C1q receptor

558

RECEPTORS ON L Y M P H O I D CELLS

[45]

receptors, other nonlymphoid viruses such as vesicular stomatitis virus (VSV) 24 (Table I) appear to utilize glycolipid or phospholipid components for cell attachment. These molecules may be responsible for the broad specificity of host cell attachment by these viruses. The virus-binding moiety of phospholipid/glycolipid receptors remains to be fully analyzed. It is anticipated that biochemical techniques such as the use of chemical cross-linking reagents will aid in the identification of other virus receptors such as CMV and measles which have less restricted lymphotropism than EBV. In addition, X-ray crystallographic analysis of viruses such as the recent studies of human rhinovirus 35and poliovirus 36may provide insights on the complementary structure of cellular recognition sites for viruses. Acknowledgment The preparation of this chapter was funded in part by National Institutes of Health Grants CA36204, CA14692, CA35048, and AI17354. 35 M. G. Rossmann, E. Arnold, J. W. Erickson, E. A. Frankenberger, J. P. Griffith, H. J. Hecht, J. E. Johnson, G. Kramer, M. Lao, A. G. Mosser, R. R. Rueckert, B. Sherry, and G. Vriend, Nature (London) 317, 145 (1985). 36 j. M. Hogle, M. Chow, and D. J. Filman, Science 229, 1358 (1985).

[45] C l q R e c e p t o r B y BERHANE GHEBREHIWET

Introduction Human Clq (Mr 460,000) is a collagen-like, structurally complex, cationic (y2 mobility) glycoprotein consisting of 18 similar but distinct polypeptide chains (6A, 6B, and 6C). 1-3 Each chain (Mr, A = 28 kDa, B = 25 kDa, and C = 24 kDa) consists of an 81-amino-acid-long, collagen-like Nterminal portion and a C-terminal globular region of 136 amino acids. 3 In normal plasma (65-75 /~g/ml) Clq circulates as a subunit of the first component of complement, C1, which is a Ca2+-dependent pentamolecular complex with a structural formula of C lqC Ir2C 1s2.4 During activation M. A. Calcott and H. J. Miiiler-Eberhard, Biochemistry 11, 3443 (1972). 2 K. B. M. Reid and R. A. Thompson, Mol. Immunol. 2,11,117 (1983). 3 K. B. M. Reid, Biochem. J. 231, 729 (1985). 4 R. J. Ziccardi and N. R. Cooper, J. Immunol. 118, 2047 (1977).

METHODS IN ENZYMOLOGY, VOL. 150

Copyright © 1987by Academic Press, Inc. All rights of reproduction in any form reserved.

[45]

Clq ~cErrog

559

of the classical pathway of complement, Clq within the C1 macromolecule functions as a recognition unit5 by virtue of its ability to recognize and bind to particle-bound IgG or IgM. 6-9 Immune complex-bound Clq then undergoes a conformational change 5,~°which in turn induces autocatalytic activation of Cir. 1~ Finally, activated Clr effects the proteolytic conversion of Cls to Cis, thus completing the activation of C1. Activated C 1 is readily controlled by C i inhibitor (Ci-INH), which firmly binds to and dissociates C|r and C|s from particle-bound Ci )2 Under these conditions, the collageneous tail of the Clq molecule becomes exposed and potentially available for interaction with Clq binding particles and cell surface Clq receptors (ClqR). More than a decade has elapsed since the existence of Clq receptors was first shown on human peripheral lymphocytes. 13 This observation was later confirmed ~4-~6and in addition, several types of cells have been shown to possess ClqR and include B lymphocytes, 14-~6T lymphocytes, ~5 Raji, WiI2WT, and Daudi c e l l s , 15,16 Molt417 null cells, monocytes, polymorphonuclear leukocytes, ~8 endothelial c e l l s , 19 fibroblasts, 2° U937 cells, 2~ and platelets, 22-24though it has yet to be determined whether the ClqR on platelets is identical to or distinct from the collagen receptor. The ClqR has been recently purified and partially characterized from the 5 H. J. Mfiller-Eberhard, in "Molecular Basis of Biological Degradative Processes" (R. D. Berlin, H. Herman, I. H. Lepow, and J. M. Tanzer, eds.), p. 65. Academic Press, New York, 1978. 6 E. Shelton, K. Yonemasu, and R. M. Stroud, Proc. Natl. Acad. Sci. U.S.A. 69, 65 (1972). 7 j. M. Kehoe and M. Faugerau, Nature (London) 224, 1212 (1969). s M. M. Hurst, J. E. Volanakis, R. B. Hester, R. M. Stroud, and J. C. Bennett, J. Exp. Med. 140, 1117 (1974). 9 A. G. Plant, S. Cohen, and T. B. Tomasi, Science 176, 55 (1972). 10 M. Golan, R. Burger, and M. Loos, Mol. Immunol. 19, 1371 (1982). it R. R. Porter, this series, Vol. 80, p. 3. 12 G. J. Arlaud, J. Gagnon, and R. R. Porter, Biochem. J. 201, 49 (1982). 13 H. B. Dickler and H. G. Kunkel, J. Exp. Med. 136, 191 (1972). 14 K. G. Sundqvist, S. E. Svehag, and R. T. Thorstenson, Scand. J. Immunol. 3, 237 (1974). 15 A. T. Sobel and V. A. Bokisch, Fed. Proc., Fed. Am. Soc. Exp. Biol. 34, 965 (1975). 16 B. Ghebrehiwet and H. J. Mtiller-Eberhard, J. lmmunol. 120, 27 (1978). 17 B. Ghebrehiwet, J. Immunol. 137, 618 (1987). is A. J. Tenner and N. R. Cooper, J. Immunol. 126, 1174 (1981). ~9B. S. Andrews, M. Shadforth, P. Cunningham, and J. S. Davis, J. Immunol. 127, 1075 (1981). z0 S. Bordin, W. P. Kolb, and R. C. Page, J. Immunol. 130, 1871 (1983). 21 j. Arvieux, A. Reboul, J. C. Bensa, and M. G. Colomb, Biochem. J. 218, 547 (1984). 22 j. p. Cazenave, S. N. Assimeh, R. H. Painer, M. A. Packham, and J. F. Mustard, J. lmmunol. 116, 162 (1976). 23 E. A. Suba and G. Csako, J. lmmunol. 117, 304 (1976). 24 E. I. B. Peerschke and B. Ghebrehiwet, J. Immunol. 138, 1537 (1987).

