Arrific icll Orgun.!
16(6):577-585, Blackwell Scientific Publications, Inc.. Boston 8 1992 International Society for Artificial Organa
Complement Activation During Low-Density Lipoprotein Apheresis A. Tridon, "J.B. Palcoux, tP. Jouanel, SM.J. Bezou, SM. Coulet, and G. Betail Lahorutoii.e d'lmmunologie, *Service d'Hkmodiulyse Pgdiatrique, tlaborutoire de Biochimie, and $Lahowtoire d'Htmarologie, CHRU Clermont-Fewand, France
Abstract: Complement system activation was investigated in two girls with familial homozygous hypercholesterolemia undergoing two monthly sessions on LA15 or LA40 (Kaneka liposorber). We determined blood levels of C3c and C3a, leukocyte counts, and plasma levels of C3c and C3a in the extracorporeal circulation device at the start of the sessions and 15 and either 60 or 120 min into them. Sequential eluates were collected from LA40 at the end ofthe sessions (O.SMNaC1, 1M hydroxylamine). Anaphylatoxin C3a increased throughout, especially with
LA40. As previously reported, C3a was trapped in the dextran column but was noticeably present in efferent plasma. Besides many proteins, nonnative complement fragments bearing C3a and C3d antigens were detected in almost all the eluates, suggesting possible in situ complement activation. Practically, complement activation induced by the first filter is a risk; long-term side effects may arise from this extracorporeal circulation device. Key Words: Complement system activation-C3 breakdown products-Dextran sulfate-Liposorber.
Two children with familial homozygous hypercholesterolemia were undergoing removal of apolipoprotein B (apoB) included in low-density lipoprotein (LDL) and very-low-density lipoprotein (VLDL) twice monthly using a plasmapharesis procedure with cascade filtration. The extracorporeal device consisted of a column of cellulose beads coated with dextran sulfate, liposorbers LA15 and LA40 (Kaneka) (Fig. 1). Specific binding of apoB occurs on this column through interaction between the negatively charged dextran sulfate and positively charged apoB (1). In addition, other proteins are adsorbed; this prompted our interest. Our main aim was to investigate complement system activation (CSA) linked to the use of this particular extracorporeal circulation (ECC) device as a criterion of biocompatibility. Until recently, apheresis specialists had paid little attention to this question since these techniques were not applied repeatedly and seldom involved reinfusion into the patient. However, with the development of frequently re-
peated selective removal procedures with return to the patient of plasma and cells (2-4), this issue now needs to be addressed. The study of CSA is only one aspect of biocompatibility, but developments in the field of hemodialysis show that it is implicated in other biological reactions (5,6). Repeated CSA causes repeated activation of cells resulting in an inflammatory response, which when recurrent has long-term side effects: relative cell deactivation with depression of natural defenses (7), chronic inflammatory syndrome (8), and immune imbalance (9). Two phenomena are implicated in CSA: depletion (linked to the adsorption capacity of the filters, here mainly liposorber) and activation (linked to the use of the plasma separator with possible involvement of the liposorber). A recent study on another system for cascade filtration of cholesterol has determined the relative extents of these two phenomena (10). CSA may occur by contact with dextran sulfate: this material is known to trigger the alternative pathway depending on its degree of polymerization and its molecular weight ( 1 1,12) and on how sulfated it is (11,13). Theoretically, any nascent C3b produced by initial fluid phase convertase will bind by transesterification to any bystander surface with exposed OH
Received February 1992; revised June 1992. Address correspondence and reprint requests to Dr. A. Tridon at Laboratoire d'Imrnunologie, FacultC de MCdecine et Pharmacie, B . P . 38, 63001 Clermont-Ferrand CEDEX, France.
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A . TRIDON ET AL.
5 78
anboagulanl plasma
FIG. 1. Extracorporeal device.
’
’
8
ALIPOSORBER adsorplion coluinn
membrane liller unil
groups. According to its biochemical surroundings (and especially the charges on neighboring substituents), surface-bound C3b will interact with B and D fractions and trigger formation of alternative C3 convertase able to cleave C3 to C3a and C3b or factor H, which favors its deactivation to intermediate (C3bi) or final (C3c and C3d) breakdown products. Antigen C3c is borne by native C3, C3b, C3bi, and C3c. Antigen C3d is borne by native C3, C3b, C3bi, and C3d. Dextran sulfate, through its free OH groups, offers available binding sites for C3b (1 1,14). In addition, Chenoweth (15,16) has postulated that the presence of negatively charged sulfate groups close to the C3b binding site favors the interaction of C3b with H, that is, its deactivation. This anticomplementary effect depends on the degree of polymerization and the sulfate titer of the dextran sulfate; it has been studied in particular by Kazatchkine et al. ( I 3,17). MATERIALS AND METHODS Patients and treatment sessions The two female patients, M and V , were 8 years old at the time of the first protocol and 10 at the time of the second. Hypercholesterolemia had been tested for because of severe familial antecedents, and they had both been undergoing repeated plasmapheresis from age 5 years until one year before the first protocol. The treatment sessions took place twice monthly and lasted about 2 h. The ECC flow rates were 60 ml/min in the blood circuit and 20 ml/min in the plasma circuit. In both protocols, samples were drawn from the ECC (Fig. I) as follows: at t,, the arterial starting point; and at different times during the session, simultaneously before the plasma separator (Site l), after the plasma separator (Site 2), and after the liposorber (Site 3). Ar/ifOrgam, Vol. 16, N o . 6, 1992
Several consecutive sessions were studied for each protocol and for each child. In Protocol 1 , the plasma separator was polyvinyl alcohol (PVA; Plasmacure, surface area 0.3 m2, Kuraray, Paris). The Liposorber was an LA 15 (1 50 ml sorbent). Sampling and t,, m,n. Three sessions per times were t o , t 1 5 child were studied. In Protocol 2, the plasma separator was polypropylene (BELCO 550, surface area 0.2 m2, Sorin Biomedica, Antony, France). The Liposorber was an LA40 (400 ml sorbent). Sampling times were f , , r , , mln, and rlZOmln.Four sessions per child were studied. The LA15 was systematically replaced by an LA40 between the two protocols for better therapeutic efficiency. Methods At each time and at each site, the following samples were taken: an EDTA tube for leukocyte counts and hematocrit, and an EDTA tube for assay of anaphylatoxin C3a, and a tube without anticoagulant for assay of C3c and C4c. Hemolytic activity was determined only for the second protocol (CH,,). The samples were annotated at each time according to their site (1, 2, or 3) (Fig. 1). The hematological parameters were determined with a Sysmex NE 8000. Assays of C3 and C4 in the form of C3c and C4c antigens were carried out using a Behring (Rueil Malmaison, France) Turbitimer. CH,, was assayed by the kinetic method (18). Assay of C3a was by radioimmunology (Amersham, Les Ulis, France). After one session per subject on the LA40, the dextran sulfate columns were rinsed with 1 L of 0.15M NaCl and then successively eluted with 1 L of 0.5M NaCl and 200 ml of 1M hydroxylamine. Fractions of 4 ml were collected. On fractions obtained with 0.5M NaCl and IM NH,OH, various proteins were sought by double immunodiffusion
