identification and characterization of opsonic fibronectin in synovial fluids of patients with...

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IDENTIFICATION AND CHARACTERIZATION OF OPSONIC FIBRONECTTN IN SYNOVIAL FLUIDS OF

PATIENTS WITH ACTIVE RHEUMATOID ARTHRITIS

JONATHAN KAY, K. FRANK AUSTEN, and JOYCE K. CZOP

A cofactor that selectively opsonizes particulate activators of the human alternative complement pathway and enhances their phagocytosis by human monocytes was identified in synovial fluids of patients with rheumatoid arthritis. The active material was present in fluids treated with protease inhibitors, was heat stable, and was unaffected by incubation with hyaluronidase. Chromatographic isolation of synovial fluid fibronectin by gelatin affinity and by immunoaffinity on antifibronectin monoclonal antibody BD4 yielded similar quantities of protein for each of 3 fluids. Synovial fluid proteins with the BD4 fibronectin epitope accounted for essentially all of the phagocytosis-enhancing activity and expressed this activity by opsonizing target activators. Additional chromatographic analyses of synovial fluid fibronectin with the BD4 epitope were carried out using Sepharose-bearing gelatin and 4 additional antifibronectin monoclonal antibodies. The opsonic materials were characterized as having 2 distinct fibronectin epitopes, which always mapped from the cell adhesive

From the Department of Medicine, Harvard Medical School, and the Department of Rheumatology and Immunology, Brigham and Women’s Hospital, Boston, Massachusetts.

Supported in part by NIH grants AI-22531, AI-22834, AI-23401, AI-23542, and HL-36110, and in part by a grant from The Peabody Foundation. Dr. Czop is an Established Investigator of the American Heart Association, and Dr. Kay i s a Postdoctoral Fellow of the Arthritis Foundation.

Jonathan Kay, MD: Instructor in Medicine; K. Frank Austen, MD: Chairman, Department of Rheumatology and Immu- nology, and Theodore Bevier Bayles Professor of Medicine; Joyce K. Czop, PhD: Associate Professor of Medicine.

Address reprint requests to Jonathan Kay, MD, Seeley G . Mudd Building, Room 607, 250 Longwood Avenue, Boston, MA 021 15.

Submitted for publication September 25, 1990; accepted in revised form January 7, 1991.

domain to the carboxyl-terminus of plasma fibronectin, but only rarely contained the gelatin binding domain.

Fibronectin is a high molecular weight glyco- protein that is ubiquitous in body fluids and tissues and is involved in various cell-contact processes (1-3). It is present in comparable concentrations in plasma from normal individuals and from patients with rheumatoid arthritis (RA) (4-7) and exhibits similar molecular size (8 ) , domain-related activities (7), and rates of metabolism and catabolism (9). Fibronectin is a major component of synovial fluid of patients with RA, present in concentrations 2-3 times higher than those in plasma and accounting for as much as 1.5% of the total joint fluid protein (5-7). Furthermore, RA synovial fluid fibronectin includes substantial quantities of fragments (7,8) which, on a weight basis, are 3-fold less active in binding gelatin than is plasma fibronectin, and 6-fold less active in binding fibrin (7).

Human monocytes phagocytose unopsonized particulate activators of the alternative complement pathway, such as zymosan particles and rabbit erythrocytes, through P-glucan receptors (10). A 180-kd opsonic fibronectin fragment (180K-opFnf) has been purified from gelatin-derived preparations of normal human plasma fibronectin (1 1); this fragment has an opsonic domain for particulate activators (1 1,12) and enhances monocyte ingestion of target particulate activators by improving contact through a separate domain for monocyte fibronectin receptors (13). Intact human plasma fibronectin expresses no phagocytosis- enhancing activity for particulate activators unless subjected to limited proteolysis (14).

Because RA synovial fluid is a particularly rich source of fibronectin fragments (7,8), this study was

Arthritis and Rheumatism, Vol. 34, No. 6 (June 1991)

KAY ET AL

undertaken to determine if molecules with opsonic function were present. RA synovial fluids were found to contain substantial amounts of target-specific phagocytosis-enhancing activity for particulate activators, and this opsonic function was expressed by molecules with defined antigenic determinants of fibronectin.