560

RECEPTORS ON LYMPHOID CELLS

[45]

lymphoblastoid B cell line Raji. 25 The receptor was solubilized from the membranes of these cells with Triton X-100 or Nonidet P-40 and purified to homogeneity using C lq-Sepharose CL-4B affinity chromatography. The surface exposed, major ClqR component is an acidic -2.4-4.2 S glycoprotein with an apparent Mr of approximately 70,000 under nonreducing conditions. Upon reduction the molecule electrophoreses on SDS-PAGE with an Mr of about 80,000-85,000. Depending upon the conditions of electrophoresis, the 70-kDa type sometimes appears as a doublet with one chain being a possible heterodimer or heteropolymer, as evidenced by two-dimensional electrophoresis under reduction (B. Ghebrehiwet, manuscript in preparation). In addition, there appears to be a 30- to 40-kDa moiety, which may represent a noncovalently linked constituent of the ClqR complex. These immunochemical parameters were recently confirmed using two IgM monoclonal antibodies (MAb), II1/D1 and II1/B5, that were produced in our laboratory 17and are presently being used to purify and characterize the C lqR. It is important to mention at the outset that during the purification procedures, considerable effort should be made to remove any possible Clq-binding cellular substances whose presence, even in minute quantities, may contribute to erroneous interpretation. These include fibronectin (Mr 440 kDa),/32-microglobulin (M~ 12 kDa), immunoglobulin, mitochondria, and DNA. This again will depend upon the cell types used (Table I). For example, Raji cells are surface immunoglobulin negative (s-Ig-) while Daudi cells are s-Ig ÷. On the other hand, Daudi cells are devoid of detectable flz-microglobulin, although most nucleated cells are presumed to express fl2-microglobulin on their surfaces as the light alloantigenically invariant chain of the major histocompatibility complex class I antigens. 26

Assay Methods Most of the assays for CIqR used in our laboratory are essentially based on a single parameter, i.e.,the abilityof the receptor to bind to the collagenous region of the C lq molecule to which C Ir2C Is2 complex is also believed to bind. Such a binding invariably inhibits the capacity of Clq to mediate the following functions: rosette formation, association with Clr2Cls2 to form C1 and C1q-dependent cellular cytotoxicity. Another property of the CIqR, which is similar to that of the plasma CIq inhibitor,27is its abilityto precipitate C Iq on solid or liquid medium in a manner B. Ghcbrchiwet, L. Silvestri,and C. A. McDeviR, J. Exp. Med. 160, 1375 (1984). H. M. Grey, R. T. Kubo, S. M. Colon, M. D. Poulik, P. CrcssweII, T. Springer, M. Turner, and J. L. Strominger, J. Exp. Med. 138, 1608 (1973). L. Silvestri,J. R. Baker, L. Roden, and R. M. Stroud, J. Biol. Chem. 256, 7383 (1981).

[45]

Clq gECEm'Og

56l

TABLE I CELL TYPES EXPRESSINGClq RECEPTORS

Ceil type

Receptors/cell (mean)

Reference

B lymphocytes T lymphocytes PMNs Null ceils Endothelial cells Fibroblasts Platelets Raji WiI2WT Daudi NC37K U937 Molt4

3 x 10s ND 3.7 x l05 ND a ND 8.4 x l06 4 x 103 1 × 106 1-2 x 106 5 x 105 ~3.5 x 10s 1.6 x 106 ND

15 15 18 18 19 20 24 15 15 15 UO b 21 17

ND: These cells have been shown to possess ClqR but the number of sites/ceil has not been determined. b UO: This is an unpublished observation (B. Ghebrehiwet). o

comparable to that of antigen-antibody interaction. These properties have been exploited to develop simple assays, some of which are described below. As is true with most integral membrane proteins, the ClqR does not stay in solution unless detergent is present. Because of this property the full potential activity of ClqR and other membrane proteins is cumbersome to estimate. It is therefore important to consider means of minimizing detergent concentration when assays that use whole cells are utilized.

Precipitation Assays Reagents Purified human Clq: Highly purified Clq is isolated according to the method of Yonemasu and Stroud. 28 Such isolated Clq is further purified by passage over a column of Con A-Sepharose (Pharmacia) equilibrated with 0.01 M veronal buffer, pH 8.0, and containing 0.5 M NaC1, 1 mM MnC12 as described.16 This procedure removes the serum C 1q inhibitor, 27 a chondroitin 4-sulfate proteoglycan which passes through the column K. Yonemasu and R. M. Stroud, J. Immunol. 106, 304 (1971).

562

RECEPTORS ON LYMPHOID CELLS

[45]

unadsorbed, while the adsorbed Clq is eluted with 10% o~-methyl-D-mannoside in equilibrating buffer. This step is important since the presence of the C 1q inhibitor which binds to the collagenous tail of C 1q16 can interfere with most of the C l q-dependent assays. The protein concentration of C 1q can be determined by measuring the optical density at 280 nm and using an extinction coefficient E 1%1 cm of 6.8. 29 Agarose: Agarose (0.8%) (Sigma Chemical Co.) is dissolved by boiling in sodium phosphate buffer, pH 7.2, containing 1.5% polyethylene glycol (PEG) 6000 (Sigma) and 0.02% sodium azide. The agarose can be used immediately or stored at 4° until used. Staining and destaining solutions: Usually Amido Black 10B or Coomassie brilliant blue (Sigma) dyes are used. Typically, 0.5 g Amido black is dissolved in a solution which consists of 500 ml H 2 0 , 4 0 0 ml 95% ethanol, and 100 ml glacial acetic acid. Destaining solution is 80 ml glacial acetic acid and 920 ml of methanol. Procedure A. The precipitation in agarose assay is fairly straightforward and is performed according to well-established, double -3° or singl e-31 immunodiffusion assays. Four milliliters of freshly boiled or melted agarose is poured onto a 2.5 × 7.5 cm glass slide and left to solidify for 5 min at 22 ° followed by 5 min at 4 °. Wells are then punched out according to the desired pattern, depending on how many samples are to be analyzed. Usually, if purified ClqR is to be used, approximately 15-30/zl (25 /zg/ml) is applied to a well that is located opposite to another well filled with 10 /xl of C l q or normal human serum (NHS). The slide is then incubated at 37 ° for 3 hr and at 22° for 24 hr. Usually, if the ClqR concentration is sufficient (25/zg/ml), a visible precipitin line will form after 24 hr. If not, the precipitin line can be visualized after staining. The procedure for staining is quite conventional; however, care should be taken not to wash with high salt buffers or solutions as these may dissolve the C l q R - C l q complex. With this in mind, the slide is washed in a slide-jar containing 75 mM NaC1 and 0.1% NaN3 for 24-48 hr, followed by washing in distilled H20 for another 24 hr. The slide is then wrapped in lens paper, pressed for 2 hr under a heavy book or wooden block, and dried for 1 hr in a 120° oven or air dried for 24 hr. After drying, the lens paper is carefully removed under running water and the slide is air dried for 30 min. The dried slide is then stained for 5 min by immersion in a slide-jar containing Amido black or Coomassie blue staining solution and then immediately destained by several immersions in methanol-glacial acetic acid mixture until all background staining has been removed (Fig. 1A). 29 K. B. M. Reid, D. M. Lowe, and R. R. Porter, Biochem. J. 130, 749 (1972). 30 O. Ouchterlony, Prog. Allergy 6, 30 (1962). 31 G. Mancini, A. O. Carbonara, and J. F. Hermans, Immunochemistry 2, 235 (1965).

