double-stranded DNA immune complexes to CR1

CLINICAL

IMMUNOLOGY

AND

33,

IMMUNOPATHOLOGY

220-23

1 f 1984)

Suramin inhibits the Binding of Complement-Fixing Antibody/Double-Stranded DNA immune Complexes to CR,’ RONALD P. TAYLOR,~AROL Departments

of Biochemistry

HORGAN, ond

Medicine,

ALISA University

Charlottesville,

Virginia

HARBIN,ANDJOSEPH BURGE? c!f‘ Viyiniu

School

of Medicine.

22908

The effects of varying concentrations of heparin and suramin on the complementmediated binding of antibody/double-stranded DNA immune complexes to red blood cells (RBCs) and Raji cells have been investigated. If the immune complexes are briefly opsonized with complement, suramin can block binding to both cell types, and heparin can block binding to RBCs. In addition, if these complexes are first allowed to bind to RBCs or Raji cells, relatively brief incubations in suramin are sufficient to cause release of the complexes from the cells’ C3b receptors. The potential clinical and diagnostic implications of these findings are discussed. C 1984 Academx Prew, Inc. INTRODUCTION

The interaction of soluble immune complexes (IC) in the circulation with complement and both fixed and circulating cells is a complex process that involves a number of distinct C3 fragments covalently bound to the IC (l-7), as well as a variety of cellular receptors for these complexes and fragments (I, 8-14). For example, the binding of C3b-coated IC to specific receptors (CR,) on red blood cells (RBCs) may be a key event which influences whether these complexes are cleared innocuously or instead deposit in the tissues and initiate inflammatory reactions in certain immune complex-mediated diseases (15 19). Raji cells have a receptor for C3b that is apparently different from CR, (20), and in addition these cells also have a receptor (CR,) that binds C3bi and C3d,g (20. 21). In view of the pathogenic potential of complement-fixing IC it would be most useful to identify specific therapeutic agents that could inhibit these binding reactions. In an effort to probe the ICK3 fragment-receptor binding reaction we have examined two drugs used in human diseases that interact with C3b: heparin and suramin (22, 23). We have chosen the antibody/double-stranded (ds) DNA IC as appropriate for this study in view of their recognized importance (24-26) in the pathogenesis of the autoimmune disease systemic lupus erythematosus (SLE). An examination of the effects of heparin and suramin on the binding andlor release of soluble, complement-coated human antibody/dsDNA IC to both RBCs and Raji cells forms the basis of this report. ’ This work was supported by NIH Grants AM 24083 and AM 3 13I I ’ To whom correspondence should be addressed: Rheumatology Division. Department of Medicine. University of Virginia School of Medicine. Charlottesville. Va. 22908. 220 OOVO- 1229184 Copyright All rights

$ I .50

B 1984 by Academx Preu. Inc. of reproduction in any form reserved

ANTIBODY/dsDNA

MATERIALS Immune

IMMUNE

COMPLEXES

221

AND METHODS

Complexes

Soluble [3H]dsDNA/anti-DNA IC were prepared with [3H]dsPM2 DNA and heat-inactivated SLE plasmas (with high titers for dsDNA) as previously described (19, 27). A number of different protocols were then followed to opsonize the IC with complement, as follows. (1) The IC were opsonized at 37°C for 5 min in a nine-fold dilution of normal human serum (NHS) as the complement source and they were then purified and isolated by sucrose density gradient ultracentrifugation (27). This procedure removes excess complement components, and the isolated complexes (we designate them as C’-treated IC) are stable, can be frozen and defrosted, and bind to RBCs and Raji cells in the absence of additional complement. (2) The [3H]dsDNA/anti-DNA IC were opsonized in a nine-fold dilution of NHS for 5 min at 37°C and were then used in the RBC or Raji cell binding studies. In some experiments EDTA was added to the samples after the 5-min incubation (to a final concentration of 0.01 M). (3) The IC were opsonized in undiluted NHS for 2 hr at 37°C (STIC). (4) The IC were allowed to bind to RBCs under the standard conditions of our RBC-CF assay (28), except NHS at a 20-fold final dilution was used as the complement source. Then, after the RBCs (containing the bound IC) were washed, 1 ml of whole autologous blood was added and, after the blood had clotted for 1 hr at 37°C the IC that were released into the supernatant were examined for their ability to bind to RBCs and Raji cells. We designate these released immune complexes as RIC. (5) C’-treated IC were treated with either buffer, or purified C3b inactivator (I), or purified H, or I + H for 3 hr at 37°C. Subsequently these soluble proteins were removed by subjecting the treated samples to another isolation via sucrose density gradient ultracentrifugation. (6) In a few cases dilutions of guinea pig serum (GPS) or other NHS dilutions were used to opsonize the complexes for periods varying between 10 min and 1 hr at 37°C. Cells RBCs were isolated from normal healthy donors as previously described (19). Raji cells were grown and isolated for the binding assays as described previously (29), except in about one-half of the experiments where the IC and the Raji cells (after washing) were resuspended in medium containing 0.02 M EDTA and 0.2% sodium azide. In no instance could we detect any significant difference in the Raji assays when the medium also contained EDTA and sodium azide. Binding