PATIENTS AND METHODS Patients. Synovial fluid was obtained from patients

with active, seropositive RA who were undergoing diagnos- tic and/or therapeutic arthrocentesis. All of the patients met the American Rheumatism Association 1987 revised criteria for RA (15). The fluids were drawn into individual plastic syringes and subjected to centrifugation at 700g for 4 minutes at 25°C; the resulting supernatant fractions were stored at -20°C.

Assessment of monocyte phagocytosis. Monolayers of normal human monocytes were prepared on siliconized glass coverslips (16) and were assessed for their phagocytic ca- pacities in RPMI 1640 medium (Gibco, Grand Island, NY) that contained 5 mM MgCl, and 10 mM HEPES, pH 7.4 (RPMI-Mg-HEPES). Erythrocytes from sheep (E") and from New Zealand white rabbits (E') were collected, washed, and stored in veronal buffered saline (17) containing 40 mM EDTA, pH 7.4. Immediately before use, the erythrocytes were washed and resuspended in RPMI-Mg-HEPES.

The preformed monolayers of monocytes were lay- ered with 0.25 ml of 5 x 107/ml E' or Es and various concentrations of synovial fluid test materials in RPMI-Mg- HEPES. After incubation for 45 minutes at 37°C in a humidified 5% CO, chamber, the monolayers were rinsed in two 50-ml volumes of RPMI 1640 medium, treated for 3 minutes with 0.84% NH,CI to lyse uningested target erythrocytes, rinsed again, fixed (161, and stained with Giemsa. The number of particles ingested by at least 300 monocytes per monolayer was determined by visual enumeration at 1,000x magnification with a light microscope, and the percentages of monocytes ingesting 1 or more particles were calculated.

Preparation of affinity columns containing gelatin, bovine serum albumin (BSA), or anti-human plasma fi- brosectin monoclonal antibody (MAb) coupled to Sepharose. Two grams of calf skin gelatin (Sigma, St. Louis, MO) was coupled with 120 ml of Sepharose 4B beads (Pharmacia, Piscataway, NJ) by cyanogen bromide activation (14). Five milligrams of BSA (ICN ImmunoBiologicals, Lisle, IL) was coupled in 0.1M phosphate buffer, pH 7.0, with 1.0 gm of activated CH-Sepharose 4B (Pharmacia) with 8 1% coupling efficiency. Mouse MAb to plasma fibronectin were raised in spinner cultures (14,181 and purified by affinity column chromatography (12) with rat MAb AHF5 anti-mouse light chain antibodies covalently linked to Sepharose by cyanogen bromide activation. Antifibronectin MAb BD4 and AB3 were each coupled in 0.1M phosphate buffer, pH 7.0, at a concentration of 6.9 milligrams of MAb per gram of activated CH-Sepharose 4B beads with coupling efficiencies of 91% and 89%, respectively. Five-milligram portions of anti-

fibronectin MAb CE9, BG8, and CPGl were each coupled with 1 gm of activated CH-Sepharose 4B beads with COU- pling efficiencies of 81%, 84%, and 82%, respectively.

Isolation and characterization of synovial fluid fibronectin Igy affinity chromatography on gelatin-Sepharose and antifibronectin MAbSepharose. Synovial fluids were prepared for chromatographic procedures by treatment with hyaluronidase (Streptornyces hyalurolyticus, type IX; Sig- ma). The hyaluronidase was dissolved in 0.094 sodium acetate buffer, pH 5.0, treated with a final concentration of 5 mM diisopropylfluorophosphate, and incubated with synovial fluid for 2 hours at 56"C, at an enzyme:fluid ratio of 150 unitdml.

Fibronectin was isolated from 1-4 ml of hyaluronidase-treated synovial fluid by prefiltration through a guard column of Sepharose 4B (1.5 x 4.0 cm) and elution with 4M urea in 0.01M phosphate buffer, pH 7.0, from a gelatin- Sepharose column (1.5 x 3.2 cm). The gelatin affinity- purified fibronectin was dialyzed against 0.001 M phosphate buffered saline (PBS), pH 7.0, and assessed for phagocytosis-enhancing activity.

For immunoaffinity purification of fibronectin, 1-4-ml samples of hyaluronidase-treated synovial fluid were prefiltered through a guard column of Sepharose 4B (1.5 x 4.0 cm) and eluted with 0.1M glycine-HC1, pH 2.5, from an antifibronectin MAb BD4-Sepharose column (1.5 x 3.4 cm). The eluted materials were neutralized with 2M Tris buffer, pH 8.0, dialyzed against 0.001M phosphate, 0.15MNaC1, pH 7.0 (PBS), and assessed for phagocytosis-enhancing activity. Chromatography was performed for individual patient samples.

