Interactions of third component of human complement (C3) with tumour cells: Evidence for presence of C3b acceptor sites and direct activation of C3 by tumour cells

Ann. lmmunol. (Inst. Pasteur) 1981, 132 O, 339-350

INTERACTIONS O F T H E OF

.'7 UMAN -.

EVIDENCE

COMPLEMENT FOR

PRESENCE

AND OF

DIRECT

COMPONENT (C3) WITH TUMOUR CELLS: THIRD

OF

C3b

ACCEPTOR

SITES

ACTIVATION

C3 B Y T U M O U R

CELLS

by M. Daveau, M. FonLaine and D. Gilbert 1 N S E R M U-78, 543, chemin de la Brel~que, 76230 Bois-Guillamne ('France)

SUMMARY The interaction of C3 with different tumour cell lines was investigated. Our results indicated that nearly all the cell lines had C3b acceptors on their membranes. The binding of C3b was visualized by the detection of this protein on the cell surface with fluoresceinated anti-C3c serum. The involvement of proteases in the activation of C3 was demonstrated: complementmembrane fluorescence occurred in the presence of EDTA but was completely abolished with ¢-anfinocaproie acid. Experiments performed with purified C3 led to the conclusion that the protease activity might be the initial event for the activation of the alternatixe pathway by tumour cells. KEY-WORDS: Complement, Tumour; C3 acceptor sites.

INTRODUCTION The interactions between tumour cells and complement have been intensively investigated [1, 9, 14, 16] but the molecular events have not been completely elucidated. The relative resistance of tumour cells to lysis by antibody and complement was related to membrane repair [10, 11]. Lymphoblastoid (RAJI) cells could activate the alternative pathway of complement [1 1 ,,,h;~h resulted in cell lysis only when the cells were incubated for a IGng time Manuscrit re~u le 11 juillet 1981, accept6 le 23 octobre 1981.

340

M. DAVEAU, M. FONTAINE AND D. GILBERT

(24 h). Theophilopoulos and Perrin [16] a t t r i b u t e d the activation of the alternative p a t h w a y to the presence of C3b receptors on the m e m b r a n e of these cells and McConnel et al. [8] a t t r i b u t e d it to Epstein-Barr virus transiormatiou of the cd:s. Recently, Schreiber el al. [12] demonstrated t h a t R A J I ceils were weak activators of t h e alternative p a t h w a y and t h a t the initial m e m b r a n e lesion did not bring immediate cell death. In this paper, we investigated the interaction of t u m o u r cells with the third component of c o m p l e m e n t (C3) and showed t h a t t u m o u r cells could activate C3 directly and bind it through an acceptor site. This phenomenon might be the first e v ent of this activation of the alternative p a t h w a y by these cells. This cellular enzyme did not inactive cell bound C3b.

MATERIALS AND METHODS Cell lines.

Eight cell lines were used in this study. RAJI and Jijoye were derived from patients with Burkitt lymphoma; IgR:,, IPCso~. Daudel from malignant melanoma (one primarv an q two metastatic lymph nodes), HEP from human epidermoid carcinoma, HeLa'fror,3 human epithelial carcinoma and RD from a rhabdomyosarcoma. Suspension cultures of these ceil lines were grown in RPMI-1640 (Flow Laboratories) medium supplemented with glutamine, 10 % foetal calf serum, antibiotics and fungizone. Cell viability was determined by trypan blue exclusion. Proteins. Anlisera.

Anti-whole . . . . . . -r..1._:,._: anti-C5 (Meioy) were pur. . . . . . .~rllm, . . . . . . . . . ~nti_c'~a . .~uu ;~ ,.J,s~,,u,,-,~umaj, chased as indicated. Anti-C3c was prepared in our laboratory. 1. ~ P r e p a r a t i o n o[ F ( a b ' ) 2 [ragmenls. - - Rabbit IgG (anti-C3c) were prepared y ammonium sulfate precipitation (40 % saturation) and by ion exchange chromatography on DEAE-cellulose (Eastman) in 0.005 M phosphate buffer, pH 6.5. Purified IgG were hydrolysed by pepsin 2 % (w/w) (Worthington) in 0.1 M sodium acetate buffer pH 4.5, for 48 h at 37 ° C. The reaction was stopped by adding 10 N NaOH until pH equalled 8. The reaction mixture was fractionated by gel filtration on AcA-34 (I. B. F.) in phosphate-buffered saline (PBS) pit 8. b