5 79
COMPLEMENT ACTIVATION IN LDL-APHERESIS with commercial antisera: anti-Ig (Silenus, Les Ulis, France), anti-C3c, ClINA, C ~ CC3PA, , fibrinogen, albumin, and apoB (Behring), anti-C3d, anti-B,-microglobulin (Nordic, Le Perray en Yvelines, France), and anti-transferrin (Dako, Trappes, France). Derivatives of C3b bearing C3c and C3d antigens were revealed by dot and blot techniques. We adapted a dot technique to the search for antigens C3c and C3d in the crude eluates and treated them with 22% polyethylene glycol (PEG), precipitating out all the derivatives of C3b except for the final C3d fragment of molecular weight 1 1,000 daltons by the method of Perrin et al. (19). The eluates were placed in contact with Immobilon in a dot apparatus (20). After washing and saturation, anti-C3c (Behring) and anti-C3d (Nordic) were laid on Immobilon. After elimination of the first antibody and washing, an antirabbit antibody labeled with peroxidase (Pasteur, Marnes la Coquette, France) was placed in contact for 1 h. Visualization was with diaminobenzidine (DAB) and H20,. The detection threshold for these two antigens was about 50 ng/ml. This antigen search was completed with a study of the electrophoretic motility of the antigen-bearing fractions using a BLOT technique. The eluates underwent electrophoretic separation on agarose under nondenaturing conditions in parallel with two controls: fresh plasma (native C3) and plasma activated with inulin (native fraction plus cleavage products). After electrophoresis, an Immobilon BLOT was performed by contact. Immunodetection was carried out under the same operating conditions as for the dot. The presence of large amounts of apoB in the 0.5 NaCl eluate did not allow formal identification of the native C3 band (these two proteins have similar beta migrations on agarose). Accordingly, this search was conducted in parallel on eluates treated with an anti-apoB (Behring). The hydroxylamine eluates were dialyzed at 4°C in phosphate-buffered saline (PBS) before blot analysis. Assays were performed on pools of eluates for each session: assay of total proteins (OD at 280 nm), assay of C3c (Behring turbitimer), and assay of anaphylatoxin C3a (RIA Amersham). RESULTS The results are presented individually in Figs. 2-5. The results obtained on the whole blood in the ECC are presented first followed by those of the plasma circuit of the ECC and then by the results for the eluates.
Whole extracorporeal blood Leukocyte count variations in sessions on LA15 and LA40 differed quantitatively but were kinetically consistent: there was a fall at I,, compared with lo(54-108% of the initial value, mean 83%) and then a rise at t,, on LA15 (100-151%, mean 130%) and LA40 (94-195%, mean 140%). These variations mainly concerned neutrophil counts (Fig. 2). There seemed to be no noteworthy variation of hematocrit , indicating that there is no hernodilution linked to the ECC priming procedure. C3c and C4c decreased moderately during the session: the results for C3c are presented in Fig. 3 . C3a increased overall, gradually over the period studied in sessions on LA15 (at fmr initial level 1.3 to 3) and strongly in sessions on LA40 (at t,,, , initial level x 3 to 36) (Fig. 4). C3a was sometimes assayed at cm on LA40, and the moderate increase observed was of the same on LA15. amplitude as the increase observed at CH,, was assayed only for the second protocol. It fell markedly from an initially normal value to a final value always less than 50%.
f,,
Extracorporeal plasma At Site 1 (with the plasma separator), we had the plasma values of the parameters studied. These could be compared with those obtained at Sites 2 (after the plasma separator) and 3 (after the liposorber) at different times ( I , ~and t60,,20). C3c and C4c had widely differing values at Sites 2 and 3: at Site 2 they were plasma values, and at Site 3 they were close to nil. The results for C3c at f,, are given in Fig. 5 . However, at t60,,20,the values of C3c and C4 were comparable at the two sites. C3a was always very high at Site 2 (>lo pglml) while at the liposorber output the observed levels were slightly higher than the plasma levels obtained sirnultaneously at Site 1. This was observed at all the times in both children with both protocols. Eluates from LA40 Numerous proteins were adsorbed on the column and eluted out (NaCI 0.15M then NaCl 0.5M then 1M hydroxylamine). Some were identified by double immunodiffusion (IDD) in the 0.5M NaCl eluates: IgG, IgA, IgM, transfenin, fibrinogen, &-microglobulin, C ~ CC, ~ CC3PA, , ClINA, and of course apoB and albumin. The quantitative results for total proteins, C ~ Cand , C3a in the three types of eluate are given in Table 1 in comparison with their overall variation during sessions. The presence of antigens C3c and C3d was demonstrated by DOT in all the eluates (Fig. 6). The final product C3d was identified in all the eluates alongside molecules bearing antigen Artif Organs, Vol. 16. No. 6, 1992
580
A . TRIDON ET A L . PN
by ul
’\
min
..,. .,
_...._. .........
,,..._....._...._....
L , I20
min.
FIG. 2. Neutrophil count variations during LA15 and LA40 sessions. Solid line, patient M; dotted line, patient V.
.
... .. .... ... . -
..
.. \
, 0
15
6C
,
min 0
15
mi”.
J20
FIG. 3. C3c variations during LA15 and LA40 sessions. Solid line, patient M; dotted line, patient V.
C3c associated or not with C3d. BLOT analysis did not always reveal antigen C3d but showed in all the eluates the presence of at least a fraction bearing C3c and corresponding to a cleavage product by comparison with an in vitro activated plasma profile. DISCUSSION We investigated CSA in this ECC from different angles: we looked at variations in C3c (parallel to those of C3a) and its fate in the liposorber via eluates. We undertook an anaphylatoxin C3a balance A r t f O ~ g a m Vol. . 16, N o . 6, I992
from its variations during sessions and its presence in the eluates. Lastly, we looked at the quantitative variations in the polynuclear neutrophil (PN) counts during sessions and endeavored to correlate these with possible CSA. The fall in C3 assayed as C3c (Fig. 3) in the children’s circulation was due first to entrapment within the liposorber as shown by the study of C3c in the ECC plasma (Fig. 5). In both protocols, this C3c depletion effect was marked at 15 min but did not occur thereafter. This is probably due to saturation like that observed much later for apoB itself (21),
COMPLEMENT ACTIVATION IN LDL-APHERESIS
5'8I
nglrnl
X I O .