[45]

Clq gEC~grog

563

A

® ®

Q

Q

NHS

ClqRl

B

1. Ctq 2. NHS 3. Clq

CIqRT

@ @

@..sE CtqRTr 4. Clq 5. NHS-E 6. Clq

CIqR]T

Fro. 1. Immunodiffusion analysis of CIqR. Concentrated pools (I and II) from C l q Sepharose affinity column (Fig. 2) were analyzed by double (A) or single (B) immunodiffusion techniques. Both plates contained 0.8% agarose in PBS, pH 7.4, 1.5% PEG, and 0.02% NAN3. Plate B contained, in addition, 25/~g/ml agarose of isolated Clq. Reproduced from Ghebrehiwet e t al. ~ by copyright permission of the Rockefeller University Press.

564

RECEPTORS ON LYMPHOID CELLS

[45]

This technique can be extended to include the single radial immunodiffusion (Fig. 1B) assay and is carried out according to the following procedure. 25,27 The agarose is boiled or melted as indicated above and 4 ml is transferred into a 10-ml test tube that is kept at 60 ° in a thermostatted water bath. Then, Clq is added to the agarose to a final concentration of 25/zg/ml, mixed thoroughly, and poured immediately onto a glass slide as indicated above. After the agar solidifies many wells can be punched out to hold purified ClqR or samples of column fractions and the slide is processed as indicated above. A precipitin ring develops after a 48-hr incubation at 4 °. The diameter of the ring is proportional to the concentration of the ClqR. 31 Procedure B. Another method of CIqR assay which is successfully used in our laboratory is the precipitation of 125I-labeled Clq in solution. For this purpose, Clq can be efficiently labeled (1/zCi//zg) by the BoRonHunter m e t h o d ) 2 Typically, 100 ~g of Clq in 0.1 M sodium borate buffer (pH 8.5) is added to 100/zCi of Bolton-Hunter reagent (New England Nuclear) and allowed to react for 30 min at 4°. The reaction mixture is then chromatographed on a column (1.2 × 21 cm) of Sephadex G-25 (Pharmacia) in PBS, pH 7.0, containing 0.1% NaN3 to separate the radiolabeled protein from free iodine. The first radioactive peak to emerge is the labeled protein. The precipitation method, which is similar in principle to that of the Clq binding a s s a y , 33'34 is carried out by incubating 50/zl of ClqR-containing samples with 50/.d 125I-labeled Clq (1/zCi//zg) and 1 ml 3% PEG-6000 in phosphate buffer, pH 7.2, containing 75 mM NaC1. The mixture is left for 2 hr on ice and centrifuged at 1500 g for 15 min. The supernatant is carefully transferred into another tube and the radioactivity of both the supernatant and the precipitate is determined. Usually 10-50 /zl of liquid remains on the sides of the tube. This can be absorbed out by inversion of the tubes on top of filter paper (Fisher) for 15 min. The total precipitable 125I-labeled C lq is obtained by addition of 1 ml of 20% trichloroacetic acid (TCA) to a control tube containing only 125I-labeled Clq. Results are expressed as percentage of TCA-precipitated 125I-labeled C lq after nonspecific or background buffer control (ClqR buffer) has been subtracted. An alternative assay would be a radioimmunoassay (RIA) performed usually with purified ClqR but equally useful when assaying fractions containing ClqR. Briefly microtiter plates (Strip Plate-8, Costar, Cambridge, MA) are coated with 50/zl ClqR (25/zg/ml) by incubation at 4° 32 A. E. BoRon and W. M. Hunter, Biochem. J. 138, 259 (1973). 33 V. E. Nydegger, P. H. Lambert, H. Gerber, and P. A. Micscher, J. Clin. Invest. 54, 297 (1974). 34 R. H. Zubler, G. Lange, P. H. Lambert, and P. A. Miescher, J. lmmunol. 116, 232 (1976).

[45]

Clq ~cErroa

565

overnight. Next, the wells are filled with PBS, pH 7.4, containing 1% BSA and 0.02% NAN3, and incubated at 37° for 2 hr to saturate excess binding sites. After three washes in PBS-BSA containing 0.05% Tween 20, 125Ilabeled Clq is added and incubated for 2 hr at 37°. Controls should include wells coated only with PBS-BSA. After incubation, the wells are washed three times and each individual well is broken off and counted. The specifically bound Clq is then determined by subtracting the nonspecifically bound 125I-labeled Clq from the total ClqR bound,

Inhibition of Rosette Formation (see also this series, Vol. 108 [7]) Reagents Glutaraldehyde-treated human erythrocytes (EhGlu): These are prepared essentially as described by Hughes-Jones. 35 Freshly obtained human erythrocytes (Eh) are treated with 100 vol of 2% glutaraldehyde in PBS at 4° for 16 hr. The cells are washed with Hanks' balanced salt solution (HBSS) and further incubated with 10 vol of HBSS containing 10 mg/ml L-lysine for 30 min at 37°. Finally the cells are washed and resuspended in HBSS at 1 × 109/ml. Clq-coated erythrocytes: These are prepared by incubating 5 x 108 EhGlu with 300/~g of hemolytically active Clq for 30 min at 22°. The excess unbound Clq is removed by centrifugation at 1000 g in HBSS, and the pellet is resuspended in 1 ml HBSS. Procedure. Before each experiment, it is necessary to test whether the prepared EhClq forms rosettes with ClqR-bearing cells. Rosette formation is assayed by incubating 2 x 107 EhClq with 5 x 105 ClqR-bearing cells (see Table I) in a total volume of 0.5 ml in HBSS for 5-10 min, followed by centrifugation for 5 min at 600 g and further incubated for 1-2 hr at 4°. The percentage of rosettes formed is determined by counting at least 200 ClqR-bearing cells under the microscope. To determine the inhibitory effect of ClqR, the EhClq cells are first pretreated with various dilutions of C 1qR containing a minimal concentration of Nonidet P-40 (usually about 0.01% or less) for 30 min at 22° and then further incubated with the C lqR-bearing ceils. The percentage of rosette inhibition can then be determined by comparing with untreated cells. In our hands a concentration of ClqR of 5 ~g/ml produces rosette inhibition of 70-80%. 25 However, the real inhibitory efficiency of the receptor may not be measured in absolute terms since the presence of detergent makes it difficult to do so especially when cells are used. The rosette assay is relatively easy to perform, though the number of 35 N. C. Hughes-Jones, Immunology 32, 191 (1977).

