Augmentation of Human Monocyte Opsonin-Independent Phagocytosis

Proc. NatL Acad. Sci. USA Vol. 78, No. 6, pp. 3649-3653, June 1981 Cell Biology

Augmentation of human monocyte opsonin-independent phagocytosis by fragments of human plasma fibronectin (particulate activators/alternative complement pathway/fragmented cold-insoluble globulin) JOYCE K. CZOP*t, JULIAN L. KADISHO§, AND K. FRANK AUSTEN*t¶ Departments of *Medicine and WPathology, Harvard Medical School; tDepartment of Rheumatology and Immunology, Brigham and Women's Hospital; and §Department of Pathology, Beth Israel Hospital, Boston, Massachusetts 02115 Contributed by K. Frank Austen, March 2, 1981

ABSTRACT Human plasma fibronectin isolated by gelatin- tein (10, 11). This protein is identical to cold-insoluble globulin affinity chromatography increases in a dose-dependent fashion the (12), which is now generally termed plasma fibronectin. The number of human monocytes that ingest particulate activators of present study demonstrates that purified human plasma fibro- the human alternative complement pathway in a fully synthetic nectin, defined with monoclonal antibody, gives rise by cleav- medium. The fibronectin effect is selective for these particulate age to a functional region that augments the capacity ofhuman activators, does not extend to particles whose ingestion is depen- monocytes to ingest particulate activators of the alternative dent upon opsonization with IgG, and is not observed with pre- complement pathway. treatment of the monocytes. Affinity chromatography with monoclonal antibody to plasma fibronectin of 440,000 daltons reveals that only 12-53% of the protein in phagocytically active gelatin- MATERIALS AND METHODS affinity-purified fibronectin preparations is bound to the antibody. Assessment of Phagocytosis by Monolayers The protein eluted after affinity chromatography with monoclonal of Monocytes. antibody ofactive preparations, which represented 1043% ofthe Monolayers of human monocytes were prepared and their protein applied, exhibits a 2- to 10-fold increment of activity per phagocytic capacities were quantitated in protein-free media as microgram of protein above the starting gelatin-affinity-purified described (3). Monolayers were incubated with 0.25 ml ofvar- material. Thus, the activity that augments the percent of human ious concentrations ofeach particle for 45 min at 370C in a CO2 monocytes ingesting particulate activators ofthe alternative path- chamber, rinsed twice in RPMI 1640 medium (GIBCO), treated way is antigenically defined as plasma fibronectin. Preparations with 0.84% NH4Cl for 4 min to lyse adherent but not ingested containing only intact 440,000-dalton fibronectin are also bound erythrocytes, and rinsed again. The monolayers were then fixed to and eluted from the monoclonal antibody, but they fail to aug- to the coverslips by sequential passage in a series of ethanol/ ment phagocytosis. When inactive 440,000-dalton plasma fibro- formaldehyde solutions (3) and stained for 4 min in 4% Giemsa nectin is subjected to limited trypsin cleavage, phagocytosis-en- solution. The number ofparticles ingested by at least 400 mono- hancing activity develops that is bound to and elutes from the cytes on each coverslip was determined by visual enumeration affinity column prepared with monoclonal antibody, thereby in- at x 1000 with a light microscope. The data are expressed as the dicating that the enhancing activity of plasma fibronectin resides percentage of monocytes ingesting one or more particles. in cleavage fragments. The target particles, namely zymosan, sheep erythrocytes (Es), rabbit erythrocytes (Er), mouse C57B[/6 erythrocytes Human monocytes possess two distinct receptors that mediate (Em), Es sensitized with rabbit 19S anti-E' (EsIgM), EsIgM bear- phagocytosis, the Fc receptors (1, 2) and the recognition unit ing fragments of the fourth and third components of comple- for particulate activators of the human alternative complement ment (EsIgMC4,3b), Es sensitized with high (1:50) and low pathway (3). Particles, such as sheep erythrocytes, which fail (1:4000) levels of rabbit 7S anti-Es (EsIgG-1:50 and E5IgG- to activate the