Biosynthesis of membrane and secreted ϵ-chains during lipopolysaccharideinduced differentiation of an IgE+ murine B-lymphoma

hfokcular

Immunology,

Vol. 22, No. 1I, pp. 1289-1296, 1985

Printed in Great Britain

0

0161-589Oj85$3.00 + 0.00 1985 Pergamon Press Ltd

BIOSYNTHESIS OF MEMBRANE AND SECRETED c-CHAINS DURING LIPOPOLYSACCHARIDEINDUCED DIFFERENTIATION OF AN IgE+ MURINE B-LYMPHOMA ROBERTO

Laboratorio

di Biologia

Molecolare,

Istituto

SITIA

Nazionale

per la Ricerca

sul Cancro,

16132 Genova,

Italy

(First received 30 January 1985; accepted in revised form 5 June 1985) Abstract-A switch variant of the I.29 murine B-cell lymphoma expressing membrane IgE and inducible by lipopolysaccharide (LPS) to increase the rate of IgE secretion was characterized. The cells (I.29 t +2) express membrane-bound IgE, and also secrete considerable amounts of IgE when grown in regular culture medium. Membrane and secreted IgE contain structurally different heavy chains. The former is constituted by a 93-kd molecule (cm), while secretory chains (cJ have an apparent mol. wt of 86,000. Both L, and es are heavily glycosylated: in the presence of tunicamycin their apparent mol. wt is reduced by approx. 35% (61 kd for c, and 56 kd for cl). Glycosylation is necessary for membrane expression and for secretion of IgE molecules. Stimulation with LPS leads to the disappearance of IgE molecules from the cell surface (determined by radioiodination) although r,-chains are still synthesized, suggesting a defective transport of membrane IgE in LPS-treated cells. The t,: c, ratio decreases upon LPS stimulation. A similar change can be observed in the messenger RNAs specific for c, and L,, possibly suggesting a major pretranslational control for L, and c, biosynthesis,

INTRODUCTION

Immunoglobulins (Ig) may serve as antigen receptors on the surface of B-lymphocytes, or, later in differentiation, as soluble effector molecules secreted by plasma cells (Vitetta and Uhr, 1975). Structural differences between m* and s heavy chains were first demonstrated for IgM (Williams et al., 1978; Vassalli et al., 1979; Kehry et al., 1980). These differences are limited to the COOH-terminal peptide of the heavy chain, which is highly hydrophobic in the m form, to permit the anchorage of the receptor molecule to the lipid bilayer, and hydrophilic (and shorter) in the s form of the p-chain. The two molecules are encoded by different mRNAs, differing at the 3’-termini (Alt et aI., 1980; Early et al., 1980; Rogers et al., 1980; Singer et aI., 1980). A similar situation was reported later for IgG (Oi et al., 1980; Rogers et al., 1981; Singer and Williamson, 1980), IgA (Sitia et al., 1982, Kikutani et al., 1981) and IgD (McCune et al., 1981; Cheng et al., 1982). Due to the paucity of IgE-bearing lymphocytes and serum IgE, rather little is known about the biochemical characteristics of murine c-chains (Ishizaka, 1973; Bennich and Bahr Lindstrom, 1974; Zajdel-Blair et al., 1981). Recently the complete nucleotide sequence of the murine (Ishida et al., 1982) and human (Nishida et al., 1982, Flanagan and

*Abbreviations: AMW, apparent mol. wt; LPS, lipopolysaccharide; kb, kilobases; kd, kilodaltons; m, membrane; PAGE, polyacrylamide gel electrophoresis; s, secreted; SDS, sodium dodecyl sulphate; Tm, tunicamycin.

Rabbitts, 1982; Max et al., 1982) C-genes have been determined. It was previously shown that IgM-bearing cells from the I.29 B-cell lymphoma (Sitia et al., 1981) can switch to IgA and, less frequently, to IgE production (Stavnezer et al., 1985). All isotypes express the same light chain (1) and the same idiotypic determinants. Restriction enzyme analyses demonstrated that the same VDJ complex expressed by I.29 p+-cells is rearranged to C, or C, by recombination events involving the switch regions (S,, S, and S,) (Stavnezer et al., 1984, 1985). Interestingly, the nucleotide sequence of the variable gene is identical in n + - and a + -cells (Klein and Stavnezer, in preparation). Furthermore, like p+- and @+-cells (Sitia et al., in preparation), IgE+ I.29 cells were found inducible to differentiate in response to LPS. The 1.29 system is thus particularly suitable to analyze and compare heavy-chain biosynthesis. In this study we report the biochemical characterization of murine E,,, and L,, as well as an analysis of their biosynthetic pathways during the LPS-induced differentiation of IgE+ I.29 cells. MATERIALS Cell

