough to detect the 200 kDa band, which is presumably present in a much lower concentration. Thus, no firm conclusion can be drawn from these experiments as to whether the 200 kDa is antigenically related to Ci-INA (possibly a high molecular weight or dimeric form of the molecule) or antigenically unrelated. One possibility is that this 200 kDa cell surface protein copurifies with CT-INA during affinity chromatography because it is complexed with CT-INA. Perhaps the 200 kDa protein is the U937 cell surface receptor for Ci-INA. If this is the case then the U937 receptor for CT-INA would be significantly larger than the 58-64 kDa Ci-INA receptor isolated from leukocyte membranes (38). Under the low ionic strength conditions used during isolation of cell surface proteins this putative Ci-INA 200 kDa protein complex may bind to the affinity column via CT-INA. The complex would then dissociate under SDS-PAGE conditions yielding the two bands seen. However, the issue of a specific interaction between the 200 kDa protein and cell surface Ci-INA cannot be resolved from the experiments presented here. Experiments are now in progress to further characterize this 200 kDa protein. In the absence of such supporting data it is still plausible to speculate that the 200 kDa protein is simply a contaminant, which happens to bind to and elute from the affinity column with CT-INA. Another possibility that was considered, but which is quite unlikely, is that the 200 kDa protein could represent a complex of CT-INA and any one of the known plasma proteases, which CT-INA has been shown to inhibit. Clr, Cls, HF, plasmin, and kallikrein are all single chain proteins with a M, of approximately 85 kDa. Upon activation, these single chain proteases are cleaved within an intrachain disulfide bond yielding a 60 kDa amino terminal fragment and a 30 kDa carboxy terminal fragment, which remain disulfide linked. The 30 kDa fragment contains the active site of the enzyme with which CT-INA interacts forming an SDS nondissociable bimolecular complex. Thus, when CT-INA protease complexes are analyzed by SDS-PAGE, the molecular weight of the complex under nonreducing conditions is approximately 185 kDa. Under reducing conditions, however, reduction of the intrachain disultide bond of the enzyme results in a 135 kDa CiINA enzyme complex and a 60 kDa enzyme-derived fragment (1, 14). In the present study the A4, of the 200 kDa U937 surface protein remained unchanged upon reduction. The precise mode of association between the U937 cell membrane and surfaceexpressed Ci-INA cannot be determined from these studies. It is possible that the low molecular weight form of CT-INA is associated intrinsically via a low molecular weight covalent modification. Such attachment of proteins to cell surfaces via fatty acids is quite common (39), and in most cases fatty acid acylation of proteins does not significantly change the apparent molecular weight as determined by SDS-PAGE (40). Using a sensitive hemolytic assay we were able to show that intact U937 cells
U937 CELLS EXPRESS FUNCTIONAL
Ci-INA
475
can inactivate exogenous human CIS, indicating that the cell surface Ci-INA is functionally active. Moreover, this CT-INA functional activity is blocked by monospecific antibody to CT-INA, and this is almost certainly due to CT-INA and not some other protein. Because we and others have never been able to demonstrate secretion of antigenic Ci-INA by U937 cells (20, 41), and since there is no significant Cf-INA functional activity in U937 cell-free supernatants as assessed by hemolytic assay (Table l), we conclude that the CT-INA activity demonstrated for intact U937 cells is due to surface CT-INA. It seems that U937 cells synthesize CT-INA, which is not secreted freely into the culture media, but rather remains tightly associated with the cell surface. This is in contrast to peripheral bloodderived monocyte/macrophage cultures, which have been shown to secrete significant quantities of Ci-INA (19). Our observations are in line with those of Malhorta and Sim (24), who reported that factor H, which is synthesized by U937 cells and is expressed on their cell surface, is not secreted. However, the underlying mechanism of this phenomenon cannot be determined from these studies. This is the first report demonstrating the presence of functional Ci-INA on the surface of any cell type. However, there is a recent abstract that indicates that Ci-INA antigen is found on the surface of cultured human umbilical vein endothelial cells (42), and our own preliminary studies suggest that Ci-INA may be present on a variety of tumor cell lines such as Raji, Daudi, and HL-60 (B. P. Randazzo, H. B. Fleit, A. P. Kaplan, and B. Ghebrehiwet, manuscript in preparation). As reported previously and as seen in Table 2, immunofluorescent analysis indicates that Ci-INA is expressed on approximately 16% of U937 cells in culture (20). PMA has been shown to alter the phenotype of U937 cells toward that of a more differentiated monocyte-like cell (43). The fact that PMA induces an increase in the percentage of U937 cells expressing surface Ci-INA indicates that CT-INA expression occurs only at certain discrete stages during the ontogeny of monocytes. It is also probable that cells more differentiated than the monoblastoid U937 may express Ci-INA at a higher frequency. The constant low percentage of U937 cells in our cultures that express surface CT-INA may represent cells that are spontaneously differentiating. Experiments are presently in progress to examine the pattern of surface Ci-INA expression during monocyte ontogeny using peripheral blood monocytes and other monoblastoid cell lines in the presence of maturation inducers. While it is clear that U937 surface Ci-INA functions as a protease inhibitor, the physiologic and or pathophysiologic role of surface Ci-INA is yet to be determined. Most nucleated cells are relatively resistant to complement lysis (44, 45). There have been a number of complement regulatory proteins described on the surface of various cells such as CR1 (27, 46), factor H (24), and HRF (47), which are believed to play some role in protecting these cells from complement lysis. All of the complement regulatory cell surface proteins described to date control complement activation at the point of C3 activation (46) or membrane attack complex assembly (47). Thus, these complement regulatory proteins likely have a relatively more important role in controlling alternative pathway activation. Our description of functional cell surface Ci-INA is the first report of a cell surface complement control protein that would function primarily to control activation of
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the classical pathway at the cell surface. Considering the important regulatory roles played by plasma CT-INA in vivo, it is possible that cell surface Ci-INA may be important in preventing autocatalytic activation of monocyte-produced C1 during secretion. We hope to assess the possibility that other cell types also express surface CT-INA. ACKNOWLEDGMENTS The authors thank Ms. Joanne Thomas for technical assistance with FACS ratory of Dr. Paul Fisher for instruction in passive immunoblotting.
