446
Eur. J . Immunol. 1978.8: 446-451
S. Aguilera and J. Ivanyi
S. Aguilera* and J. Ivanyi Department of Experimental Immunobiology, Wellcome Research Laboratories, Beckenham
Acquired heterophile antigens on the surface of human cell lines Chicken IgM antibodies raised against sheep erythrocytes (SE) reacted with a heterophile carbohydrate antigen designated as "HC". This antigen was visualized by SE rosette formation with antibody-sensitized and formaldehydefixed target cells. Cells from eight lymphoblastoid lines and ten lines of diverse histogenetic origin all expressed HC on their surface to a greater or lesser degree when grown in fetal calf serum (FCS). Lymphoblastoid cell lines, HSB2 and Namalva, lost most of their HC antigen after three cell divisions when grown in medium which contained normal human serum (NHS) instead of FCS. The acquired origin of HC was demonstrated by the conversion of HSB2-HC- into HSB2-HC' cells after incubation in the presence of FCS o r fetuin for 18 h at 37 OC. The acquisition of HC antigen depended on the concentration of fetuin as well as the time and temperature of incubation. The presence of NHS in the culture medium reduced the degree of HC incorporation. The level of HC expressed o n the cell surface was reduced by treating HSB2 cells with sodium periodate but not by proteolytic enzymes. Competitive inhibition with hog blood group substance and sheep IgG suggested similar specificity of HC on target cells which had been sensitized with either fetuin o r sheep IgM (Eur. J. Immunol. 1977.7:204). The avidity of chicken antibodies t o HC h either of these systems was several orders of magnitude lower than the binding to SE.
1 Introduction Cell surface carbohydrates are known t o play a fundamental role in various biological processes, and their chemical and immunological characterization has been t h e subject of extensive studies. It is widely assumed that these structures are products of cellular biosynthesis. However, it has also been demonstrated that in some instances membrane molecules are acquired from the surrounding environment. The acquisition of surface molecules from serum by red cells has been described for certain blood groups in various mammalian species [ 1-41, Although it has been demonstrated in man that t h e Lewis glycosphingolipid is transported in serum by lipoproteins, the precise mechanism by which it is taken u p from serum is not understood [ 51. Acquired cell surface molecules may also perform specialized functions: e.g. t h e monosialoganglioside GM1 is incorporated spontaneously into the cell surface membrane where it serves as a functional receptor for cholera toxin [6-81. It has been suggested that the sphingosine portion of t h e gangliosides partitions into t h e membrane bilayer, and that t h e oligosaccharide moiety on the cell surface accounts for the binding with the choleragen [9]. Restoration of responsiveness t o serotonin has been demonstrated after incubation of sialidase-treated stomach strips with gangliosides [ 101, and a fibroblast line which is resistant t o cytotoxicity of the plant lectin ricin exhibited increased binding and sensitivity t o ricin following incubation with the glycolipid prepared from human erythrocytes [ l l]. A glycoprotein from hog gastric mucine has been incorporated into t h e mem[I 20011
brane of guinea pig macrophages and potentiated the activity of a fetal calf serum (FCS)-derived migration inhibition factor [ 121, and uricase has been shown t o be endocytozed and built into the membrane of alveolar macrophages while preserving its enzymatic activity [ 131. It is also known that the parasite Schistosoma mansoni acquires host-specific blood group substances during its growth in animal hosts or during in vitro culture in the presence of serum and red cells. However, the exact molecular nature of the incorporated antigens has yet to be determined [ 141. Heterophile antigens with carbohydrate specificity were demonstrated recently on the surface of a Hela-derived cell line by cytotoxic antibodies from normal rabbit serum [ 151. Antibodies against heterophile carbohydrates were isolated from normal mammalian or avian sera by affinity chromatography using columns of insolubilized fetuin or mouse lymphoma cells lines [ 16, 171. These antibodies agglutinated erythrocytes from sheep and several other species but reacted also with murine spleen cells while antibodies eluted from two different lymphoma lines showed specificity probably against saccharide moieties in terminal position of the cell surface. We have reported recently that chicken IgM antibodies raised against sheep erythrocytes (SE) reacted with a heterophile antigen which is shared by the saccharide moiety of mammalian Ig [ 18, 193. The present experiments have shown that this heterophile carbohydrate designated as HC is expressed also on the cell surface of human cell lines when grown in FCS. Furthermore, the results suggested that HC is derived from fetuin in the medium and spontaneously incorporated into the cell surface membrane.
* Present address: Hospital J.J. Aguirre, Santiago, Chile. Correspondence: J. Ivanyi, Experimental Immunobiology Department, Wellcome Research Laboratories, Beckenham, Kent BR 3 3BS, GB Abbreviations: SE: Sheep erythrocytes CaSE: Chicken anti-SE serum FCS: Fetal calf serum NHS: Normal human serum HC: Heterophile carbohydrate reacting with CaSE HBGS: Hog blood group substance PBS: Phosphate-buffered saline HCR:HC rosette
2 Materials and methods 2.1 Antiserum (CaSE)
Inbred B14 chickens were injected twice a t a 14-day interval with 5 x l o 8 SE per kg body weight and bled after 7 days.
Eur. J. Inimunol. 1978.8: 446-451
Sera from several birds were pooled and absorbed with human AB erythrocytes. Two pools with similar antibody titers were used throughout thjs study. When fractions separated on Sephadex G-200 (Pharmacia, Uppsala) were tested, t h e results confirmed our previous finding [ 181 that antibodies which reacted with the heterophile antigen in the rosette assay were localized in the 1 9 S but not in the 7 S fraction. 2.2 H C rosette assay (HCR) Target cells (1 x 10 6 ) in 8 x 50 mm glass test tubes were incubated with 0.2 ml of serially diluted CaSE for 20 min at 0 OC, washed 3 times with ice-cold Hanks' solution, incubated with 0.2 ml of 4 % formaldehyde for 20 min at 0 "C and rewashed 3 times. The pelleted cells were resuspended in 0.2 ml of a suspension of washed SE ( 1 x 108/ml), centrifuged a t 200 x g for 10 min and kept at 0 OC u p t o 4 h prior t o counting rosetted and free lymphocytes. As in previous studies [ 18, 191 t h e fixation step with formaldehyde was essential since it amplified t h e reaction approximately 10-fold. Furthermore, the fixed cells could be stored at 4 OC for at least 5 days without a significant change in t h e HCR count. No HCF, were detected when CaSE was substituted with normal chicken serum. 2.3 Cell lines These were kindly provided by Drs. H. Zola and G. Christofinis (Wellcome Research Laboratories) o r purchased from Flow Laboratories (Glasgow, Scotland). All cell lines were grown in flat-bottom flasks in RMPI-1640 medium (Wellcome Reagents) supplemented with 1 0 % FCS (Wellcome Reagents) or normal human serum (NHS). The latter was separated from freshly drawn blood of healthy donors. Cell monolayers were harvested by incubation with 0.1 % trypsin in 0.05 % Versene for 10 r . i n a t 37 "C. Cells were washed 4 times in excess Hanks' solution prior t o assay. 2.4 Antigens Fetuin prepared by ammonium sulfate fractionation of FCS was purchased from Sigma Chemical Co., St. Louis, MO. Hog blood group substance (HBGS) was purified from hog gastric mucin [ 201 and human A substance (MSM) was prepared from cystic mucin [21]; both preparations were kindly provided b y Dr. C. Moreno. Sheep and human IgG were prepared by ion exchange chromatography o n DEAE-cellulose using 0.01 M potassium phosphate buffer, pH 8.0, for elution, and their purity was tested by immunoelectrophoretic analysis. Bovine IgG was obtained from Miles Laboratories (Kankakee, IL).
