GOTZE* *Department of. Immunology ... goat IgG; mAb, monoclonal antibody (ies); MAC, membrane attack complex; MHC .... The data obtained with the coating.
Immunology 1991 74 132-138
Blood dendritic cells carry terminal complement complexes on their cell surface as detected by newly developed neoepitope-specific monoclonal antibodies R. WURZNER,*t H. XU,* A. FRANZKE,* M. SCHULZE,* J. H. PETERS* & 0. GOTZE* *Department of Immunology, Georg-August-University Gottingen, Germany and tMRC Molecular Immunopathology Unit, University of Cambridge, U.K.
Acceptedfor publication 9 May 1991
SUMMARY Blood dendritic cells carry the terminal complement complex (TCC) on their surface, as detected by three monoclonal antibodies (mAb). Two of these mAb were generated by immunizing mice with the terminal complement complex, whereas the third was generated by immunizing mice with blood dendritic cells. All three mAb are directed against neoepitopes on the C9 molecule, as assessed by binding and blocking experiments and studies with several forms of denatured C9 and C9-depleted serum. Only one of these mAb binds to soluble polymerized C9. All three mAb allow the quantification of human TCC in sensitive ELISA procedures and could be used as markers for the evaluation of the functions of non-lytic TCC on dendritic cells.
have been shown to be a much more specific detector of TCC in a variety of normal and diseased tissues7 than antibodies directed against S-protein. TCC was found in human lymphoid germinal centres,8'9 where it was present on follicular dendritic cells (FDC).8 This is of importance since the MAC causes, aside from its cytolytic function, a variety of non-lethal effects in nucleated cells, including stimulation or release of inflammatory mediators and cell proliferation.'0 These functions are exhibited by the MAC without lytic effects. Identification and characterization of dendritic cells have demonstrated that this cell population is not homogeneous with respect to surface markers and anatomical sites, since resident and non-resident dendritic cells exist."I Human blood dendritic cells (BDC) were first described by von Voorhis et al.'2 They carry MHC class II antigens but lack typical markers of monocytes and lymphocytes.'2 In order to detect and characterize human immune cells carrying the terminal complement complex on their cell surface, we characterized two newly developed anti-TCC mAb (WU 7-2, WU 13-15) with respect to their epitope specificity and applied them to FACS analyses on BDC. An additional mAb (X-l 1) was obtained by immunization with an enriched BDC fraction and was first found to bind to FDC in lymphnodes. Since the binding pattern of X- 11 on tissue sections of spleen and lymphnodes was very similar to that of both anti-TCC mAb, it was suggested that this mAb might also be TCC-specific and was, therefore, included in the characterization of the anti-TCC mAb.
INTRODUCTION Classical or alternative pathway activation of the complement system results in the formation of the terminal complement complex (TCC). The membrane attack complex [MAC or C5b9(m)], the potentially membranolytic form of the TCC generated on biological membranes,' has to be distinguished from the generally non-lytic form generated in extracellular fluids in the presence of S-protein, SC5b-9.2 S-protein is identical to vitronectin3 and is one of the proteins capable of promoting cell adhesion.4 Since S-protein is also able to integrate into the membrane attack complex after generation of C5b-9(m),5 the presence of S-protein on cell surfaces could be due to S-protein alone or an adsorption of SC5b-9 or S-protein, which integrated into C5b-9(m). S-protein is also capable of binding specifically to different bacterial species6 and is, therefore, not at all a marker for the bactericidal activities of the MAC. In contrast, neoepitope-specific anti-TCC monoclonal antibodies (mAb) Abbreviations: BDC, blood dendritic cell(s); C, complement component(s); FDC, follicular dendritic cell(s); GaC, goat IgG anti-C; GIgG, goat IgG; mAb, monoclonal antibody (ies); MAC, membrane attack complex; MHC, major histocompatibility complex; MIgG, mouse IgG; NHS, normal human serum; OD, optical density; pAb, polyclonal antibody (ies); RaS, rabbit IgG anti-S protein; RIgG, rabbit IgG; TCC, terminal complement complex. Correspondence: Dr R. Wurzner, MRC Molecular Immunopathology Unit, University of Cambridge, Hills Road, Cambridge CB2 2QH, U.K.