AND METHODS

lines

I.29 t +2 cells were a king gift of Dr J. Stavnezer (Sloan-Kettering Institute, New York), IgEproducing cells arose from two independent cultures of I.29 p +-cells (IgM+, IgE- and IgA-): the former contained I.29 p+-cells seeded at a high cell density in the absence of insulin, which was originally necessary for in vitro growth of I.29 p+-cells, while the

1289

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ROBERTO SITIA

latter was composed of cells treated for 4 days with LPS and then cultured in regular medium. The same cultures also yielded IgA-producing cells. Cells from the first culture (without LPS) were cloned by limiting dilution, and seven IgE+ clones were isolated (Stavnezer et al., 1985). One of the clones (I.29 6 +2) was selected for further analyses and expanded in RPM1 1640 medium supplemented with 10% fetal calf serum (FCS) (HyClone, Logan, UT), 100 U/ml penicillin, 100 pg/ml streptomycin, 2 mM glutamine, 1 mM sodium pyruvate, 0.1 mM non-essential aminoacids (all from Flow, Milano, Italy) and 5 x lo-’ M 2-p-mercaptoethanol. LPS, purchased from Ribi (Hamilton, MT) was added to cell cultures (0.1-0.5 x 106/ml) at final concn of 5 pg/ml. Surface radioiodination 10 x lo6 viable cells were surface radioiodinated by the lactoperoxidase method as previously described in detail (Sitia et al., 1981). Biosynthetic

labeling

5-10 x lo6 viable cells were washed twice in methionine-free RPM1 1640 and labeled for 6 hr in 1 ml of the same medium supplemented with 10% dialyzed FCS, antibiotics, glutamine and 1OOpCi of [35S]methionine. Tm (E. Lilly) was used to inhibit N-linked glycosylation at a concn of 5 p g/ml. Tm was added 90 min before the addition of [35S]methionine (Sitia et al., 1984). After radiolabeling, cells were washed in ice-cold phosphate-buffered saline (PBS) and lysed on 0.5% Nonidet P40. Antagosan (1%) (Istituto Behring Spa, L’Aquila, Italy) and phenylmethylsulphonyl fluoride (2mM) were added to reduce proteolysis, and cell lysates and supernatants were spun for 15 min at 10,OOOg to remove nuclei and debris, and immunoprecipitated with monospecific antibodies and Sepharose-bound protein A (Pharmacia, Uppsala, Sweden). Total radioactivity incorporated was measured by trichloroacetic acid precipitation. Antibodies

and immunoprecipitations

Monospecific antibodies to p, CI or I.29 idiotype determinants were obtained in rabbits and tested as previously described (Sitia et al., 1981, 1982). Rabbit anti-mouse (RAM) L was a kind gift of Dr Zoltan Ovary (New York University, New York, NY). This reagent did not precipitate I*- or cc-chains from I.29 cells. A monoclonal antibody to the I.29 idiotype (Tada et al., 1981) was also used in immunoprecipitation experiments. It consisted of the ammonium sulphate (40%) precipitate of T lo-219 culture supernatants. Lysates and supernatants from radiolabeled cells were first precleared with Sepharose-bound thyroglobulin and then incubated at 4°C with 0.5-5 pg of antibodies. After more than 2 hr, 50~1 of a 1: 1 mixture of protein A-Sepharose beads and phosphate-buffered saline was added and the samples

were rotated for 3 hr or longer at 4°C. Immunoprecipitates were washed 4 times with 50mM Tris-Cl, pH 7.6, 1 M NaCl, 0.1% Nonidet P40, 1 mM phenylmethylsulphonyl fluoride, 1% Antagosan, once with 10mM Tris-Cl, pH 7.4, and then eluted by boiling for 5min in 2% SDS and 5% 2-/?-mercaptoethanol buffer (Laemmli, 1970). SDS-PAGE SDS-PAGE was performed essentially as described by Laemmli (1970). Gels were fixed, treated with either Amplify (Amersham) or DMSO-PPO (Bonner and Laskey, 1974) and exposed to Kodak XAR-5 films at -80°C. Extraction

