Immunology Letters, 32 (1992) 175 - 180 Elsevier IMLET 01775
E-rosette formation using heteropolymeric monoclonal antibodies Ronald H. Labuguen, Ronald P. Taylor and Rebecca L. Z i m m e r m a n n Department of Biochemistry, University of Virginia School of Medicine, Charlottesville, VA USA (Received 24 January 1992; accepted 12February 1992)
1. Summary We have used bispecific, cross-linked monoclonal antibodies (heteropolymers, HP) to facilitate rosette formation between human erythrocytes (E H) and dinitrophenylated sheep erythrocytes (DNP-Es) in the absence of complement. The HP contain monoclonal antibodies (mAbs) specific for both the E H C3b receptor (CRI), and the DNP group, and control experiments with homologous competing non-cross-linked mAbs and naive E H and E s confirm the specificity of the rosetting reaction. These results extend our previous studies, of HP-mediated binding of simple protein antigens to E H CR1, to complex particulate antigens and may eventually allow for the targeting and clearance from the circulation of a variety of pathogens associated with infectious disease. 2. Introduction An immunological reaction of E H which may provide defense against microorganisms was first demonstrated by Nelson in 1953 [1, 2]. He described the phenomenon of immune adherence, in which C3b-opsonized [3, 4] antibody-particulate antigen immune complexes are bound to E H via a mechanism which has since been demonstrated to involve a receptor for C3b (Complement Receptor Type 1) [5, 6], which is organized in clusters on the erythrocyte membrane [7-10]. Nelson provided Key words: Rosette formation; Heteropolymeric monoclonal antibodies; Complement receptor type 1
Correspondence to: Dr. Ronald P. Taylor, Department of Biochemistry, 6 - 14 Jordan Hall, University of Virginia, Charlottesville, VA 22908, USA.
intriguing evidence indicating that immune adherence leads to the attachment and immobilization of microorganisms onto E H, thus facilitating their phagocytosis by white blood cells. More recently, Hebert and co-workers [11, 12], followed by several other investigators [13- 16], have demonstrated a related immunologic and defensive role of E in the non-human primate model and in humans: in the circulation soluble C3b-opsonized antibody-antigen immune complexes are bound to E via CR1, and this binding reaction leads to the safe and rapid clearance of the immune complexes by the liver and spleen without removal or hemolysis of the E. In both cases binding of the substrates to EH via CRI appears to be key, because it is well known that binding of a variety of antibodies to other sites on the E H membrane leads to destruction of the E H due to intravascular hemolysis and/or direct removal of the E H from the circulation [17 - 19]. Previously we have shown that bispecific, covalently crosslinked monoclonal antibodies (heteropolymeric monoclonal antibodies, or heteropolymers, HP) (specific for E H CR 1 and target antigen) facilitate the binding of 125I-labeled target antigens (human IgG or dinitrophenylated bovine gamma globulin) to E n in vitro via CR1 under conditions that either allow or preclude complement activation . Binding is manifested in quantitative saturation curves which are in agreement with the number of CR1 per E H, and there is absolutely no hemolysis of the E even when they are saturated with HP and subjected to conditions that allow for complement activation. These preliminary results, taken in the context of the work of Nelson and Hebert [1, 2, 11, 12], indicate it may be feasible to use HP as therapeutic agents to facilitate, via CR1, the EH-mediated destruction and/or clearance of potentially pathogenic antigens in the circulation.