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TCC on blood dendritic cells MATERIALS AND METHODS
Preparation of C9-depleted serum, complement-activated serum and SC5b-9 C9-depleted serum was prepared using goat anti-C9 IgG (GaC9; Cytotech, San Diego, CA) coupled to Reacti-Gel beads, a carbonyl-diimidazole-activated support (Pierce, Oud Beijerland, The Netherlands), as described by Hissey et al.'3 The success of the C9 depletion was controlled by immunoblotting of normal and C9-depleted serum using GaC9. The complement activation of sera using boiled baker's yeast and the purification of SC5b-9 were performed as described elsewhere.'4 Isolation of human peripheral blood dendritic cells (BDC) Buffy-coats from healthy blood donors were resuspended in phosphate-buffered saline (PBS) and centrifuged through Lymphoprep (1 077 g/ml; Nycomed AS, Oslo, Norway) at I000g for 20 min. The cells at the interface were collected, washed and suspended in RPMI-1640 medium (Biochrom, Berlin, Germany), supplemented with 5% normal human serum (NHS). BDC were separated from these cells by a continuous Percoll gradient, according to Young & Steinman,'5 with minor modifications. Briefly, isotonic Percoll medium consisting of PBS, Percoll (density: 1 197 g/ml; Pharmacia, Uppsala, Sweden) and RPMI- 1640 medium was spun at 10,000 g, 40 for 25 min to generate a continuous gradient. The cells were centrifuged into this gradient at 1800 g, 4° for 25 min. The high-density fraction (1 084 g/ml) was harvested and washed with PBS/20% RPMI, after which the T lymphocytes were depleted by rosetting with sheep erythrocytes in the presence of 1% polyethylene glycol, Mr 10,000 (Merck, Darmstadt, Germany).'6 The T-cell depleted cells were then cultured at a concentration of 2-3 x 106 cells/ml in hydrophobic teflon Petri dishes (Petriperm; Heraeus, Hanau, Germany) in RPMI- 1640 medium supplemented with penicillin (100 U/ml), streptomycin (100 yg/ ml), glutamine (2 mM) and 10% normal human serum (NHS). After 2 days, the cells were collected and suspended in culture medium at a concentration of 8 x 105 cells/ml. This suspension was mixed with Sepracell-MN (Sepratech, Oklahoma City, OK) at a ratio of 1: 1 5, and then centrifuged at 1 500g, 200 for 20 min. The upper fraction was collected, washed and incubated in bacterial culture plates (Greiner, Nuirtingen, Germany) coated with human IgG (10 mg/ml in carbonate buffer, pH 10-5) for 45 min at 370 in order to remove Fc receptor-bearing cells. The non-adherent cells (BDC-enriched population) were used for immunization and FACS analyses (see below).
Production of monoclonal (mAb) and polyclonal antibodies (pAb) Polyclonal antibodies (pAb) directed against C5, C6 and C7 were raised in goats and were purified by standard methods, as already described for C7.17 Goat anti-C8 IgG (GaC8) was purchased from Cytotech. Rabbit anti-S-protein (RaS) was kindly donated by Dr K. T. Preissner, Giessen, Germany. BALB/c mice were immunized by repeated intraperitoneal injections of 30 pg SC5b-9. Fusion of spleen cells with myeloma cells and cloning of the hybridomas was performed as published elsewhere. " Hybridoma culture supernatants were screened for increased reactivity in complement activated serum by a Screening-ELISA (see below). Two positive clones (WU 7-2, WU 13-15) were recloned in culture plates and propagated as
133
ascitic cells in pristane pretreated (0-5 ml intraperitoneally) BALB/c mice. The mAb X- 1I was obtained from a BALB/c mouse, immunized with enriched human BDC, and was selected by screening hybridoma culture supernatants on cryostat sections of human lymphnodes using the Strept-ABC technique [biotinylated anti-mouse IgG (Dakopatts, Glostrup, Denmark), stteptavidin-biotin-horseradish peroxidase (Dakopatts) and the substrate 3-amino-9-ethylcarbazol (Sigma, Deisenhofen, Germany)], as described elsewhere.'8 X- 11 was further characterized because it stained FDC in cryosections of human lymphnodes. All three mAb were isolated and purified from ascitic fluids as described elsewhere."7 Isotypes were determined using a class/ subclass-specific xenoantisera kit (Nordic, Tilburg, The Netherlands). Biotinylation of pAb and mAb was performed according to standard protocols.'9 Screening-ELISA for the determination of the TCC-neoepitope
