Regulation by sulphonate groups of complement activation induced by hydroxymethyl groups on polystyrene surfaces

203

Regulation by sulphonate groups of complement activation induced by hydroxymethyl groups on polystyrene surfaces Bkatrice Montdargent*, Franqoise Maillett, Marie Paule Carreno*t, Marcel Jozefowicz*, Michel Kazatchkinet and Denis Labarre** “Laboratoire de Recherches sur /es Macromoltkxles. CNRS-.URA 502, Universit6 Paris Nord, 93430 Villetaneuse, France; +lNSERM lJ28, HGpital Broussais, 75014, Paris, France; and **Laboratoire de PhysicoChimie-Pharmacotechnic, CNRS-URA 1218, lJniversit.6 Paris Sud, 92290 Chatenay, Malabry, France

Reducing the complement-activating capacity of a polymer surface is important in improving its blood compatibility. Polystyrene surfaces bearing hydroxymethyl (CH,OH) groups activate the alternative pathway of complement. This activation depends strongly on the density of the groups. Polystyrene surfaces bearing sulphonate (SO;) groups adsorb proteins, resulting in an apparent activation. Polystyrene surfaces bearing both types of groups in close proportions are not activators in human serum, due to the adsorption of a protein of the alternative pathway, which has a protecting effect, not found when a polymer surface bearing hydroxyl groups is mixed in serum with another polymer surface bearing SO, groups. In the presence of purified proteins of alternative pathway, C3 convertase activity can be created on each of these surfaces by deposition of C3b, but their susceptibility to inactivation by regulatory proteins H and I depends on the types of chemical groups present on the surface and whether the surfaces were passivated or not before C3b deposition. Keywords: Received

Polystyrene,

complement

24 June 1992; revised

Correspondence to Professor D. Labarre, Laboratoire de PhysicoChimie-Pharmacotechnie. Centre d’Etudes Pharmaceutiques, 5 Rue J.B. Ckment, 92296 Chatenay-Malabry Cedex, France.

0142-9612/93/030203-06

Ltd

sulphonate,

21 July 19912; accepted

Reducing the complement-activating ability of artificial surfaces is important for improving biocompatibility, since activation of the complement system at the blood/ surface interface may trigger a variety of secondary adverse responses from inflammatory cells1-3. The complement-activating ability of surfaces such as zymosan and Sephadex@ may be suppressed by coupling heparin4 or carboxymethyl (CM] groups5 to the surfaces. The effect of heparin and of CM groups is mediated by their capacity to increase the susceptibility of surfacebound C3b to inactivation by the serum regulatory proteins H and 14*6.We have suggested that CM groups permit formation of a stable ternary complex on the surface between bound C3b, CM groups and H6. Sulphate groups are present in heparin, and substitution of dextran and polystyrene with sulphonate (SO,] groups provides the polymers with inhibitory properties towards complement activation7-‘. In the present study, we investigated the effects on complement activation of grafting SO; groups on a polymer backbone bearing activating hydroxymethyl (CH,OH) groups.

0 1993 Butterworth-Heinemann

activation,

haemocompatibility

21 September

1992

MATERIALS AND METHODS Polymers Polymers bearing various proportions of SO, and CH,OH groups (Figure 2) were prepared from crosslinked polystyrene beads (Biobeads@ SXZ or chloromethylated SX2 from Biorad, Ivry, France) as previously described99 lo. The sulphonated polymers were isolated as sodium salts. Chemical compositions were assessed by elemental analysis (Table 2). The beads were crushed and the submicronic particles were discarded before equilibration with the appropriate buffers for tests of complement activation. The specific surface area accessible to proteins was determined in each sample by adsorption of ‘251-labelled human serum alburning. Sephadex C25 was obtained from Pharmacia (Uppsala, Sweden], washed and crushed as previously described5.