of RNA

and northern blotting

Total cellular RNA was prepared by phenol-chloroform extraction of cells lysed with SDS, treatment with RNase-free DNase and ethanol precipitation. Aliquots of 2Opg were denatured by glyoxal and dimethyl sulphoxide, electrophoresed in 1% agarose gels (in 10 mM phosphate buffer, pH 7.0) as described by McMaster and Carmichael (1977), and blotted on diazophenylthioether paper (Seed, 1982). The blots were prehybridized for 6 hr at 42°C in 6 x SSC, 50% formamide, 50 mM phosphate, pH 7.0, 100 pgg/ml E. coli DNA, 100 pg/ml yeast RNA, 5% dextran sulphate, 0.2 M sodium azide and 1% glycine, and hybridized overnight at 42°C in the same solution (without sodium azide and glycine) containing l-3 x lo6 cpm/ml of a [“PI-nick-translated probe specific for C, (Ishida et al., 1982). After several washes in 0.1 x SSPE, 0.1% SDS at 5O”C, blots were exposed to Kodak XAR-5 films at -80°C. Immunojluorescence 5 x lo4 viable cells were attached to each single of poly+lysine-coated well multispot slides (Shandon Instruments): for cytoplasmic immunofluorescence cells were air dried, fixed in 100% methanol, soaked in PBS-FCS (l%), and stained with the appropriately diluted fluorescent antibody, while, for surface staining, the antibodies were added directly to viable cells, usually at a concn five-fold higher. Fluorescein-conjugated RAM idiotype, or RAM 6 followed by fluorescein-conjugated goat anti-rabbit Ig (a gift of U. Hgmmerling, Sloan-Kettering Institute, New York, NY) were used in this study. Densitometric

scanning of the autoradiographs

Autoradiographs were scanned at 600-nm wavelength in a Beckman DU-8 automated spectrophotometer. Several exposures of the same gel were scanned, and the relative areas of the relevant peaks were calculated and compared. RESULTS

Origin and characterization

of 1.2.9 c +-cells

As described by Stavnezer et al. (1985), IgE+ cells originated spontaneously from a culture of I.29

Biosynthesis

1

of murine

t-chains

1291

2

3

4

5

6

7

8

9

10

+

-

-

-

-

+

+

+

+

:* do-

LB:-

Fig. 1. c,,, and L, have different mol. wts. I.29 c +2 cells, cultured for 3 days in the presence (lanes 2 and 7-10) or absence (lanes 1 and 36) of LPS were either surface radioiodinated (lanes 1 and 2) or biosynthetically labeled for 6 hr with [?S]methionine in the presence (lanes 5,6,9 and 10) or absence (lanes 3, 4, 7 and 8) of Tm. Cell lysates (lanes 1, 2, 3, 5, 7 and 9) and supernatants (lanes 4, 6, 8 and 10) were immunoprecipitated with anti-t. Fluorogram of a 10% acrylamide gel. Markers are indicated by arrows and consist of phosphorylase B (92.5 kd), bovine serum albumin (69 kd), ovalbumin (46 kd) and carbonic anhydrase (30 kd).

p+-cells: the cells were cloned by limiting dilution, and one of the seven IgE+ clones isolated, clone 6 ‘2, was selected for further investigation. Over 80% of I.29 ~‘2 cells express surface IgE. This was determined by immunofluorescence, employing either anti-c or anti-I.29 idiotype reagents. The cytoplasm of methanol-fixed cells was also stained, although weakly, by anti-c. Anti-p or anti-cc reagents failed to stain I.29 c +2 cells. When cultured in the presence of LPS, I.29 c+2 cells increased their content of intracellular IgE, while surface staining was barely detectable after 24 hr of stimulation. Ig biosynthesis in control and LPS-stimulated 6 +2 cells

I.29

In order to identify m and s t-chains and analyze IgE biosynthesis in differentiating cells, I.29 c + 2 cells were cultured for 3 days in the presence or absence of LPS; aliquots of 10’ viable cells from both populations were then surface radioiodinated by the lactoperoxidase method, or biosynthetically labelled with [‘%]methionine, in the presence or absence of Tm. %-Chains could be readily identified on the surface of unstimulated cells by radioiodination (Fig. 1, lane 1). Their AMW was found to be much higher

