Cell, Vol. 5, 87-92,

May

1975,

Copyright

0 1975

by MIT

A Human Lymphoid Cell Line Secreting lmmunoglobulin G and Retaining lmmunoglobulin M in the Plasma Membrane E. Premkumar’, P. A. Singer’, and A. R. Williamson+ Department of Microbiology and Immunology U.C.L.A. School of Medicine Los Angeles, California 90024

Results

Summary A selected clone, LA 85.2, of a human iymphoid ceil line produces p, y, and light chains. The ceils secrete IgG but not IgM. Assembly of CLchains and light chains produces 8s IgM which is retained in the plasma membrane. IgM is produced at a slow rate and in lesser amounts than IgG. LA 85.2 ceils produce a plasma membrane protein which can bind to antibody-antigen precipitates. It is suggested that this protein plays a role in holding the surface igfvl in the plasma membrane. Introduction Peripheral human lymphocytes can be grown in culture and continuous cell lines can be derived from them. Epstein-Barr virus is thought to be involved in the establishment of a lymphoid cell line (Diehl et al., 1969; Steel, 1972). The cells growing in these cultures have the morphology of lymphoblasts. Each cell line usually produces small amounts of immunoglobulin which may be secreted or be retained in the cell (Lerner, McConahey, and Dixon, 1971). Human lymphoid cell lines producing two classes of heavy chain (Finegold, Fahey, and Dutcher, 1968; Takahashi et al., 1968) have been described. Cloning of lines producing two immunoglobulin classes gave lines in which each cell was producing both classes (Takahashi et al., 1969; Bloom, Choi, and Lamb, 1971). Litwin et al. (1973) described human lymphoid cell lines with both IJ and y chains on the cell surface. Recently van Boxel and Buell (1974) described six cell lines, each bearing on their surface four immunoglobulin classes (IgD, IgM, IgG, and IgA). In the present investigation we used a clone derived from cell line LA 85. This line was originated and cloned by M. Jobin and J. L. Fahey. We have shown that clone LA 85.2 cells produce both IgG and 8s IgM; the IgG is secreted and the 8s IgM is retained in the plasma membrane. We also described a membrane protein which may be involved in retaining IgM in the plasma membrane.

*Present address: Microbiological Associates, nue, Bethesda, Maryland 20014. +Present address: Department of Biochemistry, gow, Glasgow G12 EQQ, Scotland.

4733 Bethesda University

Ave-

of Glas-

lmmunoglobulins Produced by LA 85.2 Cells The biosynthesis of immunoglobulins was followed by incorporation of radioactively labeled methionine. The labeled immunoglobulins were then characterized by precipitation with specific antisera followed by dissolution of the precipitates with SDS and analysis by polyacrylamide gel electrophoresis in the presence of SDS. The immunoglobulins secreted by LA 85.2 cells were examined first. The cells were incubated with 35S-methionine for a period of 3 hr and were then separated from the incubation medium. The radioactive immunoglobulins which had been secreted into the medium during the incubation were precipitated with rabbit anti-human immunoglobulin specific for both IgM and IgG. The only labeled immunoglobulin detected was a 7s molecule which upon reduction and alkylation was shown by gel electrophoresis to contain y chains and L chains (Figure 1).

20 -

cpm (103

-

10 -

8 Figure 1. Polyacrylamide 3 Hr- Labeled Cells

16 Gel Profiles

24

of LA 85.2 Secreted

32 lg from

About 1 X 107 cells taken from log phase were suspended in 2 ml labeling media, containing 50 pCi/ml W-methionine, for a period of 3 hr. The cells were pelleted at 1000 x g for 5 min, and the labeling media treated with antiserum to human IgG and IgM. The immtmoprecipitates were reduced, alkylated, and electrophoresed on 6.7% polyacrylamide gels as described in Experimental Procedures. The markers, p, y, and L show the positions of p- and y- heavy chains, and light chain, respectively.

Cell

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In separate experiments, the biosynthetically labeled intracellular pools of immunoglobulin were examined. LA 85.2 cells were incubated with 35Smethionine at 37°C for either 3 hr or 24 hr. At the end of the incubation period the cells were recovered by centrifugation, washed, and lysed with NP40. The intracellular immunoglobulins were precipitated with antiimmunoglobulin specific for both IgM and IgG, dissolved in SDS, reduced and alkylated and analyzed on polyacrylamide gels (Figure 2).

