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Experimental

EFFECT

Cell Research 101 (1976) 383-395

OF REDUCED

PROCESSING

TEMPERATURE

AND SECRETION

(Ig) BY MOUSE

ON CELLULAR

OF IMMUNOGLOBULIN

MYELOMA

CELLS

R. BOOMHOUR and R. BAUMAL Department

of Zmmuno.!ogy, The Hospital For Sick Children, Ontario, Canada MSG IX8

Toronto,

SUMMARY A pulse-chase experimental design, in which immunoglobulin (Ig) synthesis by mouse myeloma cells could be isolated from subsequent steps in Ig processing and from secretion, was used to study the influence of reduced temperatures (22°C and 2°C) on the cellular handling of Ig and on the attainment of the completed Ig structure. The reduced temperatures blocked Ig secretion and transit of Ig from the smooth membrane fraction to the exterior of the cell, moreso at 2°C than at 22°C. Inhibition could be reversed by restoring the temperature to 37°C. Covalent assembly of heavy(H) and light (L) chains was completed inhibited at 2°C but only minimally blocked at 22°C. The block in covalent assembly was not associated with accumulation of non-covalently bonded Ig intermediates. Attachment of carbohydrate moieties to H chains was inhibited at both temperatures. It is likely that inhibition of Ig secretion at reduced temperatures results from blocks both in the cellular handling of Ig and in the attainment of its final structure.

Relatively little is known about the mechanism of secretion of immunoglobulin (Ig) molecules, apart from some experiments on the kinetics of secretion and on the lack of effect of microtubular agents (colchicine and cytochalasin) on secretion [ 1, 21. It is generally assumed that Ig is enclosed within membranous elements from the time that the heavy (H) and light (L) chains are synthesized on the polyribosomes of the rough endoplasmic reticulum until they traverse the smooth endoplasmic reticulum and Golgi complex, where assembly into H,L, occurs and carbohydrate moieties are added [3]. In contrast to the situation with hormones, neurotransmitters and vasoactive amines which are stored in secretory granules and are released to the exterior of

the cell following a stimulus, Ig molecules are not stored intracellularly but are continually released, possibly by fusion of Igcontaining vesicles with the plasma membranes. In the present study, we have examined the effect of reduced temperatures on the cellular handling of Ig and on the production of intact Ig molecules so as to elucidate the role of these factors in Ig secretion. We have utilized mouse myeloma tumors and cultured lines, which produce large amounts of homogeneous Ig, and a pulse chase experimental design, which has enabled a study of intracellular events isolated from Ig synthesis. We have found that Ig secretion by mouse myeloma cells can be reversibly inhibited at low temperatures and that the block occurs as a Exp Cdl Rr.\ 101 (1976)

384

Boomhour

and Baumal

result of changes produced both in the cellular handling of Ig and in the structure of the Ig molecules themselves. MATERIALS

AND METHODS

Mouse myeloma tumors and cultured cell lines The MPC 11 (lgGZb), MOPC 31C (IgG,) and MOPC 3 I5 (IgA) cell lines and the MOPC 104 (IgM) mouse myeloma tumor were utilized in the present studies. Cultured cells were grown in suspension using Alpha growth medium (Flow) supplemented with 15% fetal calf serum, in a humidified 37°C CO, incubator. The MOPC 104 tumor was grown subcutaneously in BALB/c mice from which isolated ceils were prepared.

Incubation of cells with radioactive amino acids, radioactive sugars and radioactive uridine For incorporation of radioactive amino acids, cells were washed twice with Eagle’s spinner medium containing l/XJth the normal amounts of valine, threonine and leucine and were resuspended at 2X IO6 cells/ml. Sixty &i of a mixture of [“*C]valine, [14C]threonine and rYZ]leucine (VTL, New England Nuclear Corp., uniformly labelled, spec. act. TO.2 Cilmmole) were added. Labelling with radioactive sugars was performed by washing myeloma cells twice in Eagle’s spinner medium lacking glucose, supplemented with either 2.50PM unlabelled D-ghrcosamine and L-fucose (rralactose labelling) or unlabelled o-galactose and Lficose (glucosamink labelling) [4]. The isotopes used were 200 &i of [SH]o-galactose and [3H]n-glucosamine (Amersham-Searle, spec. act. > 1 Cilmole). For labelhng with [*4C]uridine, exponentially growing cells in complete growth medium were supplemented with 50 &i of [3H]uridine (New England Nuclear Corp., spec. act. >5 mCi/mole).