plasma proteins of the human alternative com- 1:4000, respectively), and EsIgMC4,3b sensitized with the low plement pathway are not ingested by human monocytes. How- level of rabbit 7S anti-Es (EsIgMC4,3b-IgG) were prepared as ever, natural activators, such as rabbit or mouse erythrocytes, described (3, 8). and chemically or enzymatically modified sialic acid-deficient Isolation ofHuman Plasma Fibronectin. Plasma fibronectin sheep erythrocytes, activate the plasma proteins of the alter- was isolated from citrate- and dextran-treated plasma from in- native pathway (4) and are ingested by human monocytes in a dividual donors by affinity chromatography on gelatin-Sepha- fully synthetic protein-free medium (5). The monocyte recog- rose (13) without prior passage through a Sepharose 4B column. nition unit for activators, apart from ligand specificity, also dif- Two grams of calf skin gelatin (Sigma) was coupled to 120 ml fers from Fc receptors in its sensitivity to trypsin inactivation of Sepharose 4B beads (Pharmacia) by cyanogen bromide acti- (3, 5). The fixation ofthe third component ofcomplement, C3b, vation as modified by March et al. (14). A gelatin-Sepharose on an IgG-bearing particle or on a particulate activator of the column alternative pathway markedly enhances monocyte ingestion (2.5 x 28 cm) was sequentially washed with 0.01 M the through Fc receptors (5-8) or through the recognition unit Abbreviations: C, complement; DME medium, Dulbecco's modified for particulate activators (5, 8), respectively, whereas the pres- Eagle's medium; Em, mouse C57BL/6 erythrocytes; Er, rabbit eryth- ence of C3b on a nonactivator of the alternative complement rocytes; Es, sheep erythrocytes; E'IgG-1:50, E' sensitized with a high pathway, such as sheep erythrocytes, leads only to adherence (1:50) level ofrabbit 7S anti-E'; E'IgG-1:4000, E' sensitized with a low by human monocytes (5-8). (1:4000) level ofrabbit 7S anti-E'; E'IgM, ES sensitized with rabbit 19S Because the recognition unit for activators represents a anti-E'; E'IgMC4,3b, E'IgM bearing fragments ofthe fourth and third phagocytic mechanism that has no components ofcomplement; E'IgMC4,3b-IgG, E'IgMC4,3b sensitized requirement for IgG or com- with the low level (1:4000) of rabbit 7S anti-E'; PjNaCl, phosphate- plement, a study was undertaken ofthe possible cofactor activity buffered saline; RPMI-Mg, RPMI 1640 medium containing 5 mM ofa human protein with opsonic activity in liver slices, termed MgCl2. a2-opsonic protein (9) or opsonic a2 surface binding glycopro- ¶ To whom reprint requests should be addressed. 3649 Downloaded by guest on October 2, 2021 3650 Cell Biology: Czop et al - Proc. Natl. Acad. Sci. USA 78 (1981) sodium phosphate-buffered saline (Pi/NaCl) at pH 7.0, 8 M 160,000 daltons was purified per liter of tissue culture super- urea in 0.01 M sodium phosphate buffer at pH 7.0, and Pi/ natant. The hybridoma, designated BD4, produces anti-human NaCl containing 0.02% NaN3. A 100- to 250-ml portion of plasma fibronectin that is a mouse IgG, with K light chains as plasma was applied at 25TC to the column, which was then assessed by immunodiffusion with rabbit antisera against the washed with at least 20 bed volumes of 0.01 M Pi/NaCl con- various classes of mouse IgG and against mouse light chains taining 0.02% NaN3. The plasma fibronectin was eluted with (Miles). 4 M urea in 0.01 M phosphate at pH 7.0. Peak protein-con- Preparation of Affinity Columns Containing Monoclonal taining fractions were pooled, dialyzed at 25TC against 0.001 M Hybridoma Anti-Human Plasma Fibronectin or Nonspecific Pi/NaCl at pH 7.0, sterilized by filtration through 0.45-pum- Mouse IgG Coupled to Sepharose. Purified monoclonal hybri- pore-diameter membrane filters (Millipore), and stored at 25TC. doma antibody (3.5 mg) was coupled in PJNaCl, pH 7.0, with The amount of protein recovered in 35 fibronectin preparations 1 g of activated CH-Sepharose (Pharmacia) with 75% coupling ranged between 17 and 26 mg per 100 ml of plasma, with an efficiency. Ten milligrams of mouse IgG (Cappel) that had been average yield of about 20 mg per 100 ml of plasma. dialyzed against PjNaCl, pH 7.0, was applied to a 2-ml