For characterization of the materials with phagocytosis-enhancing activity, replicate samples of 1.0 ml or 0.5 ml of immunoaffinity-purified fibronectin with the BD4 epitope were subjected to parallel affinity chrcmatography on columns containing 0.5 ml of Sepharose beads bearing BSA, gelatin, or antifibronectin MAb CE9, BG8, AB3, or CPG1. Each of these MAb recognizes a defined epitope mapped on plasma fibronectin (18). The fall-through proteins from each of the 6 columns were collected in 1.5-ml fractions and the columns were washed with PBS. The materials retained by gelatin-Sepharose were eluted in 4M urea, whereas those retained by BSA-Sepharose and antifibronectin MAb- Sepharose were eluted in O.lM glycine-HCI, pH 2.5, and neutralized. The eluted protein fractions were collected in 1.0-ml fractions, dialyzed against PBS, and assessed with the corresponding fall-through fractions for phagocytosis- enhancing activity.

Protein determinations. Protein concentrations were determined by absorbance at 280 nm using extinction coef- ficients (1 cm/l rng/ml) of 1 .O for synovial fluids and 1.28 for fibronectin proteins (19).

RESULTS Opsonic effects of human RA synovial fluids on

monocyte phagocytosis. Synovial fluids from 7 patients with active, seropositive RA were clarified by centrifugation and screened for phagocytosis-enhancing ac-

OPSONIC FIBRONECTIN IN RA SYNOVIAL FLUIDS 689

Table 1. Effect of 2 concentrations of synovial fluid from patients with seropositive rheumatoid arthritis on monocyte ingestion of rabbit erythrocytes (E')

Mean f SD % monocytes ingesting

E'*

Patient

Synovial fluid 3.3 pl protein synovial (mg/ml) fluidhl

44 13 f 4 72 6 2 4 45 56 2 12 41 13 2 2 42 5 2 1 30 13 f 6 52 8 2 4

16.7 p1 synovial fluid/ml

21 2 7 34 .t 10

ND 58 :t 15 27 +- 10 47 It 10 41 -t 18

* Monolayers of human monocytes were assessed for E' ingestion after a 45-minute incubation with 5 x 107/ml Er in RPMI-Mg-HEPES containing 3.3 pl/ml or 16.7 pl/ml of each synovial fluid. The data are from the same 3 experiments with monocytes from different donors. In buffer alone, the percent of monocytes ingesting E' was 2 f I % . ND = not determined.

tivity for E' with human monocytes. Monolayers of monocytes were incubated for 45 minutes with 5 x 107/ml E' in RPMI-Mg-HEPES alone or E' in RPMI- Mg-HEPES containing 3.3 pl/ml or 16.7 pUml of synovial fluid, and were assessed for phagocytic activity. Incubation with each of 6 fluids from the RA patients caused an increase in the proportion of monocytes ingesting E', in a dose-related manner (Table 1) (fluid from I patient was tested at 3.3 pl, but not at 16.7 pl). With 3.3 pl of synovial fluid per ml of reaction mixture, the proportion of monocytes ingesting E' was increased from a control level of 2 .t 1% (mean 2 SD value from 3 experiments) to levels ranging from 5 5 1% to 56 5 12%. When the concentration of fluid was increased to 16.7 pllml, the proportion of ingesting monocytes ranged from 21 ? 7% to 58 2 15%. The amount of phagocytosis-enhancing activity appeared to be sample dependent, rather than related to total synovial fluid protein.

Each of the 7 synovial fluids was screened on at least 2 occasions at 16.7 pl/ml, for its effect on monocyte ingestion of E' which, unlike E', is not an activator of the human alternative complement pathway. Six of the 7 fluids failed to increase the proportion of monocytes ingesting Es from control values of 0%, and thus were selective for the activator of the alternative complement pathway. At a concentration of 16.7 pl/ml, the fluid from patient 3 increased the proportion of monocytes ingesting Es from a control level of 0 I 0% (mean k SD 4 experiments) to 59 2

19% and the proportion ingesting E' from 3 k 3% to 73 -+ 6%.