Experimental

designs

(1) Continuous labelling experiments. Myeloma cells were incubated at 37°C continuously with either iadioactive amino acids or sugars for 4 hbr with radioactive uridine for 16 h. Incorporation was stopped by immersing aliquots into an ice bath. (2) Pulse-chase experiments. Myeloma cells were pulsed at 37°C with [14C]VTL for 1 to 10 min and were then chased with a 20-fold excess of unlabelled VTL [5]. After further 1 to 3 min at 37”C, by which time the newly labelled lg chains had been chased off the polyribosomes, a portion of the cells was shifted to either 22°C (room temperature) or to 2°C (temperature of ice bath). Aliquots were removed at various times into tubes containing cold medium. In some cases, cells incubated at 22°C or 2°C were returned to 37°C and further samples were obtained. In one experiment, cycloheximide (50 pg/ml) was added 3 min after the chase, instead of shifting the temperature. Exp CellRrs I01 (1976)

Preparation of cytoplasmic and secretions

lysates

Cells were separated from the culture medium which contains the secreted Ig, by centrifugation at IO00 rpm for 5 min. Lysis was performed using 0.5% Nonidet P-40 (NP-40), in the presence of 0.1 M iodacetamide for analysis of unreduced intracellular Ig. and in the absence of this agent if reduction was to be subsequently performed. The lysates were centrifuged at lOOOOOgfor30min.

Immunologicul precipitations anti-& antisera

and

Labelled lg was precipitated from cytoplasmic lysates and secretions bv adding 10 ul of rabbit anti-mouse lg antiserum, foliowed in 30 min by 200 ~1 of sheep anti-rabbit IgG antiserum (indirect method [6]). After an overnight incubation at 4”C, the precipitates were washed twice with cold 0.02 M phosphate buffered saline (PBS) and dissolved by boihng for 1 min in 0.2 ml of PBS containing 2% sodium dodecyl sulfate (SDS). The anti-mouse Ig antisera were obtained by immunizine rabbits with nuritied MPC 11(laG2b). MOPC 31C (IgTil), MOPC jl5 (IgA) or MGPC I& (IgM) myeloma proteins and contained antibodies against mouse H and L chains. In the experiment on noncovalent assemblv. immune arecioitations were nerformed using an anti-H chain a&serum. The sheep anti-rabbit IgG antiserum was obtained from J. DeRose and Associates, Downsview, Ont.

Polyacrylamide

gel electrophoresis

Dissociated immune precipitates were analyzed for their content of H and L chains (reduced samples) and of lg intermediates (non-reduced samples), by electrophoresis on SDS containing phosphate polyacrylamide gels. In this system, non-covalent bonds are disrupted and molecular species are resolved primarily on the basis of their size. Reduced samples were analyzed on 10 cm 7.5 % gels for 4 h at 90 V. Reductions were performed by adding 0.15 M 2-mercaptoethanol for 1 h at room temnerature. followed bv 0.2 M iodoacetamide. Non-reduced samples were electrophoresed on 20 cm 5% eels for 16 h at 55 V. When MOPC 104 IgM was analised, the top 2 cm of the gel was 31% to allow IgM pentamers to enter the gel.

Radioactivity

determinations

Following electrophoresis, the gels were crushed using a Savant Autogeldivider. For 14Ccounting, fractions were collected on planchettes and counted in a Nuclear Chicago low background counter. For 3H counting, the fractions were collected in vials, scintillation fluid was added (triton, toluene, PPO) and the fractions were counted using an Intertechnique counter.

Temperature Quantitation trichloracetic precipitation

of radioactivity acid (TCA)

+

by

Aliquots of immune precipitates (10-100 ~1) were treated with cold 10% TCA for 30 min at 4°C in the presence of 10% horse serum as carrier. The solution was then filtered onto filter discs (2.5 cm in diameter, Reeve Angel) and washed with 5% TCA. The discs were dried and counted.

Fractionation of myeloma cells by sucrose gradient centrifugation Following incorporation of radioactivity, myeloma cells were washed twice in cold spinner salts, swollen in hypotonic buffer (0.01 M Tris, 0.003 M MgCl,, 0.01 M NaCl) pH 7.5 for 10 min and disrupted by six strokes of stainless steel Dounce homogenizer. The homogenate was centrifuged at 1000 rpm for 5 min to pellet nuclei and non-disrupted cells and the supernatant was applied to a discontinuous sucrose gradient consisting from top to bottom of sucrose (3 ml of 0.4 M, 3 ml of 1.4 M, 3.5 ml of 2 M and 2 ml of 2.3 M) [7]. Following centrifugation for 16 h at 38000 rpm using a Beckman L2-65 ultracentrifuge and an SW40 rotor, opalescent bands were observed at the interphase between the top two layers (smooth membrane fraction, SM) and the 1.4 M and 2 M layers (rough membrane fraction, RM). These were removed using a Pasteur pipette. The SM were pelleted at 45 000 rpm for 2 h to ensure complete separation of membranes from soluble Ig which had been released into the cytoplasmic sap as a result of membrane disruption during the homogenization. The pellet was resuspended in PBS, the membrane-associated Ig was released by lysis with 0.5 % NP-40 and the membranes were centrifuged at 100000 g for 30 min. The RM were also lyzed with 0.5% NP-40 to liberate bound Ig, spun and dialysed against PBS to remove the sucrose, which interfered with immune precipitations. Aliquots of both solubilized membrane fractions were immunologically precipitated, the immune precipitates were dissociated, reduced and analyzed on SDS-containing polyacrylamide gels.