analysis,
and the lab
REFERENCES 1. Harpel, P. C., and Cooper, N. R., J. C/in. Invest. 55, 593, 197.5. 2. Sim. R. B.. Reboul. A., Arlaud, G. J., Villiers, C. L.. and Colomb, M. G., FEBS Len. 97, Ill, 1979. 3. Ziccardi, R. J.. J. Irnmunol. 126, 1768, 1981. 4. Forbes, C. O., Pensky, J., and Ratnoff, 0. D., J. Lab. Clin. Med. 76, 809, 1970. 5. de Agostini, A.. Lijnen. H. R.. Pixley, R. A., Colman, R. W., and Schapira, M., J. C/in. Invest. 73, 1542, 1984. 6. Pixley, R. A., Schapira. M., and Colman, R. W., J. Bid. Chem. 260, 1723, 1985. 7. Gigli. I., Mason, J. W., Colman. R. W., and Austen, K. F., J. Immunol. 107, 311, 1970. 8. Harpel, P. C.. Lewin. M. F., and Kaplan, A. P., J. Bio/. Chem. 260, 4257, 1985. 9. Ratnoff, 0. D., Pensky, J., Ogston, D., and Naff. G. D., J. Exp. Med. 129, 315. 1969. 10. Aoki, N.. Moroi, M., Matsuda. M., and Tachiya, K., J. C/in. Invest. 60, 361, 1977. 11. Harpel, P. C., J. C/in. Invest. 68, 46, 1981. 12. Arlaud, G. J.. Reboul. A., Sim, R. B.. and Colomb, M. G., B&him. Biophps. Actu, 576, 151. 1979. 13. Salvesen, G. S., Catanese, J. J.. Kress, L. F., and Travis, J.. J. Biol. Chem. 260, 2432, 1985. 14. van der Graff. F.. Koedam, J. A., Griffin, J. H., and Bouma, B. N.. Biochemistry 22,486O. 1983. 15. Ziccardi, R. J., J. Zmmunol. 128, 2505, 1982. 16. Ghebrehiwet, B., Randazzo, B., and Kaplan, A. P.. C/in. Immunol. Immunopathol. 32, 201, 1984. 17. Johnson. A. M., Alper. C. A.. Rosen, R. S.. and Graig. J. M., Science 173, 553, 1971. 18. Prandini, M. H., Reboul. A., and Colomb, M. G., Biochem. J. 237, 93. 1986. 19. Bensa, J. C., Reboul, A., and Colomb. M. G., Biochem. J. 216, 385. 1983. 20. Randazzo. B. P., Dattwyler. R. J., Kaplan, A. P.. and Ghebrehiwet, B.. J. Immunol. 135, 1313. 198.5. 21. Sundstrom, C., and Nilsson, K., Int. J. Cancer 17, 565, 1976. 22. Koren, H. S., Anderson, S. J., and Larrick. J. W., Nature (London) 279, 328. 1979. 23. Anderson, C. L., and Abraham, G. N.. J. Immunol. 125, 2735. 1980. 24. Malhotra. V., and Sim, R. B.. Eur. J. Immunol. 15, 93S, 1985. 25. Schmitt, M., Mussel, H. H., and Dierich. M., J. Zmmunol. 126, 2042, 1981. 26. Pangbum, M. K.. Schreiber, R. D., and Miiller Eberhard, H. J., .I. Exp. Med. 164, 257. 1977. 27. Fearon, D. T.. Proc. Natl. Acad. Sci. USA 76, 5867, 1979. 28. Reboul, A., Arlaud, G. J., Sim, R. B., and Colomb, M. G., FEBS Lett. 79, 45. 1977. 29. Gigli, I., Porter, R. R.. and Sim, R. B., Biochem. J. 157, 541. 1976. 30. Wright, S. D., Rao, P. E., van Voorhis. W. C.. Craigmyle, L. S., lida. K., Talle, M. A., Westberg, E. F., Goldstein, G., and Silverstein, S. C.. Pvoc. Nut/. Acad. Sci. USA 80, 5699. 1983. 31. Bolton. A. E., and Hunter. W. M., Biochem. J. 133, 529, 1973. 32. Morrison, M., In “Methods in Enzymology” (S. Fleischer and L. Packer, Eds.), Vol. 32. p. 103, Academic Press, New York, 1974. 33. March, S. C., Parish, I., and Cuatrecasas, R., Anal. Biochem. 60, 149. 1974. 34. Laemmli, U. K., Nature (London) 227, 680. 1970. 35. Southern, E. M., J. Mol. Biol. 98, 503, 1975. 36. Minta, J. O., and Aziz. E., J. fmmunol. 129, 250. 1981.
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37. Gilbert, D., Peulve, P., Daveau, M., Ripoche, J., and Fontaine, M., Eur. .I. Immunol. 15, 986, 1985. 38. Chang, N. S.. Leu, R. W., and Boackle, R. J., In “Membrane Proteins” (S. C. Goheen, Ed.), p. 295, Bio-Rad Labs., 1987. 39. Schlesinger, M. J., Magee, A. I., and Schmidt, M. F. G., .I. Biol. Chem. 225, 10021, 1980. 40. Omary, M. B., and Trowbridge, I., J. Biol. Chem. 256, 4715, 1980. 41. Minta, J. 0.. and Pambrun, L., Fed. Proc. 44, 991, 1985. 42. Schmair. A. H., Farber, A., Lundberg, D., Murray, S., Kuo, A., and Cines, D. B., “Kinin 1987, Tokyo International Congress, p. 82, 1987. [Abstract 8-13-181 43. Harris, R., and Ralph, P., J. Leukocyte Biol. 37, 407, 1985. 44. Schreiber, R. D., Pangbum, M. K., Medicus. R. G., and Miiller-Eberhard. H. J.. C/in. Zmmunol. Immunoparhol. 15, 384, 1980. 45. Morgan, B. P., Imagawa. D. K., Dankert. J. R.. and Ramm, L. E., J. Zmmunol. 136, 3402, 1986. 46. Ross, G. D., In “Immunobiology of the Complement System” (G. D. Ross, Ed.), p. 87, Academic Press, San Diego, 1986. 47. Podack, E. R., In “Immunobiology of the Complement System” (G. D. Ross, Ed)., p. 130, Academic Press, San Diego, 1986. Received June 24, 1988; accepted August 16, 1988
IMMUNOLOGY
AND
Expression
IMMUNOPATHOLOGY
49,
463-477 (1988)
of Functional Cell Surface CT-Inactivator U937 Cells’*2
by
BRUCE P. RANDAZZO,*~ HOWARD B. FLEIT,~ ALLEN P. KAPLAN,* AND BERHANE GHEBREHIWET*+~ *The Department fThe Department
of Medicine, Division of Allergy, of Pathology, State University
Rheumatology, of New York,
and Clinical Stony Brook,
Immunology, and New York 11794
-We have previously shown that th_e human monocyte-like cell line U937 synthesizes Cl-INA and expresses cell surface Cl-INA. In this report we provide evidence that this surface-expressed Cl-INA is functionally active. Intact U937 cells demonstrated functional Cl-INA activity in a hemolytic assay. Th& activity was blocked when the cells were incubated with monospecific antibody to Cl-INA, and was not detectable in cellfree supematants of U937 cells. SDS-PAGE analysis of radiolabeled U937 cell surface proteins purilied by anti-Cl-INA affinity chromatography revealed two distinct bands. One protein had a M, of 105 kDa identical to plasma Cl-INA, and the second had a M, of 200 kDa. We werEunable to determine the identity of the 200 kDa protein by Western blotting with anti-Cl-INA. However, the possibility _exists that this 200 kDa molecule may represent a Cl-INA regeptor, a dimeric form of Cl-INA, or an unrelated cell surface protein with affinity for Cl-INA. Furthermore, we show that treatment of U937 cells w$h phorbol ester resulted in an increase in the percentage of cells expressing surface Cl-INA. These results suggest that U937 cells express functional cell surface Cl-INA, which could function in vivo to protect these human tumor cells from lysis by host COIIlphllent.
0 1988 Academic
Press, Inc.