specificity of mAb In all ELISA procedures the microplate was washed three times between each incubation (60 min at 22°) using PBS containing 0 05% Tween 20 (Serva, Heidelberg, Germany), which also served as incubation buffer, except for the coating step. Pre-absorption of EDTA plasma from healthy blood donors or complement-activated serum was performed in order to minimize binding of native C6 to the goat anti-C6 IgG (GaC6) capture antibody in the screening-ELISA. Therefore, the mAb WU 6-4, which binds to native C6 but not to TCC-incorporated C6, was adsorbed to microplate wells overnight at 4° (30 yg/ml in 100 p1 coating buffer comprising 68 p1 0-2 M Na2CO3, 32pl 0-2 M NaHCO3, pH 10-6). After blocking with 1% gelatin in PBS, the wells were incubated with EDTA plasma or complementactivated serum at 1:50 dilutions for 20 min to partially deplete the native C6 in these samples. The pre-absorbed samples were then transferred to other coated wells and again incubated for 20 min to increase the efficiency of depletion. For hybridoma culture supernatant screening GaC6 was adsorbed to microplate wells (20 pg/ml). After blocking the wells, either the pre-absorbed EDTA plasma or the preabsorbed complement-activated serum was added (1:50 dilution), after which undiluted hybridoma culture supernatants were introduced followed by the addition of rabbit anti-mouse IgG coupled to horseradish peroxidase (Dakopatts) at a dilution of 1:500. Biotinylated polyclonal anti-C7 IgG (GaC7 biot) together with streptavidin-horseradish peroxidase (1: 500 dilution; Dakopatts) served as positive control. Finally, 2,2'azino-di-(3-ethylbenzthiazoline) sulphonic acid (ABTS; Boehringer Mannheim, Mannheim, Germany) was introduced and the increase in the absorption at 410 nm was recorded using an automated ELISA plate reader (Dynatech, Denkendorf, Germany). Clones which reacted with complement-activated serum, but not with EDTA plasma at the 1:50 dilution, were processed further (see above).
Epitope analyses For epitope analyses, mAb derived from the immunization with SC5b-9 (WU 7-2, WU 13-15) or the mAb X- 11 were coated to microplate wells (30 pg/ml). Complement-activated serum (1: 100, final dilution) was introduced to the wells and the bound antigen was detected by biotinylated mAb (WU 7-2, WU 13-15,
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X-l 1) or pAb (GaC5, GaC6, GaC7, GaC8, GaC9, RaS) or controls [mAb anti-native C6 (WU 6-4), rabbit IgG (RIgG), goat IgG (GIgG), mouse IgG (MIgG)] at concentrations of 30 jg/ml, respectively, together with streptavidin-horseradish peroxidase (1: 500 dilution) and the substrate ABTS, as described for the Screening-ELISA. *As a modification, complement-activated serum from 33 different animals was introduced into the assay (1:12 dilution) as source of antigen and compared to a dilution of human complement-activated serum to determine the percentage of cross-reactivity of mAb. The species investigated included Arctic fox, bear, Californian sealion, camel, chicken, Congo buffalo, cow (and foetal calf), crocodile, dog, dolphin, elephant, Fennel fox, giraffe, guinea-pig, lion, mouse, oryx, pig, pigeon, puma, python, rabbit, rat, rhinoceros, sable, sable antelope, seal, sheep, tiger, tortoise, wallaby, wild boar and zebra. ELISA for the localization of the mAb-specific epitopes on the TCC Microplate wells were coated with the purified mAb WU 7-2, WU 13-15 or X-l 1 (30 jg/ml). Complement-activated serum (1:400 final concentration) was incubated separately with 1 jg GaC5, GaC6, GaC7, GaC8, GaC9, RaS, GIgG, RIgG, PBS or the coating mAb. All reaction mixtures were added to the microplate and incubated with biotinylated GaC6 (GaC6 biot) or GaC7 biot, after which the ELISA was processed as described for the Screening-ELISA. The data obtained with the coating mAb used for pre-absorption were defined as indicating 100% inhibition, the control with PBS replacing the antibodies as 0% inhibition. As a modification, NHS (1:10 dilution), C9-depleted serum or C9-depleted complement-activated serum (both 1:2 dilution) were used instead of the reaction mixtures listed above. ELISA for testing the ability of mAb to bind to coated terminal complement proteins or soluble poly-C9 Microplate wells were coated with C6, C7, C9 (all 1 jg/ml; Cytotech) or with WU 7-2, WU 13-15, X-l 1 or GaC9 (30 jig/ well). Various concentrations of poly-C9 which had been generated in the fluid phase using purified C92' were introduced only to those wells which had been coated with antibodies. Biotinylated mAb or pAb (30 jg/ml) were added after which the ELISA was processed as described for the Screening-ELISA.