0165-2478 / 92 / $ 5.00 © 1992 Elsevier Science Publishers B.V. All rights reserved
In order to investigate further the potential therapeutic use of H P , it is important to extend this methodology to complex particulate structures which were, in fact, the first substrates used by Nelson to demonstrate immune adherence [1, 2]. That is, the ability of H P to facilitate binding to E n CR1 of more relevant antigens, such as viruses and bacteria, than the smaller antigens used previously in vitro, must be demonstrated. To begin to address this question we have developed an E-rosette assay to examine the ability of HP-sensitized E n to bind large, complex antigens. In our model the target antigens, D N P - E s (which lack CR1), are bound to E n sensitized with H P containing mAbs specific for both CR1 and D N P groups. 3. Materials and Methods
3.1. Preparation o f l i P H P were prepared by individually covalently coupling two different purified anti-CR1 mAbs (HB8592 or 3D9) to a purified anti-DNA m A b (23D1) using N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) (Pharmacia AB, Uppsala, Sweden) as reported previously . 3.2. Sensitization of E M E H were isolated using standard procedures  from blood drawn from normal volunteers and reconstituted to a 50°70 (v/v) dispersion in a 107o (w/ w) solution of bovine serum albumin (BSA) (Sigma Chemical C o m p a n y , St. Louis, MO) in phosphatebuffered saline (PBS), p H 7.4 (BSA-PBS). The suspension was incubated (sensitized) with saturating levels of a cocktail of two anti-CR1 × anti-DNP H P (3D9 × 23D1 and HB8592 × 23D1) in BSAPBS for 1 h at room temperature. The EIj were washed three times with BSA-PBS to remove unbound H P and then reconstituted to 25% in BSAPBS. 3.3. D&itrophenylation of E s Solutions of varying concentrations (5 - 100 m g / m l ) of 2,4-dinitrobenzene sulfonic acid (DNTBS, Aldrich Chemical Company, Milwaukee, WI) were incubated with shaking for 176
2 - 3 h at room temperature in a 5 - 10%0 (v/v) suspension of E s in an isotonic Na2CO3-NaHCO 3 buffer ( p H 9 . 6 ) [ 2 1 - 2 3 ] . The D N P - E s were washed with BSA-PBS and then washed in Alsever's solution, reconstituted to 25% (v/v) in Alsever's solution and stored at 4°C. The D N P - E s were washed three times in BSA-PBS and reconstituted to 25% (v/v) in BSA-PBS for rosette formation. 3.4. Rosette formation Varying volume ratios of the HP-sensitized E H and the D N P - E s (both at 25 %o, v/v) were mixed to give a final volume of 50 #l, and then centrifuged for 7 min at 1000 × g . The pellets were carefully resuspended after adding 2.5 ml BSA-PBS ; 10 #I of the diluted suspension was pipetted onto a glass slide and observed at high magnification (500 × ) in a Leitz Panphot phase microscope with a Wild MPS 50 camera attachment. An individual cell (either E H or DNP-Es) was defined as part of a rosette if there was contact between it and one or more of the different cells. The D N P - E s are approximately half the size of the EH, and have a yellow tint, and thus the two cell types can be easily distinguished. In each experiment usually four or more separate fields were counted for both E H and D N P - E s, and typically a total of 60 cells were in each field, with a varying ratio of the two cells, as defined by the specific experiment. Selected fields were photographed using Kodak Ektachrome 160 slide film. 3.5.
Parallel experiments using "naive" (unsensitized) E n a n d / o r "naive" (undinitrophenylated) E s were performed to determine whether rosettes formed in the absence of H P a n d / o r the D N P group on the E s. Homologous competing monomeric mAbs to CR1 or the D N P group (in ascites fluid) were added in another set of control experiments to determine whether rosette formation occurred when CR1 sites on E n or D N P groups on E s were blocked from binding to the anti-CR1 or antiD N P moieties, respectively, of the H E This second set of experiments was controlled further by also using a mAb of irrelevant specificity, HB57 (antih u m a n IgM), also in ascites fluid.
TABLE 1 Rosetting of DNP-Es to E H sensitized with HP specific for CR1 and the DNP group (3D9x23DI and HB8592 x 23D1): influence of the input concentration of DNTBSa. DNTBS input (mg/ml)
% Es in rosettes (mean + SD (range)b)
100 75 50 25 5 0
66+19 (2579+ 9 (5074+ 9 (5049+ 9 (3015+15 ( 0 4+ 3 ( 0 -
95) 100) 90) 75) 42) 19)
a A total of 7 independent experiments were performed. A set of 5 different dinitrophenylated Es were prepared, and each set was tested with one or more different E H from normal donors. In these experiments equal numbers of human and sheep E were mixed. When "naive" EH (not treated with HP) were used, < 5% of the Es were in rosettes, b Approximate observed range for 7 different EH and 5 different DNP-Es preparations.