Effect of various treatments of RA synovial fluids on phagocytosis-enhancing activity. To determine whether activity was generated by proteases during the processing of samples, synovial fluid was aspirated from the inflamed knee joints of 3 other patients with seropositive RA (patients 8, 9, and lo), and the samples were immediately treated with a mixture of protease inhibitors (5 mM EDTA, 20 mM benzamidine, 6.7 mM phenylmethylsulfonyl fluoride, and 100 pg/ml aprotinin) o r an equivalent volume of diluent without inhibitors. Supernatant fractions were prepared by centrifugation, divided into portions, and stored at -20°C. Control and inhibitor-treated samples containing between 12.5 pl/ml and 100 pl/ml of synovial fluid in RPMI-Mg-HEPES were assessed for their effects on monocyte ingestion of 5 x 107/ml E'. For synovial fluids from each of the 3 patients, control and inhibitor-treated samples increased the proportion of monocytes ingesting E' in a similar, dose-dependent manner. Representative findings from studies using synovial fluid from patient 8 are depicted in Figure 1.

Synovial fluid samples from patients 1, 5, and 7 were each heated for 30 minutes at 56°C to inactivate the complement cascade. Untreated and heat-treated samples containing from 1.6 pl/ml to 100 pl/ml of synovial fluid in RPMI-Mg-HEPES were assessed for their effects on monocyte ingestion of 5 X 107/ml E'. The proportion of monocytes ingesting E' increased in a similar dose-dependent manner with increasing amounts of either untreated or heat-inactivated synovial fluid (Figure 2); the untreated synovial fluids failed to promote any monocyte ingestion of E'.

Characterization of the active synovial materials by column chromatography required reduction of fluid viscosity with hyaluronidase. In synovial fluids from patients 4 and 6, the effects of hyaluronidase treatment on phagocytosis-enhancing activity were determined concurrently in the same experiments that were conducted to determine the levels of activity for the native synovial fluids (Table 1). With 3.3 pl/ml and 16.7 pl/ml of hyaluronidase-treated fluid from patient 4, the mean t SD proportions of monocytes ingesting 5 x 107/ml E' were 12 k 4% and 43 2 17%, respectively, and with the same 2 concentrations of hyaluronidase-treated fluid from patient 6, the proportions were 11 5 4% and 46 f 15%, respectively. Thus, neither hyaluronidase digestion, complement inactivation by heat treatment, nor addition of protease inhib-

690 KAY ET AL

Figure 1. Dose-dependent effects on monocyte phagocytosis of rabbit erythrocytes (E') by synovial fluid from a representative rheumatoid arthritis patient (patient 8), untreated (0) or treated with protease inhibitors immediately after aspiration (0). The data are from 3 experiments with monocytes from different donors, and are plotted as the mean -C SD percent of monocytes ingesting E'.

itors changed the levels of phagocytosis-enhancing activity in synovial fluids.

Phagocytosis-enhancing activity of synovial fluid fibronectin isolated by gelatin affinity and by immunoaffinity chromatography. To determine whether fibronectin contributed to the activity of synovial fluid, hyaluronidase-treated fluids ( 1 4 ml) were subjected to column chromatography on Sepharose beads bearing gelatin or antifibronectin MAb BD4; both of these affinity columns had a capacity for B2.5 mg of human plasma fibronectin. The quantity of fibronectin isolated from a particular synovial fluid by gelatin affinity chromatography was comparable with that isolated by immunoaffinity on antifibronectin MAb BD4 (Table 2 ) and, in both cases, was <2% of the total synovial fluid protein. The concentration of synovial fibronectin defined by gelatin binding and elution ranged from 256

SYNOVIAL FLUID (p l /m l l

Figure 2. Dose-dependent effects of synovial fluids from rheumatoid arthritis patients 1 (a) and 5 (b) on monocyte phagocytosis of rabbit erythrocytes (E7, after heat inactivation of complement components (0), and on monocyte phagocytosis of rabbit erythrocytes (0) and sheep erythrocytes (E") (V) without heat treatment. The data are plotted as the percent of monocytes ingesting E' or E" in a single experiment. A separate study with synovial fluid from patient 7 gave similar results.

pglml of synovial fluid from patient 6 to as much as 840 pg/ml of synovial fluid from patient 2.