The pattern of radioactive products seen after 3 hr labeling with radioactive methionine shows, as expected, both y chain and L chain (Figure 2a). In addition to these two immunoglobulin chains, a third peak is seen running in an intermediate position between y chains and light chains. This peak (approximate molecular weight = 36,000 daltons) does not correspond to any known immunoglobulin polypeptide chain. In the course of further experiments on the biosynthesis of immunoglobulins by

5

15 0

CPm (x 10-3)

5 10 CPm (x1o-3) 0 5

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Figure 3. Polyacrylamide Gel Profile from 24 Hr-Labeled Cells-Unreduced

bb Figure 2. Polyacrylamide Ig-Reduced

10

20 Gel

Profiles

of

30 LA 65.2

Intracellular

Aliquots, at 1 x 107 cells each, taken from log phase were suspended in 2 ml labeling media, containing 50 &i/ml W-methionine, for a period of 3 hr (a) or for a period of 24 hr (b). After the labeling, the cells were pelleted at 1000 x g and lysates prepared as described in Experimental Procedures. The lysates were treated with 200 pl of antiserum to human IgG and IgM, and carrier IgG and IgM were added after 15 min at 37°C. After an additional 15 min at 37°C and overnight at 4°C the immunoprecipitates were pelleted at 3000 x g and washed three times with PBS. Reduction, alkylation, and electrophoresis on 6.7% gels were as described in Experimental Procedures.

of LA 65.2

Intracellular

IgM

(a) 1 x 10’ cells from late log phase were resuspended in labeling media containing 20% fetal calf serum and 50 pCi/ml ‘SS-methionine for a period of 24 hr. The cell lysate was prepared as described in Experimental Procedures, and 100 pg of purified antibodies to BSA was added, followed immediately with ESA at equivalence. The resulting immunoprecipitate was pelleted at 3000 x g, and the supernatant cell lysate was treated with 100 ~1 of specific antiserum to human IgM. After 15 min at 37”C, carrier IgM was added, and the immunoprecipitate thus formed was dissolved in 2% SDS, 6 M Urea, boiled 2 min, and electrophoresed on 2% polyacrylamide gels containing 0.5% agarose and 0.1% SDS in 0.1 M sodium phosphate buffer (pH 7.0). (b) Molecular weight markers consisting of radioactively iodinated mouse myeloma IgG and IgM (7s and 19s respectively) were electrophoresed under identical conditions. (c) 5 x 107 cells from late log phase were washed three times with Hank’s BSS and suspended in 1 ml PBS. 10 pg of lactoperoxidase, 0.5 mCi carrier-free 1251,and 10 ~1 of 0.03% H202 were added in order, and radioactive iodination was allowed to proceed for 5 min at 30°C. The cell lysate was prepared as described in Experimental Procedures. lmmunoprecipitation was carried out exactly as described in Figure 3a, using BSA-anti-BSA and specific antiserum for human IgM. The anti-IgM precipitate was electrophoresed on a 2% polyacrylamide gel as in Figure 3a.

Lymphoid 89

Cell Line Producing

IgG and IgM

LA 85.2 cells, more information on the nature of this new component was obtained. Biosynthetic labeling of LA 85.2 cells for 24 hr revealed the synthesis of p chains in addition to y chains, L chains and the novel 36,000 dalton component (Figure 2b). Detection of labeled ~1 chain only after prolonged incubation with radioactive amino acids suggests that p and y chain synthesis are conducted at different rates and destined for different metabolic fates. The 24 hr labeling experiment was therefore repeated in order to determine the assembly pattern of p chains in LA 85.2 cells. A modified procedure was adopted for the analysis of the labeled cells. The cells were lysed as before and then the nuclei and other cellular debris were removed by high speed centrifugation. Rabbit antiBSA and BSA were then added at equivalence to the supernatant and the resultant precipitate removed by centrifugation. This step was added with the intention of lowering the background of nonspecific absorption of radioactive proteins to the specific immunoglobulin antiimmunoglobulin precipitates. Rabbit anti-human p chain and carrier human IgM were then added to the supernatant and the resultant precipitate dissolved in SDS for analysis on a 2% composite agarose-acrylamide gel. The resultant pattern shown in Figure 3 consists of a single component at approximately 8s by comparison with 7s IgG and 19s IgM. (Figure 3b). In another similar experiment, the radioactive proteins brought down by the BSA-anti-BSA precipitation were reduced and alkylated and analyzed in the