2. ~ F l u o r e s c e i n i s o t h i o c g a n a t e - c o n / u g a l e d F ( a b ' ) 2 anli-C3e. ~ Anti-C3c F(ab')2 (20 mg/ml) was dialyzed against 0.5 M sodium bicarbonate, pH 9. Fluorescein isothiocyanate (FITC) (40 mg/ml) was added to F(ab')2 and adjusted to a final concentration of 2.5 % (w/w). The reaction mixture was shaken for 4 h at room temperature and filtered on Sephadex G-25 (Pharmacia). The FITC-F(ab')2 was dialyzed against PBS and concentrated to a 10 mg/ml final concentration. The

¢-AcA FHS FHS' FITC MW

----- ~ - a m i n o c a p r o i c a c i d . = fresh human serum. = heat inactivated serum. = fluorescein isothiocyanate. = molecular weight.

PBS R3 SDS-PAGE

= phosphate-buffered saline. = C3-depleted serum. ---- s o d i u m d o d e c y l s u l p h a t e - p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s .

DIRECT ACTIVATION OF C3 BY TUMOUR CELLS

341

number of dye molecules bound to each protein molecule (F/P ratio) wad deterF OD 495 mined spectrophotometrically, p OD 280 -- 1.37.

Preparation o/ highly purified C3. Highlv purified C3 was prepared from fresh normal human serum by (( Rivanol ,~ and euglobulin precipitation [15] followed by a reverse a~nity chromatography [3]. This preparation was free ,,, C5 as determined by immunodiffusion and gave a single precipitin line against an anti-whole human sermn immuneserum (fig. 1 b). The homogeneity of protein was demonstrated by acrylamide gel electrophoresis with sodium dodecyl sulfate (fig. 1 a). Under reducing conditions, the protein

1 2

1

2

I

i 185 K ~;>

:: -

.

-.; %

110 K

.

~

2

75 K i

m

a FIG. 1. - a) S D S - P A G E :

Imrnunochemical characleri:ation

b o[ p u r i f i e d C3.

1 = w i t h o u t mercaptoethanol; 2 ---- w i t h mercaptoethanol.

Moleculai weights are expressed in kilodaltons. b) I m m u n o - e l e c t r o p h o r e s i s w i t h an anti-whole human serum in the trough: w e l l 1 - - e u g l o b u l i n step purification; w e l l 2 = p u r i f i e d C3. Ann. Irnmunol. (Insto Past.), 132 C, n ° 3, 1981.

23

342

M. DAVEAU, M. FONTAINE AND D. GILBERT

was disrupted into two chains of 110,000 and 75,000 daltons. C3 was labelled with mI (Amersham) by the chloramine T method [5].

Polgpeptide chain analysis o/ cell bound C3. Cell bound usI-C3 was analvsed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). ~SDS-PAGE was performed according to Weber al~d Osborn's method [19] in 5 % acrylamide gel. Phosphorylase, albumin, ovalbumin, carbonic anhydrase, trypsin iiLhibitor and a-lactalbumin were used as internal markers (Pharmacia Fine Chemicals). After electrophoresis, tubes containing marker proteins were stained overnight with Coomassie blue brillant and destained. Gels containing l~sI-C3 samples were sliced into 2 mm sections and analysed for radioactivity in a gamma counter (Intertechnique).

Imm unofluorescence. In a typical experiment, 6 × 105 cells were incubated with 1 ml of fresh human serum (FHS) or 100 ~1 (1 mg/ml) of purified C3 at 370 C for 30 min. Cells were then washed three times with PBS pH 7.2, and 100 ~1 of FITC anti-C3c (diluted to 1/4) were added. The mixture was incubated for 30 min at 37 ° C. The cells were washed three times with PBS (pH 7.2) and centrifuged at 1,200 rpm, and the supernatant was discarded. Cell smears were air-dried and mounted under glycerol. Cells were observed by using a (c Zeiss )) microscope equipped with epifluorescenee and phase illumination. At least 200 cells were counted, and the percentage of positive cehs with membrane fluorescence was determined.