plasma To assess purity of the isolated plasma fibronectin, 35 Ag of fibronectin-Sepharose affinity column, and 97% of the loaded protein was incubated for 20 min at 61TC in 0.1 M Tris buffer, protein failed to bind to this column. Four milligrams ofthis fall- pH 8.0, containing 8 M urea and 1% NaDodSO4 in either the through nonspecific mouse IgG was coupled to 1 g of activated absence or presence of 100 mM dithiothreitol (Sigma) and sub- CH-Sepharose with a coupling efficiency of 80%. jected to NaDodSO4polyacrylamide gel electrophoresis (15). The protein sizing standards were human spectrin, human C3, RESULTS rabbit IgG, and ovalbumin. Effect of Fibronectin on the Ingestion of Particulate Acti- Preparation of Monoclonal Hybridoma Antibodies to Hu- vators of the Alternative Pathway by Human Monocytes. man Plasma Fibronectin. Hybridomas secreting antibodies to Monolayers of monocytes were assessed for their capacity to intact human plasma fibronectin of440,000 daltons (16, 17) were phagocytose Er at 2 x 108 per ml, zymosan at 4 x 10r particles prepared by the standard protocol of Kohler and Milstein (18). per ml, and Em at 2 x 10 per ml in the presence of various BALB/c mice were sensitized by subcutaneous immunization concentrations of plasma fibronectin derived by gelatin-affinity at two sites across the midline with 40 ,ug of homogeneous chromatography. The proportion of monocytes ingesting one plasma fibronectin emulsified in complete Freund's adjuvant or more of these activators increased in a dose-dependent fash- (Difco) and by subsequent boosters at monthly intervals with ion with increasing concentrations of the plasma fibronectin the same dosage of fibronectin emulsified in incomplete (Fig. 1A). The proportion of monocytes ingesting Er, initially Freund's adjuvant (Difco). Four days after an intravenous boost 20%, was increased to 60% with plasma fibronectin at 75 ttg/ with 40 ,ug of fibronectin, single-cell suspensions of the spleen ml, and the proportion ingesting Em and zymosan particles was cells were prepared and fused with a non-immunoglobulin-se- increased from 5% to 22% and from 20% to 49%, respectively, creting MOPC 21 plasmacytoma variant (P3-NSI-1-Ag4-1) in a with plasma fibronectin at 150 ,ug/ml. In contrast, plasma fi- 10:1 ratio with 28% (vol/vol) polyethylene glycol 1000 (Fisher) bronectin in concentrations between 40 and 150 Ag/ml had no in Dulbecco's modified Eagle's medium (DME medium; effect on the ingestion ofEsIgG-1:50 or EsIgG-1:4000 at 2 x 108 GIBCO) as described (19). The hypoxanthine/aminopterin/ per ml, or on the ingestion ofEs, EsIgMC4,3b, or E5IgMC4,3b- thymidine concentrations ofthe selective medium were 10 ,M, IgG at 2 x 108 per ml (Fig. 1B). Thus, plasma fibronectin en- 40 nM, and 15 ,uM, respectively. Positive clones were detected hanced monocyte-mediated phagocytosis of particulate activa- qualitatively by a solid-phase radioimmunoassay (20) in which 250 ng ofplasma fibronectin was adsorbed to polyvinyl chloride plates (Dynatech Laboratories, Alexandria, VA). The fibronec- A B tin-coated plates were overlaid with 25 ,ul of hybridoma supernatants, washed, and layered with goat IgG anti-mouse IgG 80- (Cappel Laboratories, Cochranville, PA) that had been radio- 70 labeled with "2I (New England Nuclear) by an iodine mono- chloride method (21). Positive clones, namely, those whose su- to contained at least twice the of control 60 pernatants radioactivity ti supernatants in which the MOPC 21 variant had grown, were 50 subcloned by limiting dilution in DME medium. 50 Hybridoma antibodies were produced by growth of the hybridoma cells at 37°C in spinner cultures containing DME me- 40 dium supplemented to 5% with calf serum (GIBCO) that had 30 been heat treated for 30 min at 56°C. The antibodies were purified from the tissue culture supernatants by affinity chroma- 20 tography on plasma fibronectin-Sepharose, prepared by cyanogen bromide coupling (14) of 1 g of human plasma fibronectin 10 to 60 ml of Sepharose 4B. A fibronectin-Sepharose column (1.5 x 8 cm) was loaded with 1 liter of cell supernatants harvested 7 days ofter inoculation and was washed with 2 liters of PJ 0 40 80 120 160 0 40O 