1+ + I::

!J

10 CPm (x1o-4)

5-

Figure 4. Polyacrylamide from LA 85.2 Cell Lysate

Gel Profile

Y

n

of BSA Anti-BSA

L

Precipitate

1 x 107 cells from late log phase were labeled in 2 ml labeling media containing 20% fetal calf serum and 50 pCi/ml 35S-methionine. The cell lysate was prepared as described in Experimental Procedures, and then treated with 100 pg purified antibodies to BSA, followed immediately with BSA at equivalence. The resulting immunoprecipitate was washed three times with PBS, dissolved in a 2% SDS, 6 M Urea solution, reduced, alkylated, and electrophoresed on 6.7% polyacrylamide gels as described in Experimental Procedures.

presence of SDS on 6.7% polyacrylamide gel (Figure 4). This analysis revealed a single major component with a molecular weight of approximately 36,000 daltons. This component appears to correspond to the novel polypeptide chain coprecipitated with immunoglobulin-antiimmunoglobulin (Figure 2). Since this polypeptide is also coprecipitated with other antigen-antibody complexes (unpublished observations), it seems highly unlikely that it is precipitated by a conventional antibody cross-reaction. We therefore advance the hypothesis that this 36,000 dalton polypeptide has the property of binding to some common feature on immunoglobulin molecules. Membrane Associated lmmunoglobulin of LA 85.2 Cells The slow rate of production of p chains and their absence from the immunoglobulin secreted by LA 85.2 cells suggested that the ,U chains might be retained in the surface membrane. This hypothesis was tested and confirmed by the use of lactoperoxidase to iodinate the proteins accessible on the outside of the plasma membrane. The labeled cells were washed, lysed with 0.7% NP40, the cytoplasmic fraction clarified by high-speed centrifugation, and the radioactive immunoglobulins were precipitated with antiserum against IgG and IgM. The precipitate was dissolved in 2% SDS, reduced, alkylated, and analyzed on a 6.7% polyacrylamide gel (Figure 5). A major peak of IJ chain is clearly seen. Despite a high background across the gel, a peak of L chain is discernible. The other major peak is at approximately 36,000 daltons, and we assume that it corresponds to the similarly sized polypeptide chain seen in the analysis of biosynthetically labeled intracellular material. The analysis illustrated in Figure 5 provides only equivocal evidence for the presence of y chains in the surface membrane. If both y chains and p chains were present in the surface membrane in a similar ratio to that seen in longterm biosynthetically labeled cytoplasmic extracts (Figure 2b), then one would be forced to say that the y chains were considerably less accessible to the lactoperoxidase iodination procedure than are the (” chains. Since y chains are being actively secreted by LA 85.2 cells, it is more logical to assume that very few y chains are present in the surface membrane at any one time and that any which are found there represent IgG molecules in the process of being secreted. In another experiment, similar to the one described above, radioactively iodinated surface proteins solubilized with NP40 were analyzed by successive precipitations, first with anti-BSA and carrier BSA, and then with anti-p chain and carrier IgM. The specific anti-p precipitate was dissolved in SDS

Cell 90

and analyzed directly, without reduction and alkylation, on a 2% polyacrylamide gel (Figure 3~). A major peak of radioactivity is seen corresponding to the position of 8s IgM. It appears that the 8s IgM seen in the biosynthetic studies (Figure 3a) is inserted into the plasma membrane without further polymerization. The minor peak of radioactively iodinated protein, having a much lower molecular weight than the 7s marker, probably represents specific precipitation by the anti-p chain antibody, for the background radioactivity, although high, appears to be uniformly distributed across the gel. The minor low molecular weight component could be either a free p chain or p L half molecules; molecular weight determination in this region of the gel is not sufficiently accurate to decide this point, and further analysis has not been carried out. The radioactively iodinated protein binding to the initial BSAanti-BSA precipitate was dissociated in SDS, reduced, and alkylated and analyzed on a 6.7% polyacrylamide gel (Figure 6). The resulting pattern is strikingly similar to that obtained by similar analyses of the biosynthetically labeled intracellular proteins. The novel peptide chain with the property of binding to immunoglobulins appears to be a component of the surface membrane. We have therefore given this component the descriptive title of “Membrane-Associated Immunoglobulin-Detaining Protein” for which we can use the acronym MAID.