Assessment of the complement-activating capacity of polymers in human serum The following buffers were used for complement assays: veronal-buffered saline (VBS); VBS containing 0.5 mM MgCl, and 0.15 mM CaCl, (VBS’+); VBS containing 10 mM EDTA (VBS-EDTA). Normal human serum Biomaterials

1993, Vol. 14 No. 3

used, since no C3~esArg antigen is found in Sl, because of adsorption of ~~a~hylatoxins to negatively charged polymersl’.

of 63 convertase activities using purified complement proteins

Assaya CH~OH CH20H

SOjNa S03Na

unit

styrem? unitTk

unit

-#CHfCHt-X

0 0

SO~NK~H&OON8 CH I 2 GOONa

S02Asp

unit

Fiure t

Structures of the elementary units of polymers.

Tabk 3

Chemicai and physicat eharecteristics of polymers

PoIymer

Sub$~tutj~~ degree mot% (*) cn&wi

PSC7 PSC6 PSSS PSC4S6 PSC6S4 PSC6S3 PSC7S2 PSC6SI PSSQ PSSAsp

so,

Developed specific surface area ~m*/rng(*~)

SO&p

66.0

0

0

66.6 0

0

:

41.6 46.5

z 4t:0

:

57.0 74.6

28.3 18.6

::

80.6 :

14.0 88.0 30.0

x 50

tx

33:5 11.3 2fl.: 25:6 21.1 ND ND

The difference bStwSen 100% and the Sum (Cl-&OH + SO;) represents unsubstitu~~ styrfme units, cross-linking units (??&of divinyfbenzenein monomer mlxtwss.] and a few restdual units bearing CFf&l groupsin the case of PSC samples. f* Developed specific surfaoa ar8a was catcutated from the amount of human serum atbumln 8dSOrbSd at saturationon the hydrated pdymffr samples, by using a value of 0.2pg cm-’ for a close-packed monolayer of albumln. The samples were weighed in a d%ssio&%dform. ND = Not determ~fl~d. *

fNHSj was obtained by allowing freshly drawn venous blood from two healthy donors to clot at room temperature for 2 h: NHS was stored at -60% until used, To assess the c~pl~e~t~a~va~ capacity of polymers, increasing amounts of polymers already equilibrated in VBS’+ were added to NHS diluted in the same buffer (final dilution of NHS in the reaction mixture: 114 (v/v)), and incubated at 37°C with gentle agitation for up to 75 min. At given intervals, samples were centrifuged at 3000 rev min-* for 5 min; supe~atants were co&c&d (St], afiquoted and stored at -80% Complement activation was measured by assessing CHSO’I or by an indirect method as described’: briefly, 150 ~1 of Sl were incubated with an excess of crushed Sephadex for 60 min at 37%. After centrifugatio~, s~pe~atants were collected (SZ)in which the concentration of CSadesArg antigen and C5adesArg antigen was measured using the radioimmunoassays {Amersham, Les Ulis, Francef. This indirect method was 3~o~a~~als

1993, VoL 14 No. 3

following buffers were used: VBS and V3SZ’ container 0.2% gelatin [GVB and GVB”‘J; half isotonic GVB and GVB” with 2.5% dextrose (DGVB and DGVB’+); DGVB supplema~ted with 0.15 mM Ca2’ and 0.5 mM NiZi (DGVB-Ni); GVB containing 4Q EM EDTA (GVBEDTA). C3, B and D were purified as described’3-‘5. Before interacting polymers with complement proteins, crushed polymer particles were either passivated with human serum diluted 1150{v/v] in GVB-EDTA for 60 min at 37°C and then with GVB’+ or equilibrated with GVB” alone. Deposition of C3b on polymer particles was performed using purified proteins as follows: first, 200 cm2 of PSCG, PSSS and PSC5S4 were incubated with 560 yg of C3,300 fig of B in 570 ~1 of GVB” containing D and 5 mM additional M$‘, in a total volume of 1.0 ml for 60 min at 37°C. The polymers were then washed twice in DGVB-Ni, incubated with 69 gg of B in 400 pl of DGVBNi containing D and 5 mM additional Mg’+, in a total volume of 0.5 ml for 30 min at 30% After washing with ice-cold DGVB-Ni, the particles wera further incubated in 200 pl of DGVBzBZ’ containing 168 pg of C3 for 45 min at 37°C. The particles were washed twice in DGVB-Ni and the second step of C3b deposition was repeated three times. The haemolytic function of poller-bound G3b was measured by the ability of C3b to consume B activity in the presence of D. Polymer particles (20 cm’] bearing C3b were incubated with 0.11jog of B and an excess of D in 280 ,ul of DGVB” containing 10 mM additional Mga+ in a total volume of 0.33 ml for various times at 37”C, and centrifuged following addition of cold DGVB-Ni. Residual haemolytic Factor B activity was determined in the supe~atant as described”. The s~~eptib~~ity of deposited C3b to inactivation by the regulatory proteins H and I was assessed by incubating 14 cm’ of polymer particles bearing C3b with a 1150dilution of human serum in GVB-EDTA for 15 min at 37% The polymers were washed twice in DGVB-Ni and assessed for residual haemolytic function of C3b, as described above.