3 Results 3.1 Demonstration by the rosette assay of a heterophile antigen (HC)o n the surface of human cell lines The HC rosette (HCR) count afte; treating washed cells with various concentrations of CaSE was taken as an arbitrary measure of t h e surface density of HC antigen. Cells from 8 lymphoblastoid lines and 10 lines of diverse histogenetic origin were examined with serial log3 dilutions of CaSE. All cell lines showed 100 % HCR using low dilutions of CaSE. Suspensions with > 50 % frequency of HCR tended t o ag-
Heterophile antigens on human cell lines
447
glutinate, and accurate counts could therefore be made only in the range of 0.5 to 50 % HCR. The differences in HC density between the various cell lines were expressed by the dilution of CaSE which gave 1 0 % HCR. This is taken as an arbitrary value which is directly related to the density of HC o n the cell surface. The cell lines varied approximately 5-fold in CaSE dilutions giving 10 % IICR (Table 1). Two T cell lines which were examined (HSB2 and MOLT-4) showed relatively higher values tha n four B cell lines. Nonlymphoid lines also varied coxsiderably without a ny regular pattern or relationship t o the cells' histogenetic origin or source. As a rule, the cells were tested when they reached the stationary phase o r confluency. However, a comparison of HSB2 cells a t the logarithmic stage ( 5 x 1 05/ml) and stationary phase ( 2 x 106/ml), respectively, showed little difference in HC density. There was no distinct specificity in the reaction with individual cell lines since absorption of CaSE with HSB2 cells ( 5 x 1 O7 cells/ml 1 :30 diluted antiserum) abolished its reactivity with several other cell lines. It was possible t o elute CaSE antibodies by incubation of CaSE-sensitized HSB2 cells a t 56 "C for 30 min or with 0.2 M glycine-HC1 buffer, pH 2.5 for 2 min. Eluted and concentrated antibodies produced an HCR titer similar t o that of the starting antiserum. The presence of HC antigen on the surface of HSB2 cells was also confirmed by immunofluorescent staining. CaSE-sensitized and formaldehyde-fixed cells were specifically stained with FITC-labeled sheep anti-chicken Ig conjugate. However, the staining was a t least 10 times less sensitive and the end-point titration less accurate when compared with the HCR assay. Further analysis of the nature and origin of HC was performed with HSB2 cells. Table 1. HC density on various human cell lines Line
Nature and origin")
HSB2 (CCRF) MOLT-4 (RPMI) 4098 (RPMI) DG75
T cell; ALL T cell; ALL B cell; healthy donor B cell; Burkitt lymphoma B cell; healthy donor B cell ; ALL B cell; healthy donor B cell; Burkitt lymphoma
Namalva 8392 (RPMI) 1778 (RPMI)
Daudi NALM/1 mou 1000
HEP-2 IleLa Floh 407 MRC-5 204 FL Amnion Flow 4000 Chang
Myeloid cell Embryonal skin and muscle Epithelial cell Cervical carcinoma Embryonal intestine Fibroblast Muscle Amnion Embryonal kidney Liver
Dilution of CaSE giving 1 0 % HCR 145
115 76 63 38 37 23 21 134
110 103 96 46 38 37 31 34 27
a) ALL = acute lymphoblastic leukemia. Cells were incubated with log3 dilutions of CaSE. 100 % HCR were found using higher concentrations of CaSE in all examined cell lines. All cell lines were cultured in 10 % FCS.
3.2 Carbohydrate nature of H C antigen Previous experiments suggested that CaSE reacted with a carbohydrate moiety of IgM from various mammalian species
Eur. J. Immunol. 1978.8: 446-451
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448
[ 181. Since the treatment of cells with sodium periodate at pH 4.5 drastically reduced the HCR reaction (Fig. l ) , we conclude that CaSE-binding molecules o n the surface of t h e HSB2 cell line were also of carbohydrate nature. Incubation of cells with the acetate buffer, pH 4.5, did not affect the reaction when compared with cells which had been incubated in phosphate-buffered saline (PBS), pH 7.4. However, treatment of cells with either neuraminidase (Sigma, 5 IU/ml) or with proteolytic enzymes, trypsin (Sigma, 5 mglml), papain (Worthington, 1.6 mg/ml) o r pronase (Koch-Light, 2 mg/ml) a t 3 7 "C for 60 min (i.e. under conditions which are known to remove other surface receptors) did not significantly diminish the extent of HCR formation (Table 2). Thus, proteins apparently do not contribute towards the structure of the CaSE-binding moiety of HC.
Table 2. Failure to remove HC from the cell surface by enzyme treatmenta) Enzyme treatment
Concentration per ml
Pronase Trypsin, Papain m 10 mM cysteine Neuraminidase None
HCR
(%I 11.18 10.04 12.28 12.26 10.68
2.0 mg 5.0 mg 1.6 mg 5 IU
a) HSBZ cells (1 x 107/ml) were incubated with various enzymes at 37 "C for 60 min. Subsequently, the cells were washed and tested by the HCR assay using 1:90 diluted CaSE. respectively, at day 3 did not show any significant difference of HC density, cells grown in NHS and harvested after longer time intervals showed a gradual loss of HC antigen. HSB2 cells from the second NHS subculture almost lost the HC, and retained only 1 % HCR (detected by high concentrations of CaSE) after further passage in NHS-containing media; (this variant has been designated as HSB2- while the FCS-cultured cells are HSB2').
100.0
10 .0
a
u
r L
u il
F
1 .(I
(1.
I I
j12001.11
2
3
4
5
LOG3 DILUTION 01. C a s t
Figure 1. Carbohydrate nature of the surface heterophile antigen. HSB2 cells were fixed with 4 % formaldehyde at 0 "C for 30 min
and washed 4 times with PBS. Then the cells were incubated either with 0.005 M sodium periodate in 0.1 M sodium acetate buffer, pH 4.5 (B), or with the buffer pH 4.5 alone (0) or in PBS, pH 7.4 (a)at 4 "c for 20 h in the dark. Following incubation, the cells were washed 3 times and tested with log3 dilutions of CaSE for HCR. 3.3 Loss of HC from HSBZ cells when grown in NHS
Preliminary experiments showed that FCS, but not NHS, blocked HCR formation when added t o CaSE prior to incubation with cell suspensions. As all cell lines listed in Table 1 were grown in the presence of 10 % FCS, we considered the possibility that HC had been acquired by the cells from FCS in the culture medium. Initially, we attempted t o remove any adsorbed surface material from HSBZ cells by (a) repeated washing (6 times), (b) incubation in medium at pH 6.5 for 30 min, and (c) overnight incubation in the absence of FCS. However, none of these procedures resulted in any change of HC density. Subsequently, washed HSB2 cells ( 2 x 105/ml) were subcultured in media containing 1 0 %of either FCS o r NHS. Cells were passaged again at a 2 x 105/ml concentration from day 4. Cells were harvested o n days 3, 4 and 5 of the first and o n day 4 of the second subculture, and examined by t h e HCR assay using six logs CaSE dilutions (Fig. 2). Although cells from the subcultures in FCS and NHS,
1 2 3 LOG3 DILUTION OF CaSE
~
5
6
Figure 2. Loss of heterophile antigen from HSB2 cells grown in N H S containing medium. HSBZ cells at a density of 0.3 x 106/ml were seeded in RPMI-1640 medium containing 10 % NHS (0) or 10 %
FCS ( 0 ) . At different times of incubation cell densities and HCR with log3 dilutions of CaSE were determined. Numbers in parentheses represent the cell counts x lo6 in NHS/FCS-containing cultures: A: day 3 (1.6/2.0); B: day 4 (2.7/2.7); C: day 5 (3.1/3.5); D: day 8, subcultured after 4 days (2.1/2.0). These results were confirmed in a similar experiment with the Namalva B lymphoblastoid cell line. The generation times of the HSB2 and Namalva lines were 2.7 and 2.2 days, respectively, and these were not significantly altered by the change of FCS for NHS in the culture medium. When the rate of HC antigen loss was plotted over t h e number of cell divisions in NHS cultures, t h e outcome for HSB2 and Namalva cells was very similar, i e . the rapid loss of HC density coincided with
Eur. J. Immunol. 1978.8: 446-451
Heterophile antigens on human cell lines
the time when the number of cells seeded into the culture had increased threefold (Fig. 3). We also attempted t o influence the growth rate by culturing HSB2' cells in t h e presence of either 1 'To CaSE o r normal chicken serum and 10 % NHS. However, no difference was observed, probably due t o the low avidity of the IgM CaSE antibodies.
449
100.0
10.0 270.(
a: 11 U
c 11 L U
w Yll.1
01
2
1 .0
0
7 311.1
4
.. 3
0
0.1 2
rn
'J 10.1
3 5 LOG3 D I L U T I O N O F C a s t
6
Figure 4. Uptake of HC antigen onto HSB2- cells by incubation in 3.. J
~~~~
I
2
3
4
N U M H t R O F C t L L D I V I S I O N S IN 10%N H S C O N T A I N I N G M L D l U M
Figure 3. The rate of loss of HC antigen from HSB2 and Namalva cell lines. HSI12 ( 0 ) (see also Fig. 2) and Namalva (0)cell lines seeded at 0.3 x lo6 cells/ml were grown in 10 % NHC-containing RPMI-1640
medium. Total cell and HCR counts were determined at subsequent days of culture.