80 120 160 NaCl, pH 7.0. The antibody was eluted with 0.1 M glycine HCl, Fibronectin, gg/ml pH 2.5, and immediately neutralized to pH 7.0 with 2 M Tris, pH 8.0. The eluted antibody was dialyzed against PJNaCl, pH FIG. 1. Effect of fibronectin dose on the capacity of human mono- ultrafiltration with XM- cytes to phagocytose various particles. (A) v, Er; e, zymosan particles; 7.0, concentrated by positive-pressure and n, Em. (B) m, E or E8IgMC4,3b; A, E8IgG-1:4000; A, E'IgMC4,3b- 50 Diaflo membranes (Amicon), and assessed for purity by IgG; and o, E8IgG-1:50. The zymosan particle-to-monocyte ratio was NaDodSOjpolyacrylamide gel electrophoresis. Ten to 20 mg approximately 2:1, whereas the erythrocyte-to-monocyte ratio for the of anti-plasma fibronectin presenting a single stained band of various intermediates was approximately 100:1. Downloaded by guest on October 2, 2021 Cell Biology: Czop et al - Proc. Natl. Acad. Sci. USA 78 (1981) 3651 tors of the alternative complement pathway without simulta- ingestion ofEr. These data indicate that the primary interaction neously affecting functions mediated by Fc and C3b receptors. of the active component in the plasma fibronectin is with the To determine if the active component in the plasma fibro- particulate activators ofthe alternative pathway and not on the nectin preparations bound to the target particles, 0.4 ml of monocytes. plasma fibronectin containing protein at 80 ugg/ml was incu- Chromatography ofFibronectin on Sepharose Coupled with bated for 20 min at 370C with Es at 2 X 108 per ml, Er at 2 x Monoclonal Hybridoma Antibodies to Intact Plasma Fibro- 108 per ml, or zymosan particles at 2 X 108 per ml. The particles nectin. The specific activities ofplasma fibronectin preparations were sedimented by centrifugation at 600 X g for 10 min at from different donors varied widely with respect to their ca- 250C, and the supernatants were tested at three dilutions for pacity to augment monocyte ingestion ofparticulate activators. their capacity to affect the ingestion of Er at 2 x 108 per ml. As determined by NaDodSO4polyacrylamide gel electropho- Adsorption of the plasma fibronectin with either zymosan par- resis, 90-95% of the protein in five plasma fibronectin prepa- ticles or Er substantially reduced the capacity of the superna- rations that were completely devoid of activity was a 440,000- tants to augment subsequent ingestion ofEr by the monocytes, dalton molecule consisting oftwo disulfide-linked polypeptides whereas adsorption with Es failed to remove any phagocytosis- of 235,000 and 210,000 daltons. In contrast, 20 plasma fibro- enhancing activity as compared to the effect of unadsorbed nectin preparations exhibiting phagocytosis-enhancing activity plasma fibronectin (Fig. 2). Thus, the active component in the contained less ofthe 440,000-dalton protein and additional pro- plasma fibronectin preparation had an affinity for the two ac- teins of 190,000, 170,000, 70,000, and 40,000 daltons. Reduc- tivators, namely zymosan particles and E , but failed to interact tion with dithiothreitol had no effect on the molecular weights with the nonactivating Es. of these additional proteins. To determine if the component Pellets of2 x 108 Er were incubated with plasma fibronectin with phagocytosis-enhancing activity in the active fibronectin at 10 gg/ml, 20 gg/ml, or 40 ,ug/ml for 20 min at 370C, washed preparations possessed fibronectin determinants, affinity chro- twice, and resuspended to 2 x 108 per ml in RPMI 1640 me- matography with a monoclonal antibody to intact plasma fibro- dium that contained 5 mM MgCl2 (RPMI-Mg). Pelleted E , ini- nectin was employed. tially suspended in RPMI-Mg, were treated in a similar fashion Intact, inactive plasma fibronectin was fully retained by the with medium alone and then resuspended in the same three BD4 hybridoma antibody and 85% of the loaded protein (875 concentrations of plasma fibronectin. Monocytes exhibited a jig) could be eluted with 0.1 M glycine HCl, pH 2.5 (Fig. 3A); dose-dependent increase in the ingestion of Er that were pre- the peak protein fractions exhibited no phagocytosis enhance- treated with plasma fibronectin and washed, comparable