X I O .
5.
x 5.
x
7
LA4n
rnl" mi".
s
,min. ,min.
LA40
I - post-filter 2
1
2
1
1
3
-
pre-1iposorter port-lrposorber
3
2
FIG. 5. C3c in ECC. Solid line, patient M; dotted line, patient V
TABLE 1. Elution rate of proteins, C ~ Cand , C3a Proteins (g) Variations during sessions Eluates NaCI O.15M NaCl 0.5M NH?OH Eluted proteins
C3c (mg)
C3a (cLg)
M
V
M
V
M
V
Loss: 15
Loss: 15
Loss: 110
Loss: 170
Production: 3.9 mg
Production: 4.5 mg
7 5.02 0.78 12.8
6.1 5.32 0.48 11.9
94 35
91 88
79 1,400
36 2,730
Traces
Traces
ND
ND
129
179
1.5 mg
3 mg
ND, not determined.
A r t f o r g u n s . Vol. 16, No. 6. 1992
A . TRIDON ET AL.
582
1
2
3
c3c
)
x
)
Y
C3D
A
c3c
C3O FIG. 6. A: Cross-dots of eluates (patient M), X, crude eluates; Y, eluates treated with PEG 22%; 1, eluates obtained with NaCl0.15M; 2, eluates obtained with NaCl 0.15M; 3, eluates obtained with hydroxylamine. Revelation with anti-C3c or antiC3d. B: Blots of eluates (patient M). 1, eluate obtained with NaCl 0.15M; 2,3,4, eluates obtained with NaCl 0.5M; 5,6,7, eluates obtained with hydroxylamine. TN, nonactivated plasma; TI, insulin-activated serum. Revelation with anti-C3c or antiC3d.
c3c B
TNT, 7 6 5 4 3 2 1
particularly as the molecular weight of C3 is high (180,000 daltons). Table 1 shows that the quantity of C3c that has disappeared matches the amount eluted from the liposorber. Most of the C3c is simply retained on the column (and washed out with 0.15M NaCl); a large amount is eluted with 0.5M NaCl by cleavage of ionic interactions, and finally a tiny amount is eluted by hydroxylamine, which also cleaves covalent interactions (22,23). Thus, the question arises of whether there is an activation of the alternative pathway of the complement system by contact with dextran sulfate and favored by the presence of large amounts of C3 in the liposorber. This question was addressed by analyzing the C3 derivatives bearing epitopes C3c and C3d contained in the eluates. In practice, we sought expression of antigens C3c and C3d by DOT and endeavored to differentiate the detected antigenic products by their electrophoretic mobilities on agarose by blot. DOT revealed the presence of the antigens concerned in all the eluates. The final product C3d was identified in all the eluates treated by precipitation with 22% PEG. The C3d, which theoretically binds covalently ArtifOrgms. Vol. 16, N o . 6 , I992
with the support, is thus mostly associated noncovalently here. This finding is consistent with the results from dialysis membrane eluates (22,23), but in this case the associated protein (and C3c) content is very low. According to Chenoweth, the C3d may break away from its support after a certain time through spontaneous hydrolysis of the ester bond (1 5). BLOT gave a pattern of fragments identified antigenically: simple washing showed several bands corresponding to native C3 and also to degradation products, the only fragments to be found in the 0.5M NaCl and hydroxylamine eluates. In conclusion, a powerful C3 deactivation process seems to be occurring within the liposorber, affecting both surface-bound and fluid phase C3b. However, in situ formation of alternative pathway convertase preceding or concomitant with this deactivation process cannot be inferred without data for C3 deactivation rates. Assay of anaphylatoxin C3a at different sites is one approach to the study of CSA in the ECC. A regular increase in the C3alevel was observed at Site 1 (corresponding to the patients’ blood circulation),
COMPLEMENT ACTIVATION IN LDL-APHERESIS moderate on LA15, and marked on LA40 (at t,,,, value found = initial value multiplied by 2.6 at 36, mean 16.3). For LA40, final values for C3a are given in the literature (21,24). Study of C3a in the plasma circuit of the ECC allows its production to be localized: it derives from the activating effect of the plasma separator. The C3a values at Site 2 were dramatically elevated (>10 pg/ml) at all the times studied and in both protocols in both patients. Different plasma separators were used in the two protocols, but both are recognized as activating the alternative pathway of the complement system (4,25,26). Their effects were apparently equivalent here and their surface areas were similar (0.2 and 0.3 m’). Generation of C3a on contact with the plasma separator was continuous during sessions. The kinetics of production of anaphylatoxin in this device were different from that observed during hemodialysis when an early peak of C3a occurred followed by a fall (5,15). The briefness of the increase in C3a is usually explained by an effect of saturation of the dialysis membrane, which becomes coated with C3b and cannot offer any more binding sites for further C3 molecules (15-17). The surface area of a plasma separator is about one-third of that of a dialysis membrane, but the volume of blood coming into contact with a plasma separator is only one-tenth of that filtered during dialysis, taking into account circulation flows and session durations. Hence, the two situations are difficult to compare. This major increase in C3a at Site 2 of the ECC (upstream of liposorber) induced a level at Site 3 (downstream of liposorber) only slightly higher than in samples simultaneously drawn from Site 1 (equivalent to blood circulation). The liposorber captured a large proportion of the C3a, which was found in the 0.5M NaCl eluates from LA40 (Table 1). The cationic charge of this anaphylatoxin accounts for its ionic interaction with dextran sulfate. The liposorber acts as an anaphylatoxin trap as already reported elsewhere (27,28). There is no effect of liposorber saturation with C3a as there is with C3c and apoB since C3a capture was observed at all the times. The very low molecular weight of this peptide (9,000 daltons) accounts for this. We undertook an anaphylatoxin C3a balance during sessions on the LA40 (Table 1). A certain amount of C3a was adsorbed on the liposorber; other unadsorbed C3a promoted a high final level in circulating blood. Overall, this corresponded to a production of 5.4 mg for patient M and 7.5 mg for patient V , that is, a C3 conversion rate of 13 and 17%. This is considerable compared with hemodialysis: C3a peaks observed on Cuprophan, the most strongly activat-
583