INTRODUCTION Human CT-inactivator (CT-INA) or a,-neuraminoglycoprotein is a plasma protease inhibitor with activity against a number of serine proteases belonging to both the complement system and the intrinsic coagulation pathway. While it is the only known plasma inhibitor of activated Cl? and ClS (l-3), Ci-INA also plays a major role in inhibiting the activities of several coagulation enzymes including factors XIIa and XIIf (HFa and HFfJ4 (4-6) and plasma kallikrein (7, 8). Inactivation of plasmin by Ci-INA in a purified system has been demonstrated, but the relative contribution of Ci-INA to the inactivation of plasmin in plasma is probably not ’ This investigation was supported by Grants AI 16337-05 and CA 41047 from the National Institute of Health, and 860742 from the American Heart Association (National Center). ’ This publication represents part of B.P.R.‘s work, performed in partial fulfillment of the Ph.D. degree in experimental pathology. 3 To whom correspondence should be addressed at the Division of Allergy, SUNY at Stony Brook, HSC T-16 Room 040, Stony Brook, NY, 11794-8161. 4 Abbreviations used: HFa, activated Hageman factor (or factor XIIa); HR, Hageman factor fragment (or factor XIIf); DMSO, dimethyl sulfoxide; EDTA, ethylenediaminetetraacetic acid; EACA, epsilon-amino-caproic acid; PMSF, phenylmethylsulfonyl fluoride; C4D GPS, C4 deficient guinea pig serum; EA, antibody sensitized sheep red blood cell; BSA, bovine serum albumin; ELISA, enzymelinked immunosorbent assay; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 463 0090-1229/88 $1.50 Copyright All rights
Q 19% by Academic Press, Inc. of reproduction in any form reserved.
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ET
AL.
significant (9-l 1). Sodium dodecyl sulfate-polyacrylamide gel electrophoretic (SDS-PAGE) analysis of Ci-INA-enzyme complexes has shown that CT-INA forms nondissociable 1: 1 stoichiometric bimolecular complexes with the enzymes it inhibits (1, 5, 12-14). In addition to its ability to inactivate serine proteases through complex formation, Ci-INA also serves as a major homeostatic component of the classical pathway of complement, since it prevents the spontaneous autocatalytic activation of the first component of complement (Cl) (15, 16). Thus, Ci-INA plays several critical roles in complement activation including stabilization of macromolecular Cl, the inactivation of Ci subcomponents, and serves as a major inhibitor of the initiation phase of the contact coagulation-kinin-forming pathways. While the majority of plasma CT-INA is probably synthesized in the liver f 17, IQ, it is becoming increasingly clear that cells of the monocyte-macrophage series represent another potential source of Ci-INA (19,20). The U937 cel1 line is believed to represent a promonocyte cell type, and manifests several features of normal human monocytes (21-23). Using the technique of metabolic labeling, we have previously shown that U937 cells synthesize Ci-INA, and demonstrated that these cells express cell surface CT-INA as assessed by fluorescence-activated cell sorter analysis (20). It has been shown recently that U937 cells express cell surface factor H (24) and the complement C3b receptor CR1 (25), both of which function to markedly enhance the proteolytic inactivation of C3b (26, 27). Thus, U937 cells express a variety of cell surface proteins, which normally serve to control complement activation and could perhaps serve to protect these cells from complement-mediated lysis. While the functional activity of U937 surfaceexpressed CR1 and factor H has been established (24), no information on the functional state of cell surface CT-INA has been reported. In this study we present evidence that CT-INA expressed on the surface of U937 cells is functionally active, and present data which show that two molecular species are affinity purified (anti-Ci-INA Sepharose 4B) from detergentsolubilized U937 membranes. The first has a molecular weight identical to serum Ci-INA and is identified in Western blots by antibody to Ci-INA. The second has a molecular weight of 200 kDa. We will discuss the possible biologic significance of this cell surface-associated multispecific enzyme inhibitor. MATERIALS
AND METHODS
Buffers and reagents. The following buffers were used: HBSS. Hanks’ balanced salt solution, pH 7.4, containing calcium (1.4 g/liter), magnesium (1.0 gi liter), and phenol red (GIBCO, Grand Island, NY); PBS, phosphate-buffered saline, consisting of 100 mM phosphate and 0.9% NaCl, pH 7.2; GVB, isotonic veronal-buffered saline, containing 0.15 mM CaC12. 0.5 m&4 MgCl?, 0.02% NaN,, and 0.1% gelatin, pH 7.2; and 4B-phorbol If-myristate 13-acetate (PMA) purchased from Sigma Chemical Co. (St. Louis, MO), dissolved in DMSO at a stock concentration of 0.3 mg/ml, and stored frozen at - 80°C. Purifiedproteins and antibodies. CT-INA was purified according to a procedure described by Reboul et al. (28), ClS was purified and activated according to the method of Gigli et al. (29). Production of monospecific polyclonal rabbit anti-
U937 CELLS EXPRESS FUNCTIONAL
Ci-INA
465
human CT-INA IgG and F(ab’), fragment has been described elsewhere (20). The antibody is monospecific by immunoelectrophoresis against normal human serum, and does not cross-react with fetal calf serum as assessed by Ouchterlony or ELISA. The cell line producing monoclonal antibody OKMl was obtained from the American Type Culture Collection (Rockville, MD) and was grown in Iscove’s modified Dulbecco’s medium supplemented with 20% FCS. OKMl has been shown to bind to the complement C3bi receptor (CR3) at a site distinct from the ligand-binding site (30). Spent culture supematants were used as the source of OKMl . Fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit Ig and FITC-conjugated goat anti-mouse Ig were purchased from Cappel Worthington Biochemicals (Malvern, PA). Guinea pig C4 and C4 deficient guinea pig serum (C4D GPS) used in the hemolytic assays were purchased from Cordis Laboratories Inc. (Miami, FL). Bovine serum albumin (fraction V powder) was purchased from Sigma Chemical Co. Cell line. The U937 cell line was propagated in suspension culture in RPM1 1640 containing 10% fetal calf serum, 100 units/ml penicillin, and 100 @/ml of streptomycin (GIBCO). It was maintained in a humidified atmosphere consisting of 5% CO2 and 95% air. Viability was assessed by trypan blue exclusion before all experiments, and was always greater than 95%. Radioiodination of proteins. Purified human serum Ci-INA was labeled with 1251by the method of Bolton and Hunter (31). Briefly, CT-INA was suspended in 0.1 A4 sodium borate buffer, pH 8.5, and 250 &i of Bolton Hunter reagent (New England Nuclear, Boston MA) was added, and the reaction proceeded for 3 hr on ice. Unbound ‘25I was removed by extensive dialysis of the protein against PBS, pH 7.4. Radioiodination of cell surface proteins. Surface radiolabeling of U937 cell membrane proteins was carried out by the lactoperoxidase-catalyzed reaction method (32). Viable U937 cells (generally, 5 x 10*-l x 109) were removed from log phase cultures, and were washed three times in HBSS. The cells were then resuspended in 1 ml of HBSS containing 65 kg of lactoperoxidase (Sigma Chemical Co.) and 1 mCi of carrier-free 1251(New England Nuclear), and the reaction mixture was kept on ice. Next, a 15+1 aliquot of a l:lO,OOO dilution of 30% H202 (Sigma Chemical Co.) in HBSS was added every 15 set until a total of five additions had been made. The radiolabeled cells were then pelleted by centrifugation, the supernatant was removed, and the cells were washed five times with HBSS. Isolation of U937 cell surface Ci-ZNA. Radiolabeled U937 cells were resuspended in 5 ml of buffer containing 10 mJ4 Na,HPO,, 20 mM NaC 1,2 m&Z EDTA, 10 mM EACA, 20 mA4 iodoacetamide, 0.5 rr&! PMSF, and 0.02% NaN,, pH 7.4 (lysis buffer). The cells were then lysed by three cycles of freezing and thawing. The cell membranes were pelleted by centrifugation for 1 hr at 30,OOOg at 4”C, and were then washed and pelleted three times in 40 ml of lysis buffer. The pelleted membranes were next solubilized in 50 ml of lysis buffer containing 1% NP-40 (v/v) and stirred for 20 hr at 4°C. The solubilized membrane proteins were separated from insoluble material by centrifugation at 30,OOOg (60 min at 4”C), and were then dialyzed against lysis buffer containing 0.1% NP-40. This material was