SDS-PAGE and immunoblotting 5 0 jg C6, C7 or C9 (all Cytotech), 2 jl NHS or complementactivated serum or 15 jl human complement lysed rabbit erythrocyte ghosts prepared according to Biesecker et al.22 were analysed by a linear gradient SDS-PAGE followed by electrophoretic transfer to nitrocellulose membranes and immunological detection, as described elsewhere.'4 FACS analyses Enriched human BDC were suspended in PBS supplemented with 0 1% (v/v) azide and 0-5% (v/v) bovine serum albumin at a concentration of 4 x I05 cells/well in U-bottomed microtitre plates (Greiner, Nirtingen, Germany). All incubations were performed in this buffer on ice. Rabbit IgG (2 mg/ml) was introduced into each well for 30 min to block non-specific binding, after which the biotinylated mAb WU 7-2, WU 13-15 or X- 11 were added for 1 hr. The microplates were centrifuged at
200 g for 1 min followed by the removal of the supernatants. After washing the cells, biotinylated sheep anti-mouse IgG (Dakopatts) pre-absorbed with rabbit IgG was added at a 1:200 dilution for 30 min followed by streptavidin coupled to fluorescein-isothiocyanate (FITC; Amersham-Buchler, Braunschweig, Germany). A panel of mAb, anti-DQ, anti-DR (both Becton-Dickinson, Heidelberg, Germany), anti-CD20 (B 1; Coulter, Krefeld, Germany), anti-CD3 (OKT3), anti-CD14 (63D3) and TIB 8 from an IgGl secreting mouse myeloma [all American Type Culture Collection (ATCC), Rockville, MD], has been used to identify BDC. The samples were analysed with a FACStar flow cytometer with CONSORT 30 software (Becton-Dickinson). Dead cells were identified by staining with propidium iodide (Calbiochem, Frankfurt/M, Germany) at a concentration of 10 jig/ml for 1 min.
RESULTS Selection of mAb with TCC neoepitope-specificity Hybridoma culture supernatants of two clones (WU 7-2, WU 13-15) were found to react with complement-activated serum but not with EDTA plasma in the Screening-ELISA. These data were confirmed when the Screening-ELISA was repeated with the purified mAb (30 jig/ml). In addition, the same reaction pattern was observed using the mAb X-l 1 (30 jg/ml). All three mAb were typed as IgGlK.
Epitope analyses of WU 7-2, WU 13-15 and X-11 and cross-
reactivites When X- 11 was used as coating antibody a high increase in optical density was obtained when biotinylated pAb directed to the components of SC5b-9 were used as detection antibodies, whereas a small increase was detected when mouse IgG, goat IgG, rabbit IgG, the mAb WU 6-4 or the mAb WU 7-2, WU 1315 or X-l 1 were used as detection antibodies (Fig. 1). Comparable results were obtained using WU 7-2 or WU 13-15 as coating antibody (not shown). When complement-activated serum from different animal species was introduced to the ELISA, the increase in optical density was less than 1-2% compared to human complementactivated serum for all three mAb and all animals species investigated, except for the orangutan where 5-3% or 4-2% increases in optical density compared to human complementactivated serum were obtained for WU 7-2 and WU 13-15 or X1 1, respectively.
Determination of the localization of the mAb-specific epitope(s) on the TCC When the same antibody was used for pre-absorption as for detection (GaC6,GaC7) or coating (WU 7-2, WU 13-15, X- 1) the development of the ELISA was completely inhibited. The results with GaC6 biot as detection pAb are shown in Fig. 2. Similar results were obtained with GaC7 biot (not shown). When complement-activated serum was preincubated with GaC9 the increase in optical density was low with all three mAb and the inhibition of SC5b-9 binding to the solid-phase mAb was calculated to be close to 100% (Fig. 2). All other pAb used for pre-absorption were only able to
TCC on blood dendritic cells 60-
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Figure 1. The mAb X- was coated onto microplate wells, complementactivated serum was added and bound antigen was detected by different biotinylated antibodies as described under epitope analyses. The increase in optical density (OD 630 nm) per minute was recorded. X- 1, WU 7-2 and WU 13-15 or WU 6-4, monoclonal antibodies directed against TCC or native C6, respectively; GaC5, GaC6, GaC7, GaC8 or GaC9, goat IgG directed against complement component C5, C6, C7, C8 or C9, respectively; RaS, rabbit IgG directed against S-protein; MIgG, GIgG or RIgG, normal mouse, goat or rabbit IgG preparation, respectively.