3.4 Acquisition o f HC from FCS or fetuin by HSB2- cells Since thz results described above indicated that HSB2' cells lost their HC when grown in NHS (i.e. in the absence of FCS), we attempted t o achieve t he converse effect by incubating HSB2- cells in the presence of FCS or fetuin. A pronounced increase in HC density was obtained by incubation of HSB2' FCS for 16 h a t 3 7 "C while incubation at 4 "C cells with 10 % failed to significantly increase the HCR count (Fig. 4). In another experiment, HC density o n HSB2- cells was increased by 24 h. and even further by 48 h incubation in the presence of fetuin at a 5 mg/ml concentration (Table 3). The effect was more pronounced if fetuin was added t o the culture medium in the absence of NHS, and 1 % FCS increased t h e HCR count significantly only when added t o medium which did not contain NHS.
3.5 Analysis of the antigenic properties of HC by rosette inhibition FCS and its main constituent fetuin were used for t h e competitive inhibition of HCR formation. By adding either of these two inhibitors a t various stages of t h e HCR test, dramatically different degrees of HCR suppression were observed (Table 4). Mixing the inhibitors with CaSE prior t o incubation with HSB2 cells resulted in 50 %inhibition of HCR at a 1 :27000 dilution of FCS o r at a 1 pg/ml concentration of fetuin (protocol A). These results are comparable when assuming an approximately 2 0 mg/ml concentration of fetuin in FCS [22]. Both inhibitors had t o be used at a 100 times higher concentration when added t o HSB2 cells which had
FCS-containing medium. HSB2- cells grown in 10 % NHS-containing medium were washed and incubated for 16 h in the presence of 10 % FCS at 37 "C (m) or 4 "C (A). Control HSBZ- cells in 10 % NHS ( O ) , and HSB2+ cells cultured in 10 % FCS (0). Table 3. Uptake of HC by incubation of HSB2- cells with fetuin or FCS Incubation of HSB2- cells at 37 "C NHS Fetuin FCS ( I ) (mg/ml) (%) 10 10 5 5 10 1 1 10
Dilution of CaSE giving 10 % HCR 24 h 48 h 4
25 45 6 19 52
6 105 360 8 83 540
Relative yield of cells (%) 24 h 48h 100 110 58 100 60
120
100
44 17
86 54 81
previously been 'sensitized' with CaSE (protocol B). Further, a n almost 10-fold increase in FCS concentration was needed for t h e inhibition of previously CaSE-sensitized and fixed cells (protocol C), and another 6-fold increase was necessary when adding FCS at the last stage of the assay together with SE (protocol D). Fetuin at a 10 mg/ml concentration was ineffective in a ny of the latter two ( C and D) protocols. These results suggest that CaSE has a considerably weaker binding to HSB2 cells than to SE. Furthermore, competitive inhibition of CaSE-binding to HSB2 cells was 100 times more effective than t h e dissociation of antibodies which were already bound. Further experiments using protocol A and various inhibitors were designed t o compare the antigenic specificity of the fetuin-derived HC on the surface of HSB2 cells with the previously described HC moiety of sheep IgM. The latter type of target cell was provided by normal sheep serum-treated chicken peripheral blood lymphocytes [ 181. The concentration of the inhibitor needed for the competitive inhibition of HCR was inversely related t o the degree of antigenic homology between the inhibitor and the surface-bound HC molecules
Eur. J. Immunol. 1978.8: 446-451
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Table 4. Competitive inhibition of HCR reaction with FCS and fetuina)
Protocol Sequence of treatment FCS dilution Fetuin concentration (mg/ml)giving HCR of HSBZ cellsb) giving HCR inhibition of: inhibition of: 1
2
3
4
9 0 %4 5 0 %
-
9000 27000
90%
50%
CaSE A
+
FA SE
0.001
0.004
1nh.d) B C
a) b)
c) d)
CaSE Inh. FA SE CaSE FA Inh. SE Inh. CaSE FA i SE
90 15
270
30
3.000
> 10.0
and Namalva had almost lost their HC antigen during culture in medium containing NHS after 3 mitotic cycles. Although several cell membrane constituents are known t o be shed from the cell surface, it appears that shedding of HC from the surface of HSB2 cells is negligible. This is substantiated by our failure to detect any material with HCR blocking activity in 10 x concentrated medium samples which were collected over a period of 5 days of HSB2 cell culture. The lack of shedding may partly explain the need for several mitotic divisions t o reduce the HC density on the cell surface.
0.280
> 10.0
Evidence for the acquired origin of HC was presented by the conversion of HSB2- cells (HC- cells from NHS-containing D >2 5 > 10.0 > 10.0 cultures) into HC' cells by incubation for 18 h a t 37 O C in the presence of FCS o r fetuin. It was surprising to find that the presence of 10 % NHS reduced the surface incorporation of HC. The most likely explanation for this effect is that Inh. = inhibitors, FCS or fetuin; FA = 4 % formaldehyde; molecules from NHS of homologous nature t o HC but of difCells were washed 3 times after each stage of the treatment. Comparison with control samples assayed in the absence of inhibitors. ferent antigenic specificity were competing with the incorCaSE was diluted 1:90 and mixed with an equal volume of inhibitor poration of HC molecules. at 0 OC for 20 min prior to addition to HSBZ cells.
(Table 5). While fetuin inhibited the sheep IgM-derived HCR at 5.8 pglml, i.e. a t a six times higher concentration than that needed for HSB2-HCR, HBGS and sheep IgG inhibited both HCR assays t o approximately the same degree. No competitive inhibition, i.e. no antigenic homology with either of the two target cell HCR was found for bovine IgG, human IgC o r human A blood group substance. The lack of inhibition with six different monosaccharides at a 2 0 mg/ml concentration confirms previous experiments [ 181 and suggests that HC antigens are likely t o be complex saccharide structures. Table 5. Similar specificity of fetuin and sheep IgM-derived HC antigens
Inhibitor added to CaSE for 20 min at Ooa)
Concentration (fig/ml)giving 50 % inhibition of HCRb) HSB2 SCLC)
HBGS Fetuin Shcep IgG Bovine IgG Human IgG Human A substanced) hlonosaccharidcse)
8.70.9 4.3 No inhibition at: No inhibition at: N o inhibition at: No inhibition at:
5.6 5.8 2.5
10 mg/ml 5 mg/ml 1 mg/ml 10 mg/ml
CaSE mixed with an equal volume of the inhibitor at various concentrations were incubated with either of the 2 cell types, and HCR were counted. Controls (100 %): HSB2: cells + CaSE 1:90 = 10.5 % HCR, SCL: cells+CaSE1:30=7,3%HCR. Chicken peripheral blood lymphocytes pre-incubated with 10 % normd sheep serum for 20 min at 0 "c. Prepared from human cystic mucin 121 1. These were: N-acetyl galactosamine; L-galactose;D-glucuronic acid; a-methyl-D-mannoside;N-acetyl glucosamine;all at a 20 mg/ml concentration. Discussion
We have demonstrated that HC o n the surface of cultured human cell lines is not biosynthesized by the cells but acquired from FCS present in the culture medium. Cell lines, HSB2