5

I-

4

-

3

-

Discussion The following picture emerges from the data presented above on studies on clone 2 of the human lymphoid cell line LA 85. The clone is synthesizing two immunoglobulin products, IgM and IgG. Only the IgG is being actively secreted, and y chains are being synthesized in larger amounts and at a greater rate than p chains. The assembly of ,Uchains and L chains goes only as far as the 8s 4-chain molecule. There is no active secretion of IgM, the assembled 8s IgM being retained in the plasma membrane. Little or no IgG is retained in the plasma membrane. The cells also synthesize a plasma membrane protein (which we have termed MAID) possessing the property of binding to antibodyantigen precipitates. This picture of LA 85.2 cells requires comment and invites speculation. Clones of human lymphoid cells producing two immunoglobulins have been described previously (Takahashi et al., 1969; Bloom et al., 1971), but without detailed biosynthetic studies of the type described here. One of the most intriguing findings in the present study is the differential fate of y chains and p chains, the former being secreted as IgG, and the latter being incorporated into the plasma membrane as 8s IgM. This recalls the detection amongst rabbit peripheral lymphocytes of cells staining with fluorescent antiimmunoglobulin reagents for cytoplasmic IgG and surface IgM (Pernis, Forni, and Amante, 1971). Such cells could be regarded as having IgM type receptors while being 3

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Figure 5. Polyacrylamide 85.2 Cells, Precipitated

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5 x 107 cells were taken from log phase and their surface proteins radioactively iodinated as described in Experimental Procedures. Lysates were treated in order with BSA-anti-BSA, and antiserum to human IgG and IgM. The anti-IgG, IgM immunoprecipitates were reduced, alkylated, and electrophoresed in 6.7% polyacrylamide gels.

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Figure 6. Polyacrylamide LA 85.2 Ceils Precipitated

1

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30

40

Gel Profiles of 1251 Surface with Anti-BSA

Proteins

of

The immunoprecipitates obtained from the anti-BSA treatment of the cell lysate from Figure 5 were also reduced, alkylated, and electrophoresed on 6.7% SDS-polyacrylamide gels as described in Experimental Procedures.

Lymphoid 91

Cell Line Producing

IgG and IgM

capable of secreting IgG. Although LA 85.2 cells do not actively secrete IgM, small amounts of IgM were detected originally in the medium of stationary phase cells (M. Jobin and J. L. Fahey, unpublished observations). Small amounts of IgM would be expected to appear in the medium due to the slow turnover of the membrane 8s IgM. Slow release of 8s IgM in the absence of active secretion of any other immunoglobulin has been described for mouse spleen cells in short term culture (Andersson, Lafleur, and Melchers, 1974). In that case, as with LA 85.2 cells, the tempo of IgM synthesis was slow. The synthesis of IgM in mouse spleen cells in short term culture is thought to represent the production of receptor antibodies on these cells. By analogy with the mouse spleen cell system, we consider the synthesis of IgM by LA 85.2 cells to be a model for the synthesis of receptor antibody. Without knowing what antigen or antigens the IgM of LA 85.2 cells can bind, we are not able to test whether it could be a functional receptor antibody. The properties of MAID lead us to speculate as to its role in the cell. We suggest that the 85 IgM is present in the plasma membrane as a complex with one or more molecules of MAID. If MAID is a membrane protein with a hydrophobic exterior, it could serve to mask the hydrophilic exterior of the IgM molecule, thus permitting the retention of the latter in the plasma membrane. The ratio of radioactivity in p chain and MAID in the steady state biosynthesis experiment (Figure 2b) is similar to the ratio of radioactivity in p chain and MAID when the latter are labeled by radioactive iodination of the cell surface (Figure 5). This tentatively suggests a balance between p chain and MAID, but it should be noted that biosynthetically labeled MAID is seen in cell lysates after short term labeling (Figure 2a) at a time when labeled p chains are not yet detectable. Consistent with a link between MAID and surface receptor antibody, MAID has not been found in a variety of high rate immunoglobulin-secreting mouse myeloma cells. Moreover, in preliminary experiments with a human lymphoid cell line producing and secreting only IgG, we have been unable to detect MAID. MAID has been detected together with surface IgM in the cells of several Ableson virus-induced lymphosarcomas (Premkumar, Singer, and Potter, unpublished observation), and a similar protein was reported by Andersson et al., (1974) in murine small splenic lymphocytes. It has been suggested by Kessler (1974) that MAID may be the Fc receptor, which has been demonstrated in a variety of ways on the surface of certain B lymphocytes. The function of the Fc receptor is unclear, but the hypothesis proposed by Ramasamy, Munro, and Milstein (1974) equates the Fc receptor