Immune adherence. C3b-coated tumour cells (107 cell/ml) were incubated (30 rain, 37 ° C) with an equal volume of human or sheep red blood cells (8 × 107 cell/ml). After incubation, rosetted cells were counted. RESULTS

Involvemenl of proleases in activation of C3 incubated with lumour cell lines. Incubation of cells with F H S (fresh h u m a n serum) and with F I T C anti-C3c induced a staining on t h e surface m e m b r a n e (fig. 2)-except for H E P and H e L a t u m o u r cells. N e a r l y 100 % of the cells were positive, b u t for I g R ~ and IPCso~ only 20 % of cells were stained (table I). No fluorescence was observed when t h e cells were incubated with F I T C anti-E3c only or with a C3-depleted serum [4]. An activation of C3 during the incubation could account for this positivity observed. In order to investigate this point, we tested three cell lines ( R A J I , Jijoye and Daudel) in other experiments. The results are summarized in table I I. When the

Fro. 2. - - Immunofluorescent staining patterns o[ C3-bearing tumour cells.

a) Incubation of R A J I cells with FHS and staining with anti-C3c FITC. b) Same experimenlal conditions with Iglq2~ cells. c) Same experimental conditions with IPCsol cells.

'rl

:; ~I

DIRECT ACTIVATION OF C3 BY TUMOUR CELLS TABLE I.

345

Demonstration of C3 deposition on different tumor cell lines. Cells

Cells a l o n e

Cells + R 3

Cells + F H S

Raji

_

_

.a_

Jijoye

-

-

+

HEP

-

-

-

Helm

-

-

-

RD

--

-

+

l)audel

-

-

+

IgR.~2

-

-

+

(*)

IPCso x

--

_

±

(*)

O n l y 2 0 % o f cells w e r e s t a i u e d . R3 = C3-depleted serum.

(*)

cells were incubated with FHS or C3, they presented a fine granular staining. They were all positive. Similar patterns were observed with RAJI cells incubated with inactivated (30 min, 56 ° C) serum (FHS'). In the same conditions, a staining decrease was noticed for Daudel cells. Furthermore, when E D T A (20 mM) and ,-aminocaproic acid (¢-AcA, 50 mM) were added to FHS, cr te C3 solutions, no fluorescence was observed. After incubation of the cells with FHS, the washing of cells with PBS conLaining 20 mM E D T A did not abolish the positivity preliminarily observed. Addition of E D T A tX\ ~i ~ f t ~,,~u) to inactivated serum did not change the results obtained with inactivated serum alone. In order to reveal a proteolytic activation of C3, three parameters were investigated: cell number, protein and prot2as~ inhibitor (¢-AcA) concentrations (table III). Only purified C3 was used in further experiTABLE II. - - Evidence for an activation of 03 by tumor cells.

Cells { : e l l s (1)

FHS'

FHS EDTA 20 mM

FHS EI)TA eAeA 20ram 50ram

C3

C3 1 mg/ml

EI)TA ~AeA

alone

FHS

Raji

_

+

+

+

_

+

+ (o)

Jijoye

-

+

NI)

ND

-

+

-

i)audel

-

+

±

•

-

+

+ + ---- i n t e n s i t y o f t h e f l u o r e s c e n c e (see t a b l e i ) ( a l l t h e cells s ~ e r e l a b e l l e d ) . (~) N u m b e r o f cells = 6 0 0 , 0 0 0 / m l . (2) O n l y 1 0 t o 1 5 % o f cells p r e s e n t e d a f a i n t s t a i n i h g . FHS' = inactivated fresh human serum.

2[)mM

50mM

346

M. DAVEAU, M. FONTAINE AND D. GILBERT TABLE III. - - I n v o l v e m e n t of proteases for the activation of C3 on R A J I cells. Intensity of the fluorescence

Nb of cells/ml

C3 1 mg/ml

Albumin

5 x 10 ~

1 ml

--

5 mM

4-4-

5 x 105

1 ml

--

20 mM

+

5 × 105

1 ml

50 m g / m l

5 mM

4-

1 x 106

1 ml

--

5 mM

4- 4-

1 ×

10 ~

1 ml

--

1 x

l0 s

1 mi

--

100 m M

1 x 10 s

1 ml

--

250 mM

cAcA

50

mM

+

-

(*) -

4- 4- 4- ± = i n t e n s i t y o f t h e f l u o r e s c e n c e (all t h e w e r e labelled.). (*) 5 % o f cells w e r e w e a k l y p o s i t i v e .

cells

ments. For a cell concentration of 1 x 10s cells/ml protein solutions were used with z-AcA concentrations varying from 5 to 250 mM. For weak concentrations (5 mM), no modificatiqn of staining was noticed, but fluorescence was strongly reduced by 50 mM ¢-AcA and completely abolished above 100 mM. If the cell concentration was decleased, the inhibition of staining was obtained for weaker concentrations of protease inhibitor. Furthermore, adding albumin (final concentration 50 mg/ml) to C3 solutions induced a decrease of fluorescence intensity when cell and C3 concehtrations were kept constant (table I I I).