~SULTS AND DISCUSSION Complement~~~tiv8ting ability of polymers in human serum Increasing amounts of polymers bearing various proportjons of SO; and CH@H groups were inoubated with NHS in the presence of VBSZ+ for 75 min at 37°C. The polymers consumed CH50 units in the serum to various extents, depending on their composition (F&ure 21: little or no consumption af CH50 was observed in the case of PSC6S3 and PSC5S4, whereas PSC4S5, PSC7S2 and PSCBSl consumed CH50 with increasing relative efficiency in a dose-dependent fashion The complement-activating ability of the polymers was also assessed by the ability of

Regulation of complement

A

activation: 6, Nontdafgent

et al.

A

1000 Polymer surface

2000

area [cm21 1000

Figure2 Complement consumption by polymers in NHS, assessed by measuring residual CH50 units in serum Sl. Increasing amounts of polymers Y-v, PSC’I; V-V, PSC8Si; O-6, PSC7S2; O-0, PSC4S5; !I--[I1, PSC6S3; A-A, PSC5S4 were incubated in t ml of NHS diluted l/4 &iv) in VBS2+ for 75 min at 37X. CH50 units in serum incubated in the absence of polymers were given the value of 100%.

serum that had been incubated with the polymer (Sl) to generate C3a or C5a in the presence of an excess of Sephadex, a Potent activator of the alternative complement pathway. As seen inFigure 3, the complement-activate ability of the polymers, as assessed using this indirect method, only manifested itself above a concentration threshold: in addition, PSC4S5 exhibited the highest apparent complement-activating ability, followed by PSC5S4 and PSC7S2. Similar results were obtained by measuring C3a or CEa antigen in S2. The discrepancy between the results obtained using CH50 measurements and the indirect assay of CJafC5a generation by Sephadex in Sl, e.g. for polymer PSC5S4, indicates that the polymer did not consume G3 or any protein that may have a limiting role in CH50 activity and suggested that it had adsorbed or consumed a component which had a limiting role in alternative pathway activation by Sephadex, i.e. 3 or D, Since the relative ability of polymers to adsorb 3 or D followed the order of increasing content in SO; groups, the adsorbed alternative pathway protein was more likely to be Factor D, which is more positively charged than B’?. The analysis of the resuits depicted in Figure& indicates that the presence of SO, groups resulted in an inhibition of the activating potential of EIpolymer bearing CH,OH groups: thus, PSCBSl, which is substituted for 80% with CH,OH and 14% with SO, exhibited a lower complement-activating ability than PSC?, which is substituted for 85% with CH@H and bears no SO;. In the case of PSC5S4, which is similarly substituted with C&OH and SO<, no complement activation occurred in serum, suggesting that the inhibiting properties of SO; counterbalanced the activating potential of CH,OH groups.

Polymer surface

0

b

area (cm21

2000

1000 Polymer surface area [cm21

Rgure 9 Apparent complement consumption by polymers as assessed by a, C3adesArg and b, CSadesArg generation upon incubation of St with Sephadex@. Increasing amounts of polymers U-0, PSC4S5; A-A, PSC5S4; CL-D, PSC6S3; O-O, PSC7S2 were incubated in f ml of NHS diluted 114 @iv) in VBS2+ far 75 min at 37°C. Supernatants Sl were collected and incubated with an exc#ss of crushed Sephadefl far 60 min at 37°C. The total (100%) represents the cantrol serum incubated in the absence of polystyrene derivatives, then activated by Sephadex*. The values are the mean of at least two determinations, s.d. 110%.