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applied to an anti-CT-INA aftinity column, which was constructed as previously described (20, 33), and equilibrated with the same buffer used for dialysis (starting buffer). The column was washed with starting buffer until the radioactivity in the effluent was negligible. The specifically bound material was then eluted with a linear NaCl concentration gradient consisting of 50 ml of starting buffer and 50 ml of the same buffer containing 1 M NaCl. The eluted material was pooled, dialyzed against starting buffer, and concentrated using an Amicon ultrafiltration cell fitted with a UM-10 membrane (Amicon Corp., Danvers, MA). In some experiments the radiolabeled detergent-solubilized U937 membrane proteins, prepared as described above, were applied onto an anti-albumin Sepharose 4B column in an attempt to remove nonspecifically adsorbing proteins. The membrane proteins were loaded onto the column, and the column was washed with 10 bed volumes of starting buffer. Material that passed through this column unbound was then applied onto the anti-CT-INA column as described above. SDS-PAGE analysis of U937 cell surface Ci-INA. The material eluted from the affinity column was subjected to 8% SDS-PAGE using the method described by Laemmli (34). Samples to be reduced were boiled for 3 min in sample buffer containing P-mercaptoethanol at a final concentration of 1%. Each gel was run with prestained high molecular weight markers (Bethesda Research Laboratories, Gaithersburg, MD) and radiolabeled human serum CT-INA. After electrophoresis, the gel was fixed in a mixture of 7% acetic acid, 40% methanol in H,G, vacuum dried, and autoradiographed at -80°C using Kodak XAR film (Kodak, Rochester, NY). Western blot analysis affinity chromatography.
of U937 membrane
proteins
isolated
by anti-Cl-INA
U937 cells (5 x lo*) were surface radiolabeled using the lactoperoxidase method. The membrane proteins were solubilized in NP-40 and applied onto an anti-Ci-INA aflinity column as described above. The radiolabeled material eluted from the affinity column with a NaCl gradient was pooled, concentrated by ultrafiltration, and subjected to 6% SDS-PAGE. One lane containing prestained rainbow molecular weight markers (Amersham, Arlington Heights, IL) was included in all gels to estimate molecular weights and to evaluate the efficiency of transfer. Following electrophoresis, the proteins were passively transferred to nitrocellulose paper (pore size 0.2 pm, Schleicher and Schuell Inc., Keene, NH) using a method described by Southern (35). Briefly, the gel was placed on a piece of blotter paper on a flat piece of glass. The blotter paper was made continuous with a reservoir containing a buffer consisting of 8 mM Tris base (Sigma Chemical Co.) and 58 nuV glycine. The nitrocellulose paper was placed in contact with the gel with a piece of filter paper placed over it. An approximately 3-in. layer of paper towel was placed over the filter paper, and a heavy book was placed on top. The transfer was carried out for 4 days at room temperature. After transfer, the nitrocellulose paper was incubated for 60 min at 37°C in 0.1% BSA (w/v) in PBS with 0.05% Tween 20 (v/v). Next, the paper was incubated for 2 hr at 37°C in PBS-Tween with polyclonal rabbit IgG anti-CT-INA at approximately 1 kg/ml. The paper was washed for 90 min with PBS-Tween, and was then incubated for 1 hr at 37°C with alkaline phosphatase-conjugated affmity-purified
U937 CELLS EXPRESS FUNCTIONAL
Ci-INA
467
goat anti-rabbit IgG (Cappel Corp.) diluted 1: 1000 in PBS-Tween. After a 90-min wash in PBS-Tween and a brief wash in HzO, a 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium-based substrate in Tris buffer (Kirkegaard and Perry Labs. Inc., Gaithersburg, MD) was added and incubated at 37°C until purple bands appeared on the paper. After the nitrocellulose paper had air dried, it was autoradiographed at - 80°C using Kodak XAR film (Kodak). Determination of Ci-ZNA functional activity. CT-INA functional activity was assayed using a C4 dependent hemolytic assay essentially as described by Minta and Aziz (36). Briefly, we first established that 10 CHsO units of C4 would reconstitute the maximal hemolytic activity of 200 ~1 of a 1:40 dilution of C4 deficient guinea pig serum (C4D-GPS) in GVB when incubated with 100 l.~l of antibody sensitized sheep erythrocytes (EA) at 1 x 109/ml. Next, we determined that preincubation of 20 ng of ClS with 10 CHS, units of C4 for 45 min at 37°C prior to addition of the diluted C4D-GPS caused a reduction in C4 dependent lysis to approximately 10% of maximal. Finally, we established that preincubation of varying doses of purified human serum Ci-INA with the 20 ng of Ci could cause a dose-dependent inhibition of Cl<-induced C4 destruction. To assay the functional activity of U937 cell surface CT-INA, various numbers of U937 cells were suspended in GVB and 20 ng of ClS was added. After adjusting the volume of each sample to 0.2 ml with GVB, the suspensions were incubated for 2.5 hr at 4°C with gentle rocking. The cells were then pelleted by centrifugation at 8OOg, and 0.175 ml of the supernatant was removed from each sample and transferred to a separate tube. Next, 10 CHso units of C4 were added to each supernatant, and the mixture was further incubated for 45 min at 37°C. Finally, 200 ~1 of a 1:40 dilution of C4D-GPS and 100 l.~l of EAs at 1 x 109/ml were added to each tube. After a l-hr incubation at 37”C, each tube was centrifuged at 8OOg and the percentage of lysis of each sample was determined by assaying for free hemoglobin in the supernatant spectrophotometrically at 412 nm. Fluorescence-activated cell sorter analysis of 11937 cells with and without PMA-induced differentiation. U937 cells were cultured in plastic bacteriological culture dishes at 5 x lo5 cells/ml in a total volume of 4 ml of RPM1 1640 with 10% FCS, with or without PMA at a final concentration of 160 ti. After 2 days in culture, cells were gently scraped from the plastic dishes with a Teflon policeman, washed three times in HBSS, counted in the presence of trypan blue, and resuspended in HBSS containing 1% BSA and 0.02% NaN3 (HBSS-BSA). Then, 1 x lo6 U937 cells in HBSS-BSA were incubated either with rabbit F(ab’), antiCi-INA (100 kg/ml) or OKM 1 monoclonal antibody (2 pglrni) for 30 min at 4°C. After incubation, the cells were washed three times with HBSS-BSA. Cells stained for CT-INA were incubated with FITC-conjugated goat anti-rabbit IgG at 40 pg/ml while cells stained with OKMl were incubated with FITC-conjugated goat anti-mouse Ig at 50 p&ml in HBSS-BSA for 30 min at 4°C. After incubation, the cells were washed three times in HBSS-BSA and were resuspended at 1 x LO6 cells/ml in HBSS-BSA with 1% formalin. Flow microfluorometry was performed using an EPICS V fluorescence-activated cell sorter (FACS, Coulter, Hialeah, Fla). Data are shown as the log of integrated green fluorescence gated on forward
468
RANDAZZO
ET AL.