566

RECEPTORS ON LYMPHOID CELLS

[45]

rosettes formed may vary from experiment to experiment. This variability is probably due to the number of active C I q molecules that are bound per erythrocyte. It is therefore useful to estimate the optimal number of Clq molecules that should be bound per cell in order to obtain maximal rosetting. Under optimal conditions 50-70% rosettes can be obtained when cultured cells such as Raji or U937 cells are used. z5

Inhibition of Clq Hemolytic Function Reagents GVB: Veronal-buffered saline, pH 7.4, containing 0.15 mM CaCI2,0.15 mM MgC12, and 0.1% gelatin Clq-depleted serum (ClqD): This is prepared by passage of I0 mM EDTA-containing normal human serum over an anti-C lq-Sepharose CL-4B column or IgG-Sepharose column) 6 The excluded material is concentrated to original volume, dialyzed against veronal-buffered saline, and finally the ClqD is reconstituted with Ca 2÷ and Mg 2÷ by addition of 20/zl/ml C 1qD of a stock solution of 0.3 M CaCI2 and 1 M MgCI2. The reconstituted ClqD is then aliquoted into small volume (0.1 ml) and stored at -80°). Clq: Purified as described elsewhere 28 (5/zg/ml in GVB buffer) EA: Sheep erythrocytes are washed three times and resuspended in GVB at 1 x 109/ml and EDTA is added from a stock solution (0.2 M EDTA, pH 7.2) to a final concentration of 20 mM. Then, an equal volume of an appropriate dilution of anti-E antibody (A) (Cordis Laboratories) is added dropwise, with constant mixing and incubated for 15 min at 37 °. The cells are then washed and resuspended at 5 × 10S/ml in GVB. With commercial antibody, the optimal dilution for sensitization of sheep E is usually 1/1000 to 1/2000 for IgG and 1/1201/240 for IgM Procedure. The optimal amount of Clq that causes maximal lysis is assayed in the following manner. Various amounts of Clq, ranging from 10 to 1000 ng, are incubated with 20/zl of ClqD in a total volume of 100/zl GVB for 60 min at 37°. Then to the reaction mixture is added 600/xl of GVB and 100/~1 of EA (5 x 10S/ml) and the incubation is continued for 60 min at 37°. The volume of the reaction mixture is then brought to 1 ml with cold GVB and centrifuged, and the amount of hemoglobin released into the supernatant determined spectrophotometrically at 412 nm. The total releasable hemoglobin is measured by lysing 100/~1 of EA with 900/zl of H20 and is used as a 100% reference. The percentage of Clq hemolytic ~s W. S. Kolb, L. M. Kolb, and E. R. Podack, J. lmmunol. 122, 2103 (1979).

[45]

Clq RECEPTOR

567

activity is plotted against the amount of Clq added and the optimal amount of Clq hemolytic activity is determined. This amount of Clq (usually - 2 0 - 5 0 lag) is then incubated with buffer or purified ClqR or ClqR-containing samples for 60 rain at 37° and for an additional 60 min at 37° with 100/zl EA diluted 1 : 10 with GVB. This step is necessary in order to dilute the detergent-containing reaction mixtures before addition of the EAs. The percentage inhibition is determined by comparison with the amount of hemoglobin released in tubes with untreated Clq.

Other Functional Assays Other ClqR functional assays such as the Clq-dependent cellular cytotoxicity ~6can be performed. However, such an assay, albeit reliable, is time consuming. Another assay that could be useful in the assay of ClqR is the inhibition of ~2SI-labeled Clq binding to ClqR-bearing cells. The binding of human Clq to ClqR-bearing cells is assayed by incubating 2 x 106 cells with concentrations of 125I-labeled Clq ranging from 0 to 60 tzg/ 0.3 ml of PBS containing 90 mM NaC1 and 0.5% gelatin at 4°. After incubation, 150/~1 of the reaction mixture is layered onto 30% sucrose in PBS in a 1.5-ml microfuge tube and centrifuged for 90 see at 12,000 g. The cell pellet-containing portion of the microfuge tube is cut off with a razor blade or, better yet, with a dog nail-clipper and the specifically bound ~25Ilabeled Clq is determined. Nonspecific 1251trapping or binding can be determined by incubating irrelevant cells (e.g., sheep red blood cells) under the same conditions. The amount of specifically bound Clq is plotted against the concentration of Clq used and thus the saturating concentration can be determined. This amount of 125I-labeled Clq is then incubated with or without various concentrations of ClqR for 15 min at 37° followed by another 60 min with 2 x 10 6 ClqR-bearing cells at 4°, making sure that the detergent concentration is properly diluted before the cells are added. The amount of inhibition of 125I-labeled Clq binding can be determined by comparison with untreated Clq.

ELISA Assay Using Monoclonal Antibody to ClqR In our hands, the assays described above have been satisfactory. Currently our method of choice is an enzyme-linked immunosorbent assay (ELISA) in which alkaline phosphatase- or horseradish peroxidase-conjugated MAb III/D1 or II1/B5 is used. The two monoclonal antibodies II1/ D1 and III/B5 do not cross-react with any of the Clq-binding proteins such as Ig, fl2-microglobulin, fibronectin, collagen, or DNA. Their preparation and purification are discussed below.