ing membrane (5,6,15), correspond in adults to a maximum conversion of C3 of 10%. The main undesirable effect of CSA is the appearance of anaphylatoxins C3a along with some C5a (16,17). This activates leukocytes. We observed leukocytal dynamics, mainly involving PNs, closely analogous to that described for dialysis; prompt leukopenia was followed by recovery (Fig. 2). In hemodialysis, the leukopenia is concomitant with C3a peaks and is usually attributed to the effect of C5. This activates the PNs and promotes adherence to endothelial cells (4,5,16,29) and leukostasis in the pulmonary vascular bed (5,16) though this last point is controversial (30). Thus, a granulocyte adherence increment and an increased expression of glycoprotein Mol (equivalent of CDI la-CD18) are transiently observed during hemodialysis (17,31,29) parallel to CSA as evidenced by anaphylatoxin C3a assay and leukopenia. Recovery of PNs with overshoot is attributed to the return of marginated cells with bone marrow cell contribution (5). The transient nature of the leukopenia is not fully understood; it might be explained by the briefness of the CSA as mentioned above (5,154, but certain authors have postulated that the C5a continues to be produced throughout the session and that the PNs cease to respond to C5a because of down-regulation of their C5a receptors (5,7). The leukopenia-CSA relationship is not currently considered to be unequivocal and seems to be affected by other factors (32,33). Our results on the liposorber are consistent with this; the leukocytal kinetics closely resembled that observed in hemodialysis whereas the anaphylatoxin production kinetics differed markedly. In addition, leukocyte activation, particularly of PNs, is increasingly considered as depending partly on CSA (32,34)and possibly connected with contact with membranes, modulated by adsorbed proteins. We had previously shown that the production of leukotriene B, during hemodialysis occurred independently of anaphylatoxin generation on various membranes (35). Increase in cell adhesiveness is one of the phenomena linked to cell activation; it does not necessarily correspond to an increase in the expression of adhering molecules but more likely to a conformational modification of these molecules after phosphorylation (36). Thus, the common PN kinetics in hemodialysis and ECC may result more from cell activation than a direct effect of CSA. This could amplify the effect through production of C5a, an inducer of leukocytal activation. The proteins found in the 0.5M eluates of LA40 were numerous and varied: IgG, IgA, IgM, transferrin P,-microglobulin, fibrinogen, C ~ C C, ~ C and , Arrif Organs, Vol. 16, N O . 6 , 1992
A . TRIDON ET AL. CILNA. Ionic interactions between these proteins and dextran sulfate may be assumed. The presence of IgG and of complement system molecules raises the problem of a possible activation of the classical complement pathway in the liposorber. This cannot be ruled out since the occurrence of antidextran IgG was rather high (37), and also small amounts of dextran might have sensitized the patient despite the dextran trap of the liposorber. Work is being done to see whether the classical pathway is involved in the marked CSA phenomenon demonstrated here. We have seen that a complement system activation occurred in the extracorporeal circulation device described here, linked essentially to the use of a strongly activating plasma separator. The liposorber eliminated only part of the anaphylatoxins generated, and the anaphylatoxin levels at the end of the session were still high in the children’s circulation. Risk of long-term side effects may be incurred here, and it is evidently important to avoid using a plasma separator that activates the complement system. This study reveals leukocytal dynamics comparable to that observed during dialysis while the kinetics of anaphylatoxin production were markedly different. Comparison of the two systems of extracorporeal filtering is of fundamental interest and calls for further work. Acknowledgments: W e thank Kaneka (Mitsuhi a n d Co., 37 av P d e Serbie, 75008 Paris) for financial support a n d Mr. Eglizot for technical assistance a n d GIBM (PR Dereix, CHRU Clermont F d , France).
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in chronic dialysis patients. A m J Kidney Dis 1987;9: 38 1-95. 8. Docci D, Bilancioni R, Baldrati L, Capponcini C , Turci F, Feletti C. Elevated acute phase reactants in hemodialysis patients. Clin Nephrol 1990;34:88-91. 9. Shaldon S, Koch K , Dinarello C. Interleukin 1 activation and acute phase response during hemodialysis. Contrih NephvoI 1988;6 1 18-26. 10. Wurzner R , Schuff-Werner P, Franzke A, Nitze R, Opperman M, Armstrong W, Eisenhauer T, Seidel D, Gotze 0. Complement activation and depletion during LDL-apheresis by heparin-induced extracorporeal LDL-precipitation. Eirr J CIin Invest 1991;21:288-94. I I . Burger R, Hadding U , Schorlemmer HU, Brade V, BitterSuermann D. Dextran sulphate: a synthetic activator of C3 via the alternative pathway. Immuno/ogy 1975;29:549-55. 12. Pangburn M. Analysis of recognition in the alternative pathway of complement. J . Immirnol 1989;142:2766-70. 13. Mauzac M, Maillet F, Jozefonvicz J, Kazatchkine M. Anticomplementary activity of dextran derivatives. Biomateriuls 1985;6:6 1-3. 14. Pangburn M. Analysis of the mechanism of recognition in the complement alternative pathway using C3b-bound low molecular polysaccharides. J Immunol 1989; 142:2759-65. 15. Chenoweth D. Complement activation during hemodialysis: clinical observations, proposed mechanisms and theoretical implications. Artif Organs 8:281-7. 16. Chenoweth D. Complement activation in extracorporeal circuits. Ann N Y Arud Sci 1987;516:306-13. 17. Kazatchkine M, Carreno M. Activation of the complement system at the interface between blood and artificial surfaces. Biomurerials 1988;9:30-5. 18 Lachman PJ, Hobart M. Complement technology. In: Hundhook of experirnentul immunology. Oxford: Raven Press, 1978:l-23 (Weir A, ed. Immunochemistry; vol 5). 19 Perrin L, Lamberg P, Miesher P. Complement breakdown products with systemic lupus erythematosus and patients with membranoproliferative or other glomerulonephritiis. J CIin Invest 1975;56:165-76. 20. Alric M, Cheyvialle D, Renaud M. Cross blot and cross dot system: a high performance system for the detection of antigen-antibody complexes on nitrocellulose. Anal Biocliern 1986;155:328-34. 21. Schooneman F, Ziegler 0, Briquel M, Streiff F. Efficacitt de 2 systemes d’epuration du cholesterol. Ann Med Inl 1990;I4 1:600-3. 22. Cheung A, Parker C, Janatova J. Analysis of the complement C3 fragments associated with hemodialysis membranes. Kidney Int 1989;35:576-88. 23. Gachon AMF, Mallet J, Tridon A, Deteix P. Analysis of proteins eluted from hemodialysis membranes. J Biomater Sci Polymer Edn 1991;2:263-76. 24. Schooneman F, Marchand M. Vigneron C , Streiff F. Evaluation du C3a et C5a dans les procedures d’hemaphkrese. ’4nn Med Int 1990;7:597-9. 25. Omokawa S , Malchesky P, Goldcamp J , Savon S, NosC Y. lmmunomodulating effects of serum-material interactions. J Biomed Muter Res 1991;25:621-36. 26. Omokawa S , Malchesky P , Suzuki T , Uchida N , Nose: Y. Surface area effects on efficiency and biocompatibility in experimental membrane plasmapharesis. Arrif Organs 1989;13:78-86. 27. Yokoyamd S , Kawaguchi T, Kojima T, Hayashi R, Satani M, Yamamuto A. Treatment of hypercholesterolemia by chemical adsorption of lipoproteins with dextran sulfate cellulose [Abstract]. Artif Organs 1987;11:334. 28. Yamamoto A, Kojima S, Hatanaka K. Long term experience and assessment of LDL apharesis in Japan [Abstract]. Artif Organs 1991;15:336. 29 Aljama P, Martin Malo A, Garin J, Torres A, Castillo D, Fuentes M, Gomez J. Granulocyte adherence changes: an index of biocompatibility. Kidney Inr 1988;33:68-72.