In the experiments on covalent assembly, the amount of HzLz present following electrophoresis on SDS gels was quantitated by measuring the areas of all the Ig peaks on the gels and the amount of HzLz was exmessed as a percentage. In the exueriments using radioactive sugars, the-amount of Hz, formed after 3 h at 37°C was quantitated by area measurement following electrophoresis, and the amounts subsequently formed following temperature shifts were expressed relative to this. In the subcellular fractionation experiments, the amount of Ig in the SM and RM fractions were also obtained by area measurement following electrophoresis. These areas were expressed as per-

c?2"C-I

MOPC

385

k37OC-I

31C CELLS

6

cpmx lo-*. q , Intracellular Ig; q , secreted Ig. Effect of alterations in temperature on the secretion of immunoglobulin. 50X 106 IgG, producing MOPC 31C cells were pulsed for 5 min at 37°C with 60 &i of [14C]valine, threonine and leucine and chased for 3 min at 37°C with a 20-fold excess of these unlabelled amino acids. One-third of the sample was kept at 37”C, one-third was shifted to 22°C and one-third was shifted to 2°C. After 3 h, the cell suspensions were cooled and the cells were separated from the secretions by low speed centrifugation. Cytoplasmic lysates were prepared and both lysates and secretions were immunologically precipitated. Aliquots of the immune precipitates were treated with 10% TCA and counted.

Fig. 1. Ordinate:

centages of the amount of Ig in the SM and RM fractions and in the secretion. All areas were traced out using a polar planimeter.

Electron microscopy SM and RM fractions were fixed for 3 h at 4°C with 2 % glutaraldehyde and then with 1% osmium tetroxide for 1 h at 4°C. The fractions were embedded in Spurr blocks, sections were cut with a Porter-Blum ultra-

Table 1. Effect of alterations

in temperature on protein and immunoglobulin synthesis by mouse myeloma cells Temp. “C

Measurementofareas of immunoglobulin peaks

Z"C4

effect on Ig secretion

Total TCA (cpm)

Ig @pm)

$ cpm)

2 10 15 20

2700 6500 19300 51 900

0 0 200 3 300

ii l:o 6.4

:i 37 40

280 166 700 500 424 400 435 600

22 13 400 600 33 200 39 ooo

8.1 8.0 7.8 8.9

5 X 106IgG, producing MOPC 3 1C myeloma cells were incubated for 30 min with 3 Ki of r14Clvaline, r”Clthreonine and [14C]leucine at ‘the temperatures shown. Cytoplasmic lysates were prepared and samples were precipitated with TCA before and after immunological precipitation with anti-MOPC 3 1C antiserum. ExpCellRes

101 (1976)

386

Boomhour and Baumal

Fig. 2. Abscissa: time (mitt); ordinate: cpmx IO-*. Kinetics of Ig secretion at different temperatures. 100x IO0 Ig&, producing MPC 11 culture myeloma cells were used for a pulse chase experiment as outlined in fig. 1, except that cell aliquots were taken at various times after the chase. At 150 min, a portion of the cells at 22°C and 2°C were returned to 37°C and samples were taken for a further 90 min. The cells were separated from the culture medium, containing the secreted Ig, by low speed centrifugation. The secreted Ig was immunologically precipitated, ahquots of the dissociated immune precipitates were treated with 10% TCA and counted.

microtome (MT-2, Savant Inst.) and collected on copper grids. Electron microscopy was performed using a Phillips Model 300 electron microscope at 60 kV.

RESULTS Secretion of Ig by mouse myeloma cells at different temperatures

The effect of temperature changes on Ig secretion was determined in pulse chase experiments. IgG, producing MOPC 31C cells were incubated for 5 min at 37°C with [‘“ClVTL and chased at 37°C for 3 min with unlabelled VTL. The chase stopped further inExp Cell Rrs 101 (1976)

corporation of label but allowed synthesis of unlabelled Ig to proceed [S]. One aliquot of cells was maintained at 37°C while the remainder were placed at 22°C or 2°C. Following a further 3 h of incubation, the cells were separated from the supernatant medium and lysed. Intra- and extra-cellular Ig were immunologically precipitated with anti-MOPC 31C antiserum and aliquots of the immune precipitates were treated with TCA and counted (fig. 1). The amount of intracellular Ig was greatest at 2°C and least at 37°C. In contrast, there was little Ig secreted at 2°C while over 50% was secreted at 37°C. The results at 22°C were intermediate. Therefore, Ig secretion was markedly inhibited at 2°C and partially inhibited at 22°C. In the foregoing pulse-chase experiment, the reduction in temperature following the chase also reduced Ig synthesis. The inhibitory effect of temperature reduction on Ig synthesis was determined by pulsing IgG producing MOPC 31C cells for 30 min with [14C]VTL at different temperatures. Cytoplasmic lysates were prepared and samples were treated with TCA before and after immunological precipitation (table I). Incorporation of label into TCA-precipitable radioactivity was greatest at 40°C and fell progressively to 2°C. Above 2O”C, a relatively constant percentage of the label was incorporated into Ig. If continual synthesis of Ig is required for secretion to occur, the inhibition of secretion at 2°C and at 22°C could have resulted from decreased Ig synthesis at these temperatures. This possibility was tested by performing a pulse chase experiment at 37°C and adding cycloheximide 3 min after the chase. This drug freezes nascent polypeptides on polyribosomes, thus inhibiting Ig synthesis. The kinetics of Ig secretion were identical in the presence or absence