and Inhibition

Assays

Binding of complement opsonized antibody/dsDNA immune complexes to RBCs was performed following our published procedures (27). Typically 100 ~1 of the complement-treated [3H]dsDNA/anti-DNA IC was mixed with 100 ~1 of a 25% suspension of washed RBCs. After an incubation of 20 min at 37°C the RBCs

222

TAYLOR

ET

AL..

were pelleted by centrifugation at 8OOOg for 2 min and 100 ~1 of supernatant wab counted for tritium, to determine the amount of IC bound to the RBCs. In the Raji cell assay 100 ~1 of the IC was mixed with 50 ~1 of Raji cells (2 x lo6 cells) and after an incubation of 20 min at 37°C the sample was centrifuged and binding was determined by counting one-half of the supernatant for tritium. In these experiments spontaneous precipitation of the IC was measured by centrifuging them in the absence of RBCs or Raji cells. In almost all cases less than 10% of the IC precipitated under these conditions, and all reported values for binding are corrected for this low level of “background” precipitation. In the inhibition assays the complement-treated IC were mixed with solutions containing varying concentrations of heparin or suramin, and after a IO-min incubation binding to the RBCs or Raji cells was determined. In a few cases the drugs were added to the IC before complement opsonization (see below). In this case the drugs and IC were first mixed, and after 10 min the complement source (a dilution of serum) was added. In no C’CISCdid we incubate the drugs with serum before addition of the IC. In the release experiments. cells containing known amounts of bound IC were washed and then resuspended in buffers containing varying concentrations of heparin or suramin. After 1-2 hr at 37°C the samples were centrifuged, the supernatants were counted, and the percentage of released tritium was determined. Control experiments included release studies in gelatinVerona1 buffer (GVB+‘, <50/o release), and in solutions containing DNase (>95% release). Sodium suramin, USP, was purchased from F. B. A. Pharmaceuticals, Mobay Corporation, New York. RESULTS Studies with Freshly

Prepared

Complexes

and C-Treated

IC

If moderate amounts of heparin or suramin are first added to the antibody! dsDNA IC before they are opsonized with complement, there is little detectable binding of the IC to RBCs or Raji cells (Fig. 1). These results are not surprising because it is well known that both of these drugs inhibit a number of reactions in the complement activation cascade (22-23. 30-33). Figure 2 indicates that if the complexes are opsonized with complement b&w addition of the drug (i.e., C’-treated IC were used). there is also a marked reduction in binding to both RBCs and Raji cells in the presence of suramin. However, in this case although heparin inhibits RBC binding, it has only a marginal effect on Raji cell binding. We have also determined that if cells containing bound C’-treated IC are washed and then incubated in varying concentrations of the drugs, suramin is quite effective at causing the release of the complexes from both types of cells (Fig. 3). but once again heparin is effective only in releasing complexes bound to RBCs. These dose-response experiments also indicate that for a given concentration, suramin is a more potent agent than heparin for both inhibiting binding and causing release of the complexes (Figs. 2 and 3). For these reasons the remaining experiments were done with suramin only. We next compared two sources of complement in these experiments, NHS and

ANTIBODY/dsDNA

Drug

Concentration.

mg/mt

Drug

IMMUNE

Drug

Concentration,

223

COMPLEXES

Concentration.

mg/ml

mglml

FIG. I. Effect of varying concentrations of heparin (0) and suramin (0) on the complement-mediated binding of antibody/[3HJdsDNA IC to RBCs (A and B) and Raji cells (C). The drugs were added to the immune complexes ca. 10 min before complement was added. In A and C, NHS was used as the complement source, and GPS was used in (B). In some cases error bars were omitted for clarity.

GPS (Table 1). The results indicate that the IC opsonized with GPS are not bound by Raji cells. In addition, we find that suramin is a more effective inhibitor of binding of the IC to human RBCs when NHS is used for opsonization than when GPS is used. It also should be noted that even a relatively low input of NHS is sufficient to opsonize the IC. Incubation of the antibody/dsDNA IC for 5 min at 37°C in a final concentration corresponding to a U-fold dilution of NHS gave 59% binding to Raji cells and 82% binding to RBCs. In this particular experiment binding to the washed Raji cells was determined in buffers containing EDTA and sodium azide in order to prevent the release of I and H (20,34) and the possible generation of C3bi.