angle light scatter and log of 90” light scatter. In each case, control histograms were obtained for cells stained in the absence of primary antibody, and were subtracted from appropriate experimental samples to account for nonspecific staining. RESULTS Isolation of surface-expressed CT-INA. Elution of the radiolabeled detergentsolubilized U937 membrane proteins from the Sepharose 6B-anti-Ci-INA affinity column was accomplished with a linear NaCl gradient. As the elution profile in Fig. 1 shows, one single peak of radioactive material was eluted under these conditions. The first, unadsorbed peak contained a total of approximately 3.3 1 x IO6 cpm of radiolabeled U937 membrane proteins, while a total of 76,370 cpm were contained in the second peak, which was eluted with the NaCl gradient. Thus, the specifically bound and eluted material represents approximately 2.3% of the total detergent-solubilized surface-labeled U937 membrane proteins. SDS-PAGE analysis of surface-expressed CT-INA. The material eluted from the anti-Ci-INA affinity column was concentrated, and was then analyzed by 8% SDS-PAGE and autoradiography. As shown in Fig. 2, the U937-derived material contains two distinct proteins with different molecular weights. One migrates with an apparent M, that is similar to that of the radiolabeled human serum CT-INA, which is 105 kDa, and the other has an apparent molecular weight of approximately 200 kDa. No shift in mobility was observed for either the 105 kDa or 200
600 i 500.
l\+
I\
.Y.400+ / * - 200c” 3 100- i1 z I 30 20 5 8 roi! + 0 * 0
.“.
+\+ \*\ \+ \
16o . .-I50 t - -{40 +\+ \ +\ . i 130 I /20 +. 14: ~ +-•. ‘* j ‘0 +-+, 1. .. . . . .C’*-+.+-+.~ /. ‘* *llfllll ; .~~ ~ : .~.~ .2?2d 0 50 6” 70 80 90 400 FRACTIONS
-FIG. I. Affinity chromatography of solubilized U937 cell membrane proteins on an antiCl-INA-Sepharose 6B affinity column. U937 cells (5 x 10’) were surface labeled with 12’1 by lactoperoxidase catalyzed iodination, and lysed by freeze thawing in 5 ml of buffer consisting of 10 mM Na2HP0,, 20 m&f NaCl, 2 mM EDTA, 10 mM EACA, 20 mM iodoacetamide, 0.5 mM PMSF, and 0.02% NaN,, pH 7.4 (lysis buffer). The membrane fraction was isolated by centrifugation at 30,OOOg and was solubilized in lysis buffer made 1% with NP-40. The membrane proteins were then apphed onto an anti-Cl-INA-Sepharose 6B affinity column equilibrated with the above buffer made 0.1% with NP-40. After washing, the specifically bound protein was eluted with a linear NaCl gradient and 3.5-m] fractions were collected. This figure shows a typical elution profile from a series of four independent experiments.
U937
CELLS
A
EXPRESS
B
Ci-INA
FUNCTIONAL
C
469
D
200
(kD)
66
43
FIG:~. SDS-PAGE analysis of U937 cell surface-expressed CT-INA. The material eluted from the anti-Cl-INA-Sepharose 6B afftnity column was dialyzed against starting buffer, concentrated by ultrafiltration, and run on an 8% SDS-PAGE gel. The gel wa_s fixed, dried, and autoradiographed. Lanes A and B contain purified, radiolabeled human serum Cl-INA run under nonreducing (A) and reducing (B) conditions. The band in each lane toward the bottom of the figure is at the level of the dye front. Lanes C and D contain the isolated U937 membrane proteins run under nonreducing (C) and reducing (D) conditions.
kDa band when the U937-derived material was electrophoresed under reducing conditions. In experiments in which the radiolabeled detergent-solubilized U937 membranes were first passed over anti-albumin Sepharose, subsequent antiCi-INA affinity chromatography still isolated a 105 kDa and 200 kDa band (data not shown). Western blotting of radiolabeled detergent-solubilized U937 membrane proteins with anti-CT-ZNA. Western blotting was performed on radiolabeled deter-
gent-solubilized U937 membrane proteins post anti-Ci-INA affinity chromatography to determine if anti-CT-INA would identify the same bands seen by SDSPAGE in the experiment described for Fig. 2. After SDS-PAGE, proteins were transferred to nitrocellulose and blotted using the same polyclonal rabbit antiCi-INA IgG antibody used in the affinity column described above. In Fig. 3 lanes A to D on the left are the nitrocellulose paper of the Western blot. Lanes A and B contain purified serum CT-INA run under nonreducing (A) and reducing (B) conditions. A single band with a M, of 105 kDa is seen in each lane. Lanes C and D contain U937 membrane proteins run unreduced (C) and reduced (D). Under these conditions, only a single band with a M, of 105 kDa identical to that of native human serum Ci-INA is seen in each lane, Lanes E and F on the right side of the figure are an autoradiogram of the Western blot shown on the left side of the figure. The bands seen in lanes E and F correspond to the position of lanes C and D in the blot, which contain the radiolabeled U937 membrane proteins. Thus, a 200 kDa band is present on the nitrocellulose, in addition to the 105 kDa band, yet
470
RANDAZZO
A
ET AL.
6
FIG,~. Western blot of surface radiolabeled detergent-solubilized U937 membrane proteins with anti-Cl-INA. U937 cells were radiolabeled using the lactoperoxidase method. Membrane proteins were solubilized in lysis buffer (see Materials and Methods) containing the detergent NP-40. The membrane proteins were then subjected to affinity chromatography on anti-CT-INA-Sepharose 4B. Specifically bound proteins were eluted from the affinity column with a linear NaCl gradient, The eluted material was subjected to 6% SDS-PAGE and transfemed passively to nitrocellulose paper. The nitrocellulose paper was then blotted with rabbit anti-CT-INA, followed by alkaline phosphataseconjugated goat anti-rabbit lg. Finally. an appropriate chromogenic substrate was added. After air drying, the blot was autoradiographed. Lanes A through D are the nitrocellulose paper of the Western blot. Lanes A and B contain purified human serum Ci-INA run under nonreducing (A) and reducing (B) conditions. Lanes C and D contain lJ937 membrane proteins run under nonreducing (C) and reducing (I)) conditions. Lanes E and F are an autoradiogram of the Western blot. The bands that are seen correspond in position to lanes C and D in the Western blot containing the U937 membrane proteins.