The incorporation of HC by HSB2- cells depended on the concentration of FCS o r fetuin as well as the time and temperature of incubation. Similar observations were made for the acquisition of the Lewis glycolipid by erythrocytes: erythrocytes became agglutinable by anti-Leb serum after incubation at 31 O C for 5 min with 5 pg o r for 1 8 h with 0.01 p g glycolipid, while at 4 'C, incubation with 60 pg glycolipid for 2 h was required [5]. Studies with the choleragen receptor showed that cells incubated with tritiated GM1 took u p about 4 % of the labeled ganglioside regardless of its initial coqcentration, and that the uptake at a rapid rate lasted for 4 8 h [ 81. In comparison with the latter data, it appears that HC is acquired by HSB2- cells less avidly since a significant increase of HC density was observed only after 1 8 h of culture at 37 O C , whereas incubation with FCS o r fetuin for 2 h at 37 "C o r for 20 h at 4 OC was ineffective. However, neither the mechanism by which HC is incorporated into the cell surface membrane nor the exact structure of the acquired molecules could be fully explained on the basis of these experiments. Although competitive inhibition of HCR was achieved with microgram amounts of fetuin, acquisition of HC by HSB2- cells required incubation with as much as 5 mg/ml concentration of fetuin. A similar 1000-fold difference was found between the HCR inhibitory and HC incorporation-inducing concentrations of FCS. We cannot distinguish whether these results reflect the different quantitative thresholds of the tw o reactions or whether the membrane incorporation of HC is mediated by a distinct molecular form or complex of fetuin which was present in the used fetuin preparation and FCS in small amounts. Previous studies have indicated that a lipid constituent could be important [5, 9, 141. Incubation of human lymphocytes with fetuin has been shown t o inhibit mitogen-induced lymphocyte transformation [ 231 and increase E rosette formation [24]. On the other hand, treatment of human lymphocytes with fetuin glycopeptides blocks E rosette formation [25]. Although the authors did not offer any explanation for the paradox that E rosette formation is inhibited by the fetuin glycopeptide but not by 2 5 % FCS, it is plausible t o assume that the E rosette-related structure could be a 'hidden' saccharide epitope. In view of our finding that native fetuin o r FCS strongly inhibited the HCR assay and that HSB2 cells cultured in either FCS or
Eur. J . Immunol. 1 9 7 8 . 8 : 446-451 NHS did n o t show E rosette formation (unpublished data) we conclude that t h e HC antigen in o u r experiments and t h e previously reported fetuin-mediated effects o n E rosette formation are probably unrelated phenomena. The present and previous results [ 181 suggest that H C antigen from fetuin and mammalian Ig, respectively, are detected b y IgM but not by IgC chicken antibodies. There are various examples from o t h e r systems where IgM antibodies against saccharide o r glycolipid antigens were found, and it has been suggested that they represent a n i m m u n e response against 0-galactosyl groups [ 2 6 , 271. Fetuin is a glycoprotein with 4 8 000 mol. wt. containing 26 % saccharides (galactose, mannose, glucosamine, galactosamine and sialic acid) which are organized in three units each of a b o u t 3 5 0 0 mol. w t . [ 2 2 , 281. Our failure t o inhibit HCR with various monosaccharides suggests that t h e H C epitope structure involves a complex polysaccharide chain. Competitive inhibition experiments with fetuin, HBGS and sheep IgG a t various stages of the rosette assay indicated that a close hl3mology exists between t h e HC antigenic properties of fetuin and the saccharide moiety of sheep IgG. The reaction of CaSE obviously results from fortuitous cross-reactivity and promiscuous distribution of o n e of t h e S E antigens within a wide varrety of glycoproteins, Consequently, it is hardly surprising that the avidity of binding of CaSE t o either fetuin or sheep IgG was m u c h lower than the binding t o SE. Moreover, the low binding avidity of CaSE t o H C also explains t h e requirement for t h e fixation step in t h e rosette assay. Although CaSE-derived anti-HC antibodies react w i t h red cells of several mammalian species [ 181, their distinct specificity is revealed by a lack of reaction with human red cells and h u m a n or bovin. IgG. This may a t least partially b e d u e t o t h e presence in chickens of h u m a n A group and Forsman-like substances [29, 301. It has been reported t h a t chicken serum asialoglycoproteins contain a n N-acetyl glucosamine terminal residue, but lack galactose which is present in glycoproteins o f a variety of mammalian species [ 3 I]. It appears that chicken antibodies against mammalian red cells can distinguish carbohydrate determinants in glycoproteins which share a high degree of homology in chemical composition [ 321, and their usefulness in this respect deserves further exploration. Our demonstration that cells can incorporate molecules from the surrounding medium into their surface membrane m a y have important theoretical implications. I t is possible t h a t such acquired molecules perform a functional role in growth regulation o r differentiation and serve a s receptors for exogenous factors such as hormones, viruses, toxins o r immunoregulatory factors. T h e latter possibility seems pertinent i n view of recent reports o n t h e presence of carbohydrate-rich material with Ia antigenic properties i n mouse serum 1331. The avidity of binding and rate of HC incorporation could reflect the structural properties o r specific acceptor sites in the membrane o f various cell types. Further w o r k along these lines may reveal useful markers for cell subsets a n d their stages of differentiation o r malignant transformation.
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5 References 1 Stormont, C., Proc. Nat. Acad. Sci. US 1949. 3 5 : 232.
2 Rendel, J., Neimann-Sorensen, A. and Irwin, M. R., Genetics 1954. 39: 396. 3 Sneath, J. S. and Sneath, P. H. A., Nature 1955. 176: 172. 4 Andersen, E., Ann. N Y A c a d . Sci. 1962. 9 7 205. 5 Marcus, D. M. and Cass, L. E., Science 1969. 164: 553.
6 Cuatrecasas, P., Biochemistry 1973. 1 2 : 3558.
7 Holmgren, J., Lonnroth, I., Mansson, J.-E. and Svennerholm, L., BOC. Nat. Acad. Sci. US 1975. 72: 2520. 8 Moss, J., Fishman, P. H., Manganiello, V. C., Vaughan, M. and Brady, R. W., Proc. Nut. Acad. Sci. US 1976. 73: 1034. 9 Bennett, V. and Cuatrecasas, P., in Cuatrecasas, P. and Greaves, M. F. (Eds.), Receptors and Recognition, Series B, The Specificity and Action o f Animal, Bacterial and Plant Toxins, Chapman and Hall Ltd., London 1976, p. 3. 1 0 Woolley, D. W. and Gommi, B. W., Boc. Nat. Acad. Sci. US 1965. 5 3 : 959. 11 Hughes, R. C. and Gardas, A., Nature 1976. 264: 63. 1 2 Fox, R. A., MacSween, J. M. and McGuire, R. L., Scand. J. Immunol. 1 9 7 6 . 5 : 941. 1 3 Theodore, J., Acvedo, J. C. and Robin, E. D., Science 1972. 178: 1302. 14 Clegg, J. A,, i n Porter, R. and Knight, J. (Eds.), Parasites in the Immunized Host: Mechanisms o f Survival, CIBA Found. Symp. 25, ASP Amsterdam 1974, p. 161. 15 Kennett, R. H., Fairbrother, T., Hampshire, B. and Bodmer, W. F., Tissue Antigens 1976. 8: 21. 16 Sela, B. A., Wang, J. L. and Edelman, G. M., Proc. Nat. Acad. Sci. U S 1975. 72: 1127. 1 7 Sela, B. A. and Edelman, G. M., J. Exp. Med. 1977. 145: 443. 18 Ivanyi, J., Strudwick, L. and Makings, C., Eur. J. Immunol. 1977.
7: 204. 1 9 Ivanyi, J., Cell. Immunol. 1977. 2 9 : 159. 20 Moreno, C. and Kabat, E. A., J. Immunol. 1969. 102: 1363. 21 Schiffman, G., Kabat, E. A. and Thompson, W., Biochemistry 1964. 3: 113. 22 Spiro, R. G., J. Biol. Chem. 1960. 235: 2860. 2 3 Yachnin, S., J. Exp. Med. 1975. 1 4 1 : 242. 24 Gupta, S., Goel, Z. and Grieco, M. H., h t . Arch. Allergy Appl. Immunol. 1976. 5 2 : 273. 25 Boldt, D. H. and Armstrong, J. P., J. Clin. Invest. 1976. 5 7 : 1068. 26 Uhlenbruck, G., Pardoe, G. 1. and Bud, G. W. G., 2. immunitatsforsch. Exp. Klin. Immunol. 1969. 138: 423. 27 Tsai, C.-M., Zopf, D. A,, Wistar, R. and Ginsburg, V., J. Immunol. 1976. 117: 717. 28 Spiro, R. G. and Bhoyroo, V. D., J . Biol. Chem. 1974. 249: 5704. 29 Hyde,R. R., Am. J. Hyg. 1928. 205: 1928. 30 Springer, G. F., in Neter, E. and Milgrom, F., Tne Immune System and Infectious Diseases. 4th Int. Convoc. Immunol. Buffalo, NY, Karger, Basel 1975, p. 202. 31 Lunney, J. and Ashwell, G., Proc. Nor. Acad. Sci. US 1976. 73: 341. 32 Clamp, J. R., in Putnam, F. W. (Ed.), The Hasma Proteins, vol. 2. Academic Press, New York 1975, p. 163. 33 Parish, C. R., Jackson, D. C. and McKenzie, I. F. C., Zmmunogenetics 1976. 3 : 455.
We wish to thank Dr. G. Christofinis, Department of Virology, f o r the cultured cell lines. and Drs. Moreno and Shand for reading the manuscript.
Note added in proof: Since submitting the manuscript for publication, it has come to our notice that aquisition of a serum-derived heterophile antigen (HM Ag) by cultured cell lines has been reported by hie, R.F., Irie, K. and Morton, D.L.,J. Nut. Cancer Inst. 1974. 5 4 : 1545, and Tissue Antigens 1978.11: 265.
Received January 4, 1978; in revised form March, 29, 1978.
Received June 6, 1978.