Temperature effect on Ig secretion 22*c

37°C

CYTOPLASM

CYTOPLASM

SECRETION

387

b212I5 SECRETION

SECRETION

1 3 2 I L

10

20

30

40

50

60

70

L

fraction no.; ordinate: cpmx lO-2. Immunoglobulin species produced and secreted at different temperatures. 100x 106IgM producing MOPC 104 tumor cells were used for a pulse chase experiment as outlined in fig. 1. One-fifth of the cells were maintained at 37°C two-fifths each were shifted to 22 and 2°C. respectively. After another 24 h, one-half of the cells at 22°C and 2°C were shifted back to 37°C

Fig. 3. Abscissa:

40

50

LO

70

and incubation was continued for another 60 min. The cells were then separated from the culture medium by low speed centrifugation and lysed in the presence of 0.1 M iodoacetamide. The cytoplasmic lysates and secretions were immunologically precipitated and the dissociated immune precipitates were analysed by electrophoresis on 5% SDS containing polyacrylamide spacer gels.

another 90 min. There was a rapid restoration of secretion in both cases. In some instances, secretion in the restored samples caught up to the cells maintained at 37°C (fig. 2, top). In other cases, Ig secretion accelerated rapidly with the shift in temperaKinetics of Ig secretion by myeloma ture but by 240 min had not caught up to the cells at different temperatures cells maintained at 37°C (fig. 2, bottom). In A pulse-chase experiment was performed a few instances, secretion by the cells as above using the IgG producing MPC 11 restored to 37°C achieved a level greater cells. At various times after the chase, cell than in the cells maintained throughout at samples were taken and the amount of Ig 37°C. Therefore, these studies showed that the kinetics of Ig secretion were altered at secreted was quantitated by immunological precipitation and TCA determination of the low temperatures but they could be rapidly secretions. At 37”C, the labelled Ig was restored by returning the cells to 37°C. gradually secreted between 30 and 150 min after it had been synthesized (fig. 2) [lo]. Identification of Ig species produced Secretion was completely inhibited at 2°C and secreted by myeloma cells at (fig. 2, bottom) and occurred with markedly different temperatures reduced kinetics at 22°C (fig. 2, top). At Pulse chase experiments were performed 150 min after the pulse and chase, a portion using IgG, IgA and IgM producing myeloma of the cells at 2°C and 22°C were returned cells. Following the pulse and chase at to 37°C and Ig secretion was followed for 37”C, the cells were divided into 5 aliquots, of cycloheximide [9]. Therefore, continued Ig synthesis was not required for Ig secretion in this experiment and by inference, the reduced Ig synthesis in the cold also would not adversely affect Ig secretion.

388

Boomhour

Fig. 4. Electron

and Baumal

microscopy of rough and smooth membrane fractions prepared from mouse myeloma cells. IgG, producing MOPC 31C cells were swollen, homogenized and spun on a discontinuous sucrose gradient, as described in the Materials and Methods. The opalescent bands containing the rough and smooth membrane fractions were recovered, pelleted by

centrifugation for 4 h at 25000 t-pm and the pellets were examined by electron microscopy. (a) Rough Membrane Fraction (R&f), the arrows indicate RM present as both membrane sheets and membrane vesicles; (b) Smooth Membrane Fraction (SM), the arrows indicate SM, Golgi (G), lysosomes (L) and some RM. x35000.