4. Results P r e l i m i n a r y c a l i b r a t i o n s with different inputs o f D N T B S indicate t h a t it is i n d e e d necessary to label the E s with D N P groups in o r d e r to facilitate rosette f o r m a t i o n (Table 1). A t a sufficiently high in-
p u t o f D N T B S the m a j o r i t y o f the D N P - E s are f o u n d in rosettes, b u t at lower D N T B S i n p u t s a d o s e - r e s p o n s e in the g e n e r a t i o n o f D N P - E s with a sufficient n u m b e r o f D N P groups to f o r m rosettes is evident. T h e n a t u r e o f the rosettes v a r i e d c o n s i d e r a b l y with the r a t i o o f E H a n d D N P - E s a n d the p a r t i c u l a r E n d o n o r . H o w e v e r , tight a s s o c i a t i o n b e t w e e n the different cells c o u l d easily be d e m o n s t r a t e d (Fig. 1). N o rosette f o r m a t i o n o c c u r r e d when: (1) unsensitize~l h u m a n e r y t h r o c y t e s a n d / o r u n d i n i t r o p h e n y l a t e d sheep e r y t h r o c y t e s were used; or (2) CR1 receptors a n d D N P g r o u p b i n d i n g sites were b l o c k e d with h o m o l o g o u s c o m p e t i n g m o n o m e r i c m A b s as d e t a i l e d a b o v e (results not shown). T h e a b s e n c e o f rosette f o r m a t i o n in the c o n t r o l e x p e r i m e n t s illust r a t e s t h e specificity o f the H P a n d d e m o n s t r a t e s the p a r t i c i p a t i o n o f b o t h CR1 r e c e p t o r s a n d D N P groups in rosette f o r m a t i o n . We p e r f o r m e d the E-rosette assay using mixtures c o n t a i n i n g (1) even ratios o f h u m a n to sheep cells, (2) a slight excess o f h u m a n cells, a n d (3) a slight excess o f sheep cells (Fig. 1 a n d Table 2). A s w o u l d be a n t i c i p a t e d , when one o f the cells is in slight excess, o n l y a b o u t o n e - q u a r t e r o f t h a t cell p o p u l a -
Fig. 1. Rosette formation using heteropolymer-sensitized E H and DNP-E s with (a) even ratio of heteropolymer-sensitized E H to DNP-Es, (b) slight excess of heteropolymer-sensitized EH, and (c) slight excess of DNP-E s. A slightly different magnification was used in (a) as compared to (b) and (c). 178
TABLE 2 Dependence of percentage of E in rosettes on the ratio of EH to Es used in the E-rosette assaya. 070E in mixture
Even ratio, EH:Es Excess EH Excess Es
070E in rosettes
EH (mean -+SD (range))
Es (mean _+SD (range))
50 80 20
50 20 80
47_+ 8 (28-71) 22 _+ 9 (12- 37) 63 _+11 (36 - 84)
66+_19 (25-95) 69 _+19 (33 - 83) 28 +_19 (15 - 40)
a A total of 7 independent experiments were performed. In these experiments Es were dinitrophenylated using a 100 mg/ml solution of DNTBS. tion is found in rosettes, but the majority of the other (minority) cell type is present in rosettes. For equal numbers of ceils, close to half of each cell type is in a rosette. Thus, under these conditions the avidity of binding between the cells caused by the cross-linking H P is reasonably high. The wide range of rosettes seen in different experiments (Tables 1 and 2) undoubtedly is influenced by factors such as the range and number of CR1 per E• from a given blood donor [26, 27] and the degree of CR1 clustering on the cells [7 - 10].
5. Discussion The present results concur with our previous research which demonstrated that H P may be used to attach soluble antigens to the CR1 receptor of E H without requiring the activation of complement . The success of rosette formation using H P indicates that the binding potential of the H P can be extended to a large and complex particulate antigen. Our previous studies with protein antigen substrates were done at very high E n concentrations (corresponding approximately to typical hematocrit levels) which would have tended to stabilize binding by mass action. Our new findings indicate that the simultaneous binding of H P to both the CR1 of E n and the D N P groups on the E s must be very strong since the majority of cells are found in rosettes even after the high dilution required for microscopic examination. Sheep erythrocytes are approximately half the size of human erythrocytes and are much larger than almost all potentially pathogenic antigens in the circulation. Also, due to their high concentration in the circulation, the potential ratio of E n to
circulating particulate pathogens should be greater than 1000:1 in virtually all cases, which would clearly correspond to E H at extreme excess, compared to our prototype experiments. Thus, based on the present results, this condition of extreme E H excess at normal hematocrit levels should lead to HP-mediated binding to E n CR1 of the majority of a circulating particulate pathogen, if mAbs to the pathogen are available for construction of the H E The potential use of this methodology as a therapy will also require that En-bound pathogens then be cleared from the circulation. Our preliminary results in squirrel monkeys indicate that E-bound DNP-bovine g a m m a globulin is cleared , and the next step in this research will require demonstration that particulate antigens bound to E via H P in vivo are also removed from the circulation.
Acknowledgements We would like to thank Dr. J. David Deck, Mr. W. Carlton White, and Mr. Craig J. Reist for their assistance. This research was supported by N I H Grant AR41072 and by the Howard Hughes Undergraduate Research Program in the Biomedical Sciences at the University of Virginia.
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