Monocytes were assessed for their ingestion of 5 x 107/ml E' in RPMI-Mg-HEPES alone and in buffer containing either increasing concentrations of the 2 fibronectin preparations or the unfractionated synovial fluid used as starting material. At the highest concentration tested (65 pg/ml), gelatin-derived fibronectin increased the proportion of monocytes ingesting E' to a mean of 13%, which represented the same level of enhancement effected by 3 pg/ml (interpolated) of the MAb BDUerived fibronectin (Figure 3). With the MAb BD4-purified fibronectin at a concentration of 40 pg/ml, the proportion of monocytes ingesting E' was 63%; in comparison, with a 1,600-pg/ml concentration of starting synovial fluid, gelatin fall-through, and

Table 2. Levels of fibronectin isolated from synovial fluid by column Chromatography on gelatin or on antifibronectin monoclonal antibody (MAb) BD4

Fibronectin (pg/ml)*

Patient Gelatin-Sepharose MAb BDkSepharose

2 840 790 4 559 568 6 256 238

~~ ~

* Data are expressed as pg of fibronectin per ml of synovial fluid, calculated using an extinction coefficient of 1.28.

OPSONIC FIBRONECTIN IN RA SYNOVIAL FLUIDS 69 1

MAb BD4 effluent, the proportion of monocytes ingesting E' was 61%, 49%, and 14%, respectively. The fibronectin isolated by gelatin affinity chromatography was approximately 6-fold and 22-fold less active than the fibronectin isolated by immunoaffinity chromatography on MAb BD4, for synovial fluids from patients 1 and 6, respectively. Thus, fibronectin with the BD4 epitope was present in synovial fluids from patients 1 and 6 and accounted for a greater proportion of the phagocytosis-enhancing activity than synovial fluid fibronectin with the gelatin binding domain.

To confirm the requirement for the BD4 epitope on the biologically active fibronectin fragment, 2 ml of synovial fluid from patient 4 and 4 ml of synovial fluid from patient 6 were chromatographed on antifibronectin MAb BD4-Sepharose, and 1.22 mg and 905 pg of fibronectin protein, respectively, were obtained. The unfractionated and eluted fibronectin proteins increased the proportion of monocytes ingesting 5 x 107/ml E' in a dose-related manner, whereas the fall- through materials (which contained >98% of the synovial fluid protein), at 800 pg/ml, had no effect (Figure

I I I 0 i000 2000 3000

b)

Figure 3. Dose-dependent effects on monocyte phagocytosis of rabbit erythrocytes (E') by synovial fluid from rheumatoid arthritis patient 6 (a) and by fibronectin proteins eluted from gelatin- Sepharose (0) and from antifibronectin monoclonal antibody BD4- Sepharose (A) (b). The data are from 2 experiments with monocytes from different donors in which duplicate determinations were made, and are plotted as the mean and range percent of monocytes ingesting E'.

c b,

t c

M 1 200 400 600 800 jl ) 20 40 60 80

PROTE/N Ipg/rn/ J

Figure 4. Dose-dependent effects on monocyte phagocytosis of rabbit erythrocytes (E') by synovial fluids from rheumatoid arthritis patients 4 (0) and 6 (A) (a) and by proteins in antifibronectin monoclonal antibody BD4 column effluents (b) and eluates (c). The data are from 2 experiments with monocytes from different donors, and are plotted as the mean and radge percent of monocytes ingesting E'. Separate studies with synovial fluids from patients 11, 12, and 13 gave similar results.

4). With synovial fluid from patient 4, the proportion of monocytes ingesting E' was increased to 50% with 39 @g/ml (interpolated) of the fibronectin proteins with the BD4 epitope and approached a mean plateau level of 62% with an input of 46 pg/ml. With synovial fluid from patient 6, the proportion of monocytes ingesting E' was increased to 50% with 43 pg/ml (interpolated) of the fibronectin with the BD4 epitope and approached 60% with 68 pglml. Thus, on a weight basis, fibronectin proteins with the BD4 epitope purified from 2 synovial fluids exhibited comparable levels of phagocytosis-enhancing activity, whereas the proteins lacking the BD4 epitope in the 2 fluids were inactive.