COMPLEMENT ACTIVATION IN LDL-APHERESIS 30. Terakoa S , Sugawara M, Kitano Y , Hoshino T , Takahashi M. Minagawa Y, Naganuma S , Sanaka T, Mineshima M, Era K , Honda H, Fuchinoue S , Agishi S, Ota K. Microscopic observation of leukocyte kinesis in the vascular bed during hernodialysis using the rabbit ear chamber technique. Znt J Artif Organs 1989;12:229-33. 31. Arnaout A, Hakirn R, Todd R , Dana N , Colten H. Increased expression of an adhesion-promoting surface glycoprotein in the granulocytopenia of hemodialysis. N En81 J Med 1985;3 12:457-62. 32. Wauters JP, Markert M. Aspects nouveaux de la biocompat ibilite des membranes d’htmodialyse. Actrcal Nephrol 1990.
33. Wiegmann TB, MacDougall ML, Diederich DA. Dialysis leu-
34. 35. 36.
37.
kopenia, hypoxemia, and anaphylatoxin formation: effect of membrane, bath, and citrate anticoagulation. Am J Kidney Dis 1988;11:418-24. Markert M, Waridel P, Heierli C , Wauters JP. Neutrophil functions during hemodialysis. Conrrib Nephrol 1988;62: 99-108. Tridon A, Albuisson E, Deteix P, Marques Verdier A , Gaillard G , Betail G, Baguet JC. Leukotriene B, in hemodialysis. Artif Organs 1990;14:387-98. Patarroyo M. Leucocyte adhesion in host defense and tissue injury. Clin Imrnunol Zrnrnunopathol 1991;60:333-48. Maillet F, Kazatchkine MD. Specific antibodies enhance alternative complement pathway activation by cuprophane. Nephrol Dial Transplant 1991;6: 193-7.
Artif Organs, Vol. 16, No. 6 , 1992
16(6):577-585, Blackwell Scientific Publications, Inc.. Boston 8 1992 International Society for Artificial Organa
Complement Activation During Low-Density Lipoprotein Apheresis A. Tridon, "J.B. Palcoux, tP. Jouanel, SM.J. Bezou, SM. Coulet, and G. Betail Lahorutoii.e d'lmmunologie, *Service d'Hkmodiulyse Pgdiatrique, tlaborutoire de Biochimie, and $Lahowtoire d'Htmarologie, CHRU Clermont-Fewand, France
Abstract: Complement system activation was investigated in two girls with familial homozygous hypercholesterolemia undergoing two monthly sessions on LA15 or LA40 (Kaneka liposorber). We determined blood levels of C3c and C3a, leukocyte counts, and plasma levels of C3c and C3a in the extracorporeal circulation device at the start of the sessions and 15 and either 60 or 120 min into them. Sequential eluates were collected from LA40 at the end ofthe sessions (O.SMNaC1, 1M hydroxylamine). Anaphylatoxin C3a increased throughout, especially with
LA40. As previously reported, C3a was trapped in the dextran column but was noticeably present in efferent plasma. Besides many proteins, nonnative complement fragments bearing C3a and C3d antigens were detected in almost all the eluates, suggesting possible in situ complement activation. Practically, complement activation induced by the first filter is a risk; long-term side effects may arise from this extracorporeal circulation device. Key Words: Complement system activation-C3 breakdown products-Dextran sulfate-Liposorber.
Two children with familial homozygous hypercholesterolemia were undergoing removal of apolipoprotein B (apoB) included in low-density lipoprotein (LDL) and very-low-density lipoprotein (VLDL) twice monthly using a plasmapharesis procedure with cascade filtration. The extracorporeal device consisted of a column of cellulose beads coated with dextran sulfate, liposorbers LA15 and LA40 (Kaneka) (Fig. 1). Specific binding of apoB occurs on this column through interaction between the negatively charged dextran sulfate and positively charged apoB (1). In addition, other proteins are adsorbed; this prompted our interest. Our main aim was to investigate complement system activation (CSA) linked to the use of this particular extracorporeal circulation (ECC) device as a criterion of biocompatibility. Until recently, apheresis specialists had paid little attention to this question since these techniques were not applied repeatedly and seldom involved reinfusion into the patient. However, with the development of frequently re-
peated selective removal procedures with return to the patient of plasma and cells (2-4), this issue now needs to be addressed. The study of CSA is only one aspect of biocompatibility, but developments in the field of hemodialysis show that it is implicated in other biological reactions (5,6). Repeated CSA causes repeated activation of cells resulting in an inflammatory response, which when recurrent has long-term side effects: relative cell deactivation with depression of natural defenses (7), chronic inflammatory syndrome (8), and immune imbalance (9). Two phenomena are implicated in CSA: depletion (linked to the adsorption capacity of the filters, here mainly liposorber) and activation (linked to the use of the plasma separator with possible involvement of the liposorber). A recent study on another system for cascade filtration of cholesterol has determined the relative extents of these two phenomena (10). CSA may occur by contact with dextran sulfate: this material is known to trigger the alternative pathway depending on its degree of polymerization and its molecular weight ( 1 1,12) and on how sulfated it is (11,13). Theoretically, any nascent C3b produced by initial fluid phase convertase will bind by transesterification to any bystander surface with exposed OH
Received February 1992; revised June 1992. Address correspondence and reprint requests to Dr. A. Tridon at Laboratoire d'Imrnunologie, FacultC de MCdecine et Pharmacie, B . P . 38, 63001 Clermont-Ferrand CEDEX, France.