this 200 kDa protein does not appear to be identified by anti-CT-INA in the Western blot, while the 105 kDa protein is. Demonstration of the functional activity of U937 cell surface CT-INA. The Ci-INA functional activity of intact U937 cells was assessed by a hemolytic assay, which utilizes the ability of Ci-INA to inactivate Cl? and thus prevent CIS-induced destruction of C4. The results of a representative experiment are shown in Fig. 4. Maximal or 100% C4 dependent hemolysis for these experiments is defined as the amount of EA lysis caused by C4D GPS reconstituted with an optimal dose of C4 as described under Materials and Methods. It can be seen that preincubation of this C4 dose with ClS causes destruction of C4 and reduced C4 dependent hemolysis to 8% of maximal in this experiment. Incubation of increasing amounts of intact U937 cells with the ClJ prior to the addition of the C4 caused a dose-dependent inhibition of C Is-induced destruction of C4, as is apparent from the increase in C4 dependent hemolysis. Neutralization of U937 cell surface CT-INA activity by anti-CT-INA. To ascertain that the CT-INA activity observed with intact U937 cells was in fact due to a protein antigenically related to plasma Ci-INA, we performed experiments in which U937 cells were incubated with exogenous ClS in the presence or absence of monospecific, polyclonal IgG anti-Ci-INA at 100 p&ml during the entire 2.5-h]
U937 CELLS EXPRESS FUNCTIONAL
: ,L 0
5
10
U937
CELL
NUMBER
471
Ci-INA
15
20
(x~O-~)
FIG. 4. Inactivation of exogenous ClS by intact U937 cells. The CT-INA functional activity-of intact U937 cells was assessed by a C6dependent hemolytic assay that utilizes the ability of Cl-INA to inactivate Cl?., and thus prevent ClGinduced destruction of C4. Intact U937 cells ranging from 0 to 20 X lo6 were incubated _with 20 ng of ClS for 2.5 hr at 4°C in GVB. The cells were pelleted by centrifugation and the Cl-INA activity of each supematant was assayed by adding 10 CH, units of C4 and incubating for 45 min at 37°C. Finally, C4D GPS diluted 1:40 and EA were added to each sample and the residual CCdependent hemolytic activity of each sample was determined by measuring the amount of free hemoglobin released into the supematant. These data represent a typical experiment and each point is the mean 2 SD of triplicate determinations.
incubation at 4°C. In Fig. 5, the CT-INA activity of 1 x IO7 U937 cells as determined by C4 hemolytic assay is shown in the bar second from the left labeled “Cl5 + U937,” and is taken as maximal activity. All data in the figure are calculated as a relative percentage of the maximal C4 dependent hemolysis. Thus,
FIG. 5. Neutralization of U937 cell surface CT-INA activity by anti-Ci-INA. CT-INA activity as assessed by inactivation of exogenous ClS was determined for 1 X 10’ U937 cells using the C4dependent hemolytic assay, and this activity is designated as 100% (bar labeled “Cl: + U937 cells”). The bar labeled “ClS + U937 cells + anti-Cl-INA” shows th_e effect of incubating the cells with exogenous ClS in the prese_nce of monospecitic polyclonal anti-Cl-INA at 100 &ml. The bars labeled “Cl? and “ClS + anti-Cl-INA” show the activity of the exogenous ClS in the absence of U937 cells in the presence and absence of antibody, respectively. Data are expressed as the mean 2 SD of triplicate determinations.
472
RANDAZZO
ET AL.
in the absence of U937 cells, exogenous ClS reduces the C4 dependent lysis to 18% of maximal as shown in the figure by the bar on the far left. Addition of anti-Ci-INA causes a significant (P < 0.005) decrease in the Ci-INA activity of U937 cells. The bar on the far right represents a control, in which the effect of the antibody on ClS destruction of C4 was examined in the absence of U937 cells. Assay of U937 supernatants for Ci-INA, and C4 functional activity. These experiments were performed in an effort to establish that the CT-INA activity demonstrated by intact U937 cells in the previous experiments was in fact due to cell surface CT-INA, and not due to secretion of Ci-INA by the cells. Thus, we incubated cells in GVB under conditions indentical to those used in the previous experiments, and then assayed the cell-free supernatants for various functional activities. The first line in Table 1 is a buffer control and shows that the exogenous ClS has reduced C4 dependent lysis to 23.4% of the amount obtained in the absence of exogenous CIS. The second line shows that iqcubation of U937 supernatant with the CIS does not cause an increase in C4 dependent lysis compared to that observed for the buffer control. Thus, there is no detectable CT-INA functional activity in the cell-free supernatant under these experimental conditions. Since any addition of C4 to the assay system by the U937 cells would give results indistinguishable from CT-INA activity, it was important to assay U937 supernatants for C4 functional activity. The last two lines of the table show that the U937 supernatant has no significant C4 activity. Effect of PMA treatment on expression of surfuce Ci-INA by U937 cells. The effect of a 2-day treatment of U937 cells with PMA on the expression of Ci-INA was studied by immunofluorescent analysis. As seen in Table 2 treatment of U937 cells with PMA (160 nM) caused an increase in the percentage of cells expressing surface CT-INA. There was also a shift in the mean peak channel of positively TABLE 1 ASSAY OF U937 CELL-FREE SUPERNATANT FOR CT-INA, AND C4 ACTIVITY ___-U937 supematant
Reaction mixture ClS
C4D-GPS
+ + -
+
EA f
t t
t t
$6 Lybis
23.4 t 0.85 18.9 2 1.4 0 L 0.4 -+ + 2.4 t 1.6 --___ -.-. ~~~~~-..- --.. ~-~~ -~ ~~. -~~~~~~ ~~~ Note. U937 cells were incubated in 0.2 ml of GVB for 2.5 hr at 4°C. Next, the cells were pelleted by centrifugation and 175 p,l of supematant were removed from each sample. To assay for CT-INA functional activity (data shown by the first pair of lines in the table), 175 ~1 of the supematant or buffer as control, was incubated with 20 ng of Clsfor 45 min at 37°C. Following the incubation, 10 CH,, units of C4 were added and the mixture was incubated an additional 45 min at 37°C. Next, 200 )LI of a 1:40 dilution of C4D GPS was added to the mixture along with 100 ~1 of EA (1 x lo9 cells/ml). The mixture was incubated (3X, 60 min), centrifuged, and the percentage of lysis was determined by assaying the supematant for free hemoglobin spectrophotometrically. To assay for C4 functional activity, 175 ~1 of U937 supematant or buffer was added to C4D GPS and EAs as above, and incubated (60 min, 37°C). after which time the mixture was centrifuged and the percentage of hemoIysis was determined. Data are expressed as the mean * SD of triplicate determinations. + +
+ + -
c4
U937
CELLS
EXPRESS
FUNCTIONAL
473
Ci-INA
TABLE2 EFFECT
--__ Antibody
anti-Ci-INA OKMl -~
OF
PMA
TREATMENT
OF
U937 CELLS ON SURFACE
PMA concentration 0 160 0 160
(nM)
EXPRESSION
OF
CT-INA % Positive cells 15.3 36.4 8.3 19.4
f 2 ” 2
1.2* 0.9* 1.5** 1.2**
Note. U937 cells were examined for cell surface CT-INA and CR3 using immunofluorescence and FACS analysis. U937 cells were cultured for 48 hr in the absence or presence of PMA (160 nM) prior to immunofluorescent staining. These data represent the mean 2 SD of three separate experiments. *P
cells following PMA treatment. An experiment examining the effect of PMA on expression of U937 CR3 (C3bi receptor) as detected by the OKMI monoclonal antibody was included as a control to show that PMA-induced differentiation had occurred. PMA treatment has been shown by others to increase the number of U937 cells staining with OKMl (37). The results shown in Table 2 confirm this observation. staining
DISCUSSION The data presented here indicate that U937, a human monocyte-like cell line, expresses functional Ci-INA as a cell surface protein. We have shown previously (20) that U937 cells synthesize CT-INA, and since there is not antigenic crossreactivity between our anti-human CT-INA antibody and fetal calf serum, in which the U937 cells are grown, it is clear that the cell surface-expressed CT-INA is being synthesized by the cells and is not absorbed from the culture media. However, it is important to state at this point that all of our studies have been done with U937 cells cultured in the presence of unheated FCS, since we find that culture of the cells in heat-inactivated FCS causes a significant decrease in U937 CT-INA synthesis, as well as expression of cell surface Ci-INA (B. P. Randazzo, H. B. Fleit, A. P. Kaplan, and B. Ghebrehiwet, unpublished data). Thus, FCS, and specifically some as yet undetermined heat labile factor(s) in FCS, seems to be important in the regulation of Ci-INA synthesis and surface expression in this experimental system. Isolation of Ci-INA antigen from detergent-solubilized U937 membranes by affinity chromatography yields two species: one migrates identically with native serum Ci-INA in SDS-PAGE, while the second has a M, of approximately 200 kDa (Fig. 2). Neither of these two proteins were adsorbed to an irrelevant antibody affinity column (data not shown), thus lending support to a specific interaction of the 105 kDa and 200 kDa protein with the anti-Ci-INA affinity column. While both of these species were isolated by affinity column with anti-CT-INA antibody raised to human serum Ci-INA, the 200 kDa protein was not recognized by the same antibody in a Western blot (Fig. 3). This may indicate that the 200 kDa molecule is not antigenically related to Ci-INA. However, the intensity of the 200 kDa band in the autoradiogram (Fig. 3) is approximately one order of
474
RANDAZZO
ET AL.