Temperature effect on Zg secretion

389

into SM and RM fractions by sucrose gradient ultracentrifugation. The degree of membrane separation achieved was assessed in three ways. (A) Electron microscopic examination showed that the SM fraction contained primarily smooth membrane vesicles with some contamination by Golgi (G), lysomes (~5)and rough membrane vesicles (fig. 4, bottom). The RM fraction contained rough membranes present as membrane sheets and vesicles (fig. 4, top). (B) [3H]uridine incorporation into RNA was used as a marker for rough endoplasmic reticulum (RM fraction). Of the total TCAprecipitable counts applied to the gradient, 81% were located in the RM fraction. Eleven percent of the counts were at the top of the gradient, likely due to the presence of free monosomes. Only 8% of the label was located in the SM fraction and this may have been caused either by free monosomes which had descended into the SM fraction, or by minimal contamination with rough endoplasmic reticulum, as shown in the electron micrographs (C). The sugar galactose has been reported to be added to H chains of Ig primarily in the smooth endoplasmic reticulum and Golgi [ll, 121. Myeloma cells were therefore labelled with galactose to determine the extent of separation of SM and RM fractions. It was found that equal amounts of galactose-labelled Ig were present in SM and RM fractions. This may indicate that transitional endoplasmic reticulum, containing galactose-labelled Ig, Effect of alterations in temperature migrates with the RM rather than the SM on Zg transport through fraction. Therefore, the three techniques myeloma cells used showed enrichment of SM and RM (A) Fractionation of myeloma cells into fractions in the appropriate subcellular smooth and rough membranes and assess- organelles. (B) Distribution of Zg in smooth and ment of degree of separation. In order to determine whether reduced temperatures rough membrane fractions of myeloma cells interfered with intracellular transport of Ig at different temperatures. A pulse-chase molecules, myeloma cells were separated experiment was performed using the IgGzb

one-fifth was maintained at 37°C while twofifths were shifted to either 2°C or 22°C. Following a 26 h incubation at these temperatures, one-half of the cells at 2°C and 22°C were shifted back to 37°C and incubation was carried out for another hour. The cells were then cooled and separated from the supernatant medium and lyzed. Cytoplasmic lysates and secretions were immunologically precipitated and analyzed by electrophoresis on SDS-containing polyacrylamide gels. Data are shown only for the IgM producing MOPC 104 tumor cells (fig. 3). At 2°C and 22°C there were large amounts of intracellular pZL2, and PL and free L chains, precursors in the biosynthesis of IgM. There was no intracellular accumulation of IgM polymers at these low temperatures. At 37°C there were only small amounts of intracellular Ig intermediates (fig. 3, top). No Ig was secreted at 2°C only a small amount was secreted at 22”C, while large amounts of IgM pentamers, monomeric IgM and free L chains were secreted at 37°C (fig. 3, bottom). The samples shifted from 2°C and 22°C to 37°C began to secrete Ig, the intracellular IgM content decreased and the gel electropherograms were similar to those of the cells maintained at 37°C (fig. 3, right hand panels). These studies confirmed the kinetic charts of fig. 2 and demonstrated the intracellular and secreted Ig species at the various temperatures.

Exp Cd Res 101 (1976)

390

Boomhour und Baumal

Table 2. Effect of alterations in temperature on the transit of irnrnunoglohulirls through the rough and smooth membrane fractions of‘myeloma cells

Temp. (“C)

Secretion (%)

Smooth membranes (%o)

Rough membranes (96)

Ig ratio smooth/rough membranes

31 22 2

97 36 9

2; 52

2 35 39

0.5 0.8 1.3

100~ IO6 IgC,, producing MPC I I myeloma cells were used for a pulsedhase experiment as outlined in fig. I. After 3 h of incubation at the various temperatures, the cells were separated from the supematant medium by low speed centrifugation, swollen disrupted by Dounce homogenization and applied to the discontinuous sucrose gradient described in the Materials and Methods. The SM and RM fractions were obtained, lysed with NP40 and aliquots of the lysates and secretions were immunologically precipitated, reduced and analysed by electrophoresis on IO cm 7.5 % SDS polyacrylamide gels. The areas of the H and L chain peaks on the gel electropherograms were measured using a polar planimeter. The amount of labelled Ig in each of these fractions is expressed as a percentage of the total.

producing MPC 11 cells, as outlined in fig. 1. Following incubation for 3 h at the various temperatures, the cells were pelleted, swollen in hypotonic buffer, disrupted and separated by sucrose gradient centrifugation into SM and RM fractions. The amount of labelled membrane bound Ig in these fractions and of secreted Ig was determined by immunological precipitation and SDS polyacrylamide gel electrophoresis. The area of the H and L chain peaks were measured and these values were converted into percentages (tables 2, 3). At 37”C, 97% of the Ig was secreted, 1% was in the SM and

2% in the RM fraction. At 2°C only, 9% of the Ig was secreted, 52% was in the SM and 39% in the RM fraction. The distribution of Ig at 22°C was intermediate to 2°C and 37°C; 36% was secreted, 29 % was in the SM and 35% in the RM fraction (table 2). The Ig ratio (SMIRM) was 0.5 at 37”C, 0.8 at 22°C and 1.3 at 2°C. Therefore, there was relatively more Ig in the SM fraction at the lower temperatures and this was likely due to a block in its transit to the exterior of the cell. In an additional experiment, the cells which had been shifted to 2°C were returned to 37°C and the distribution of Ig in

Table 3. Effect of alterations in temperature on the transit of immunoglobulins through the rough and smooth membrane fractions of myeloma cells

Temp. “C

Secretion m

Smooth membrane (%I

Rough membrane (%)