Effect of mixlug poiymeN activation in serum

on complement

The experiments depicted in Figure 2 suggested a mutual antagonistic role of SO, and CH,OH groups, when these

are expressed on the same polymer backbone. We therefore investigated whether mixing an activating Bicmaterials

1993,

Vol. 14 No. 3

206

Regulation

of complement

activation:

B. Montdargent

et al.

polymer-bearing hydroxyl [OH) group (i.e. Sephadex) with an SO,-bearing polymer (PSSg) inhibited Sephadexinduced activation in serum. The effect of PSSg was compared with that of a polymer-bearing equivalent amounts of aspartic acid sulphonamide groups (PSSAsp). The addition of anionic group-bearing polymers did not inhibit complement activation induced by Sephadex; rather, a slight decrease in residual CH50 units in the presence of mixed polymers was observed (Figure 4). These observations indicate that the inhibitory effect of SO, groups on the complement activation by an OHbearing polymer required the immediate proximity of both types of chemical determinants.

Investigation of the role of (SO;) groups in modulating C3 convertase activity by using purified complement proteins To investigate the mechanisms by which the proximity of SO, groups may downregulate complement activation induced by OH groups on the same polymer backbone, we examined the formation and stability of the C3 convertase, using purified complement proteins and polymers bearing different amounts of SO, and CH,OH groups. The polymers were PSC6 (no SO, groups), PSS5 (no CH,OH groups) and PSC5S4 (equivalent amounts of SO; and CH,OH groups). C3b was deposited on the three types of polymers as previously described. The function of surface-bound C3b was then assessed by its ability to consume Factor B in the presence of D. Polymers bearing C3b exhibited an equivalent ability to consume Factor B activity [Figure 5), indicating that SO, groups did not inhibit the function of C3b on a CH,OH polymer in the absence of serum regulatory proteins. Furthermore, SOY-bearing polymers without CH,OH permit formation of a functional C3 convertase on surface-bound C3b. Figure 5 also shows that SO,bearing polymers adsorb B activity to some degree and that adsorption of B by sulphonate groups may be prevented by passivation of the polymer surface with diluted serum in EDTA. Passivation of polymers with diluted serum in EDTA did not, however, alter the haemolytic function of surface-bound C3b. The role of SO, groups in modulating the susceptibility of surface-bound C3b to the serum regulatory proteins H and I was then investigated by assessing the haemolytic function of C3b deposited on polymers following incubation with serum-EDTA. The presence of SO, groups in the proximity of CH,OH groups on polymer PSC5S4 protected surface-bound C3b from inactivation (?$ble 2). This effect of SO, groups was, however, suppressed if polymers had been passivated by pre-incubation with serum diluted in EDTA before C3b deposition on the surface, suggesting that passivation results in a capacity of polymers bearing SO, to adsorb regulatory proteins. In the latter situation, we speculated that the inhibitory effect of the regulatory proteins in serum was mediated by the binding of H to both C3b and SO,, as previously suggested in the case of CM groups on other polymer surface8.

CONCLUSIONS Taken together, these data demonstrate that the presence sulphonate groups in the immediate vicinity of OH

of

Biomaterials

1993, Vol. 14 No. 3

[I

q

-u D

I

0

5

_& 10

PSS9

amount

15

(mg)

C

u------Q 0

I

I

5

10

PSSAsp

amount

n 15

(mg)

Effect of mixing polymers on complement activation in serum. Increasing amounts of a, PSS9 or b, PSSAsp were added to O-O, 0 mg; A-A, 4 mg; or q,8 mg of Sephadex@, before incubating the polymers in 1 ml of human serum diluted l/4 (v/v) in VBW for 60 min at 37°C. After centrifugation, the amount of residual CH50 units was measured in the supernatants. The total (100%) represents the control serum incubated in the absence of polymers. Figure 4

groups on the same polymer backbone modulates the activating function linked to the hydroxyls. Studies using purified complement proteins indicated that the modulating function of SO, groups may be inhibitory or enhancing towards the complement-activating ability of the polymer, depending on whether it has been passivated or not before exposure to complement proteins. The

Regulation

of complement

activation:

6. Mont&went

207

et al.