magnitude less than that of the 105 kDa band. Assuming that the two molecules radiolabel equally well (which may not be the case), then the concentration of the 200 kDa species is significantly lower than that of the 105 kDa in this experiment. Since the intensity of the 105 kDa band is only moderate in the Western blot (Fig. 3) it is quite possible that the Western blot is not sensitive enough to detect the 200 kDa band, which is presumably present in a much lower concentration. Thus, no firm conclusion can be drawn from these experiments as to whether the 200 kDa is antigenically related to Ci-INA (possibly a high molecular weight or dimeric form of the molecule) or antigenically unrelated. One possibility is that this 200 kDa cell surface protein copurifies with CT-INA during affinity chromatography because it is complexed with CT-INA. Perhaps the 200 kDa protein is the U937 cell surface receptor for Ci-INA. If this is the case then the U937 receptor for CT-INA would be significantly larger than the 58-64 kDa Ci-INA receptor isolated from leukocyte membranes (38). Under the low ionic strength conditions used during isolation of cell surface proteins this putative Ci-INA 200 kDa protein complex may bind to the affinity column via CT-INA. The complex would then dissociate under SDS-PAGE conditions yielding the two bands seen. However, the issue of a specific interaction between the 200 kDa protein and cell surface Ci-INA cannot be resolved from the experiments presented here. Experiments are now in progress to further characterize this 200 kDa protein. In the absence of such supporting data it is still plausible to speculate that the 200 kDa protein is simply a contaminant, which happens to bind to and elute from the affinity column with CT-INA. Another possibility that was considered, but which is quite unlikely, is that the 200 kDa protein could represent a complex of CT-INA and any one of the known plasma proteases, which CT-INA has been shown to inhibit. Clr, Cls, HF, plasmin, and kallikrein are all single chain proteins with a M, of approximately 85 kDa. Upon activation, these single chain proteases are cleaved within an intrachain disulfide bond yielding a 60 kDa amino terminal fragment and a 30 kDa carboxy terminal fragment, which remain disulfide linked. The 30 kDa fragment contains the active site of the enzyme with which CT-INA interacts forming an SDS nondissociable bimolecular complex. Thus, when CT-INA protease complexes are analyzed by SDS-PAGE, the molecular weight of the complex under nonreducing conditions is approximately 185 kDa. Under reducing conditions, however, reduction of the intrachain disultide bond of the enzyme results in a 135 kDa CiINA enzyme complex and a 60 kDa enzyme-derived fragment (1, 14). In the present study the A4, of the 200 kDa U937 surface protein remained unchanged upon reduction. The precise mode of association between the U937 cell membrane and surfaceexpressed Ci-INA cannot be determined from these studies. It is possible that the low molecular weight form of CT-INA is associated intrinsically via a low molecular weight covalent modification. Such attachment of proteins to cell surfaces via fatty acids is quite common (39), and in most cases fatty acid acylation of proteins does not significantly change the apparent molecular weight as determined by SDS-PAGE (40). Using a sensitive hemolytic assay we were able to show that intact U937 cells
U937 CELLS EXPRESS FUNCTIONAL
Ci-INA
475
can inactivate exogenous human CIS, indicating that the cell surface Ci-INA is functionally active. Moreover, this CT-INA functional activity is blocked by monospecific antibody to CT-INA, and this is almost certainly due to CT-INA and not some other protein. Because we and others have never been able to demonstrate secretion of antigenic Ci-INA by U937 cells (20, 41), and since there is no significant Cf-INA functional activity in U937 cell-free supernatants as assessed by hemolytic assay (Table l), we conclude that the CT-INA activity demonstrated for intact U937 cells is due to surface CT-INA. It seems that U937 cells synthesize CT-INA, which is not secreted freely into the culture media, but rather remains tightly associated with the cell surface. This is in contrast to peripheral bloodderived monocyte/macrophage cultures, which have been shown to secrete significant quantities of Ci-INA (19). Our observations are in line with those of Malhorta and Sim (24), who reported that factor H, which is synthesized by U937 cells and is expressed on their cell surface, is not secreted. However, the underlying mechanism of this phenomenon cannot be determined from these studies. This is the first report demonstrating the presence of functional Ci-INA on the surface of any cell type. However, there is a recent abstract that indicates that Ci-INA antigen is found on the surface of cultured human umbilical vein endothelial cells (42), and our own preliminary studies suggest that Ci-INA may be present on a variety of tumor cell lines such as Raji, Daudi, and HL-60 (B. P. Randazzo, H. B. Fleit, A. P. Kaplan, and B. Ghebrehiwet, manuscript in preparation). As reported previously and as seen in Table 2, immunofluorescent analysis indicates that Ci-INA is expressed on approximately 16% of U937 cells in culture (20). PMA has been shown to alter the phenotype of U937 cells toward that of a more differentiated monocyte-like cell (43). The fact that PMA induces an increase in the percentage of U937 cells expressing surface Ci-INA indicates that CT-INA expression occurs only at certain discrete stages during the ontogeny of monocytes. It is also probable that cells more differentiated than the monoblastoid U937 may express Ci-INA at a higher frequency. The constant low percentage of U937 cells in our cultures that express surface CT-INA may represent cells that are spontaneously differentiating. Experiments are presently in progress to examine the pattern of surface Ci-INA expression during monocyte ontogeny using peripheral blood monocytes and other monoblastoid cell lines in the presence of maturation inducers. While it is clear that U937 surface Ci-INA functions as a protease inhibitor, the physiologic and or pathophysiologic role of surface Ci-INA is yet to be determined. Most nucleated cells are relatively resistant to complement lysis (44, 45). There have been a number of complement regulatory proteins described on the surface of various cells such as CR1 (27, 46), factor H (24), and HRF (47), which are believed to play some role in protecting these cells from complement lysis. All of the complement regulatory cell surface proteins described to date control complement activation at the point of C3 activation (46) or membrane attack complex assembly (47). Thus, these complement regulatory proteins likely have a relatively more important role in controlling alternative pathway activation. Our description of functional cell surface Ci-INA is the first report of a cell surface complement control protein that would function primarily to control activation of
476
RANDAZZO
ET AL.