with a proreceptor molecule-that is, a site in the membrane to accommodate the receptor antibody, empty proreceptor sites being revealed as Fc receptors. It seems to us that MAID could fulfill both of these functions. Experimental

Procedures

Cell Cultures The cell line LA 85 was derived from the peripheral blood lymphocytes of a healthy adult and grown in RPMt 1640 Special media (RPM1 1640 with 2 x amino acids and 3 x vitamins added), containing 20% fetal calf serum. Clone 2 cultures were derived by single-cell cloning technique from the parent LA 85 cell line and grown in suspension culture in RPM1 1640 Special media. Cell Labellng Biosynthetlcally 1 x 10’ cells were labeled biosynthetically by suspension in RPM1 1640 Special media (2 ml) lacking methionine and containing 20% fetal calf serum. 50 @i/ml XsS-methionine (Amersham-Searle) was added to the labeling media, and incubation at 37°C under a 5% COZ atmosphere was allowed for either 3 hr or 24 hr. Cells were taken from mid-log phase for the 3 hr labeling experiments and from late log phase for the 24 hr labeling experiments. Cell viability was greater than 90% throughout the experiments. Labeling of Surface Proteins Cell surface proteins were radioactively iodinated using the lactoperoxidase method (Marchalonis, Cone, and Santer, 1971). 5 x 107 cells were prepared and washed three times in Hanks’ BSS (Balanced Salt Solution) and suspended in 1 ml PBS (0.05 M sodium phosphate, pH 7.2, 0.14 M NaCI). 10 pg of lactoperoxidase (Sigma Chemical Co.) was added, along with 0.5 mCi carrierfree 1251(Amersham-Searle). The reaction was allowed to proceed for 5 min at 30°C after the addition of 10 ~1 0.03% hydrogen peroxide, and terminated by the addition of 10 vol of cold Hanks’ BSS. Cell lysates were prepared as described below. Analysis of Biosynthetic lmmunoglobulln or MAID The cells were lysed in 0.5 ml 0.7% Nonidet P-40 (Shell Chemical Co.) for 15 min on ice, and the nuclei and other cellular matter pelleted at 10,000 x g for 30 min. The lysates were treated with 200 gl of antiserum to Human IgG and IgM (Meloy Laboratories), or 100 pg purified antibodies to BSA (bovine serum albumin) for a period of 15 min at 37’C followed by addition of carrier IgG and IgM, or BSA, at equivalence. A further 15 min incubation at 37°C followed by overnight at 4’C was included to assure complete immunoprecipitate formation. The growth media was treated with antiserum in a likewise manner for the analysis of secreted immunoglobulins. The resulting immunoprecipitates were washed three times with PBS, dissolved in a 2% SDS (Sodium dodecyl sulfate) 6 M urea solution, and boiled for 2 min. Where indicated, the dissolved immunoprecipitates were reduced with 50 mM DTT (dithiothreitol) for 30 min at 37”C, followed by alkylation with a 3x molar excess of iodoacetamide under the same conditions. Polyacrylamide

Gel Electrophoresls

The reduced immunoprecipitates were electrophoresed on 6.7% polyacrylamide gels containing 0.1% SDS in 0.1 M sodium phosphate buffer (pH 7.0). The unreduced immunoprecipitates were electrophoresed with the same buffering solution on 2% acrylamide gels containing 0.5% agarose (Sigma Chemical Co.) as stabilizing agent. Gel slices were dissolved in concentrated ammonium hydroxide and counted in Bray’s scintillation liquid. Molecular weight markers consisted of radioactively iodinated human IgG and IgM myeloma proteins.

Cell 92

Acknowledgments This

work

was supported

Received

February

by an NIH Grant

to Dr. John

L. Fahey.

21, 1975

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