577
A . TRIDON ET AL.
5 78
anboagulanl plasma
FIG. 1. Extracorporeal device.
’
’
8
ALIPOSORBER adsorplion coluinn
membrane liller unil
groups. According to its biochemical surroundings (and especially the charges on neighboring substituents), surface-bound C3b will interact with B and D fractions and trigger formation of alternative C3 convertase able to cleave C3 to C3a and C3b or factor H, which favors its deactivation to intermediate (C3bi) or final (C3c and C3d) breakdown products. Antigen C3c is borne by native C3, C3b, C3bi, and C3c. Antigen C3d is borne by native C3, C3b, C3bi, and C3d. Dextran sulfate, through its free OH groups, offers available binding sites for C3b (1 1,14). In addition, Chenoweth (15,16) has postulated that the presence of negatively charged sulfate groups close to the C3b binding site favors the interaction of C3b with H, that is, its deactivation. This anticomplementary effect depends on the degree of polymerization and the sulfate titer of the dextran sulfate; it has been studied in particular by Kazatchkine et al. ( I 3,17). MATERIALS AND METHODS Patients and treatment sessions The two female patients, M and V , were 8 years old at the time of the first protocol and 10 at the time of the second. Hypercholesterolemia had been tested for because of severe familial antecedents, and they had both been undergoing repeated plasmapheresis from age 5 years until one year before the first protocol. The treatment sessions took place twice monthly and lasted about 2 h. The ECC flow rates were 60 ml/min in the blood circuit and 20 ml/min in the plasma circuit. In both protocols, samples were drawn from the ECC (Fig. I) as follows: at t,, the arterial starting point; and at different times during the session, simultaneously before the plasma separator (Site l), after the plasma separator (Site 2), and after the liposorber (Site 3). Ar/ifOrgam, Vol. 16, N o . 6, 1992
Several consecutive sessions were studied for each protocol and for each child. In Protocol 1 , the plasma separator was polyvinyl alcohol (PVA; Plasmacure, surface area 0.3 m2, Kuraray, Paris). The Liposorber was an LA 15 (1 50 ml sorbent). Sampling and t,, m,n. Three sessions per times were t o , t 1 5 child were studied. In Protocol 2, the plasma separator was polypropylene (BELCO 550, surface area 0.2 m2, Sorin Biomedica, Antony, France). The Liposorber was an LA40 (400 ml sorbent). Sampling times were f , , r , , mln, and rlZOmln.Four sessions per child were studied. The LA15 was systematically replaced by an LA40 between the two protocols for better therapeutic efficiency. Methods At each time and at each site, the following samples were taken: an EDTA tube for leukocyte counts and hematocrit, and an EDTA tube for assay of anaphylatoxin C3a, and a tube without anticoagulant for assay of C3c and C4c. Hemolytic activity was determined only for the second protocol (CH,,). The samples were annotated at each time according to their site (1, 2, or 3) (Fig. 1). The hematological parameters were determined with a Sysmex NE 8000. Assays of C3 and C4 in the form of C3c and C4c antigens were carried out using a Behring (Rueil Malmaison, France) Turbitimer. CH,, was assayed by the kinetic method (18). Assay of C3a was by radioimmunology (Amersham, Les Ulis, France). After one session per subject on the LA40, the dextran sulfate columns were rinsed with 1 L of 0.15M NaCl and then successively eluted with 1 L of 0.5M NaCl and 200 ml of 1M hydroxylamine. Fractions of 4 ml were collected. On fractions obtained with 0.5M NaCl and IM NH,OH, various proteins were sought by double immunodiffusion
5 79
COMPLEMENT ACTIVATION IN LDL-APHERESIS with commercial antisera: anti-Ig (Silenus, Les Ulis, France), anti-C3c, ClINA, C ~ CC3PA, , fibrinogen, albumin, and apoB (Behring), anti-C3d, anti-B,-microglobulin (Nordic, Le Perray en Yvelines, France), and anti-transferrin (Dako, Trappes, France). Derivatives of C3b bearing C3c and C3d antigens were revealed by dot and blot techniques. We adapted a dot technique to the search for antigens C3c and C3d in the crude eluates and treated them with 22% polyethylene glycol (PEG), precipitating out all the derivatives of C3b except for the final C3d fragment of molecular weight 1 1,000 daltons by the method of Perrin et al. (19). The eluates were placed in contact with Immobilon in a dot apparatus (20). After washing and saturation, anti-C3c (Behring) and anti-C3d (Nordic) were laid on Immobilon. After elimination of the first antibody and washing, an antirabbit antibody labeled with peroxidase (Pasteur, Marnes la Coquette, France) was placed in contact for 1 h. Visualization was with diaminobenzidine (DAB) and H20,. The detection threshold for these two antigens was about 50 ng/ml. This antigen search was completed with a study of the electrophoretic motility of the antigen-bearing fractions using a BLOT technique. The eluates underwent electrophoretic separation on agarose under nondenaturing conditions in parallel with two controls: fresh plasma (native C3) and plasma activated with inulin (native fraction plus cleavage products). After electrophoresis, an Immobilon BLOT was performed by contact. Immunodetection was carried out under the same operating conditions as for the dot. The presence of large amounts of apoB in the 0.5 NaCl eluate did not allow formal identification of the native C3 band (these two proteins have similar beta migrations on agarose). Accordingly, this search was conducted in parallel on eluates treated with an anti-apoB (Behring). The hydroxylamine eluates were dialyzed at 4°C in phosphate-buffered saline (PBS) before blot analysis. Assays were performed on pools of eluates for each session: assay of total proteins (OD at 280 nm), assay of C3c (Behring turbitimer), and assay of anaphylatoxin C3a (RIA Amersham). RESULTS The results are presented individually in Figs. 2-5. The results obtained on the whole blood in the ECC are presented first followed by those of the plasma circuit of the ECC and then by the results for the eluates.