the classical pathway at the cell surface. Considering the important regulatory roles played by plasma CT-INA in vivo, it is possible that cell surface Ci-INA may be important in preventing autocatalytic activation of monocyte-produced C1 during secretion. We hope to assess the possibility that other cell types also express surface CT-INA. ACKNOWLEDGMENTS The authors thank Ms. Joanne Thomas for technical assistance with FACS ratory of Dr. Paul Fisher for instruction in passive immunoblotting.
analysis,
and the lab
REFERENCES 1. Harpel, P. C., and Cooper, N. R., J. C/in. Invest. 55, 593, 197.5. 2. Sim. R. B.. Reboul. A., Arlaud, G. J., Villiers, C. L.. and Colomb, M. G., FEBS Len. 97, Ill, 1979. 3. Ziccardi, R. J.. J. Irnmunol. 126, 1768, 1981. 4. Forbes, C. O., Pensky, J., and Ratnoff, 0. D., J. Lab. Clin. Med. 76, 809, 1970. 5. de Agostini, A.. Lijnen. H. R.. Pixley, R. A., Colman, R. W., and Schapira, M., J. C/in. Invest. 73, 1542, 1984. 6. Pixley, R. A., Schapira. M., and Colman, R. W., J. Bid. Chem. 260, 1723, 1985. 7. Gigli. I., Mason, J. W., Colman. R. W., and Austen, K. F., J. Immunol. 107, 311, 1970. 8. Harpel, P. C.. Lewin. M. F., and Kaplan, A. P., J. Bio/. Chem. 260, 4257, 1985. 9. Ratnoff, 0. D., Pensky, J., Ogston, D., and Naff. G. D., J. Exp. Med. 129, 315. 1969. 10. Aoki, N.. Moroi, M., Matsuda. M., and Tachiya, K., J. C/in. Invest. 60, 361, 1977. 11. Harpel, P. C., J. C/in. Invest. 68, 46, 1981. 12. Arlaud, G. J.. Reboul. A., Sim, R. B.. and Colomb, M. G., B&him. Biophps. Actu, 576, 151. 1979. 13. Salvesen, G. S., Catanese, J. J.. Kress, L. F., and Travis, J.. J. Biol. Chem. 260, 2432, 1985. 14. van der Graff. F.. Koedam, J. A., Griffin, J. H., and Bouma, B. N.. Biochemistry 22,486O. 1983. 15. Ziccardi, R. J., J. Zmmunol. 128, 2505, 1982. 16. Ghebrehiwet, B., Randazzo, B., and Kaplan, A. P.. C/in. Immunol. Immunopathol. 32, 201, 1984. 17. Johnson. A. M., Alper. C. A.. Rosen, R. S.. and Graig. J. M., Science 173, 553, 1971. 18. Prandini, M. H., Reboul. A., and Colomb, M. G., Biochem. J. 237, 93. 1986. 19. Bensa, J. C., Reboul, A., and Colomb. M. G., Biochem. J. 216, 385. 1983. 20. Randazzo. B. P., Dattwyler. R. J., Kaplan, A. P.. and Ghebrehiwet, B.. J. Immunol. 135, 1313. 198.5. 21. Sundstrom, C., and Nilsson, K., Int. J. Cancer 17, 565, 1976. 22. Koren, H. S., Anderson, S. J., and Larrick. J. W., Nature (London) 279, 328. 1979. 23. Anderson, C. L., and Abraham, G. N.. J. Immunol. 125, 2735. 1980. 24. Malhotra. V., and Sim, R. B.. Eur. J. Immunol. 15, 93S, 1985. 25. Schmitt, M., Mussel, H. H., and Dierich. M., J. Zmmunol. 126, 2042, 1981. 26. Pangbum, M. K.. Schreiber, R. D., and Miiller Eberhard, H. J., .I. Exp. Med. 164, 257. 1977. 27. Fearon, D. T.. Proc. Natl. Acad. Sci. USA 76, 5867, 1979. 28. Reboul, A., Arlaud, G. J., Sim, R. B., and Colomb, M. G., FEBS Lett. 79, 45. 1977. 29. Gigli, I., Porter, R. R.. and Sim, R. B., Biochem. J. 157, 541. 1976. 30. Wright, S. D., Rao, P. E., van Voorhis. W. C.. Craigmyle, L. S., lida. K., Talle, M. A., Westberg, E. F., Goldstein, G., and Silverstein, S. C.. Pvoc. Nut/. Acad. Sci. USA 80, 5699. 1983. 31. Bolton. A. E., and Hunter. W. M., Biochem. J. 133, 529, 1973. 32. Morrison, M., In “Methods in Enzymology” (S. Fleischer and L. Packer, Eds.), Vol. 32. p. 103, Academic Press, New York, 1974. 33. March, S. C., Parish, I., and Cuatrecasas, R., Anal. Biochem. 60, 149. 1974. 34. Laemmli, U. K., Nature (London) 227, 680. 1970. 35. Southern, E. M., J. Mol. Biol. 98, 503, 1975. 36. Minta, J. O., and Aziz. E., J. fmmunol. 129, 250. 1981.
U937
CELLS
EXPRESS
FUNCTIONAL
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37. Gilbert, D., Peulve, P., Daveau, M., Ripoche, J., and Fontaine, M., Eur. .I. Immunol. 15, 986, 1985. 38. Chang, N. S.. Leu, R. W., and Boackle, R. J., In “Membrane Proteins” (S. C. Goheen, Ed.), p. 295, Bio-Rad Labs., 1987. 39. Schlesinger, M. J., Magee, A. I., and Schmidt, M. F. G., .I. Biol. Chem. 225, 10021, 1980. 40. Omary, M. B., and Trowbridge, I., J. Biol. Chem. 256, 4715, 1980. 41. Minta, J. 0.. and Pambrun, L., Fed. Proc. 44, 991, 1985. 42. Schmair. A. H., Farber, A., Lundberg, D., Murray, S., Kuo, A., and Cines, D. B., “Kinin 1987, Tokyo International Congress, p. 82, 1987. [Abstract 8-13-181 43. Harris, R., and Ralph, P., J. Leukocyte Biol. 37, 407, 1985. 44. Schreiber, R. D., Pangbum, M. K., Medicus. R. G., and Miiller-Eberhard. H. J.. C/in. Zmmunol. Immunoparhol. 15, 384, 1980. 45. Morgan, B. P., Imagawa. D. K., Dankert. J. R.. and Ramm, L. E., J. Zmmunol. 136, 3402, 1986. 46. Ross, G. D., In “Immunobiology of the Complement System” (G. D. Ross, Ed.), p. 87, Academic Press, San Diego, 1986. 47. Podack, E. R., In “Immunobiology of the Complement System” (G. D. Ross, Ed)., p. 130, Academic Press, San Diego, 1986. Received June 24, 1988; accepted August 16, 1988