568

RECEPTORS ON LYMPHOID CELLS

[45]

Reagents Preparation and purification of MAb: In general, the production of monoclonal antibodies is based on the method of K6hler and Milstein. 37 Several well-established modifications 3s-4° exist, however. The III/D1 and II1/B5 monoclonal antibodies, both of which have been characterized as IgM with h light-chain specificity, 17 are produced according to a strategy that can be summarized as follows: Five CD-1 mice [Crl; CD-1 (ICR) BR strain; Charles River, Wilmington, MA] are first immunized with 107 viable Raji cells suspended in 0.5 ml sterile HBSS and 0.5 ml complete Freund's adjuvant (diluted 1:2 with incomplete Freund's adjuvant) at three different sites (two intramuscular and one subcutaneous) every 2 weeks for 6 weeks (other mice strains such as BALB/c-AnNcrlBr are used in most immunization procedures and can be used for this purpose). After 6 weeks, the mice are trial bled from the retrobulbar sinus using a Pasteur pipet (usually 100/zl blood is collected). The serum is separated by clotting in a glass test tube (2 hr at 22°), centrifuged (15 min at 1600 g), and analyzed for precipitating antibody by the Ouchterlony method with purified CIqR or analyzed by ELISA. The mouse selected for fusion is then allowed to rest for 1-2 weeks before receiving a final intravenous injection (tail vein) of 5 × l07 Raji cells in 0.25 ml sterile HBSS 4 days before fusion. The production of mouse hybridomas is initiated by mixing 107 hyperimmune spleen cells with 5 × 107 of the HAT-sensitive [HAT: DMEM with FCS containing 0.1 mM hypoxanthine, 0.8/zM aminopterin, and 10 /zM thymidine (Sigma)] P3 x 63 Ag8.653 mouse myeloma cells in log phase. The cell mixture is centrifuged for 10 min at 400 g, washed in DMEM two times, and after removal of the supernatant 1 ml of a 50% PEG-1000, pH 8.5 (PEG: polyethylene glycol, Mr 1000; Sigma), is added dropwise over a period of 1 min and the cells are agitated gently for 90 sec at 22 °. To this cell mixture the following is added: 1 ml of DMEM over 1 min and 49 ml of DMEM at a rate of 5 ml/min. The cells are then centrifuged and resuspended in 50 ml HAT medium containing 20% FCS and distributed into 96-well fiat-bottomed microtiter plates at 0.2 ml of cell suspension per well and cultured at 37° in an atmosphere of 95% air and 5% CO2. The cells are fed every 3 days with HAT medium until visible clones are formed (usually 2-3 weeks) and subsequently grown into HT medium and finally into DMEM with 20% FCS. 37 G. K6hler and C. Milstein, Nature (London) 256, 495 (1975). 3s G. Galfr6 and C. Milstein, this series, Vol. 73, p. I. 39 S. Fazekas de St. Groth and D. Sheidegger, J. Immunol. Methods 35, 1 (1980). R. H. Kennett, T. J. McKearn, and K. B. Bechtol, eds., "Monoclonal Antibodies." Plenum, New York, 1980.

[45]

C l q I~CEPTOR

569

Detection o f anti-ClqR-producing hybridomas: The supernatants of the hybridomas obtained are then tested using ELISA assays for the presence of antibodies that (1) bind to Raji cells coated onto microtiter plates, (2) react with purified ClqR, and (3) fail to bind to Raji cells presaturated by incubation with c-Clq 17 (collageneous portion of Clq obtained by pepsin digestion). 4~ This method of approach leads to the identification of II1/D1 and II1/B5, both of which isotype as IgM h and are positive in all three assays. The II1/D1 is cloned twice by the method of limiting dilution42 and subsequently injected (107 cells/mouse) into the peritoneal cavities of syngeneic CD-1 nu × nu mice (Charles River, MA) and after 10-15 days the ascites fluid is collected. The ascites fluid is immediately centrifuged (800 g for 15 min) to remove cells and large aggregates and filtered through cotton wool (about I-2 cm wool loosely packed into a 5- or 10-ml microcolumn or syringe barrel) to remove fibrin clots, DNA, and cell fragments. Finally the ascites fluid is centrifuged at 45,000 g for 1 hr to remove aggregates. The antibody is concentrated by precipitation with 50% ammonium sulfate. It should be noted, however, that ammonium sulfate precipitation may denature some MAb, especially IgM, and should be avoided as much as possible. The precipitate is dissolved in equilibrating buffer consisting of 0.1 M Tris/HC1, pH 8.0, 0.15 M NaCI, and 0.02% NAN3, and applied to a column (2.5 x 100 cm) of Sepharose 6B, BioGel A5M (Bio-Rad), or Ultrogel AcA 22 (LKB). The gel filtration method is a modification of that used by Jehanli and Hough, 43 who purified human IgM to homogeneity by filtration on Ultrogel AcA 34 followed by ion-exchange chromatography on DEAE-Sepharose CL-6B. Elution is carded out at a flow rate of 20 ml/hr and 2-ml fractions are collected. The IgM-containing fractions are identified by double immunodiffusion with rabbit anti-mouse IgM (Boehringer Mannheim Biochemicals) or by ELISA using a mouse immunoglobulin identification kit (Boehringer Mannheim Biochemicals) containing IgM (Fc), IgGl, IgG2a, IgG2b, IgG3, as well as r and h light-chain-specific antibodies. The ELISA is performed according to the manufacturer's specifications. Fractions containing IgM are pooled, concentrated by ultrafiltration, dialyzed against 0.05 M NaEHPO4/citric acid, pH 6.8, 43 and applied to a column (4.5 x 20 cm) of DEAE-32 or DEAE-Sepharose CL-6B (Pharmacia) equilibrated with the same buffer. Under these conditions the IgM is bound to the ion exchanger and is eluted with a linear gradient between 400 ml starting buffer and 400 ml of 0.1 M NaEHPO4/citric acid, pH 5.0, at 4t K. B. M. Reid, Biochem. J. 155, 5 (1976). 42 I. Lefkovits and H. Waldmann, "Limiting Dilution Analysis of Cells in the Immune System." Cambridge Univ. Press, London and New York, 1979. 43 A. Jehanli and D. Hough, J. Immunol. Methods 44, 199 (1981).