to that ment of Er ingestion by human monocytes. When a 968-,ug observed in the ingestion ofEr incubated together with plasma portion of a fibronectin preparation containing substantial fibronectin and monocytes. Under both conditions, the pro- amounts of phagocytosis-enhancing activity was applied to the portion of monocytes ingesting one or more E , initially 5%, BD4 hybridoma antibody column, 53% of the loaded protein increased to 20%, 42%, and 80% with plasma fibronectin at 10 was unbound, whereas 38% ofthe loaded protein and all of the ,g/ml, 20 ,ug/ml, and 40 ,ug/ml, respectively. Monolayers of phagocytosis-enhancing activity eluted with the glycine buffer monocytes pretreated with plasma fibronectin at 10-300 ,ug/ (Fig. 3B). The specific activity of the material eluted from the ml at either 25°C or37°C and washed did not exhibit augmented

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0 1:8 1:4 1:2 Fraction Dilution of adsorbed FIG. 3. Affinity chromatography with monoclonal antibody of fi- supernatants bronectin preparations lacking (A) or containing (B) phagocytosis-enhancing activity. The wash was carried out with Pj/NaCl, pH 7.0, and FIG. 2. Effect of fibronectin before (0) and after adsorption with the elution (arrows) with 0.1 M glycine HCl, pH 2.5. Column fractions ES (i), zymosan particles (s), or Er (v) on the capacity to augment sub- of both samples were assayed for protein by A2N (o) and assessed for sequent ingestion of one or more Er by monolayers of human their capacity to affect the ingestion of one or more Er (A) by human monocytes. monocytes from the same donor. Downloaded by guest on October 2, 2021 3652 Cell Biology: Czop et al - Proc. Natl. Acad. Sci. USA 78 (1981) BD4 hybridoma antibody column was twice that of the starting ingestion of E' were each augmented when the monocytes and material. Upon immunodiffusion with a polyclonal rabbit anti- Erwere incubated with fibronectin treated for 10 minwith tryp- serum to intact plasma fibronectin, the proteins that failed to sin (Fig. 4). In three separate studies, trypsin treatment oftwo bind to the BD4 monoclonal antibody showed a line of identity other batches ofphagocytically inactive fibronectin resulted in with the proteins retained by and eluted from the monoclonal the appearance ofphagocytosis-enhancing activity oftwo to four affinity column. Three other inactive fibronectin preparations times baseline ingestion at 10 min followed by apparent trypsin remained inactive after affinity chromatography on BD4 anti- inactivation of the newly formed active component. When two fibronectin, and in consecutive experiments the specific activ- phagocytically active fibronectin preparations were similarly ities of glycine eluates of 20 different active fibronectin prep- treated with trypsin, the 20-min samples exhibited only 10% arations increased to 2-10 times the specific activity ofthe initial and 23% of the initial phagocytosis-enhancing activity. material. The activity of the peak fractions, defined by the pro- To determine if the trypsin-generated material was recog- tein concentration required to increase by 50% the absolute nized by BD4 anti-fibronectin, the reaction mixtures, after neu- number of monocytes ingesting one or more Er, ranged be- tralization with Trasylol, were applied to the BD4 anti-fibro- tween 30 and 135 ,ug/ml. nectin affinity column. After a 10-min incubation with trypsin, Columns containing nonspecific mouse IgG coupled to Seph- about 30% of the starting material was no longer bound by the arose failed to retain any proteins when either phagocytosis- monoclonal antibody to fibronectin and acquired no phagocytic enhancing or nonenhancing plasma fibronectin was applied. All activity, whereas the protein eluting with glycine increased the of the phagocytosis-enhancing activity of the fibronectin prep- proportion of monocytes ingesting Er from 5% to 17% when a arations eluted with the breakthrough fibronectin protein with- concentration of 30 ,ug/ml was tested. Trypsin digestion for 20 out a change in the specific activity for the three preparations min decreased the phagocytic activity of the protein retained