Table 2 Residual C3b activity (expressed %) on polymers following incubation with human serum diluted in EDTA as a source of regulatory proteins H and I Polymer

PSC6

PSS5

PSC5S4

Unpassivated Passivated

53 41

77 10

100 0

Passivated and unpassivated polymerswere coated with C3b and interacted with serum EDTA for 15 min at 37°C. Residual C3b haemolytic activity was then assessed by interacting the CSb-bearing polymers with B and D. The total (100%) represents C3b haemolytic activity on polymer bearing C3b which had not been exposed to serum EDTA after C3b deposition.

60

30

a

Time

90

(min)

outcome, in terms of the group on the activating inferred solely from the within purified systems relevance for the design surfaces for biomedical

modulating effect of a chemical capacity of a surface cannot be study of molecular interactions in vitro. Our results may be of of non-complement-activating applications.

ACKNOWLEDGEMENTS This work was supported by Centre National de la Recherche Scientifique and Groupement d’Inter6t Public ‘Therapeutiques Substitutives’. The secretarial assistance of Habiba El Ayachi is gratefully acknowledged.

b

Time

(min)

I

I

30

60 Time

90

(min)

Figure 5 Haemolytic function of C3b on polymers bearing SO, and/or CH,OH groups. Polymers were PSC6 bearing no SO;, a, PSS5 bearing no CH,OH, b, or PSC5S4 bearing equivalent amounts of SO; and CH,OH, c. Polymers were either equilibrated with buffer (open symbols) or passivated with diluted serum in EDTA (closed symbols) before being interacted with complement proteins. Polymers were then either coated with C3b (triangles) or not (circles) before being interacted with 6 and D. The figure represents the rate of consumption of Factor B haemolytic activity by the polymers.

Cazenave, J.P., Davies, J.A., Kazatchkine, M.D. and Van Aken, W.G., Blood-surface interactions, in Biological Principles Underlying Hemocompatibility With Artificial Materials Elsevier, Amsterdam, The Netherlands, 1986 Jahns, G., Haeffner-Cavaillon, N., Nydegger, U.E. and Kazatchkine, M.D., Complement activation and secondary cellular responses at the interface between blood and artificial surfaces, CRC Critical Reviews in Biocompatibility, (Ed. D.F. Williams) 1992 [in press] Muller-Eberhard, H.J., Molecular organization and function of complement system, Ann. Rev. Biochem. 1988, 57, 321-347 Kazatchkine, M.D., Fearon, D.T., Silbert, J.E. and Austen, K.F., Surface-associated heparin inhibits zymosaninduced activation of the human alternative pathway by augmenting the regulatory action of the control proteins on particle-bound C8b.I. Exp. Med. 1979,130,1202-1215 Carreno, M.P., Labarre, D., Jozefowicz, M. and Kazatchkine, M.D., The ability of Sephadex to activate human complement is suppressed in specifically substituted functional Sephadex derivatives, Mol. Immunol. 1988, 25,165-171 Carreno, M.P., Labarre, D., Maillet, F., Jozefowicz, M. and Kazatchkine, M.D., Regulation of the human alternative complement pathway: formation of a ternary complex between factor H, surface-bound C8b and chemical groups on non activating surfaces, Eur. 1. Immunol. 1989, 19,2145-2150 Maillet, F., Petitou, M., Choay, J. and Kazatchkine, M.D., Structure-function relationships in the inhibitory effect of heparin on complement activation: independency of the anticoagulant and anti-complementary sites on the heparin molecule, Mol. Zmmunol. 1988, 25, 917-923 Mauzac, M., Maillet, F., Jozefonvicz, J. and Kazatchkine, M.D., Anticomplementary activity of dextran derivatives, Biomaterials 1985, 6, 81-63 Montdargent, B., Labarre, D. and Jozefowicz, M., InterBiomaterials

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Regulation of complement activation: f3. ~o~tdargefft

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