570

RECEPTORS ON LYMPHOID CELLS

[45]

a flow rate of 100 ml/hr. 43 Residual proteins are removed by washing with a solution consisting of 0.1 M Na2HPOa/citric acid, pH 5.0, containing 0.4 M NaC1. 43The IgM-containing fractions are identified as described above, concentrated by ultrafiltration, and stored at 4° in the presence of 0.02% NaNa. MAb in general and IgM in particular are extremely susceptible to denaturation under repeated freezing and thawing and should be avoided. Conjugation of MAb with alkaline phosphatase: Conjugation of MAb with alkaline phosphatase or horseradish peroxidase has been described in this series by Engvall. 44 Briefly, calf-skin alkaline phosphatase (Sigma) is dissolved in 3.4 M (NH4)2SO4 such that the final concentration of enzyme is 2 mg/ml. Then, 0.3 ml of the enzyme is added to 0.1 ml of 2 mg/ml antibody in PBS, pH 7.2, and the mixture dialyzed overnight against 2 liters of PBS. The dialyzed enzyme-antibody mixture is then cross-linked by addition of glutaraldehyde (25% aqueous solution, Sigma) diluted in PBS to a final concentration of 0.2%. Conjugation is allowed to proceed for 3 hr at 22 ° or until the initially colorless solution becomes straw colored. The conjugated MAb is finally dialyzed against PBS, pH 8.0, and stored at 4 ° in the presence of I mg/ml bovine serum albumin. Procedure. ELISA using intact cells attached to microtiter plates: When intact cells are used, the microtiter plate wells are first incubated with 50 ~1 of a 10/zg/ml poly (L-lysine) (PLL) solution in PBS, pH 7.2, for 30 min at 37°. After removal of the excess PLL solution by aspiration, 105 cells in 50/.d PBS are added and the plates centrifuged in a Beckman model J-6B centrifuge for 5 min at 800 g using Beckman JS 4.2 microplate carriers (Beckman Instruments). Then, 50/.d of 0.5% glutaraldehyde in cold PBS is added and the plates incubated for 15 min at 37°. After incubation the cells are washed with PBS by filling the wells and flicking out the excess buffer. The wells are then filled with 100 mM glycine in 0.1% BSA solution for 30 min at 37° to block the glutaraldehyde and again washed with PBS. To each well, 50/~1 of an appropriate dilution of alkaline phosphatase-conjugated anti-ClqR MAb (with II1/D1 we use a 1/50 dilution) is added. After a 1-hr incubation at 37°, 50/~1 of a 1 mg/ml (in 10% diethanolamine buffer, pH 9.8) substrate solution ofp-nitrophenyl phosphate is added to each well. When the reaction is completed (about I hr at 37°) it is stopped by the addition of 2 M NaOH (one-quarter volume of mixture). At alkaline pH, the amount of p-nitrophenol formed is determined at 410 nm in a MicroElisa Autoreader (Dynatech). Controls for specificity are always included by reacting the ClqR-bearing cells with nonimmune IgM [or F(ab')2 in the case of IgG MAb] or by including irrelevant cells such as human or sheep erythrocytes. 44 E. Engvall, this series, Vol. 70, p. 419.

[45]

Clq RECEPTOR

571

Although we have used the alkaline phosphatase-conjugated MAb successfully, care should be taken when using polymorphonuclear lymphocytes since they contain high levels of alkaline phosphatase. An appropriate control for cellular phosphatase would therefore be reaction of the cells directly with the substrate and subtraction of the result from experimental wells. Peroxidase-conjugated MAb can be used effectively, especially when cultured tumor lines are used as targets, but myeloid cells and macrophages possess high levels of endogenous peroxidase and it should be avoided or an alternative method sought. ELISA with C l q R coated to plates: Purified ClqR or ClqR in column fractions can be analyzed by coating microtiter plates as follows. Samples are diluted in coating buffer which consists of 15 mM NaCO3, 35 mM NaHCO3, and 0.02% NAN3, pH 9.6, and 50/zl are coated onto each well of a microtiter plate which is incubated for 2 hr at 37°. Irrelevant antigens such as BSA or irrelevant antibody or nonimmune Ig are included as controls for specificity. After removing the excess unbound protein, a 0.1% solution (w/v) of BSA and 100 mM glycine in coating buffer is added to each well and incubated for 1 hr at 37°. The excess BSA is removed and the wells refilled with 50 /~1 of appropriately diluted (in PBS, pH 7.4, containing 0.05% Tween 20) alkaline phosphatase-conjugated anti-ClqR MAb and further incubated for 1 hr at 37°. After incubation the wells are washed two to three times with a solution containing 10 mM Tris-HC1, pH 8.0, and 0.05% Tween 20 and two times with distilled H20. Finally, 50 /zl of p-nitrophenyl phosphate (1 mg/ml) in 10% diethanolamine buffer, pH 9.8, is added, incubated for 1 hr, and the color developed read at 410 nm immediately or the reaction stopped by addition of 2 M NaOH and kept at 4° until read. Purification of C lqR Reagents Clq-Sepharose 4B. Highly purified Clq 28is coupled to Sepharose CL4B according to the method described by March et al. 45 Briefly, 50 ml of packed Sepharose 4B (Pharmacia) is washed extensively with deionized, distilled water and resuspended in 50 ml of 5 mM potassium phosphate tribasic buffer, pH 12.5 (1114.2 g K2HPO4" 3H20 and 140 g KOH/liter) and stirred in an ice bath kept in a fume hood for 15 min. Then, 7 g of CNBr dissolved in 3.5 ml of N,N-dimethylformamide or acetonitrile (Sigma) is quickly added and the mixture stirred for 5 min. The Sepharose 45 S. C. March, I. Parish, and P. Cuatrecasas, Anal. Biochem. 60, 149 (1974).

572

RECEPTORS ON LYMPHOID CELLS

[45]

beads are then washed in a Biichner filter funnel in the fume hood with 1 liter of water followed by 1 liter of coupling buffer consisting of 8 g NaHCO3, and 29.2 g NaCl/liter of H20, pH 9.0. The CNBr-activated Sepharose is mixed in a wide-mouth, 250-ml plastic bottle with 50 ml of human Clq (2 mg/ml) previously dialyzed against coupling buffer, and the mixture is left for 16 hr at 4° with continuous gentle stirring. Next, the slurry is placed in a B(ichner funnel again and dried. The amount of protein coupled to the Sepharose is determined by assaying the protein content 46 in the filtrate. The Clq-Sepharose is then washed with 1 liter of PBS, pH 7.0, and resuspended in 50 ml of 0.2 M ethanolamine adjusted to pH 8.0, and allowed to react for 2 hr at 22 ° in order to block the unreacted sites on the Sepharose beads. The slurry is washed by filtration with 1 liter of 5 mM sodium phosphate buffer, pH 7.5, containing 0.5 mM EDTA, 150 mM NaCI, and 0.02% NaN3 and poured into a 4.5 x 20 cm column. Before each use, the Clq-Sepharose column is tested for its binding activity by taking 0.5 ml of packed beads and incubating them (I hr at 4°) with 0.2 ml of monospecific, polyclonal antiserum to Clq 21 which is produced in a rabbit by multiple injection of 100/zg purified Clq in Freund's complete adjuvant (diluted to one-quarter with incomplete adjuvant) at three sites: one subcutaneous and two intramuscular every 2 weeks for 6 weeks. After incubation the mixture is centrifuged (500 rpm, 5 rain) and the remaining anti-Clq activity of the supernatant tested by Ouchterlony analysis. Since such a Clq-Sepharose can bind Clr or Cls in the presence of Ca 2÷ it is assumed that the Clq is bound to the Sepharose by a site which is different than the Clr2Cls2 and/or ClqR-binding site.