Ig ratio smooth/rough membrane

37 2 2437

90 0 35

3 75 30

7 25 35

0.4 3.0 0.9

A pulse-chase experiment was performed using the IgGzb producing MPC 11 myeloma cells, as outlined in fig. 1. The amount of labelled Ig in the SM and RM fractions and in the secretions at 37°C. at 2°C and at the shifted temperature was calculated. Erp Cell Rcs 101 (IY76)

Temperature effect on Zg secretion

391

Fig. 5. Abscissu: fraction no.; or&are: cpmx lo-*. Effect of alterations in temperature on the covalent assembly of immunoglobulins. 10O~loB IgG, producing MOPC 31C cells were pulsed for If mm at 37°C with 60 &i of [‘*C]valine, [‘Tlthreonine and [‘Tjleucine and chased for 1 min at 37°C with a 20fold excess of these unlabelled amino acids. One-third

of the cells were kept at 37”C, whereas one-third/ each were shifted to 22 and 2°C. Cells were removed at 3, 12, and 25 min after the pulse and chase, chilled and pelleted. Cytoplasmic lysates were prepared in the presence of 0.1 M iodoacetamide, immunologically precipitated and analysed by electrophoresis on 20 cm 5 % SDS polyacrylamide gels.

SM, RM and secretion was determined (table 3). At 37°C 90% of the Ig was secreted, 3 % was in the SM fraction and 7 % was in the RM fraction. At 2°C no Ig was secreted, 75% was in the SM and 25% in the RM fraction. In the cells which were shifted from 2°C back to 37°C 35 % of the Ig was secreted, 30% was in the SM and 35% in the RM fraction. The Ig ratio (SM/ RM) was 0.4 at 37”C, 3.0 at 2°C and 0.9 in the cells shifted from 2°C to 37°C. Therefore, the accumulation of Ig in the SM fraction at 2°C could be reversed by restoring the temperature of the cells to 37°C.

cells. The cells were pulsed for only lf min at 37°C to show as much Ig intermediates as possible, and chased for 1 min at 37°C. They were then divided into 3 portions, one remaining at 37°C and one shifted to each of 22°C and 2°C. Cell aliquots were obtained at various times, lysates were prepared, immunologically precipitated and analysed without reduction on SDS polyacrylamide gels (fig. 5). In the cells maintained at 37”C, covalent assembly progressed rapidly and the amount of HzL2 increased from 20% to 72 % of all the Ig intermediates present over the 25 min time course of the experiment (fig. 5, right hand). In the cells shifted to 2°C covalent assembly was completely inhibited and the amount of HzLz present at 3 min (15 %) did not differ from that present at 25 min (fig. 5, left panel). At 22”C, the amount of H,L, increased 3-fold from 17% at 3 min to 47% at 25 min (fig. 5, middle panels). These studies showed that covalent

Effect of alterations in temperature on the covalent assembly of Zg by myeloma cells The effect of reduced temperatures on the rate and completeness of covalent assembly of Ig was determined in pulse chase experiments using IgG, producing MOPC 31C

Exp Cd Res 101 (1976)

392

oI?

Boomhour and Baumal

,

,

,

,

(

1

0

,

(

60

70

25mm

10

20

i0

A0

50

0

10

20

JO

fraction no.; ordinate: cpmx lo-*. Effect of alterations in temperature on non-covalent assembly of immunoglobulins. A pulse-chase experiment was performed using MOPC 31C myeloma cells

Fig. 6. Abscissa:

40

50

b0

70

0

,o

20

30

40

50

b0

70

as outlined in fig. 5. The immune precipitations were performed using antiserum possessing only anti-H chain specificity.

assembly was completely inhibited at 2°C covalent assembly and immune precipitations were performed using anti-H chain but only partially inhibited at 22°C. antiserum (fig. 6). No free L chains were Effect of alterations in temperature demonstrable on the gels at any of the temon the non-covalent assembly of Ig peratures either early on or late following by myeloma ceils the pulse and chase (fig. 6, top and bottom). In previous studies, it was shown that This indicated that large amounts of nonimmunologic precipitation of labelled covalently bonded H2L2, H2L or HL were myeloma cell cytoplasmic lysates with not present at the low temperatures and anti-H or anti-L chain antisera, followed by that the block in covalent assembly at these SDS polyacrylamide gel electrophoresis, temperatures was not associated with acwas a valid method for studying non- cumulation of non-covalently bonded Ig covalent assembly of Ig [5]. Precipitation of intermediates. lysates prepared from IgG-producing cells with anti-H chain antiserum failed to show Effect of alterations in temperature free L chains on the gels, which would have on the addition of carbohydrate been seen if non-covalently bonded H2L2, moieties to H chains HzL or HL were present. This indicated The effect of alterations in temperature on that there was no large pool of these non- the addition of carbohydrate moieties to H covalently bonded molecules, implying that chains was determined by incubating IgG,non-covalent assembly was rapidly fol- producing MOPC 3 1C myeloma cells with lowed by covalent assembly. A pulse chase [3H]glucosamine for 3 h, in order to saturate experiment was carried out to study the the intracellular pool of this labelled sugar. effect of alterations in temperature on non- An aliquot of cells was examined for sugar