Functional characterization of synovial fluid fibronectin with the BD4 epitope. To determine which fibronectin domains were present on synovial fluid proteins with phagocytosis-enhancing activity, synovial fluids from 3 more patients with seropositive RA (patients 11, 12, and 13) were each chromatographed on antifibronectin MAb BD&Sepharose, followed by parallel affinity chromatography on BSA-Sepharose, gelatin-Sepharose, or antifibronectin MAb CE9-, BG8-, AB3-, or CPGl-Sepharose. The location of the epitopes on human plasma fibronectin which are recognized by each of the MAb is shown in Figure 5. The fall-through and eluted fibronectin proteins from each of the second columns were assessed, in dose- response experiments, for their effects on the propor-

692 KAY ET AL

AB3 CE9 BD4 + 4

A * V I

S

N h Figure 5. Schematic diagram of intact plasma fibronectin, showing the location of the epitopes recognized by 5 antifibronectin monoclonal antibodies. The arrangement of functional domains within the intact molecule is symbolically shown as the N-terminus (N-A), gelatin binding (El), 18-kd link between the gelatin binding and cell adhesive domains (O), cell adhesive domain (-), and high-affinity heparin binding domain (0). A subunit chain difference is depicted by an asterisk above the a chain.

tion of monocytes ingesting 5 x 107/ml E'. For synovial fluid from patient 11, 28 pg/ml of the fibronectin proteins that failed to bind to BSA-Sepharose or gelatin-Sepharose increased the percentages of monocytes ingesting E' to 51% and 47%, respectively; 28 pghl of the fall-through fibronectin proteins from MAb BG&, AB3-, and CPG1-Sepharose had no effect. When the corresponding eluted fractions were assessed at a constant dilution of 1:4, the percentages of monocytes ingesting E' were increased to 5296, 50%, and 48% by fibronectin with the BG8, AB3, and CPGl epitopes, respectively; a 1:4 dilution of the materials eluted from BSA- and gelatin-Sepharose had little phagocytosis-enhancing activity (Figure 6a).

For synovial fluid from patient 12, 65 pghl of the fibronectin proteins that failed to bind to BSA- Sepharose or gelatin-Sepharose increased the percentages of monocytes ingesting E' to 39% and 25%, respectively; 65 pglml of the fall-through fibronectin proteins from MAb C E S , BG&, AB3-, and CPGl- Sepharose had no activity. When the corresponding eluted fractions were assessed at a constant dilution of 1:2, the percentages of monocytes ingesting E' were increased to 3596, 31%, 31%, and 44% by fibronectin with the CE9, BG8, AB3, and CPGl epitopes, respectively; a 1:2 dilution of the materials eluted from BSA- and gelatin-Sepharose had no phagocytosis-enhancing activity (Figure 6b).

At 31 pg/ml of the fall-through fibronectin proteins and a 1:2 dilution of the eluted samples, synovial fluid from patient 13 was similar in its phagocytosis- enhancing activity to synovial fluid from patient 12 (results not shown). Although the effluent of affinity chromatography with MAb CE9 increased the per-

701 60 *;

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0 8 60 @ 50

40

30

20

10

0

2

T T

- + - + - + - + - + - + 8SA G E L C E 9 B G 8 AB3 CPGl

AFF/N/ TY MA TRlX Figure 6. Effects on monocyte phagocytosis of rabbit erythrocytes (E') by fibronectin proteins with monoclonal antibody (MAb) BD4 epitopes from synovial fluids from rheumatoid arthritis patients I 1 (a] and 12 (b), after affinity chromatography on separate bovine serum albumin (BSA)-Sepharose, gelatin-Sepharose, and antifibronectin MAb CE9-, BG8-, AB3-, and CPGl-Sepharose columns. The histograms depict the percent of monocytes ingesting E' for the effluents (stippled bars) and the eluates (diagonally hatched bars) from each of the 6 columns. The data are from 2 experiments with different monocyte donors, and are plotted as the mean and range. Two separate studies with synovial fluid from patient 13 and monocytes from different donors gave results similar to those for patient 12.


centage of monocytes ingesting E' to 29% in patient 11, the fall-through for patients 12 and 13 had little or no activity.

Mechanism by which synovial fluid fibronectin proteins with the BD4 epitope enhance monocyte ingestion of E'. To determine whether synovial fluid fibronectin fragments bound firmly enough to the target (E') or to the monocytes to allow pretreatment followed by washing, the phagocytosis enhancement achieved when the fragment was present throughout the assay was compared with that achieved when it was present only during pretreatment of one of the cellular components. Monocytes were pretreated for 35 minutes at 37°C with increasing concentrations of fibronectin proteins with the BD4 epitope, rinsed in two SO-ml volumes of RPMI 1640 medium, and assessed for their phagocytosis of 5 x 107/ml E' in RPMI-Mg-HEPES. Alternatively, monocytes were pretreated with buffer for 35 minutes at 37"C, rinsed, and assessed for their phagocytosis of 5 x 107/ml E' that had been pretreated for 35 minutes at 25°C with the same doses of fibronectin proteins and washed by sedimentation at 600g. As a positive control, monocytes were assessed for their phagocytosis of 5 x 107/ml E' in the presence of the same amounts of fibronectin protein throughout the assay.