Eur. J . Immunol. 1978.8: 446-451
S. Aguilera and J. Ivanyi
S. Aguilera* and J. Ivanyi Department of Experimental Immunobiology, Wellcome Research Laboratories, Beckenham
Acquired heterophile antigens on the surface of human cell lines Chicken IgM antibodies raised against sheep erythrocytes (SE) reacted with a heterophile carbohydrate antigen designated as "HC". This antigen was visualized by SE rosette formation with antibody-sensitized and formaldehydefixed target cells. Cells from eight lymphoblastoid lines and ten lines of diverse histogenetic origin all expressed HC on their surface to a greater or lesser degree when grown in fetal calf serum (FCS). Lymphoblastoid cell lines, HSB2 and Namalva, lost most of their HC antigen after three cell divisions when grown in medium which contained normal human serum (NHS) instead of FCS. The acquired origin of HC was demonstrated by the conversion of HSB2-HC- into HSB2-HC' cells after incubation in the presence of FCS o r fetuin for 18 h at 37 OC. The acquisition of HC antigen depended on the concentration of fetuin as well as the time and temperature of incubation. The presence of NHS in the culture medium reduced the degree of HC incorporation. The level of HC expressed o n the cell surface was reduced by treating HSB2 cells with sodium periodate but not by proteolytic enzymes. Competitive inhibition with hog blood group substance and sheep IgG suggested similar specificity of HC on target cells which had been sensitized with either fetuin o r sheep IgM (Eur. J. Immunol. 1977.7:204). The avidity of chicken antibodies t o HC h either of these systems was several orders of magnitude lower than the binding to SE.
1 Introduction Cell surface carbohydrates are known t o play a fundamental role in various biological processes, and their chemical and immunological characterization has been t h e subject of extensive studies. It is widely assumed that these structures are products of cellular biosynthesis. However, it has also been demonstrated that in some instances membrane molecules are acquired from the surrounding environment. The acquisition of surface molecules from serum by red cells has been described for certain blood groups in various mammalian species [ 1-41, Although it has been demonstrated in man that t h e Lewis glycosphingolipid is transported in serum by lipoproteins, the precise mechanism by which it is taken u p from serum is not understood [ 51. Acquired cell surface molecules may also perform specialized functions: e.g. t h e monosialoganglioside GM1 is incorporated spontaneously into the cell surface membrane where it serves as a functional receptor for cholera toxin [6-81. It has been suggested that the sphingosine portion of t h e gangliosides partitions into t h e membrane bilayer, and that t h e oligosaccharide moiety on the cell surface accounts for the binding with the choleragen [9]. Restoration of responsiveness t o serotonin has been demonstrated after incubation of sialidase-treated stomach strips with gangliosides [ 101, and a fibroblast line which is resistant t o cytotoxicity of the plant lectin ricin exhibited increased binding and sensitivity t o ricin following incubation with the glycolipid prepared from human erythrocytes [ l l]. A glycoprotein from hog gastric mucine has been incorporated into t h e mem[I 20011
brane of guinea pig macrophages and potentiated the activity of a fetal calf serum (FCS)-derived migration inhibition factor [ 121, and uricase has been shown t o be endocytozed and built into the membrane of alveolar macrophages while preserving its enzymatic activity [ 131. It is also known that the parasite Schistosoma mansoni acquires host-specific blood group substances during its growth in animal hosts or during in vitro culture in the presence of serum and red cells. However, the exact molecular nature of the incorporated antigens has yet to be determined [ 141. Heterophile antigens with carbohydrate specificity were demonstrated recently on the surface of a Hela-derived cell line by cytotoxic antibodies from normal rabbit serum [ 151. Antibodies against heterophile carbohydrates were isolated from normal mammalian or avian sera by affinity chromatography using columns of insolubilized fetuin or mouse lymphoma cells lines [ 16, 171. These antibodies agglutinated erythrocytes from sheep and several other species but reacted also with murine spleen cells while antibodies eluted from two different lymphoma lines showed specificity probably against saccharide moieties in terminal position of the cell surface. We have reported recently that chicken IgM antibodies raised against sheep erythrocytes (SE) reacted with a heterophile antigen which is shared by the saccharide moiety of mammalian Ig [ 18, 193. The present experiments have shown that this heterophile carbohydrate designated as HC is expressed also on the cell surface of human cell lines when grown in FCS. Furthermore, the results suggested that HC is derived from fetuin in the medium and spontaneously incorporated into the cell surface membrane.
* Present address: Hospital J.J. Aguirre, Santiago, Chile. Correspondence: J. Ivanyi, Experimental Immunobiology Department, Wellcome Research Laboratories, Beckenham, Kent BR 3 3BS, GB Abbreviations: SE: Sheep erythrocytes CaSE: Chicken anti-SE serum FCS: Fetal calf serum NHS: Normal human serum HC: Heterophile carbohydrate reacting with CaSE HBGS: Hog blood group substance PBS: Phosphate-buffered saline HCR:HC rosette
2 Materials and methods 2.1 Antiserum (CaSE)
Inbred B14 chickens were injected twice a t a 14-day interval with 5 x l o 8 SE per kg body weight and bled after 7 days.
Eur. J. Inimunol. 1978.8: 446-451
Sera from several birds were pooled and absorbed with human AB erythrocytes. Two pools with similar antibody titers were used throughout thjs study. When fractions separated on Sephadex G-200 (Pharmacia, Uppsala) were tested, t h e results confirmed our previous finding [ 181 that antibodies which reacted with the heterophile antigen in the rosette assay were localized in the 1 9 S but not in the 7 S fraction. 2.2 H C rosette assay (HCR) Target cells (1 x 10 6 ) in 8 x 50 mm glass test tubes were incubated with 0.2 ml of serially diluted CaSE for 20 min at 0 OC, washed 3 times with ice-cold Hanks' solution, incubated with 0.2 ml of 4 % formaldehyde for 20 min at 0 "C and rewashed 3 times. The pelleted cells were resuspended in 0.2 ml of a suspension of washed SE ( 1 x 108/ml), centrifuged a t 200 x g for 10 min and kept at 0 OC u p t o 4 h prior t o counting rosetted and free lymphocytes. As in previous studies [ 18, 191 t h e fixation step with formaldehyde was essential since it amplified t h e reaction approximately 10-fold. Furthermore, the fixed cells could be stored at 4 OC for at least 5 days without a significant change in t h e HCR count. No HCF, were detected when CaSE was substituted with normal chicken serum. 2.3 Cell lines These were kindly provided by Drs. H. Zola and G. Christofinis (Wellcome Research Laboratories) o r purchased from Flow Laboratories (Glasgow, Scotland). All cell lines were grown in flat-bottom flasks in RMPI-1640 medium (Wellcome Reagents) supplemented with 1 0 % FCS (Wellcome Reagents) or normal human serum (NHS). The latter was separated from freshly drawn blood of healthy donors. Cell monolayers were harvested by incubation with 0.1 % trypsin in 0.05 % Versene for 10 r . i n a t 37 "C. Cells were washed 4 times in excess Hanks' solution prior t o assay. 2.4 Antigens Fetuin prepared by ammonium sulfate fractionation of FCS was purchased from Sigma Chemical Co., St. Louis, MO. Hog blood group substance (HBGS) was purified from hog gastric mucin [ 201 and human A substance (MSM) was prepared from cystic mucin [21]; both preparations were kindly provided b y Dr. C. Moreno. Sheep and human IgG were prepared by ion exchange chromatography o n DEAE-cellulose using 0.01 M potassium phosphate buffer, pH 8.0, for elution, and their purity was tested by immunoelectrophoretic analysis. Bovine IgG was obtained from Miles Laboratories (Kankakee, IL).