Purification Procedure Step 1: Membrane Solubilization. The successful purification of any receptor depends upon an appropriate solubilizing detergent and buffering system. There are several types of detergents that have proved successful in solubilizing membrane receptors in their functional state. Our laboratory has successfully utilized the nonionic detergents Triton X-100 or Nonidet P-40 (NP-40) in phosphate buffer to solubilize the ClqR from lymphocytes,47 Raji cells, and U937 cells 25 (these cultured cell lines can be obtained from the American Type Culture Collection, Rockville, MD). Phosphate buffer was chosen instead of Tris since phosphate is more efficient in dissociating the electrostatic interactions of intermolecular 46 O. H. Lowry, H. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). 47 B. Ghebrehiwet and M. Hamburger, J. lmmunol. 129, 157 (1982).

[45]

Clq RECEPTOR

573

forces in cell membranes.48 Briefly, 1 x 109 cultured cells that have been surface iodinated by the lactoperoxidase-catalyzed reaction method49 are mixed with 101° unlabeled cells and washed three times with 100 ml of 10 mM sodium phosphate buffer, pH 7.5, containing 150 mM NaCI, 2 mM EDTA, 10 mM e-amino caproic acid (EACA; Sigma), 2 mM phenylmethylsulfonyl fluoride (PMSF, freshly dissolved in dimethyl formamide; Sigma), 1 /zM pepstatin, and 1 mg/ml soybean trypsin inhibitor (Sigma) (especially when polymorphonucleates or monocytes are used) and 0.02% NAN3. Cell membranes are then prepared by a combination of freezethawings (frozen at -80 ° for 1 hr and thawed at 37°) and sonication for 5 min, in a sonifier cell disrupter W140 (Ultrasonics, Plainview, NY). The membrane fragments are then washed two times in 100 ml at 45,000 g in the same buffer for 30 min and the membrane fragments are finally solubilized in 100 ml of the same buffer containing 1% NP-40 and using 50 mM sodium phosphate instead of 10 mM (to dissociate the electrostatic interactions). After incubation for 12 hr at 4° with constant but gentle stirring, the solubilized membrane proteins are freed from insoluble material by centrifugation at 45,000 g for 60 min at 4°. The supernatant containing the solubilized membrane proteins is carefully removed and precleared sequentially with 2 ml each of packed gelatin-agarose, protein A Sepharose CL-4B, and IgG-protein A-Sepharose CL-4B by incubation for 4 hr each at 4°. Each of the affinity matrices is commercially available (Pierce Chemical Co., Rockford, IL). The total protein concentration is then determined by the detergent-compatible Bicinchoninic Protein assay reagent5° (BCA Protein Assay Reagent, Pierce). Step 2. Clq-Sepharose Affinity Chromatography. The solubilized membrane solution is dialyzed against 10 mM sodium phosphate buffer, pH 7.5, containing 2 mM EDTA, 90 mM NaC1, 10 mM EACA, 2 mM PMSF, 1/~m pepstatin, 0.02% NAN3, and 0.1% NP-40. Only half of the solution is applied to the Clq-Sepharose column in order to maximize the purification efficiency and to avoid overloading of the column. The column is then washed with three bed volumes or until reading at 280 nm (or radioactivity) reaches background and the bound proteins are eluted with a linear NaC1 concentration gradient with 100 ml of starting buffer and 100 ml of same buffer containing 1 M NaC1 at a flow rate of 10 ml/hr. Fractions (2 ml/tube) containing Clq-binding activity are identified by one of the assay methods described above. We have used this method of A. C. Dey, R. Sheilagh, R. L. Rimsay, and I. R. Senical, Biochemistry 110, 373 (1981). o M. Morrison, this seres, Vol. 32, p. 103. ~0 p. K. Smith, R. I. Krohn, G. T. Hermanson, A. K. Mallia, F. H. Gartner, M. D. Provenzano, E. K. Fujimoto, N. M. Goeke, B. J. Olson, and D. C. Klenk, Anal. Biochem. 150, 76 (1985).

574

RECEPTORS

ON

LYMPHOID

[45]

CELLS

purification to isolate the C l q R from the lymphoblastoid cell line Raji. 25 Two Clq-reactive peaks which eluted at approximately 22 and 30 mmho/ cm, respectively, are usually obtained, suggesting that Raji cells and possibly other cells may possess two species of receptors with different affinities or charge heterogeneity (Fig. 2) although Scatchard plot analysis of C l q m or MAb ~7 binding data have not suggested the existence of two population of receptors. Figure 3 shows a representative preparation. Occasionally a much more homogeneous preparation which stains with Coomassie blue as a single band is obtained without any further purification (Fig. 4). This depends probably upon the amount of gelatin and protein A used to preclear the membrane solution. Step 3. Affinity Chromatography on Con A - S e p h a r o s e . The C lq-reactive pools from the Clq-Sepharose affinity column are concentrated and dialyzed against 10 mM sodium phosphate buffer, pH 8, containing 2 mM EDTA, 150 mM NaC1, 10 mM EACA, 2 mM PMSF, 0.02% NaN3, and 0.1% NP-40 and then applied to a column of Con A-Sepharose (2.5 x 40 cm) (Sigma) equilibrated with the same buffer. After washing with 300 ml of equilibration buffer the adsorbed C lqR is eluted with a 10% solution of

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FIt~. 2. Purificationof Raji CIqR. Proteins of solubilized Rajicell proteins were chromatographed on Clq-Sepharose 4]3 afl~ty column (4.5 x 20 cm). After extensivewashing the absorbed proteins were eluted by NaCI concentration gradient. Two ClqR reactive pools were obtained, each of which was further repurified by mTmitychromatography on Con ASepharose. Reproducedfrom Ghehrehiwet et al. msby copyrightpermission of the Rockefeller University Press.