Inclusion of the fibronectin protein throughout the assay or only during prior opsonization of E' resulted in similar, dose-dependent increases in monocyte phagocytosis, whereas pretreatment of monocytes before the addition of E' did not produce such an increase (Figure 7). At the highest concentration tested, 60 pdml of the affinity-purified protein increased the proportion of monocytes ingesting E' from 0% to 4%, 55%, and 57% for pretreated monocytes, monocytes in complete mixtures of E' and protein, and monocytes incubated with pre-opsonized E', respectively. Thus, the fibronectin proteins with the BD4 epitope enhanced phagocytosis by a mechanism in- volving direct opsonization of E' and subsequent binding to monocytes for ingestion.

DISCUSSION The demonstration in human rheumatoid arthri-

tis synovial fluid of forms of fibronectin that are opsonic for particulate activators identifies a novel in vivo compartmental source of such activity. Of the 13 fluids studied, only 1 also promoted monocyte ingestion of E', thereby focusing attention on opsonic

80 Lul I c L

70

Figure 7. Dose-dependent effects on monocyte phagocytosis of rabbit erythrocytes (E') by fibronectin proteins with monoclonal antibody BD4 epitopes from synovial fluids from rheumatoid arthritis patients 4 (a) and 6 (b), when introduced simultaneously with E' (A) and when used to pretreat monocytes (0) or E'(A). The data are plotted as the percent of monocytes ingesting E' from single experiments. Two separate complete studies of each synovial fluid with monocytes from different donors gave similar results.

activity distinct from cofactors such as those derived from the complement system. This distinction is fun- damental inasmuch as particulate activators, by defi- nition, recruit the alternative complement pathway (20). Fibronectin in RA synovial fluid is distinguished not only by its elevated levels (5-7), but also by its content of proteolytic fragments (7,8).

Several lines of evidence indicate that fibronectin fragments with the BD4 epitope are the major, and possibly the only, proteins with phagocytosis-enhancing activity for particulate activators. In- activation of heat-labile activating complement proteins at 56°C for 30 minutes (Figure 2) or for 2 hours during hyaluronidase treatment failed to affect the levels of phagocytosis-enhancing activity for particulate activators obtained with native fluids. After affinity chromatography, synovial fluid fibronectin with the gelatin binding domain was, on a weight basis,

KAY ET AL

6-22-fold less active than synovial fluid fibronectiri with the BD4 determinant (Figure 3). Immunochemi- cal depletion of fibronectin proteins with the BD4 epitope from synovial fluids of patients with active RA (Figure 4a) yielded proteins that had no phagocytosis- enhancing activity (Figure 4b). Inasmuch as immuno- chemical analysis of human plasma proteins with several antifibronectin MAb revealed no proteins with the BD4 epitope other than fibronectin (18), it can be concluded that the opsonic synovial fluid fragment was derived from fibronectin.

The 3 RA synovial fluids phenotyped by se- quential MAb affinity chromatography each contained phagocytosis-enhancing fibronectin fragments that did not bind to gelatin (Figure 6). In each of these fluids, essentially all of the phagocytosis-enhancing activity of the fibronectin proteins with the BD4 epitope was subsequently removed by affinity chromatography on antifibronectin MAb BG&, AB3-, or CPG 1-Sepha- rose, thereby indicating that the active components had 2 distinct and defined epitopes for fibronectin.

Each of the 5 MAb used in this study maps to a different domain of fibronectin, and none has detect- able specificity for plasma proteins other than fibronectin (18). The BD4 epitope is located within the cell adhesive domain; the BG8 epitope is located within the heparin binding domain; the AB3 epitope, which is unique to the a chain of plasma fibronectin, is located between the heparin binding domain and the interchain disulfide bonds; and the CPGl epitope is located at the carboxyl-terminus of both fibronectin subunit chains (Figure 5) . Thus, on a molecular basis, the opsonic forms of RA synovial fluid fibronectin span from the BD4 epitope within the cell adhesive domain to the carboxyl-terminus. For each of the 3 synovial fluids analyzed, varying levels of phagocytosis-enhancing activity were exhibited by fibronectin bearing the CE9 and BD4 epitopes (Figure 6). MAb CE9 maps to an epitope which is within 61,000 daltons of the amino-end of the BD4 epitope and is contained on an 18-kd polypeptide located immediately adjacent to the carboxy-end of the gelatin-binding domain (18). The variable expression of the CE9 epitope on opsonic forms of synovial fibronectin was, in all likelihood, governed by the location of CE9 in a region of fibronectin that is particularly sensitive to proteolytic cleavage. However, inclusion of the CE9 epitope on opsonic fragments beyond the amino-end of the BD4 epitope was not essential for phagocytosis-enhancing activity.