Whole extracorporeal blood Leukocyte count variations in sessions on LA15 and LA40 differed quantitatively but were kinetically consistent: there was a fall at I,, compared with lo(54-108% of the initial value, mean 83%) and then a rise at t,, on LA15 (100-151%, mean 130%) and LA40 (94-195%, mean 140%). These variations mainly concerned neutrophil counts (Fig. 2). There seemed to be no noteworthy variation of hematocrit , indicating that there is no hernodilution linked to the ECC priming procedure. C3c and C4c decreased moderately during the session: the results for C3c are presented in Fig. 3 . C3a increased overall, gradually over the period studied in sessions on LA15 (at fmr initial level 1.3 to 3) and strongly in sessions on LA40 (at t,,, , initial level x 3 to 36) (Fig. 4). C3a was sometimes assayed at cm on LA40, and the moderate increase observed was of the same on LA15. amplitude as the increase observed at CH,, was assayed only for the second protocol. It fell markedly from an initially normal value to a final value always less than 50%.
f,,
Extracorporeal plasma At Site 1 (with the plasma separator), we had the plasma values of the parameters studied. These could be compared with those obtained at Sites 2 (after the plasma separator) and 3 (after the liposorber) at different times ( I , ~and t60,,20). C3c and C4c had widely differing values at Sites 2 and 3: at Site 2 they were plasma values, and at Site 3 they were close to nil. The results for C3c at f,, are given in Fig. 5 . However, at t60,,20,the values of C3c and C4 were comparable at the two sites. C3a was always very high at Site 2 (>lo pglml) while at the liposorber output the observed levels were slightly higher than the plasma levels obtained sirnultaneously at Site 1. This was observed at all the times in both children with both protocols. Eluates from LA40 Numerous proteins were adsorbed on the column and eluted out (NaCI 0.15M then NaCl 0.5M then 1M hydroxylamine). Some were identified by double immunodiffusion (IDD) in the 0.5M NaCl eluates: IgG, IgA, IgM, transfenin, fibrinogen, &-microglobulin, C ~ CC, ~ CC3PA, , ClINA, and of course apoB and albumin. The quantitative results for total proteins, C ~ Cand , C3a in the three types of eluate are given in Table 1 in comparison with their overall variation during sessions. The presence of antigens C3c and C3d was demonstrated by DOT in all the eluates (Fig. 6). The final product C3d was identified in all the eluates alongside molecules bearing antigen Artif Organs, Vol. 16. No. 6, 1992
580
A . TRIDON ET A L . PN
by ul
’\
min
..,. .,
_...._. .........
,,..._....._...._....
L , I20
min.
FIG. 2. Neutrophil count variations during LA15 and LA40 sessions. Solid line, patient M; dotted line, patient V.
.
... .. .... ... . -
..
.. \
, 0
15
6C
,
min 0
15
mi”.
J20
FIG. 3. C3c variations during LA15 and LA40 sessions. Solid line, patient M; dotted line, patient V.
C3c associated or not with C3d. BLOT analysis did not always reveal antigen C3d but showed in all the eluates the presence of at least a fraction bearing C3c and corresponding to a cleavage product by comparison with an in vitro activated plasma profile. DISCUSSION We investigated CSA in this ECC from different angles: we looked at variations in C3c (parallel to those of C3a) and its fate in the liposorber via eluates. We undertook an anaphylatoxin C3a balance A r t f O ~ g a m Vol. . 16, N o . 6, I992
from its variations during sessions and its presence in the eluates. Lastly, we looked at the quantitative variations in the polynuclear neutrophil (PN) counts during sessions and endeavored to correlate these with possible CSA. The fall in C3 assayed as C3c (Fig. 3) in the children’s circulation was due first to entrapment within the liposorber as shown by the study of C3c in the ECC plasma (Fig. 5). In both protocols, this C3c depletion effect was marked at 15 min but did not occur thereafter. This is probably due to saturation like that observed much later for apoB itself (21),
COMPLEMENT ACTIVATION IN LDL-APHERESIS
5'8I
nglrnl
X I O .
X I O .
5.
x 5.
x
7
LA4n
rnl" mi".
s
,min. ,min.
LA40
I - post-filter 2
1
2
1
1
3
-
pre-1iposorter port-lrposorber
3
2
FIG. 5. C3c in ECC. Solid line, patient M; dotted line, patient V
TABLE 1. Elution rate of proteins, C ~ Cand , C3a Proteins (g) Variations during sessions Eluates NaCI O.15M NaCl 0.5M NH?OH Eluted proteins
C3c (mg)
C3a (cLg)
M
V
M
V
M
V
Loss: 15
Loss: 15
Loss: 110
Loss: 170
Production: 3.9 mg
Production: 4.5 mg
7 5.02 0.78 12.8
6.1 5.32 0.48 11.9
94 35
91 88
79 1,400
36 2,730
Traces
Traces
ND
ND
129
179
1.5 mg
3 mg
ND, not determined.
A r t f o r g u n s . Vol. 16, No. 6. 1992
A . TRIDON ET AL.
582
1
2
3
c3c
)
x
)
Y
C3D
A
c3c
C3O FIG. 6. A: Cross-dots of eluates (patient M), X, crude eluates; Y, eluates treated with PEG 22%; 1, eluates obtained with NaCl0.15M; 2, eluates obtained with NaCl 0.15M; 3, eluates obtained with hydroxylamine. Revelation with anti-C3c or antiC3d. B: Blots of eluates (patient M). 1, eluate obtained with NaCl 0.15M; 2,3,4, eluates obtained with NaCl 0.5M; 5,6,7, eluates obtained with hydroxylamine. TN, nonactivated plasma; TI, insulin-activated serum. Revelation with anti-C3c or antiC3d.