120-
Coating antibody WU13-15
WU7-2
GaC8 WU GaC7 WU GskC9 7-2' 13-15
Figure 3. Human complement-lysed rabbit erythrocytes (15 P1/lane) were subjected to SDS-PAGE and transferred to nitrocellulose membranes. Lanes (b) and (d) were incubated with biotinylated WU 7-2 or WU 13-15, lanes (a), (c) and (e) with biotinylated GaC8, GaC7 or GaC9, respectively. Transferred proteins were detected by biotinylated pAb (10 pg/mi in PBS supplemented with 1% gelatin for 2 hr) in combination with streptavidin-horseradish peroxidase at a 1: 500 dilution. The blots were developed using the peroxidase substrate 4-chloro- 1 -naphthol. For abbreviations see Fig. 1.
inhibit SC5b-9 binding to the solid phase to less than 30% with the exception of pAb anti-S which caused an inhibition of up to 57% (Fig. 2). When C9-depleted serum served as antigen the increase in optical density was less than 5% for all three mAb compared to NHS, regardless of the polyclonal antibody used for detection. Complement-activated C9-depleted serum yielded an increase in optical density of less than 10% compared to non-depleted complement activated serum.
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Figure 2. pAb and the mAb anti-TCC were preincubated with complement-activated serum. The amount of TCC which was still able to bind to the coated mAb, [TCC]X, was determined as described under ELISA for the localization of the mAb-specific epitopes on the TCC. GaC7 biot (not shown) or GaC6 biot served as detecting reagents. The high amounts of bound TCC in reaction mixtures which originated from preincubation with PBS, [TCC]PBS, were set to 0% inhibition, whereas the low amounts of bound TCC in reaction mixtures which originated from preincubation with the same mAb which was used for coating, [TCC]mAb, were set to 100% inhibition. The percentage of inhibition was calculated as follows:
[TCC]PBS- [TCC] x l00 [TCCIPBS [TCC]mAb GIgG and RIgG inhibited to less than 2% with all three mAb. For inhibition (%)
abbreviations
see
Fig.
1.
=
Binding patterns of mAb to terminal complement proteins and to poly-C9 The reaction pattern of WU 13-15 with membrane-bound TCC present on erythrocyte ghosts was similar to that of WU 7-2 and comparable to that of GaC9 (Fig. 3). The mAb X-1 1 was not tested. When purified terminal complement proteins were used in SDS-PAGE, all three mAb detected purified C9 but not C6 or C7 after immunoblotting. This was deduced from a single stained band at the position of C9 (68,000 MW) and a complete absence of bands when purified C6 or C7 had been applied (not shown). Using NHS or complement-activated serum only very weakly stained bands at positions of 68,000 or 155,000 MW, representing monomeric or dimeric C9, respectively, were observed with all three mAb (not shown). In ELISA procedures, WU 7-2 and WU 13-15 reacted with less than 5% of adsorbed C7 and all three mAb with 15-20% of coated C9 but not with C6 when the increases in optical density were compared to those obtained with GaC7, GaC9 or GaC6,
respectively. When a poly-C9 preparation was introduced in various concentrations to mAb-coated wells no increase in optical density was observed with WU 13-15 or WU 7-2; X-1 1, however, was able to bind poly-C9 in a dose-dependent fashion
(Fig. 4).
R. Wurzner et al.
136 60-
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Figure 4. All three mAb anti-TCC as well as GaC9 were coated and different concentrations of poly-C9 were added, after which bound antigen was detected by GaC9 biot. The increase in optical density (OD 630 nm) per minute was recorded. For abbreviations see Fig. 1.