tested. Thus, the component mediating augmented phagocy- by the affinity column to baseline levels without altering the tosis shares immunologic determinants with intact inactive ratio of the unbound to bound proteins from that of the 10-min plasma fibronectin. trypsin sample. Thus, the phagocytosis-enhancing component Generation of Phagocytosis-Enhancing Activity by Trypsin in the fibronectin preparations is a proteolytically derived form Digestion of Intact Plasma Fibronectin. Multiple forms of fi- of fibronectin that can be inactivated by continued digestion bronectin that are antigenically identical to the intact 440,000 with trypsin. dalton molecule circulate in normal human plasma (22) and can be generated from the intact molecule in vitro by plasmin (22, DISCUSSION 23) or trypsin (24, 25) digestion. To determine if fragmented The capacity of human monocytes to ingest particulate activa- fibronectin contained phagocytosis-enhancing activity, 1.5 mg tors of the human alternative complement pathway in a fully of intact plasma fibronectin that failed to augment E' ingestion synthetic protein-free medium is augmented in a dose-response was incubated at 37°C with 10 jig of affinity-purified trypsin fashion by the addition ofhuman plasma fibronectin isolated by (3). Portions were removed at various time intervals, diluted gelatin-affinity chromatography (Fig. 1A). The fibronectin effect 1:5 in cold RPMI-Mg containing 2 ,ug ofTrasylol, and assessed is selective for ingestion of the particulate activators and does at a concentration of 75 ,g/ml for a capacity to augment the not extend to particles whose ingestion is dependent upon op- ingestion of Er by two different monocyte preparations. The sonization with IgG. Further, fibronectin does not induce the ingestion capacities of monocytes with low and high baseline ingestion ofparticles that adhere to monocytes through the C3b receptors and does not alter the C3b-mediated augmentation of an IgG-dependent phagocytic response (Fig. 1B). Two lines of evidence indicate that fibronectin interacts directly with the particulate activators to facilitate augmented phagocytosis by human monocytes. Pretreatment of the particulate activators with fibronectin followed by washing augments ingestion to the same extent as when fibronectin is present during the interaction of the particle with human monocytes, and adsorption IA 401 of fibronectin with activating particles removes the capacity of b. the preparation to augment the ingestion of particulate activa- ax tors by human monocytes (Fig. 2). Because monocytes pre- ._. treated with fibronectin and then washed do not have an in- C6.)0 creased capacity to ingest particulate activators, it is apparent Ca A\\ that the fibronectin function requires binding to the target par- so as to 0 20[ ticles. Whether that binding alters the surface augment directly its interaction with the monocyte recognition unit for I \b .-o activators, or whether, like C3b, fibronectin serves as a cofactor that facilitates phagocytosis by interacting with a second monocyte site is unknown. Fibronectin preparations obtained by gelatin-affinity chromatography and adjusted to contain the same protein concen- 0 10 20 30 trations differed by as much as 50-fold in their phagocytosis- Trypsin reaction time, min enhancing activity. NaDodSO4polyacrylamide gel electrophoresis of inactive plasma fibronectin revealed a major 440,000- FIG. 4. Generation of phagocytosis-enhancing activity from gela- dalton protein that reduced to 235,000- and 210,000-dalton frag- tin-affinity-purified phagocytically inactive plasma fibronectinby lim- ments. The active contained not the 440,000- ited treatment with trypsin. Samples were removed after various time preparations only intervals, treated with Trasylol, and assessed with fibronectin at 75 dalton protein but also fragments of 190,000, 170,000, 70,000, ,ug/ml for their capacity to augment ingestion of one or more Er by and 40,000 daltons as analyzed in unreduced gels, and reduction monocytes of two different donors. generated the 235,000- and 210,000-dalton fragments of the Downloaded by guest on October 2, 2021 Cell Biology: Czop et al. Proc. Natl. Acad. Sci. USA 78 (1981) 3653 440,000-dalton protein without altering the other components. 