c3c B
TNT, 7 6 5 4 3 2 1
particularly as the molecular weight of C3 is high (180,000 daltons). Table 1 shows that the quantity of C3c that has disappeared matches the amount eluted from the liposorber. Most of the C3c is simply retained on the column (and washed out with 0.15M NaCl); a large amount is eluted with 0.5M NaCl by cleavage of ionic interactions, and finally a tiny amount is eluted by hydroxylamine, which also cleaves covalent interactions (22,23). Thus, the question arises of whether there is an activation of the alternative pathway of the complement system by contact with dextran sulfate and favored by the presence of large amounts of C3 in the liposorber. This question was addressed by analyzing the C3 derivatives bearing epitopes C3c and C3d contained in the eluates. In practice, we sought expression of antigens C3c and C3d by DOT and endeavored to differentiate the detected antigenic products by their electrophoretic mobilities on agarose by blot. DOT revealed the presence of the antigens concerned in all the eluates. The final product C3d was identified in all the eluates treated by precipitation with 22% PEG. The C3d, which theoretically binds covalently ArtifOrgms. Vol. 16, N o . 6 , I992
with the support, is thus mostly associated noncovalently here. This finding is consistent with the results from dialysis membrane eluates (22,23), but in this case the associated protein (and C3c) content is very low. According to Chenoweth, the C3d may break away from its support after a certain time through spontaneous hydrolysis of the ester bond (1 5). BLOT gave a pattern of fragments identified antigenically: simple washing showed several bands corresponding to native C3 and also to degradation products, the only fragments to be found in the 0.5M NaCl and hydroxylamine eluates. In conclusion, a powerful C3 deactivation process seems to be occurring within the liposorber, affecting both surface-bound and fluid phase C3b. However, in situ formation of alternative pathway convertase preceding or concomitant with this deactivation process cannot be inferred without data for C3 deactivation rates. Assay of anaphylatoxin C3a at different sites is one approach to the study of CSA in the ECC. A regular increase in the C3alevel was observed at Site 1 (corresponding to the patients’ blood circulation),
COMPLEMENT ACTIVATION IN LDL-APHERESIS moderate on LA15, and marked on LA40 (at t,,,, value found = initial value multiplied by 2.6 at 36, mean 16.3). For LA40, final values for C3a are given in the literature (21,24). Study of C3a in the plasma circuit of the ECC allows its production to be localized: it derives from the activating effect of the plasma separator. The C3a values at Site 2 were dramatically elevated (>10 pg/ml) at all the times studied and in both protocols in both patients. Different plasma separators were used in the two protocols, but both are recognized as activating the alternative pathway of the complement system (4,25,26). Their effects were apparently equivalent here and their surface areas were similar (0.2 and 0.3 m’). Generation of C3a on contact with the plasma separator was continuous during sessions. The kinetics of production of anaphylatoxin in this device were different from that observed during hemodialysis when an early peak of C3a occurred followed by a fall (5,15). The briefness of the increase in C3a is usually explained by an effect of saturation of the dialysis membrane, which becomes coated with C3b and cannot offer any more binding sites for further C3 molecules (15-17). The surface area of a plasma separator is about one-third of that of a dialysis membrane, but the volume of blood coming into contact with a plasma separator is only one-tenth of that filtered during dialysis, taking into account circulation flows and session durations. Hence, the two situations are difficult to compare. This major increase in C3a at Site 2 of the ECC (upstream of liposorber) induced a level at Site 3 (downstream of liposorber) only slightly higher than in samples simultaneously drawn from Site 1 (equivalent to blood circulation). The liposorber captured a large proportion of the C3a, which was found in the 0.5M NaCl eluates from LA40 (Table 1). The cationic charge of this anaphylatoxin accounts for its ionic interaction with dextran sulfate. The liposorber acts as an anaphylatoxin trap as already reported elsewhere (27,28). There is no effect of liposorber saturation with C3a as there is with C3c and apoB since C3a capture was observed at all the times. The very low molecular weight of this peptide (9,000 daltons) accounts for this. We undertook an anaphylatoxin C3a balance during sessions on the LA40 (Table 1). A certain amount of C3a was adsorbed on the liposorber; other unadsorbed C3a promoted a high final level in circulating blood. Overall, this corresponded to a production of 5.4 mg for patient M and 7.5 mg for patient V , that is, a C3 conversion rate of 13 and 17%. This is considerable compared with hemodialysis: C3a peaks observed on Cuprophan, the most strongly activat-
583
ing membrane (5,6,15), correspond in adults to a maximum conversion of C3 of 10%. The main undesirable effect of CSA is the appearance of anaphylatoxins C3a along with some C5a (16,17). This activates leukocytes. We observed leukocytal dynamics, mainly involving PNs, closely analogous to that described for dialysis; prompt leukopenia was followed by recovery (Fig. 2). In hemodialysis, the leukopenia is concomitant with C3a peaks and is usually attributed to the effect of C5. This activates the PNs and promotes adherence to endothelial cells (4,5,16,29) and leukostasis in the pulmonary vascular bed (5,16) though this last point is controversial (30). Thus, a granulocyte adherence increment and an increased expression of glycoprotein Mol (equivalent of CDI la-CD18) are transiently observed during hemodialysis (17,31,29) parallel to CSA as evidenced by anaphylatoxin C3a assay and leukopenia. Recovery of PNs with overshoot is attributed to the return of marginated cells with bone marrow cell contribution (5). The transient nature of the leukopenia is not fully understood; it might be explained by the briefness of the CSA as mentioned above (5,154, but certain authors have postulated that the C5a continues to be produced throughout the session and that the PNs cease to respond to C5a because of down-regulation of their C5a receptors (5,7). The leukopenia-CSA relationship is not currently considered to be unequivocal and seems to be affected by other factors (32,33). Our results on the liposorber are consistent with this; the leukocytal kinetics closely resembled that observed in hemodialysis whereas the anaphylatoxin production kinetics differed markedly. In addition, leukocyte activation, particularly of PNs, is increasingly considered as depending partly on CSA (32,34)and possibly connected with contact with membranes, modulated by adsorbed proteins. We had previously shown that the production of leukotriene B, during hemodialysis occurred independently of anaphylatoxin generation on various membranes (35). Increase in cell adhesiveness is one of the phenomena linked to cell activation; it does not necessarily correspond to an increase in the expression of adhering molecules but more likely to a conformational modification of these molecules after phosphorylation (36). Thus, the common PN kinetics in hemodialysis and ECC may result more from cell activation than a direct effect of CSA. This could amplify the effect through production of C5a, an inducer of leukocytal activation. The proteins found in the 0.5M eluates of LA40 were numerous and varied: IgG, IgA, IgM, transferrin P,-microglobulin, fibrinogen, C ~ C C, ~ C and , Arrif Organs, Vol. 16, N O . 6 , 1992
A . TRIDON ET AL. CILNA. Ionic interactions between these proteins and dextran sulfate may be assumed. The presence of IgG and of complement system molecules raises the problem of a possible activation of the classical complement pathway in the liposorber. This cannot be ruled out since the occurrence of antidextran IgG was rather high (37), and also small amounts of dextran might have sensitized the patient despite the dextran trap of the liposorber. Work is being done to see whether the classical pathway is involved in the marked CSA phenomenon demonstrated here. We have seen that a complement system activation occurred in the extracorporeal circulation device described here, linked essentially to the use of a strongly activating plasma separator. The liposorber eliminated only part of the anaphylatoxins generated, and the anaphylatoxin levels at the end of the session were still high in the children’s circulation. Risk of long-term side effects may be incurred here, and it is evidently important to avoid using a plasma separator that activates the complement system. This study reveals leukocytal dynamics comparable to that observed during dialysis while the kinetics of anaphylatoxin production were markedly different. Comparison of the two systems of extracorporeal filtering is of fundamental interest and calls for further work. Acknowledgments: W e thank Kaneka (Mitsuhi a n d Co., 37 av P d e Serbie, 75008 Paris) for financial support a n d Mr. Eglizot for technical assistance a n d GIBM (PR Dereix, CHRU Clermont F d , France).
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