224

TAYLOR

ET AL.

FIG. 2. Effect of heparin (0) and suramin (0) on the binding of preopsonized IC (C’-treated IC) to RBCs (A) and Raji Cells (B). The drugs were added to the opsonized IC, and 10 min later the cells were added.

Studies with “Aged”

Complexes

It is now well recognized that if IC covalently labeled with C3b and either bound to RBCs or free in solution are incubated for varying time periods in serum, much of the bound C3b on these complexes is degraded to either C3bi or C3d,g (35-38). We performed the next series of experiments to determine how this “aging” process might affect the recognition of these altered immune complexes by RBCs and Raji cells, and how suramin could perturb the binding reaction. Table 2 summarizes the results of experiments on IC at different stages of this “aging” process. We can distinguish these complexes by virtue of both their ability to bind to RBCs and Raji cells and the potential of suramin to inhibit this binding. As we have noted, immune complexes in group A (briefly opsonized with dilute NHS) bind very well to both RBCs and Raji cells, and the binding is eliminated by suramin. If a C’-treated IC is treated with purified I and H. and then the I and H are removed by sucrose gradient ultracentrifugation (the treated C’-treated IC is pelleted, and then redispersed in buffer) it can be seen that for these IC (group B) binding to RBCs is markedly diminished, but there is no significant loss in binding to the Raji cells. In addition, suramin still effectively blocks binding of these complexes to both cell types (Table 2). If the complexes are exposed to NHS for prolonged periods, however, the results are quite different (STIC, group C). For example, binding to RBCs is

ANTIBODY/dsDNA

0

05

IMMUNE

COMPLEXES

1Q 1.5 Concentratton.

20 mglml

25

Drug

1.0 I.5 Concentration,

20 mg/mb

25

Drug

0-

225

FIG. 3. Release of C’-treated IC from either RBCs (A) or Raji Cells (B) by heparin (0) or suramin (0). The C’-treated IC were tirst bound to the cells. After washing, the drugs were added and the samples incubated for 1 hr at 37”C, and then the 3H counts released into the supematant were measured. When the incubation time for the RBCs was decreased to 15 min, essentially identical results were obtained.

reduced considerably (~30%), but in this case although the complexes still bind to Raji cells, this binding is only slightly inhibitable by suramin. We also examined samples that were released from RBCs after I hr of clotting at 37°C (RIC). These complexes do not rebind to RBCs at all, but they do bind to Raji cells, and again suramin has only a small (if any) effect on the binding. Other Control Experiments In other experiments neither heparin nor suramin had any detectable effect on the binding between antibodies and dsDNA. For example, we found that the binding of PM2 [3H]dsDNA in C’-treated IC was 98% in the Farr assay (39) in the presence and absence of the drugs. Also, in a few studies *251-heat aggregated IgG was used as a model IC, and we found that its complement-mediated binding to RBCs could also be inhibited by suramin (results not shown). DISCUSSION The most important finding in the present work is the observation that suramin can prevent the binding of freshly opsonized antibody/dsDNA IC to both RBCs and Raji cells, and it can also release the majority of the complexes from the cells after they are bound. We believe that the major complement component on

226

TAYLOR

ET AL.

TABLE I EFFECT OF COMPLEMENT SOURCEAND SURAMIN ON ‘IH~ BINUIN~ ok PK~WSONILEI) [‘H]dsDNA: ANTI-DNA IC TO RBCs AND RAJI CELLS -__--~-

Complement source and concentration

~~

.~~~

? [‘HIDNA bound ~-~-.-~-~..--.~~---..-~~~._ -~~--Kaji cell binding

-___~--. RBC binding .-- Suramin

+ Suramin”

Suramin

GPS120h

96’ 95 82

hl XI 32

0 (I 0

4 I) (I

GPSllh GPSIld NHS/20b NHSIlh

90 90 92 55

65 50 0 0

(1 0 71 67

0 0 f!

t Suramin

17

” Final concentration of 2.5 mgiml. b Sample was opsonized in diluted complement source (GPSIZO means a 20-fold dilution of GPSl for lo-30 min at 37°C. ’ Multiple entries represent the results for independent experiments with freshly prepared IC preparations. ’ Opsonized for 2 hr at 31°C.