5. Czop, J. K., Fearon, D. T. & Austen, K. F. (1978) Proc. Natl. The possibility that fragmentation of intact plasma fibronectin Acad. Sci. USA 75, 3831-3835. was essential to the generation of the phagocytosis-enhancing 6. Huber, H., Polley, M. J., Linscott, W., Fudenberg, H. H. & Mfiller-Eberhard, H. J. (1968) Science 162, 1281-1283. principle was validated by establishing the antigenic relation- 7. Ehlenberger, A. G. & Nussenzweig, V. (1977)J. Exp. Med. 145, ship of the activity to plasma fibronectin (Fig. 3) and by gen- 357-371. erating the activity by proteolysis of inactive, intact plasma fi- 8. Czop, J. K. & Austen, K. F. (1980)J. Immunol. 125, 124-128. bronectin (Fig. 4). Affinity chromatography with monoclonal 9. Blumenstock, F. A., Saba, T. M. & Weber, P. (1978)J. Reticu- antibody to plasma fibronectin revealed that all the protein in loendothel. Soc. 23, 119-134. four inactive preparations and only 12-53% ofthat in 21 differ- 10. Scoville, W. A., Saba, T. M., Blumenstock, F. A., Bernard, H. & Powers, S. R., Jr. (1978) Ann. Surg. 188, 521-529. ent active preparations was bound. The eluted protein from the 11. Saba, T. M., Blumenstock, F. A., Scoville, W. A. & Bernard, H. inactive preparations had no phagocytosis-enhancing activity, (1978) Science 201, 622-624. whereas that from the active preparations exhibited a 2- to 10- 12. Blumenstock, F. A., Saba, T. M., Weber, P. & Laffin, R. (1978) fold increment over the specific activity applied. Thus, the J. Biol. Chem. 253, 4287-4291. phagocytosis-enhancing activity is defined as plasma fibronectin 13. Engvall, E., Ruoslahti, E. & Miller, E. J. (1978) J. Exp. Med. but is not the 440,000-dalton intact plasma protein (Fig. 3). 147, 1584-1595. 14. March, S. C., Parikh, I. & Cuatrecasas, P. (1974) Anal. Biochem. The observation that fibronectin as defined antigenically can 60, 149-152. be isolated from plasma in various states of fragmentation (22) 15. Weber, K. & Osborn, M. (1969)J. Biol. Chem. 244, 4406-4412. that resemble those achieved in vitro by plasmin (22, 23) or 16. Mosesson, M. W., Chen, A. B. & Huseby, R. M. (1975) Biochim. trypsin (24, 25) cleavage has functional implications. It is fibro- Biophys. Acta 386, 509-524. nectin cleavage (Figs. 3 and 4) that gives rise to the activity that 17. Chen, A. B. & Mosesson, M. W. (1977) Anal. Biochem. 79, increases the percentage ofhuman monocytes ingesting partic- 144-151. 18. Kohler, G. & Milstein, C. (1975) Nature (London) 256, 495-497. ulate activators of the alternative pathway. In earlier studies 19. Gefter, M. L., Margulies, D. H. & Scharff, M. D. (1977) Somatic demonstrating fibronectin augmentation of the phagocytic ac- Cell Genet. 3, 231-236. tivity ofKupffer cells in rat liver slices (9, 26, 27) or rat peritoneal 20. Brown, C. A., Corey, K. & Colvin, R. B. (1979)J. Immunol. 123, macrophages (28), gelatin-coated oil droplets (9, 26) or gelatin- 2102-2107. coated latex beads (27, 28) were used and the presence ofhep- 21. Helmkamp, R. W., Goodland, R. L., Bale, W. F., Spar, I. L. arin was required. Because both gelatin (13, 29) and heparin & Mutschler, L. E. (1960) Cancer Res. 20, 1495-1500. 22. Chen, A. B., Amrani, D. L. & Mosesson, M. W. (1977) Biochim. (30) have affinity for plasma fibronectin, it is possible that the Biophys. Acta 493, 310-322. phagocytosis-enkancing region of the fibronectin molecule was 23. Jilek, F. & Hormann, H. (1977) Hoppe-Seyler's Z. Physiol. uncovered during these assays or that the formation ofcleavage Chem. 358, 133-136. fragments was augmented. 24. Ruoslahti, E., Hayman, E. G., Kuusela, P., Shively, J. E. & Engvall, E. (1979)J. Biol. Chem. 254, 6054-6059. This work was supported by Grants AI-07722, AI-10356, and RR- 25. Sekiguchi, K. & Hakomori, S.-I. (1980) Proc. Natl. Acad. Sci. 05669 from the National Institutes ofHealth and in part by a grant from USA 77, 2661-2665. 26. Saba, T. M., Filkins, J. P. & DiLuzio, N. R. (1966)J. Reticuloen- the New England Peabody Home for Crippled Children. dothel. Soc. 3, 398-414. 27. Molnar, J., Gelder, F. 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