Immunochemical characterizalion of lumour cell bound C3b. l~5I-labelled C3 (0.5 rag) was incubated in PBS for 30 rain at 37 ° C in a suspension of 4 x 106 R A J I and Daudel cells, giving a final volume of 0.5 ml. After centrifugation, the pellets were washed four times and then incubatcd in 1 ml of 8 M urea solution containing 1 % Triton-X100. The solution was concentrated to 0.2 ml and prepared for electrophoresis; 50 ~1 of radioactive samples were incubated with 50 [zl of a solution of 2 % SDS with or without 1 % mercaptoethanol. Figure 3 shows the distribution of radioactivity into the gels after electrophoresis. Arrows indicated the mobility of native C3 and of its polypeptide chains (~ = 110,000 and $ - 75,000 daltons). It clearly appeared t h a t $ chain was poorly labelled. In non-reducing conditions, no radioactive band corresponding to C3 mobility was found in solubilized membrane samples, but only high molecular weight components were detected, indicating complexation of r'~I C3 with membrane components. Reduced samples corroborated these results. No radioactivity was found at the position of ~ chain, but higher radioactive polypeptides were recovered, indicating a strong fixation of

DIRECT ACTIVATION OF C3 BY TUMOUR CELLS IC3

347

~C3

a

;t

b ==. o ~.,

-5

,5

(N

,_r 2

4

6

GEL LENGTH cm

GEL LENGTH cm

ap

aa

It

c

d

1! =E

:E

¢1. g

U

¢9 0

O3 0

,ira

-5

~-, a3 ¢N

5

n

_.J 2

4

;, 6

GEL LENGTH cm FIG.

3.

--

;

I 2

i 4

I6

GEl. LENGTH cm

Distribution o/ radioaclivily (1~5I, 10a cpm) in the gels a/ler eleclrophoresis.

Arrows indicated the m o b i l i t y of native C3 and of its polypeptide chains. Samples of 125I-C3 (a) and cell-bound 1=5I-C3b (b) w i t h o u t reducing agent. Samples of 1=5I-C3 (c) and solubilized m e m b r a n e (d) with 10 % ME. Gels were cut ill 2 m m slices and counted in a g a m m a counter.

chain with m e m b r a n e components. No radioactivity was present under the position of ~ chain, indicating whole integrity of cell-bound C3b.

Immune adherence. Purified C3 (0.5 mg) was allowed to interact with R A J I and Daudel cells, as described above. Fixation of C3 was monitored by immunofluorescence. One volume of 10 ~ C3b-coated t u m o u r cells was tested for i m m u n e adherence. In all cases, no immune adherence phenomenon was observed.