3 Results 3.1 Demonstration by the rosette assay of a heterophile antigen (HC)o n the surface of human cell lines The HC rosette (HCR) count afte; treating washed cells with various concentrations of CaSE was taken as an arbitrary measure of t h e surface density of HC antigen. Cells from 8 lymphoblastoid lines and 10 lines of diverse histogenetic origin were examined with serial log3 dilutions of CaSE. All cell lines showed 100 % HCR using low dilutions of CaSE. Suspensions with > 50 % frequency of HCR tended t o ag-
Heterophile antigens on human cell lines
447
glutinate, and accurate counts could therefore be made only in the range of 0.5 to 50 % HCR. The differences in HC density between the various cell lines were expressed by the dilution of CaSE which gave 1 0 % HCR. This is taken as an arbitrary value which is directly related to the density of HC o n the cell surface. The cell lines varied approximately 5-fold in CaSE dilutions giving 10 % IICR (Table 1). Two T cell lines which were examined (HSB2 and MOLT-4) showed relatively higher values tha n four B cell lines. Nonlymphoid lines also varied coxsiderably without a ny regular pattern or relationship t o the cells' histogenetic origin or source. As a rule, the cells were tested when they reached the stationary phase o r confluency. However, a comparison of HSB2 cells a t the logarithmic stage ( 5 x 1 05/ml) and stationary phase ( 2 x 106/ml), respectively, showed little difference in HC density. There was no distinct specificity in the reaction with individual cell lines since absorption of CaSE with HSB2 cells ( 5 x 1 O7 cells/ml 1 :30 diluted antiserum) abolished its reactivity with several other cell lines. It was possible t o elute CaSE antibodies by incubation of CaSE-sensitized HSB2 cells a t 56 "C for 30 min or with 0.2 M glycine-HC1 buffer, pH 2.5 for 2 min. Eluted and concentrated antibodies produced an HCR titer similar t o that of the starting antiserum. The presence of HC antigen on the surface of HSB2 cells was also confirmed by immunofluorescent staining. CaSE-sensitized and formaldehyde-fixed cells were specifically stained with FITC-labeled sheep anti-chicken Ig conjugate. However, the staining was a t least 10 times less sensitive and the end-point titration less accurate when compared with the HCR assay. Further analysis of the nature and origin of HC was performed with HSB2 cells. Table 1. HC density on various human cell lines Line
Nature and origin")
HSB2 (CCRF) MOLT-4 (RPMI) 4098 (RPMI) DG75
T cell; ALL T cell; ALL B cell; healthy donor B cell; Burkitt lymphoma B cell; healthy donor B cell ; ALL B cell; healthy donor B cell; Burkitt lymphoma
Namalva 8392 (RPMI) 1778 (RPMI)
Daudi NALM/1 mou 1000
HEP-2 IleLa Floh 407 MRC-5 204 FL Amnion Flow 4000 Chang
Myeloid cell Embryonal skin and muscle Epithelial cell Cervical carcinoma Embryonal intestine Fibroblast Muscle Amnion Embryonal kidney Liver
Dilution of CaSE giving 1 0 % HCR 145
115 76 63 38 37 23 21 134
110 103 96 46 38 37 31 34 27
a) ALL = acute lymphoblastic leukemia. Cells were incubated with log3 dilutions of CaSE. 100 % HCR were found using higher concentrations of CaSE in all examined cell lines. All cell lines were cultured in 10 % FCS.
3.2 Carbohydrate nature of H C antigen Previous experiments suggested that CaSE reacted with a carbohydrate moiety of IgM from various mammalian species
Eur. J. Immunol. 1978.8: 446-451
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448
[ 181. Since the treatment of cells with sodium periodate at pH 4.5 drastically reduced the HCR reaction (Fig. l ) , we conclude that CaSE-binding molecules o n the surface of t h e HSB2 cell line were also of carbohydrate nature. Incubation of cells with the acetate buffer, pH 4.5, did not affect the reaction when compared with cells which had been incubated in phosphate-buffered saline (PBS), pH 7.4. However, treatment of cells with either neuraminidase (Sigma, 5 IU/ml) or with proteolytic enzymes, trypsin (Sigma, 5 mglml), papain (Worthington, 1.6 mg/ml) o r pronase (Koch-Light, 2 mg/ml) a t 3 7 "C for 60 min (i.e. under conditions which are known to remove other surface receptors) did not significantly diminish the extent of HCR formation (Table 2). Thus, proteins apparently do not contribute towards the structure of the CaSE-binding moiety of HC.
Table 2. Failure to remove HC from the cell surface by enzyme treatmenta) Enzyme treatment
Concentration per ml
Pronase Trypsin, Papain m 10 mM cysteine Neuraminidase None
HCR
(%I 11.18 10.04 12.28 12.26 10.68
2.0 mg 5.0 mg 1.6 mg 5 IU
a) HSBZ cells (1 x 107/ml) were incubated with various enzymes at 37 "C for 60 min. Subsequently, the cells were washed and tested by the HCR assay using 1:90 diluted CaSE. respectively, at day 3 did not show any significant difference of HC density, cells grown in NHS and harvested after longer time intervals showed a gradual loss of HC antigen. HSB2 cells from the second NHS subculture almost lost the HC, and retained only 1 % HCR (detected by high concentrations of CaSE) after further passage in NHS-containing media; (this variant has been designated as HSB2- while the FCS-cultured cells are HSB2').
100.0
10 .0
a
u
r L
u il
F
1 .(I
(1.
I I
j12001.11
2
3
4
5
LOG3 DILUTION 01. C a s t
Figure 1. Carbohydrate nature of the surface heterophile antigen. HSB2 cells were fixed with 4 % formaldehyde at 0 "C for 30 min
and washed 4 times with PBS. Then the cells were incubated either with 0.005 M sodium periodate in 0.1 M sodium acetate buffer, pH 4.5 (B), or with the buffer pH 4.5 alone (0) or in PBS, pH 7.4 (a)at 4 "c for 20 h in the dark. Following incubation, the cells were washed 3 times and tested with log3 dilutions of CaSE for HCR. 3.3 Loss of HC from HSBZ cells when grown in NHS
Preliminary experiments showed that FCS, but not NHS, blocked HCR formation when added t o CaSE prior to incubation with cell suspensions. As all cell lines listed in Table 1 were grown in the presence of 10 % FCS, we considered the possibility that HC had been acquired by the cells from FCS in the culture medium. Initially, we attempted t o remove any adsorbed surface material from HSBZ cells by (a) repeated washing (6 times), (b) incubation in medium at pH 6.5 for 30 min, and (c) overnight incubation in the absence of FCS. However, none of these procedures resulted in any change of HC density. Subsequently, washed HSB2 cells ( 2 x 105/ml) were subcultured in media containing 1 0 %of either FCS o r NHS. Cells were passaged again at a 2 x 105/ml concentration from day 4. Cells were harvested o n days 3, 4 and 5 of the first and o n day 4 of the second subculture, and examined by t h e HCR assay using six logs CaSE dilutions (Fig. 2). Although cells from the subcultures in FCS and NHS,
1 2 3 LOG3 DILUTION OF CaSE
~
5
6
Figure 2. Loss of heterophile antigen from HSB2 cells grown in N H S containing medium. HSBZ cells at a density of 0.3 x 106/ml were seeded in RPMI-1640 medium containing 10 % NHS (0) or 10 %
FCS ( 0 ) . At different times of incubation cell densities and HCR with log3 dilutions of CaSE were determined. Numbers in parentheses represent the cell counts x lo6 in NHS/FCS-containing cultures: A: day 3 (1.6/2.0); B: day 4 (2.7/2.7); C: day 5 (3.1/3.5); D: day 8, subcultured after 4 days (2.1/2.0). These results were confirmed in a similar experiment with the Namalva B lymphoblastoid cell line. The generation times of the HSB2 and Namalva lines were 2.7 and 2.2 days, respectively, and these were not significantly altered by the change of FCS for NHS in the culture medium. When the rate of HC antigen loss was plotted over t h e number of cell divisions in NHS cultures, t h e outcome for HSB2 and Namalva cells was very similar, i e . the rapid loss of HC density coincided with
Eur. J. Immunol. 1978.8: 446-451
Heterophile antigens on human cell lines
the time when the number of cells seeded into the culture had increased threefold (Fig. 3). We also attempted t o influence the growth rate by culturing HSB2' cells in t h e presence of either 1 'To CaSE o r normal chicken serum and 10 % NHS. However, no difference was observed, probably due t o the low avidity of the IgM CaSE antibodies.
449
100.0
10.0 270.(
a: 11 U
c 11 L U
w Yll.1
01
2
1 .0
0
7 311.1
4
.. 3
0
0.1 2
rn
'J 10.1
3 5 LOG3 D I L U T I O N O F C a s t
6
Figure 4. Uptake of HC antigen onto HSB2- cells by incubation in 3.. J
~~~~
I
2
3
4
N U M H t R O F C t L L D I V I S I O N S IN 10%N H S C O N T A I N I N G M L D l U M
Figure 3. The rate of loss of HC antigen from HSB2 and Namalva cell lines. HSI12 ( 0 ) (see also Fig. 2) and Namalva (0)cell lines seeded at 0.3 x lo6 cells/ml were grown in 10 % NHC-containing RPMI-1640
medium. Total cell and HCR counts were determined at subsequent days of culture.