The finding that some gelatin affinity-derived

fibronectin also had phagocytosis-enhancing activity, but less than the BD4-derived fibronectin from the same RA synovial fluid at similar protein concentrations (Figure 3), again suggested that fragments containing additional amino-terminal portions of fibronectin beyond that provided by the BD4 epitope were not essential for activity, and further implied that the carboxyl-terminal ends of most fragments obtained by gelatin affinity chromatography lacked epitopes necessary for opsonic activity. Taken together, the findings indicate that the novel fibronectin fragments with opsonic activity for particulate activators are generated by cleavage from the amino-terminus.

As with the gelatin binding plasma-derived fragment, 180K-opFnf (12), the synovial fluid fibronectin fragments with the BD4 epitope were opsonic via binding to the target particles (Figure 7), to provide a link to the fibronectin receptors on monocytes (12,21- 23). The opsonic domain is the functional determinant that permits 180K-opFnf to distinguish asialylated microbial and sialic acid-deficient erythroid particulate activators (E') from fully sialylated nonactivators such as Es (24), and then provides the domain for interaction with the monocyte (12).

Hyposialylated proteins have been found in the sera of patients with RA and may be involved in the pathogenesis of synovitis. Rabbits immunized with asialylated rabbit IgG in Freund's complete adjuvant developed serum anti-IgG, while those immunized with untreated rabbit IgG did not. When challenged with an intraarticular injection of asialylated rabbit IgG, rabbits previously immunized with that antigen developed a histologically evident inflammatory synovitis (25).

IgG rheumatoid factor (IgG-RF), isolated from the sera of 5 patients with RA, had 1657% the sialic acid content of the non-RF IgG from the same individuals. Their IgM-RF contained <25% the sialic acid content reported for normal IgM (26). In plasma and synovial fluid of patients with RA, fibronectin is often a predominant component of cryoprecipitable immune complexes (27). RF activity is associated with fibronectin immunoreactivity in 2.5% polyethylene gly- col precipitates from RA patient sera (28). Opsonic forms of fibronectin that recognize such hyposialylated materials may provide a physiologic mechanism of rapid clearance in the microenvironment of the joint or may contribute to inflammation by initiating such monocyte responses as the release of lysosomal en- zymes (29) and/or the generation of leukotrienes B, and C4 (30).


Proteolytic cleavage of plasma fibronectin ex- poses other biologic activities not found in the intact molecule. These activities include inhibition of endothelial cell growth in culture (31,32), binding to low- sulfated heparin sulfate (33), enhancement of morpho- logic transformation of fibroblasts by Rous sarcoma virus (34), initiation of fibroblast DNA synthesis (39, chemoattraction of monocytes (36-38), adhesion of neutrophils to endothelial cells (39), and stimulation of T helperhnducer (CD4+, 4B4+, 2H4-) cell proliferation (40). The plasma fibronectin fragments that act in concert with anti-CD3 antibodies to stimulate T helperhnducer cell mitogenesis contain the cell adhesive domain, including the BD4 epitope, and adjacent regions of the fibronectin molecule. Similar to the opsonic fibronectin fragments, those fragments that stimulate T helperhducer cell proliferation also display variable cleavage from the N-terminus; some express the CE9 epitope of plasma fibronectin, while others lack the CE9 epitope and still contain the heparin binding domain (40). The presence of these fragments in RA synovial fluid suggests a mechanism by which limited proteolysis could regulate both monocyte phagocytosis and lymphocyte activation.

ACKNOWLEDGMENTS We thank Dr. Julian L. Kadish for providing us with

the monoclonal antifibronectin antibodies and Drs. Ronald J. Anderson, Jean M. Jackson, and Michael E. Weinblatt for supplying the synovial fluids.

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identification and characterization of opsonic fibronectin in synovial fluids of patients with...

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