348

M. D~,VEAU, M. FONTAINE AND D. GILBERT

DISCUSSION We chose an experimental procedure which allowed to visualize the direct binding of C3 by cell membranes This method using fluorescent anti-C3 F(ab')~. permitted to compart the different ways of C3 binding by tumour cells. Nearly all the cell lines tested showed a deposit of C3 on their membrane when incubated with FHS. A peripheral staining was observed. Only H E P and HeLa were negative. These data showed t h a t probably most of the tumour cells could activate the complement system. In order to investigate this last point, three cell l i n e s - - R A J I , Jijoye and Daudel--were used in further experiments. EDTA (which inhibits the classical and alternative complement pathways) was added to the tumour cells mixture. The binding of C3b to the cell membrane was not inhibited by EDTA but only decreased. The cells were able to activate C3 directly. This phenomenon was confirmed by using purified C3. When the cells were incubated with purified C3, patterns of fluorescence similar to those obtained with FHS, EDTA were observed. The fixation of C3 was inhibited only when EDTA and ¢-AcA were added to the medium, indicating the presence of a protease activity. This activity was confirmed by varying the concentrations of the reagents. Activation of C3 was enhanced by increasing the cell concentration and decreasing the inhibitor concentration. Moreover, the addition of albumin to C3 solution inhibited the fixation of C3. ~5I-C3 was used to determine whether tumour cells bound C3 through an acceotor or a receptor _ . . . .sit~._ . . . . . . .If. C_~h, ~.~n~.~+~a . . . . . . . . . by e~,-~. ~. . . . activity, bound to its receptor site, it would be eluted from the membrane. On the contrary, if C3b bound to an acceptor site in the membrane through its labile binding site (l,. B. S.), it would not be eluted from the membrane since it would form a covalent bond with a component of the membrane [ 7]. Data reported in figure 3 clearly indicated t h a t C3 bound to tumour cell membrane through its labile binding site when it was directly activated by tumour cells. Furthermore, these experiments showed t h a t cell=bound C3b was no longer cleaved by tumour cell proteases since no light fragment was observed in the gels. We could conclude t h a t C3b receptors were not responsible for the activation of alternative pathway and that the weak activation of alternative pathway by R A J I cells (measured by Schreiber et al. [12]) could not be related to a degradation of C3b by the proteases liberated by the cells. This protease activity could be initial event of the alternative pathway activation by tumour cells. This hypothesis is in good agreement with the results of McConnel el al. [8] and Schulz el al. [13]. McConnel found t h a t the cell transformation with EBV enhanced the ability of the cells to activate the alternative complement pathway, and Schulz el al. reported an enhancement of protease activity in EBV + cells. We never found an immune adherence phenomenon when C3 was deposited directly onto tumour cells, though C3b was fixed in the membrane

DIRECT ACTIVATION OF C3 BY TUMOUR CELLS

349

through its labile binding site and therefore exhibited its receptor sites for CR1 and CR2 [2]. This lack of immune adherence phenomenon might be due to hidden immune adherence sites on C3b or to a weak deposition of C3b as mentioned by Kessler and Lobuglio [5]. Verbrugh el al. [18] found t h a t the encapsulation of bacteria interfered with the process of C3 fixation, and Wilkinson et al. [20] suggested t h a t opsonically active C3 molecules were not exposed at the true external surface of the bacterium. The tumour cells which are resistant to complement-dependent lysis and particularly R A J I cells which are weak activators of the alternative p a t h w a y [12] could be related to encapsulated bacteria.

RI~SUMI~ INTERACTIONS DU C3 HUMAIN ET DES CELLULES TUMORALES " PRI~SENCE DE SITES ACCEPTEURS ET ACTIVATION DIRECTE DE C 3 PAR LES CELLULES TUMORALES

L'interaetion du composant C3 avec diff6rentes lign6es de cellules tumorales a 6t6 6tudi6e. Nous pouvons ainsi visualiser sur presque toutes les lign6es test6es la pr6sence de sites accepteurs pour le C3b. La liais, :t du C3b est d~montr6e par la pr6sence de cette prot6ine sur la membrane eellulaire au moyen d ' r ~ immuns6rum marqu6 par la f l u o r e ~ i n e anti-C3c. L'activation du C3 se fair par d~s prot6ases lib6r6es par les cellules. En effet, la fluorescence de membrane persiste en pr6sence d'EDTA, mais est . . . . ~ v T~t~ll~ ,.,.,.~. . ~rf~_ aVt~C e ~ ; - ~ l t l l l l l•l O l S i : i ~ / l -"~" Olq"~uK;n. aJ,~ ~.llJ~a " ~ .,~La,.,,~ . . . . t o m pi~t enaent a. l j'l J.l-l l'd ""1 . -i i-U.:l t l ~"" r~o tu6es avee du C3 purifi6 nous am~nent ~ penser que l'aetivit6 prot~asique pourrait ~tre c( l'6v~nement initial )~ de raetivation de la vole alterne par les cellules tumorales. MOTS-CLI'S

"

Compl6ment, Tumeur ; Sites accepteurs de C3. ACKNOWLEDGMENTS

The authors wish to thank Doctor F. Picard for the generous gift of tumour cell lines and Mme M. M. Fontaine for reviewing the manuscript. The technical assistance of Mme L. Dumouchel (Centre R6gional de Transfusion Sanguine, 76230 Bois-Guillaume) is gratefully acknowledged.

REFERENCES D. B., LACHMANN, P. J. & ]t/[CCONNEL, I., Activation of the alternative complement pathway by lymphoblastoid cell lines derived from patients with Burkitrs lymphoma and infections mononucleosis. Cell. lmmunol., 1976, 28, 98-109. [2] DIERICH, M. P. • BOKISCH, V. A., Receptor-binding site on C3 and C3b. J. lmmunol., 1972, 118, 2145-2150.