BINDING OF [3H]dsDNA/ANrt-DNA METHOD OF OPSONIZATION

IC WITH

TO

TABLE 2 RBCs AND RAJI C~I.I.S:

COMPLEMENT

.~ND INHIBITORY

% [‘HIDNA --____ RBC binding Type of IC Group A I. Opsonize in NHSi20 for 5 min 2. Cl-treated IC Group B’ I. C’-treated IC + I+H Group C 1. STIC 2. RIC

EFFECTS EFFECTS

rn VAKIATION IN THL OF SUKAMIN

bound Raji cell binding -~-~Suramin + Suramin”

- Suramin

+ Suramin”

97” 89 79 86

0 4 0 0

77 78 62 68

I! 2 I’I 6

28

0

77

0

30 12

0 0

73 62

(3 17

0 0

0 0

54 7x

57 t,7

U Final concentration of 2.5 mg/ml. b Multiple entries represent the results of independent experiments. c C’-treated IC were treated with I and H for 3 hr, and then the treated IC were isolated by sucrose density gradient ultracentrifugation.

ANTIBODY/dsDNA

IMMUNE

COMPLEXES

227

these IC (group A, Table 2) is C3b, because the complexes are prepared under conditions (brief incubations in relatively dilute NHS) that should tend to prevent significant degradation of C3b to C3bi and C3d,g. In addition, we have previously demonstrated that C3b (and not C4b) is apparently the most important component in the binding of opsonized antibody/dsDNA IC to RBCs (40). Whether these complexes (group A, Table 2) also contain significant quantities of C3bi and C4b cannot be determined by the present work. Treatment of C’-treated IC with purified I and H should generate immune complexes containing C3bi (group B, Table 2) (1, 6, 41). In fact, we find that RBC binding is decreased, and this finding is in agreement with other reports that suggest that RBCs can bind C3bi, albeit with lower avidity (13, 20). It should be noted that the binding of these complexes (group B, Table 2) to Raji cells is still quite strong, and we believe this may involve both the C3b receptor of the Raji cell (different from CR,) and the CR, of the Raji cell. It also can be seen that suramin does inhibit binding of immune complexes of group B to both RBCs and Raji cells. The long-term aging of the immune complexes (group C, Table 2) must generate mostly C3d,g because although RBC binding is abrogated, Raji cell binding remains quite strong. We have noted that suramin does not inhibit binding of these immune complexes to Raji cells. These results therefore suggest that suramin can bind to both C3b and C3bi, and that this binding has the effect of inhibiting the binding of C3b and C3bi labeled IC by RBCs and Raji cells. However, when the bound C3b/C3bi is degraded down to C3d,g, the binding of suramin is eliminated, and it can no longer inhibit the Raji cell binding that is presumably via CR,. The inability of suramin to inhibit in this case is not due to the competitive binding of suramin by other serum proteins (32) in the undiluted NHS (used to prepare aged complexes). In other control studies we have found that incubation of the antibody/dsDNA IC in undiluted NHS forjldsr 10 min gave binding to RBCs and Raji cells of 55 and 62%, respectively. In the presence of suramin this binding (possibly involving C3b, C3bi, and C3d,g) was reduced to 0 and 37%, respectively, and this indicates that suramin can inhibit binding even in undiluted NHS (Table 1). One other possible explanation for the effects of suramin on RBCs must be considered. It is known that structurally related compounds such as trypan blue block CR, of RBCs (42) and this could explain the ability of suramin to prevent binding of complement-fixing IC to RBCs. However, as we have noted, when GPS is used as a complement source, the inhibitory effects of suramin with respect to RBC binding are reduced significantly (Table 1). In addition, our evidence (see below) suggests that the C3b receptor of Raji cells is different from CR,, yet suramin also can inhibit binding of C3b- or C3bi-coated complexes to Raji cells. Thus, it is likely that the direct binding of suramin to C3b and/or C3bi (provided by NHS) on the IC is responsible for most of its inhibitory effects with respect to RBC binding. We wish to emphasize that in the experiments displayed in Table 2 we have attempted to prepare complement-opsonized antibody/dsDNA IC which can reasonably be identified as having only one major C3 fragment. Clearly if these

228

TAYLOR

ET

Al..