Temperature effect on Ig secretion Table 4. Effect of alterations in temperature on the amount of carbohydrate incorporated into immunoglobulin Cellular Amount of [3Hlglucosamine labelled Ig after 3 h at 37°C Pulse [W]amino acids for 10 min at 37°C and chase for 5 min at 37°C Amount of glucosamine labelled Ig after Zt h at 2°C

%

Intra (1.47)=100

IgG, producing MOPC 31C cells were labelled for 3 h with [3H]glucosamine and then used for a pulse-chase experiment with [‘Q&no acids as outlined in fig. 1. The purpose of the [W]amino acids was to serve as an internal standard to correct for sampling errors. After a further 2f h, the cells were separated from the secreted Ig by low speed centrifugation, lysed and both intra and extracellular Ig were immunologically precipitated and analysed on SDS polyacrylamide gels. The areas of the carbohydrate-labelled Ig peaks were measured using a polar planimeter, and expressed as a percentage of the amount of intracellular Ig present at 3 h. The figures in parentheses are the actual measured areas obtained.

labelled Ig by immunological precipitation of lysed cells followed by SDS polyacrylamide gel electrophoresis. The Ig was quantitated by measurement of the H2L2 area by polar planimetry and was assigned a value of 100% (table 4). The remainder of the cells were centrifuged, washed and pulsed at 37°C for IO min with [14C]VTL. Following a 5 min chase at 37°C the cells were divided into three aliquots, one being kept at 37°C while the others were shifted to 22°C and 2°C. The purpose of introducing the [14C]VTL was as a control for subsequent sampling errors. The cells were incubated for a further 24 h and the amounts of sugar labelled intra- and extra-cellular Ig were determined by immunologic precipita-

393

tion, SDS polyacrylamide gel electrophoresis and area measurement of the H,L, peaks. These amounts were compared with the amount of sugar labelled Ig present at 3 h in order to determine whether additional sugar became attached to the Ig at the altered temperatures (table 4). It was found that at 37”C, 2.7 times more glucosamine was present on the Ig molecules 2$ h after labeling with [14C]VTL. In contrast, either negligible or no sugar was added to Ig at 22°C and at 2°C. Therefore, these studies showed that addition of carbohydrate moieties to H chains was temperature dependent. DISCUSSION The purpose of the present study has been to explore the effect of reduced temperatures on the cellular processing and secretion of Ig molecules by mouse myeloma cells. A number of investigators have studied Ig secretion by rabbit lymphocytes at reduced temperatures using a continuous labelling design and concluded that inhibition of secretion was a consequence of decreased synthesis [ 13, 141. The pulsechase experimental design used in the present study enabled intracellular handling and secretion to be studied at the altered temperatures after Ig synthesis had occurred at the optimal temperature of 37°C. Moreover, since cycloheximide inhibited Ig synthesis but did not affect Ig secretion, it was concluded that continued synthesis of Ig was not necessary for secretion. However, continued synthesis of other cellular constituents, such as the postulated transport protein which fixes L chains to the endoplasmic reticulum, could conceivably be required for secretion [ 151. The kinetics of Ig secretion at 37°C by mouse lymphoid and myeloma cells have Exp CellRrs

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been previously described with results similar to those reported here [9]. Following a 15 to 30 min lag, Ig appears in the secretion of normal lymphoid cells in a time ordered manner (i.e. the first Ig made is secreted) and of myeloma cells in a random fashion (i.e. there is a mixing of old with newly synthesized molecules). The kinetics of Ig secretion were markedly altered at low temperatures and a number of mechanisms affecting both the cellular handling of Ig and the Ig molecules themselves were likely responsible. Temperature reduction may have two effects on the myeloma cells themselves. Firstly, secretion has been shown in other systems to require metabolic energy. For example, amylase secretion from the pancreas depends upon maintenance of oxidative phosphorylation, while glycolysis acts as the main energy source for insulin secretion [16, 171. Work in our laboratory has indicated that 2,4-dinitrophenol, an inhibitor of oxidative phosphorylation, completely blocks Ig secretion by myeloma cells. The block in Ig secretion at the reduced temperatures could have been due to inhibition of energy production and decreased cellular metabolism. Nevertheless, cell viability was retained since secretion was rapidly restored when the cells were returned to 37°C. A second possible mechanism resulting in inhibition of Ig secretion may reside in the effect of temperature changes on the physical state of the lipid components of cell membranes. Lipidcontaining membranes are normally in a fluid state at physiological temperatures. With temperature reduction, a point is reached (transition temperature) at which the lipids became more ordered, form a crystalline-gel and act as an impermeable barrier to the migration of molecules. Ig molecules are enclosed within the mem-