[1] BUDZKO,

350

M. DAVEAU, M. FONTAINE AND D. GILBERT

[3] FONTAINE,M. & PtIVAT, C., A study of the breakdown of the third component of human complement (C3). Ann. lmmunol. (Insl. Pasteur), 1979, 130 C, 349-366. [4] FONTAINE, M., JOISEL, F. • DUMOUCHEL, L., Preparation of an R3 reagent (serum depleted of the third component of human complement (C3)) by immunoadsorption. Application to the hemolytic assay of human C3. J. immunol. Methods, 1980, 33, 145-158. [5] HUNTER, W. R., Radioimmunoassay, in ,~ Handbook of experimental immunology-immunochemistry ~, (D. M. Weir), 1, (p. 171), Blackwell Sci. Publ., Oxford, 1973. [6] KESSLER, H B. & LOBUGLIO, A. F., Interaction of monocytes with tumor cells coated with complement with or without antibody. Cell. Immunol., 1980, 49, 352-359. [7] LAW, S. K., LICHTENBERG, N. M. & LEVlNE, R. P., Evidence for an ester linkage between the labile binding site of C3b and receptive surfaces. 3. Immunol., 1979, 123, 1388-1394. [8] McCONNEL, I., KLEIN, G., LINT, T. F. & LACHMANN,P. J., Activation of the alternative complement pathway by human T B cell lymphome lines is associated with Epstein-Barr virus transformation of the cells. Europ. 3. lmmunol., 1978, 8, 453-458. [9] OKADA,N. & OKADA, H., Activation of complement by spontaneous leukemic cells of A K R mice. Int. J. Cancer, 1978, 22, 282-287. [10] SCHLAGEa, S. I., OHANXAN, S. H. & BoHsos, T., Metabolic requirement for hormone-induced resistance to antibody-complement mediated killing of tumor cells. J. In:munol., 1977, 119, 789-794. [lll SCHLA6En, S. I., OHANIAN, S. H. ~[; BORSOS, T., Identification of lipids associated with the ability of tumor cells to resist humoral immune attack. d. Immunol., 1978, 120, 472-480. [12] SCHREIBER, R. D., PANGBURN, M. K., MEDICUS, l:{. G. & MULLEREBERHARD, ~-I. J., RAJI cell injury and subsequent lysis by the purified cytolytic alternative pathway of human complement. Clin. lmmunol. lmmunopali~., 1980, 15, 384-396. [13] SCHULZ,T., DIERICH, M. P., YEFENOF, E. t~ KLEIN, G., C3 activated prote~ases on human iymphobiastoid ceils super infected with EBV. Cell. Immunol., 1980, 51, 168-172. [14] SHIMBO, T., YATA, J. & OKADA, H.. Non specific activation o[ complement by leukemic cells. Int. 3. Cancer, 1978, 22, 422-425. [15] STEINBUCK,M., Isoiement de la ~lC-globuline et ~tude de sa transformation en ~lA-globuline. Vox Sang. (Basel), 1964, 9, 96-98. [16] THEOPHILOPOULOS,A. N. & PERRIN, L. H., Binding of components o[ the properdin system to cultured human lymphoblastoid ceils and ~ lymphocytes. 3. exp. Med., 1976, 143, 271-289. [17] ThEOPHILOPOULOS,A. N. & PERRIN, L. H., Lysis of human cultured lymphoblastoid cells by cell-induced activation of the properdin pathway. Science, 1977, 195, 878. [18] VERBRUGH,H. A., VAN DIJK, W. C., VAN ERNE, M. E., PETERS, 1={.,PETERSOl~, P. K. & VERHOEF, J., Quantitation of the third component Gf human cemplement attached to the surface of opsonized bacteria: opsonindeficient sera and phagocytosis-resistant strains, bt[ect. Immun., 1979, 26, 808-812. [19] WEBEH, K. & OSBORN, M., The reliability of molecular weight determination by dodecyl st'.lfate polyacrylamide gel electrophoresis. 3. biol. Chem., 1969, 244, 4406-4412. [20] WILKINSON, B. J., SISSON, S. P., KIM, Y. 8¢ PETEBSON, P. K., Localization of the third component of complement on the cell wall of encapsulated Staphylococcus aureus M. Implications for the mechanism of resistance to phagocytosis. In/ect. Immun., 1979, 26, 1159-1163.