[45]

Clq l~CEm'oa

1

575

2

94K--

68K 43K 26K--

FIG.3. Ten percentpolyacrylamidegel in sodiumdodecylsulfateof ClqR. Afterconcentration using an Amiconultrafdtrationdevice fittedwith a YM-5 membrane, a sample was taken from pool II of an affinitychromatography(similarto Fig. 2) and analyzedby SDSPAGE and autoradiographyunder reducing(lane 2) and nonredueing(lane 1) conditions.

a-methyl-D-mannoside in the same buffer. The ClqR-containing fractions are pooled, concentrated, and stored at -80 °.

Purification of ClqR Using MAb MAb raised against ClqR can be efficiently used to purify Clq from detergent-solubilized cell membranes. The two monoclonal antibodies II1/D1 and II1/B5 are known to react positively in a dot blot (not shown) and interact predominantly with the 70-kDa moiety when analyzed by Western blot (Fig. 5). We have used the II1/D1 MAb for immunoaffinity purification of Raji cell ClqR. Since large amounts (of the order of 10-30 mg) of MAb are required for making an immunoaffinity column, II1/D1 from ascites fluid is used. Cyanogen bromide-activated Sepharose CL-4B is used to construct these immunoadsorbent columns (see above). The II1/D1-Sepharose 4B immunoadsorbent column (1.5 x 10 cm) is washed

576

RECEPTORS ON LYMPHOID CELLS

I

[45]

2

94K

68K

43K

30K

20K .

.

.

.

Fro. 4. Ten percent polyacrylamide gel in sodium dodecyl sulfate of purified ClqR. Purified ClqR eluted from Sepharose CL-4B was electrophoresed and visualized by Coomassie blue staining.

in PBS, pH 7.4, containing 150 mM NaCI, 10 mM EACA, 2 mM PMSF, 0.02% NAN3, and 0.1% NP-40. Then, approximately 25-50 ml of the solubilized membrane proteins is added very slowly so that equilibration between the receptor and the immobilized antibody can be achieved. The column is then washed slowly (at a rate of 5 ml/hr) with 20-40 ml of buffer-containing detergent (NP-40) to remove nonspecitically adsorbed proteins. Elution of specifically bound receptor is achieved by 0. I M glycine, pH 2.5, containing 0.1% NP-40 and 1-ml fractions are collected at a flow rate of 20 ml/hr. The eluates are immediately neutralized by addition of 50/zl of 1 N NaOH to prevent denaturation of the receptor. After elution the immunoadsorbent column is washed with equilibrating buffer until the pH of the eluate returns to pH 7.4.

[45]

Clq RECEPTOR 1

94K

-

2

577 3

,

68K-

43K . . . .

30K-

20K..

,

FI6.5. Detection of CIqR by Western blotting. Solubilized membrane solution from Raji cells was electrophoresed on a 10% polyacrylamide gel (SDS-PAGE). The eluted proteins were then transferred onto a nitrocellulose paper (Scb.leicherand Schuell, Keene, NH), after which the transferred proteins probed with alkaline phosphatase-conjugated MAb (lane 1, II1/D1; lane 2, II1/B5; lane 3, irrelevant antibody). The antibody-antigen complex was visualized by a substrate composed of 10 parts 0.1 M "Iris buffer, pH 9.5, 1 part BCIP (5bromo-4-chloro-3-indolyl phosphate), and 1 part nitroblue tetrazolium (Kirkegaard, Galthersburg, MD).

Yield and Purity. The best yield of ClqR from 101° Raji cells differs from one purification to another but usually is of the order of 100-200 lzg. The preparation after C l q-Sepharose affinity chromatography appears to be 60-70% homogeneous for pool I and 90-95% for pool II when analyzed on SDS-PAGE in the presence or absence of reducing agents.25 A second band of Mr -30,000-40,000 under both reducing and nonreducing conditions is usually seen and varies with the cell line and labeling conditions. 17 Stability. Purified ClqR is reasonably stable provided that repeated freezing and thawing is avoided. However, it has to be kept in solutions

578

RECErrORS ON LYMPHOID CELLS

[45]

containing 0.05-0.1% NP-40 and 0.02% NaN3 since irreversible aggregation may occur in the absence of detergent.

Properties of Human ClqR Recent data obtained in our laboratory seem to indicate the complexity of this receptor. The major band of the ClqR appears to be a singlechain glycoprotein of Mr -70,000. Upon reduction, this band electrophoreses as a weakly stained 80,000-85,000 Da molecule and seems to yield at least two other bands approximately 50,000 and 60,000 Da, respectively. In addition, we have always observed a 30,000-40,000 Da single-chain band which may represent a noncovalently linked constituent of the ClqR complex. While this structural description seem to justify what is observed both in SDS-PAGE and two-dimensional electrophoretic analyses, the precise structure of the molecule will have to wait until conclusive structural data is obtained. The function and significance of ClqR both in health and disease has become more apparent over the last few years. The receptor has been shown to mediate cellular cytotoxicity, 16inhibit collagen-induced platelet aggregationz2-24 enhance phagocytosis of C3b/iC3b coated, 51 or IgG opsonized 52target particles, modulate synthesis of Ig by B cells,53 inhibit ILl production by B cells, 54 and suppress the proliferation of tumor cell lines. 55 The ubiquity and widespread distribution of the ClqR makes one predict that it may be involved in different types of immunoregulatory functions which in turn may play significant roles both in health and disease. Acknowledgments Some of the experiments reported here were supported by Grants CA-41047 from the National Cancer Institute and 86-0742 from the American Heart Association. The skillful technical assistance of Austin Chen, Deirdra McKibbin-Vanghan, and Priscilla Munoz, and the excellent secretarial help of Karen Abramowski is acknowledged.

51 j. Sorvillo, I. Gigli, and E. Pearlstein, J. lmmunol. 316, 1023 (1986). 52 D. A. Boback, T. A. Gaither, M. M. Frank, and A. J. Tenner, J. Immunol. 138, 1150 (1987). 53 A. J. Tenner, R. Young, A. Malbran, A. S. Fauci, and J. L. Ambrus, Fed. Proc., Fed. Am. Soc. Exp. Biol. 46, 1195 (1987). 54 G. S. Habicht, G. Beck, and B. Ghebrehiwet, J. Immunol. 138, 2593 (1987). 55 B. Ghebrehiwet, G. Beck, and G. Habicht, Clin. Res. 35, 456A (1987).