3.4 Acquisition o f HC from FCS or fetuin by HSB2- cells Since thz results described above indicated that HSB2' cells lost their HC when grown in NHS (i.e. in the absence of FCS), we attempted t o achieve t he converse effect by incubating HSB2- cells in the presence of FCS or fetuin. A pronounced increase in HC density was obtained by incubation of HSB2' FCS for 16 h a t 3 7 "C while incubation at 4 "C cells with 10 % failed to significantly increase the HCR count (Fig. 4). In another experiment, HC density o n HSB2- cells was increased by 24 h. and even further by 48 h incubation in the presence of fetuin at a 5 mg/ml concentration (Table 3). The effect was more pronounced if fetuin was added t o the culture medium in the absence of NHS, and 1 % FCS increased t h e HCR count significantly only when added t o medium which did not contain NHS.
3.5 Analysis of the antigenic properties of HC by rosette inhibition FCS and its main constituent fetuin were used for t h e competitive inhibition of HCR formation. By adding either of these two inhibitors a t various stages of t h e HCR test, dramatically different degrees of HCR suppression were observed (Table 4). Mixing the inhibitors with CaSE prior t o incubation with HSB2 cells resulted in 50 %inhibition of HCR at a 1 :27000 dilution of FCS o r at a 1 pg/ml concentration of fetuin (protocol A). These results are comparable when assuming an approximately 2 0 mg/ml concentration of fetuin in FCS [22]. Both inhibitors had t o be used at a 100 times higher concentration when added t o HSB2 cells which had
FCS-containing medium. HSB2- cells grown in 10 % NHS-containing medium were washed and incubated for 16 h in the presence of 10 % FCS at 37 "C (m) or 4 "C (A). Control HSBZ- cells in 10 % NHS ( O ) , and HSB2+ cells cultured in 10 % FCS (0). Table 3. Uptake of HC by incubation of HSB2- cells with fetuin or FCS Incubation of HSB2- cells at 37 "C NHS Fetuin FCS ( I ) (mg/ml) (%) 10 10 5 5 10 1 1 10
Dilution of CaSE giving 10 % HCR 24 h 48 h 4
25 45 6 19 52
6 105 360 8 83 540
Relative yield of cells (%) 24 h 48h 100 110 58 100 60
120
100
44 17
86 54 81
previously been 'sensitized' with CaSE (protocol B). Further, a n almost 10-fold increase in FCS concentration was needed for t h e inhibition of previously CaSE-sensitized and fixed cells (protocol C), and another 6-fold increase was necessary when adding FCS at the last stage of the assay together with SE (protocol D). Fetuin at a 10 mg/ml concentration was ineffective in a ny of the latter two ( C and D) protocols. These results suggest that CaSE has a considerably weaker binding to HSB2 cells than to SE. Furthermore, competitive inhibition of CaSE-binding to HSB2 cells was 100 times more effective than t h e dissociation of antibodies which were already bound. Further experiments using protocol A and various inhibitors were designed t o compare the antigenic specificity of the fetuin-derived HC on the surface of HSB2 cells with the previously described HC moiety of sheep IgM. The latter type of target cell was provided by normal sheep serum-treated chicken peripheral blood lymphocytes [ 181. The concentration of the inhibitor needed for the competitive inhibition of HCR was inversely related t o the degree of antigenic homology between the inhibitor and the surface-bound HC molecules
Eur. J. Immunol. 1978.8: 446-451
S. Aguilera and J. Ivanyi
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Table 4. Competitive inhibition of HCR reaction with FCS and fetuina)
Protocol Sequence of treatment FCS dilution Fetuin concentration (mg/ml)giving HCR of HSBZ cellsb) giving HCR inhibition of: inhibition of: 1
2
3
4
9 0 %4 5 0 %
-
9000 27000
90%
50%
CaSE A
+
FA SE
0.001
0.004
1nh.d) B C
a) b)
c) d)
CaSE Inh. FA SE CaSE FA Inh. SE Inh. CaSE FA i SE
90 15
270
30
3.000
> 10.0
and Namalva had almost lost their HC antigen during culture in medium containing NHS after 3 mitotic cycles. Although several cell membrane constituents are known t o be shed from the cell surface, it appears that shedding of HC from the surface of HSB2 cells is negligible. This is substantiated by our failure to detect any material with HCR blocking activity in 10 x concentrated medium samples which were collected over a period of 5 days of HSB2 cell culture. The lack of shedding may partly explain the need for several mitotic divisions t o reduce the HC density on the cell surface.
0.280
> 10.0
Evidence for the acquired origin of HC was presented by the conversion of HSB2- cells (HC- cells from NHS-containing D >2 5 > 10.0 > 10.0 cultures) into HC' cells by incubation for 18 h a t 37 O C in the presence of FCS o r fetuin. It was surprising to find that the presence of 10 % NHS reduced the surface incorporation of HC. The most likely explanation for this effect is that Inh. = inhibitors, FCS or fetuin; FA = 4 % formaldehyde; molecules from NHS of homologous nature t o HC but of difCells were washed 3 times after each stage of the treatment. Comparison with control samples assayed in the absence of inhibitors. ferent antigenic specificity were competing with the incorCaSE was diluted 1:90 and mixed with an equal volume of inhibitor poration of HC molecules. at 0 OC for 20 min prior to addition to HSBZ cells.
(Table 5). While fetuin inhibited the sheep IgM-derived HCR at 5.8 pglml, i.e. a t a six times higher concentration than that needed for HSB2-HCR, HBGS and sheep IgG inhibited both HCR assays t o approximately the same degree. No competitive inhibition, i.e. no antigenic homology with either of the two target cell HCR was found for bovine IgG, human IgC o r human A blood group substance. The lack of inhibition with six different monosaccharides at a 2 0 mg/ml concentration confirms previous experiments [ 181 and suggests that HC antigens are likely t o be complex saccharide structures. Table 5. Similar specificity of fetuin and sheep IgM-derived HC antigens
Inhibitor added to CaSE for 20 min at Ooa)
Concentration (fig/ml)giving 50 % inhibition of HCRb) HSB2 SCLC)
HBGS Fetuin Shcep IgG Bovine IgG Human IgG Human A substanced) hlonosaccharidcse)
8.70.9 4.3 No inhibition at: No inhibition at: N o inhibition at: No inhibition at:
5.6 5.8 2.5
10 mg/ml 5 mg/ml 1 mg/ml 10 mg/ml
CaSE mixed with an equal volume of the inhibitor at various concentrations were incubated with either of the 2 cell types, and HCR were counted. Controls (100 %): HSB2: cells + CaSE 1:90 = 10.5 % HCR, SCL: cells+CaSE1:30=7,3%HCR. Chicken peripheral blood lymphocytes pre-incubated with 10 % normd sheep serum for 20 min at 0 "c. Prepared from human cystic mucin 121 1. These were: N-acetyl galactosamine; L-galactose;D-glucuronic acid; a-methyl-D-mannoside;N-acetyl glucosamine;all at a 20 mg/ml concentration. Discussion
We have demonstrated that HC o n the surface of cultured human cell lines is not biosynthesized by the cells but acquired from FCS present in the culture medium. Cell lines, HSB2