branous elements of the cell throughout their intracellular transport. either free within the cisternal space. associated with the membranes via hydrophobic interactions, or embedded in the lipid layer of the membranes via the Fc portion of the H chain. Transport might involve lateral diffusion along or through the lipid layer of the endoplasmic reticulum. Reduced temperatures, which increase the phase transition of the membranes and decrease their fluidity, would be expected to inhibit Ig transport and secretion. Moreover, postGolgi vesicles are assumed to migrate to and fuse with the plasma membrane of the cell, leading to release of Ig to the exterior via reverse pinocytosis [3]. These postGolgi vesicles may be pinched off from the Golgi zone of the smooth endoplasmic reticulum. By interfering with the fluidity of lipid layers, reduced temperatures may block the “pinching-off’ process and the subsequent fusion of these vesicles with the plasma membranes. In addition to these cellular events affecting the handling of Ig molecules, the reductions in temperature also prevented the Ig from attaining its final structure. Firstly, covalent assembly of H and L chains was slightly inhibited at 22°C but at 2°C it was completely blocked. The blocked assembly was not associated with accumulation of large amounts of noncovalently bonded Ig intermediates. Restoring the temperature to 37°C led to a rapid restoration of Ig assembly. Secondly, Ig molecules are glycoproteins and their intracellular transit coincides with the addition of carbohydrate moieties to H and rarely to L chains [ 18, 191. The importance of carbohydrate attachment for transport and secretion is not completely clear. Melchers used 2-deoxy-o-glucose to inhibit glycosylation and showed that this agent

Temperature effect on Ig secretion inhibited Ig transport from the polyribosomes and rough to the smooth endoplasmic reticulum [20]. He postulated that addition of glucosamine was necessary for the initial stages of intracellular transport, and that it served as a “tag” to identify those molecules destined for transport and secretion from those which were not. On the other hand, Ig was not inhibited by deoxyglucose in its transit from smooth endoplasmic reticulum to the exterior of the cell, indicating that addition of galactose, fucose and sialic acid residues were not required for secretion. Two other observations point against a role for carbohydrate in secretion; carbohydrate-free L chains are secreted by mouse myeloma cells, and variant myeloma cells producing intact H chains with carbohydrate residues, but no L chains, fail to secrete the H chains [21]. The present studies have shown that addition of the bridge sugar glucosamine to H chains is inhibited at both 22°C and 2°C. Sugars are added through the action of membrane bound glycosyl transferases and lowered temperatures would be expected to affect the action of these enzymes. If glycosylation is needed for intracellular transport and/or secretion, the reduced temperatures may have inhibited secretion by inhibiting glycosylation. In summary, inhibition of Ig secretion by myeloma cells at reduced temperatures likely occurs as a result of the cumulative effect of a number of processes on the cel-

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lular on handling of Ig and on attainment by the Ig molecules of their final form. This work was supported by the Medical Research Council of Canada and was submitted in partial fulfillment for the M.Sc. degree in the Institute of Immunology, University of Toronto.

REFERENCES 1. Melchers, F, Biochem j 119 (1970) 765. 2. Parkhouse, R M E & Allison, A C, Nature new biol235 (1972) 220. 3. Uhr, J W, Cell immunol 1 (1970) 228. 4. Melchers, F & Anderson. J. Transulant rev 14 (1973) 76. 5. Baumal, R, Potter, M & Scharff, M D, J exp med 134 (1971) 1316. 6. Horowitz, M S & ScharlT, M D, Fundamental techniques in virology, p. 297. Academic Press, New York (1969). 7. Melchers, F, Biochemistry 10 (1971) 653. 8. Laskov. R. Lanzerotti. R & Scharff. M D., J cell biol56 (1971) 327. 9. Baumal, R & Scharff, M D, Transplant rev 14 (1973) 163. 10. Scharff, M D, Shaniro. A L & Ginsbere. B. Cold Spring Harbor symp quant bio132 (1%8)235. 11. Zaaurv. D J, Uhr. J W, Jamieson. J D & Palade. G E,j cell bio146 (1970)‘52. 12. Uhr, J W & Schenkein, I, Proc natl acad sci US 66 (1970) 952. 13. Helmreich, E, Kern, M & Eisen, H N, J biol them 237 (1962) 1925. 14. Sidorova, E U, Bull exp biol med USSR 72 (1971) 1415. 15. Schubert, D & Cohn, M, J mol biol38 (1968) 273. 16. Schramm, M, Ann rev biochem 36 (1967) 152. 17. Levine, R, New Eng j med 283 (1970) 522. 18. Melchers, F & Knopf, P M, Cold Spring Harbor symp quant biol 32 (1967) 255. 19. Schenkein, I & Uhr, J W, J cell bio146 (1970) 42. 20. Melchers, F, Biochemistry 12 (1973) 1471. 21. Morrison, S L & Schartf, M D, J immunol 144 (1975) 655. Received January 26, 1976 Accepted March 17, 1976

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