FACS analyses Enriched human BDC analysed by FACS consisted of two populations, as detected by dot-plots of side scatter (granulation) versus forward scatter (size). The low side scatter fraction showing non-granulated cells contained about 60-70% of the cells. They were CD3-positive and CD20-positive (not shown). The high side scatter fraction with moderate granulation contained about 30-40% of the cells. The mAb X-1 1, WU 7-2 and WU 13-15 reacted selectively with this high side scatter fraction as representatively shown for X-1 1 in Fig. 5. This fraction was also strongly MHC class II antigen positive, as shown for the mAb anti-DQ, but negative for anti-CD14, antiCD3 and anti-CD20 (Fig. 5).
DISCUSSION Two mAb derived from a mouse which had been immunized with SC5b-9 (WU 7-2, WU 13-15) and one mAb derived from a mouse immunized with enriched BDC (X-1 1) were found to be directed against an antigen which was present in complementactivated serum but not in normal human plasma at 1:50 dilutions. In addition, this antigen contains the complement proteins C5, C6, C7, C8, C9 and S-protein. All three mAb are, therefore, directed against neoepitopes on the TCC. Epitope analyses using all combinations of the three mAb revealed a pattern of reciprocal inhibition. It is, therefore, most probable that the epitopes are adjacent, overlapping or identical. Inhibiting studies with GaC9 and the comparison of staining patterns on complement-lysed erythrocytes revealed that the epitopes of all three mAb are located on the C9 moiety within the TCC, as has been shown for all neoepitope-specific anti-TCC mAb described so far.23 The non-reactivity of mAb with C9-depleted serum in native or complement-activated form not only confirmed the location on C9 but also excluded a recognition of C5b-8 in contrast to the neoepitope-specific mAb aElI which cross-reacts with activated C~a within this complex.24 Despite
Fluorescence intensity
Figure 5. Reaction of BDC with the mAb X-l 1. Human peripheral blood cells were depleted of erythrocytes, T cells and monocytes as described. Within the remaining population BDC were gated according to forward versus side scatter and were clearly distinguished from lymphocytes. BDC were stained with the mAb X-l 1 and plotted as overlay histograms with mAb anti- (a) HLA-DQ, (b) CD 14, (c) CD3 and (d) CD20. Note that the scale of the y-axis in frame (b) has been extended to include up to 200 events (cells) rather than only 100 as in frames (a), (c) and (d). It is shown that BDC reacted with X- 1 and antiHLA-DQ but were negative for monocyte/macrophage, T-and B-cell markers, as indicated by the negative control (C) with the irrelevant mAb TIB 8, IgG, K (ATCC) in frame (a).
this cross-reaction, the latter mAb, kindly provided by Dr Mollnes, Oslo, Norway also showed a reciprocal inhibition with all three mAb in epitope analyses (data not shown). The three mAb characterized here were able to bind to their epitopes when C9 alone was adsorbed to microplates or subjected to SDSPAGE followed by transfer to nitrocellulose membranes, although to a much lower extent then GaC9, excluding the possibility that other terminal proteins may contribute to the epitopes detected. Weak reactions of anti-TCC mAb with C9 adsorbed onto nitrocellulose membranes25 or with microplateadsorbed C9 in ELISA procedures26 have been described. We suggest that the change of the conformation of C9 during purification, adsorption to microplates, incubation in SDS/ urea, application to SDS-PAGE and/or electrotransfer to nitrocellulose membranes leads to an unfolding of C9 similar to that observed during MAC- or poly-C9-formation,27 creating neoepitopes. Bands at 68,000 MW and 155,000 MW were detected in human serum before and after complement activation by all three mAb and by GaC9. They most likely represent monomeric and dimeric C9 and demonstrate that the epitopes of the three mAb are not destroyed during SDS solubilization and/or immunoblotting, as has been shown for the epitope of the anti-TCC mAb bC5.25 In ELISA procedures a weak cross-reactivity to microplatecoated C7 was observed for WU 7-2 and WU 13-15 but not for X-l 1, which may be due to the high degree of homology in the amino acid sequences of C9 and C728 and conformational changes of the proteins during the coating step. In addition, X11 but not the other two mAb reacted with in vitro generated
137
TCC on blood dendritic cells Table 1. Reaction patterns of WU 7-2. WU 13-15 and X- I
Method
Antigen
WU 7-2
WU 13-15
X-1 1
ELISA
SC5b-9 Coated C9 Coated C7 Poly-C9 C9-depleted serum
+++*
+++
+++
+ (+) -
+ (+) -
+
Ery-ghostst C9 Activated NHSt
++ + + +
+++ + +
Western blot
++
ND + +
* The strengths of the reactions are summarized as follows: + ++, very strong reaction; + +, strong reaction; +, moderate reaction; (+), weak reaction; -, no reaction; ND, not determined. t Human complement-lysed rabbit erythrocytes. t Complement-activated human serum.