The incorporation of HC by HSB2- cells depended on the concentration of FCS o r fetuin as well as the time and temperature of incubation. Similar observations were made for the acquisition of the Lewis glycolipid by erythrocytes: erythrocytes became agglutinable by anti-Leb serum after incubation at 31 O C for 5 min with 5 pg o r for 1 8 h with 0.01 p g glycolipid, while at 4 'C, incubation with 60 pg glycolipid for 2 h was required [5]. Studies with the choleragen receptor showed that cells incubated with tritiated GM1 took u p about 4 % of the labeled ganglioside regardless of its initial coqcentration, and that the uptake at a rapid rate lasted for 4 8 h [ 81. In comparison with the latter data, it appears that HC is acquired by HSB2- cells less avidly since a significant increase of HC density was observed only after 1 8 h of culture at 37 O C , whereas incubation with FCS o r fetuin for 2 h at 37 "C o r for 20 h at 4 OC was ineffective. However, neither the mechanism by which HC is incorporated into the cell surface membrane nor the exact structure of the acquired molecules could be fully explained on the basis of these experiments. Although competitive inhibition of HCR was achieved with microgram amounts of fetuin, acquisition of HC by HSB2- cells required incubation with as much as 5 mg/ml concentration of fetuin. A similar 1000-fold difference was found between the HCR inhibitory and HC incorporation-inducing concentrations of FCS. We cannot distinguish whether these results reflect the different quantitative thresholds of the tw o reactions or whether the membrane incorporation of HC is mediated by a distinct molecular form or complex of fetuin which was present in the used fetuin preparation and FCS in small amounts. Previous studies have indicated that a lipid constituent could be important [5, 9, 141. Incubation of human lymphocytes with fetuin has been shown t o inhibit mitogen-induced lymphocyte transformation [ 231 and increase E rosette formation [24]. On the other hand, treatment of human lymphocytes with fetuin glycopeptides blocks E rosette formation [25]. Although the authors did not offer any explanation for the paradox that E rosette formation is inhibited by the fetuin glycopeptide but not by 2 5 % FCS, it is plausible t o assume that the E rosette-related structure could be a 'hidden' saccharide epitope. In view of our finding that native fetuin o r FCS strongly inhibited the HCR assay and that HSB2 cells cultured in either FCS or
Eur. J . Immunol. 1 9 7 8 . 8 : 446-451 NHS did n o t show E rosette formation (unpublished data) we conclude that t h e HC antigen in o u r experiments and t h e previously reported fetuin-mediated effects o n E rosette formation are probably unrelated phenomena. The present and previous results [ 181 suggest that H C antigen from fetuin and mammalian Ig, respectively, are detected b y IgM but not by IgC chicken antibodies. There are various examples from o t h e r systems where IgM antibodies against saccharide o r glycolipid antigens were found, and it has been suggested that they represent a n i m m u n e response against 0-galactosyl groups [ 2 6 , 271. Fetuin is a glycoprotein with 4 8 000 mol. wt. containing 26 % saccharides (galactose, mannose, glucosamine, galactosamine and sialic acid) which are organized in three units each of a b o u t 3 5 0 0 mol. w t . [ 2 2 , 281. Our failure t o inhibit HCR with various monosaccharides suggests that t h e H C epitope structure involves a complex polysaccharide chain. Competitive inhibition experiments with fetuin, HBGS and sheep IgG a t various stages of the rosette assay indicated that a close hl3mology exists between t h e HC antigenic properties of fetuin and the saccharide moiety of sheep IgG. The reaction of CaSE obviously results from fortuitous cross-reactivity and promiscuous distribution of o n e of t h e S E antigens within a wide varrety of glycoproteins, Consequently, it is hardly surprising that the avidity of binding of CaSE t o either fetuin or sheep IgG was m u c h lower than the binding t o SE. Moreover, the low binding avidity of CaSE t o H C also explains t h e requirement for t h e fixation step in t h e rosette assay. Although CaSE-derived anti-HC antibodies react w i t h red cells of several mammalian species [ 181, their distinct specificity is revealed by a lack of reaction with human red cells and h u m a n or bovin. IgG. This may a t least partially b e d u e t o t h e presence in chickens of h u m a n A group and Forsman-like substances [29, 301. It has been reported t h a t chicken serum asialoglycoproteins contain a n N-acetyl glucosamine terminal residue, but lack galactose which is present in glycoproteins o f a variety of mammalian species [ 3 I]. It appears that chicken antibodies against mammalian red cells can distinguish carbohydrate determinants in glycoproteins which share a high degree of homology in chemical composition [ 321, and their usefulness in this respect deserves further exploration. Our demonstration that cells can incorporate molecules from the surrounding medium into their surface membrane m a y have important theoretical implications. I t is possible t h a t such acquired molecules perform a functional role in growth regulation o r differentiation and serve a s receptors for exogenous factors such as hormones, viruses, toxins o r immunoregulatory factors. T h e latter possibility seems pertinent i n view of recent reports o n t h e presence of carbohydrate-rich material with Ia antigenic properties i n mouse serum 1331. The avidity of binding and rate of HC incorporation could reflect the structural properties o r specific acceptor sites in the membrane o f various cell types. Further w o r k along these lines may reveal useful markers for cell subsets a n d their stages of differentiation o r malignant transformation.
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5 References 1 Stormont, C., Proc. Nat. Acad. Sci. US 1949. 3 5 : 232.
2 Rendel, J., Neimann-Sorensen, A. and Irwin, M. R., Genetics 1954. 39: 396. 3 Sneath, J. S. and Sneath, P. H. A., Nature 1955. 176: 172. 4 Andersen, E., Ann. N Y A c a d . Sci. 1962. 9 7 205. 5 Marcus, D. M. and Cass, L. E., Science 1969. 164: 553.
6 Cuatrecasas, P., Biochemistry 1973. 1 2 : 3558.
7 Holmgren, J., Lonnroth, I., Mansson, J.-E. and Svennerholm, L., BOC. Nat. Acad. Sci. US 1975. 72: 2520. 8 Moss, J., Fishman, P. H., Manganiello, V. C., Vaughan, M. and Brady, R. W., Proc. Nut. Acad. Sci. US 1976. 73: 1034. 9 Bennett, V. and Cuatrecasas, P., in Cuatrecasas, P. and Greaves, M. F. (Eds.), Receptors and Recognition, Series B, The Specificity and Action o f Animal, Bacterial and Plant Toxins, Chapman and Hall Ltd., London 1976, p. 3. 1 0 Woolley, D. W. and Gommi, B. W., Boc. Nat. Acad. Sci. US 1965. 5 3 : 959. 11 Hughes, R. C. and Gardas, A., Nature 1976. 264: 63. 1 2 Fox, R. A., MacSween, J. M. and McGuire, R. L., Scand. J. Immunol. 1 9 7 6 . 5 : 941. 1 3 Theodore, J., Acvedo, J. C. and Robin, E. D., Science 1972. 178: 1302. 14 Clegg, J. A,, i n Porter, R. and Knight, J. (Eds.), Parasites in the Immunized Host: Mechanisms o f Survival, CIBA Found. Symp. 25, ASP Amsterdam 1974, p. 161. 15 Kennett, R. H., Fairbrother, T., Hampshire, B. and Bodmer, W. F., Tissue Antigens 1976. 8: 21. 16 Sela, B. A., Wang, J. L. and Edelman, G. M., Proc. Nat. Acad. Sci. U S 1975. 72: 1127. 1 7 Sela, B. A. and Edelman, G. M., J. Exp. Med. 1977. 145: 443. 18 Ivanyi, J., Strudwick, L. and Makings, C., Eur. J. Immunol. 1977.
7: 204. 1 9 Ivanyi, J., Cell. Immunol. 1977. 2 9 : 159. 20 Moreno, C. and Kabat, E. A., J. Immunol. 1969. 102: 1363. 21 Schiffman, G., Kabat, E. A. and Thompson, W., Biochemistry 1964. 3: 113. 22 Spiro, R. G., J. Biol. Chem. 1960. 235: 2860. 2 3 Yachnin, S., J. Exp. Med. 1975. 1 4 1 : 242. 24 Gupta, S., Goel, Z. and Grieco, M. H., h t . Arch. Allergy Appl. Immunol. 1976. 5 2 : 273. 25 Boldt, D. H. and Armstrong, J. P., J. Clin. Invest. 1976. 5 7 : 1068. 26 Uhlenbruck, G., Pardoe, G. 1. and Bud, G. W. G., 2. immunitatsforsch. Exp. Klin. Immunol. 1969. 138: 423. 27 Tsai, C.-M., Zopf, D. A,, Wistar, R. and Ginsburg, V., J. Immunol. 1976. 117: 717. 28 Spiro, R. G. and Bhoyroo, V. D., J . Biol. Chem. 1974. 249: 5704. 29 Hyde,R. R., Am. J. Hyg. 1928. 205: 1928. 30 Springer, G. F., in Neter, E. and Milgrom, F., Tne Immune System and Infectious Diseases. 4th Int. Convoc. Immunol. Buffalo, NY, Karger, Basel 1975, p. 202. 31 Lunney, J. and Ashwell, G., Proc. Nor. Acad. Sci. US 1976. 73: 341. 32 Clamp, J. R., in Putnam, F. W. (Ed.), The Hasma Proteins, vol. 2. Academic Press, New York 1975, p. 163. 33 Parish, C. R., Jackson, D. C. and McKenzie, I. F. C., Zmmunogenetics 1976. 3 : 455.
We wish to thank Dr. G. Christofinis, Department of Virology, f o r the cultured cell lines. and Drs. Moreno and Shand for reading the manuscript.
Note added in proof: Since submitting the manuscript for publication, it has come to our notice that aquisition of a serum-derived heterophile antigen (HM Ag) by cultured cell lines has been reported by hie, R.F., Irie, K. and Morton, D.L.,J. Nut. Cancer Inst. 1974. 5 4 : 1545, and Tissue Antigens 1978.11: 265.
Received January 4, 1978; in revised form March, 29, 1978.
Received June 6, 1978.