complexes are incubated for intermediate time periods with different amounts of NHS, it should probably be possible to generate complexes containing significant amounts of at least two, and possibly all three major fragments. Some of the IC described in Table 1 (IC opsonized in NHS for 10 min) do indeed show “intermediate” properties between the groups of Table 2. On the C3b Receptor

of Raji Cells

Several new experiments reported in the present work provide further evidence that the C3b receptor of Raji cells is in fact distinct from CR, of RBCs. First, although antibody/dsDNA complexes opsonized with GPS do bind to RBCs, they do not bind to Raji cells. In addition, although heparin can effectively inhibit the binding of C’-treated IC to RBCs, its inhibitory effect on Raji cells is negligible. On the other hand, suramin is an effective inhibitor for both cell types. This suggests that suramin and heparin bind to different sites on C3b, and that the site recognized by Raji cells is not effectively blocked by heparin. We are also confident that the Raji cells are binding the group A complexes (Table 2) via C3b because even when these complexes are briefly opsonized with low concentrations of NHS (to prevent the action of I and H) and are added to Raji cells in an EDTA-azide medium (to prevent release of I and H from the cells) (20, 34) binding is still observed. On the C3b Receptor

of RBCs

We wish to emphasize that there is no longer any ambiguity about the existence of the CR, (C3b receptor) on human RBCs (1, 10). Fearon’s work conclusively demonstrated their existence on RBCs as well as in a number of other cells (10). The affinity of this receptor for C3b dimers as well as the number of CR, per RBC has been determined independently by two groups using binding isotherm analyses (17,43). The use of specific antibodies to CR, has also allowed additional determinations of their number per RBC (17, 18, 44). Several lines of evidence indicate that for a lqariety of IC, binding to human RBCs does require complement. For example, the binding of antibody/ [3H]dsDNA IC to human RBCs is abrogated (binding drops to less than 10% from 80%) if the complement source is heat inactivated or if complement fixation is inhibited by EDTA (28). More recently, we have demonstrated that the addition to antibody/[3H]dsDNA IC of appropriate amounts of purified samples of the first four components of complement (which allows complement activation to the C3b stage), enables these IC to bind to RBCs (40). We have also found that the binding of 1251-labeled heat-aggregated IgG to human RBCs is reduced from 10% to less than 1% if complement activation is inhibited by 0.01 M EDTA (45). Medof and co-workers have demonstrated that the binding of ““I-bovine serum albumin (BSA)/anti-BSA immune complexes to human RBCs also manifests an absolute requirement for fresh complement. They found binding values dropped from 60% to less than 2% if complement activation was prevented (35). Cornacoff et itI. have done similar experiments and they also reported that the binding of immune

ANTIBODY/dsDNA

IMMUNE

COMPLEXES

229

complexes to human or baboon erythrocytes (known to contain the CR, receptor) is complement dependent (46). Several studies from the groups cited above indicate that when RBCs from species lacking CR, are examined (e.g., sheep, rabbits, or guinea pigs) no immune complex binding can be demonstrated (19, 35, 46). We do note that recently Virella et al. (47) did report some lower level noncomplement-mediated binding of certain IC to human RBCs, which they suggested may be due to Fc receptors. However, in all the IC systems we have cited, we believe that the vast majority of IC binding must be C3b mediated, because when the CR,-C3b/IC interaction is prevented, net binding typically drops from 60-90% to less than 10% (19, 28, 35, 46). Finally, although the biological function of the CR, of RBCs is still not established, we note that there is increasing interest in the possibility that it does play a crucial role in immune complex clearance (15-19). Potential Applications

and Clinical Implications

At the present time a number of circulating immune complex assays are based on the binding of complement-coated IC to certain cells (11). Clearly the use of suramin at appropriate concentrations may be useful in identifying and distinguishing binding based on C3b and C3bi (suramin inhibitable) and binding due to C3d,g (not inhibitable by suramin). The use of reagents such as suramin may also aid in understanding how other cells interact with immune complexes simultaneously via Fc and C3 receptors (1, 8), as suramin could be used for specific inhibition of C3b and C3bi binding. There is a continuing interest in understanding how the dynamics of the RBC/ complement/immune complex reaction affects the fate of immune complexes (1518). For example, if immune complexes are bound in the circulation to RBCs via CR,, then it is possible that suramin could be used to facilitate their release and quantitation. A number of fixed cells also contain CR, (48, 49), and it is also possible that suramin could be used to release bound complexes from tissues in diseases in which the deposition of complement-fixing immune complexes is suspected. Finally, we wish to point out that suramin has been used therapeutically in the treatment of a number of diseases (23). Its concentration in the circulation can in fact approach levels of ca. 0.1-0.5 mg/ml (23), which, according to our in vitro studies, is sufficient to significantly inhibit the binding of C3b-coated immune complexes to CR,. Whether these suramin levels might therefore cause a clinical problem for patients in whom circulating immune complexes are suspected should be considered. ACKNOWLEDGMENT We thank Ms. Kathi Wilson for technical assistance.

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IMMUNE

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231

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