soluble polymerized C9. From these data we conclude that X-1 1 reacts with a different neoepitope on the C9 moiety from WU 72 and WU 13-15. All epitopes are present in the fluid phase TCC as well as on membranes and are, therefore, not dependent on the formation of high molecular weight C9 polymers since the latter are absent in SC5b-9.' The finding that an anti-TCC mAb reacts with fluid phase SC5b-9 but not with poly-C9 has not been described before. It appears possible that the epitope in question may be dependent on a conformational change induced in C9 by its integration into the SC5b-9 complex' and that this conformation differs from that present in poly-C9. The properties of our three mAb are compiled in Table 1. Sensitive ELISA, based on neoepitope-specific mAb which are able to detect TCC in EDTA plasma have been reviewed.23 The ELISA based on our two IgGI ,K anti-TCC mAb WU 13-15 and X-11 are of comparable sensitivity with the other three published assays which are based on IgG2 (a and b) mAb (data not shown), as calculated for the ELISA using WU 7-2.'4 The ELISA procedures established in our laboratory using different mAb anti-TCC can be used to quantify TCC in human specimens but are not able to measure TCC in complementactivated serum of other species, since even that of the orangutan only yielded an increase of 5% in optical density compared to the human one. Other anti-TCC mAb cross-react with guinea-pig and/or sheep and/or rabbit26 when applied to heterologous TCC consisting of human C5b-7 and animal C8 and C9. It is surprising that the anti-TCC mAb X- I1 was generated after immunization with BDC. However, the first mAb ever described reacting with a neoepitope on C9 was also not obtained after immunization with a purified form of TCC but with collagenase-digested normal human glomerular basement membrane in order to produce mAb directed towards the latter.29 Blood dendritic cells (BDC), which represent about 0-1% of peripheral blood mononuclear cells, have been shown to be potent antigen-presenting cells since they induce a much stronger lymphocyte proliferation in the one-way mixed lymphocyte reaction than freshly obtained monocytes or cultured macrophages.30 Using FACS analyses, well characterized mAb
directed against TCC-incorporated C9 were found to bind to viable BDC. The epitopes recognized by these antibodies have not been found on other proteins or cells of the human blood, as investigated by SDS-PAGE and immunoblot or FACS analyses, respectively. Cross-reactions of the mAb with cellular structures on blood dendritic cells other than TCC, albeit still possible, appear unlikely since at least two different epitopes are involved. Therefore, we conclude that BDC carry TCC on their surface. Complement activation probably occurs on the membrane itself, Although this cannot yet be confirmed since our three mAb cannot distinguish between fluid-phase generated and then membrane-trapped SC5b-9 and the membrane-generated SC5b-9(m) into which S-protein is integrated after complement activation.5 None of the mAb anti-TCC or anti-S described so far is able to distinguish between both forms.7 However, concomitant deposition of IgM, activated C3 products and TCC on FDC has been described which makes a complement activation on the membrane via the classical pathway more likely.8 BDC were found to be viable despite the presence ofTCC on their surface, even after several days of culture (not shown). It remains to be examined ifthis is due to an increased resistance or recovery from complement membrane attack'0 and if elimination or shedding of TCC complexes'0 occurs together with ongoing complement activation. Since membrane-bound but non-lytic TCC has been shown to exhibit manifold biological functions,'0 its presence on the surface may play a role in the differentiation or function of the carrying cells.
ACKNOWLEDGMENTS The authors are grateful to Dr T. E. Mollnes, Oslo, Norway for donating an anti-TCC monoclonal antibody and to Dr K. T. Preissner, Giessen, Germany for sending a polyclonal anti-S antibody. The authors wish to thank Ms B. A. Fernie for providing sera from different animal species and Drs P. J. Lachmann and M. J. Hobart for fruitful discussions and advice in preparing the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 236, projects B5 and B6; SFB 330, projects C6 and BlI; and fellowship WU 165/2-1).
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