(N 93 kd) than that of the other murine heavy chains (p = 82 kd; tl = 69 kd; y = 65 kd; 6 = 68-70 kd). Interestingly, &,-chains could not be detected on the surface of LPS-stimulated I.29 E + 2 cells (Fig. 1, lane 2) although the same amount of trichloroacetic acid precipitable radioactivity (2 x 10’ cpm) from control and LPS-treated cells was subjected to immunoprecipitation. Immunoprecipitation with monoclonal anti-I.29 idiotype yielded the same results (data not shown). The results of the biosynthetic labeling experiments are shown in Fig. 1 (lanes 3-10). An abundant band of 85-87 kd could be isolated from the supernatants of both unstimulated (Fig. 1. lane 4) and LPS-treated (Fig. 1, lane 8) cells. Its exclusive presence in culture supernatants suggested that this band consisted of ca. Two heavy-chain bands were precipitated by anti-6 from the lysates of both control (Fig. 1, lane 3) and LPS-treated (Fig. 1, lane 7) cells. The major intracellular band (Q had an AMW of 77-80 kd and probably consisted of partially glycosylated precursors to cs, and, to a lesser extent, to t,. A minor band of -88 kd was also present in Fig. 1 (lanes 3 and 7). Although its mobility was slightly slower than that of t,-chains (85-87 kd) it was not possible to determine whether this band consisted of a partially

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ROBERTO SITIA

glycosylated precursor to cm or cs. Figure 1 (lane 7) also contained a minor band of 58 kd, which possibly consisted of partially glycosylated c-chains (see below). When N-linked glycosylation was inhibited by Tm, only one major band, of 56-61 kd, was identifiable in the lysates of control (Fig. 1, lane 5) or LPS-treated (Fig. 1, lane 9) cells. This band must contain both the unglycosylated forms oft, and E,, which could not be resolved in this gel (the two molecules could be easily resolved in longer gels, see below). The faint doublet visible in the 77-79-kd region could represent either partially glycosylated e,,-chains, or more likely the heavy-chain-binding protein that frequently coprecipitates with anti-Ig in I.29-derived and other B-cell lines (Haas and Wabl, 1983; Sitia et al., 1984). Unglycosylated cs molecules could not be secreted by Tm-treated cells (see lanes 6 and 10). This was particularly evident in (Fig. 1, lane lo), where traces of glycosylated L, (86-kd) chains, probably originated from molecules which escaped the Tm block, could be identified, while nothing in the mol. wt range of 58,000-60,000 was present. In summary, LPS-treated cells increased the overall IgE synthesis about two-fold (as determined by gel scanning) [compare also the more intense I-chain bands in Fig. 1 (lanes 7-9)J but expressed much less (if any) membrane-associated IgE, as shown by the radioiodination experiments. c,-Chains were, however, synthesized by LPS-treated cells although at a lower rate than in unstimulated cells. This was shown in Fig. 2, where the anti-Id immunoprecipitates from Tm-treated control (lane 1) and LPS-stimulated (lane 2) cells were analyzed on longer gels. The lighter band could be identified as unglycosylated c,-chains: its intensity increased significantly in LPS-treated cells. As in other isotypes the AMW of unglycosylated c,-chains was higher (61,000) than that of its counterpart (56,000). Glycosylation is necessary for membrane expression as well as for secretion of IgE As demonstrated above (see Fig. 1) the secretion of IgE molecules was totally inhibited by Tm. To determine whether unglycosylated mIgE could be expressed on the cell surface, I.29 t +2 cells were treated with pronase (2 mg/ml) for 20 min at 37°C to remove mIgE, washed, cultured in the presence or absence of 2 pg/ml Tm and stained for various times with fluorescent anti-Id. As evident from Fig. 3b Tm inhibited the reexpression of mIgE by pronasetreated cells. Furthermore, I.29 t +2 cells cultured in the presence of Tm (without pronase treatment) could not be stained on the cell surface after 24 h (Fig. 3a). D&erent mRNAs encode c,- and ES-chains To analyze the messenger RNAs encoding t,- and t,-chains, 20-pg aliquots of total cellular RNA, isolated from the same cell populations that were bio-

Fig. 2. Characterization of unglycosylated L,- and c,-chains. I.29 t +2 cells were cultured for 3 days with (lane 2) or without (lane 1) LPS, labeled for 6 hr with [“Slmethionine in the presence of 4pgg/ml Tm, lysed and immunoprecipitated with monoclonal anti-I.29 Fluorogram of 11% acrylamide

idiotype (Tada et al., 1981). gels. Markers as in Fig. 1.

synthetically in Fig. 1 (Fig. 4b) and Fig. 2 (Fig. 4a), were blotted and the blots hybridized with a probe specific for C, (Ishida et al., 1982). Three bands of 3.5, 2.9 and 1.9 kb were present in both control (Fig. 3, lanes 1 and 3) and LPS-stimulated (Fig. 3, lanes 2 and 4) cells. However, densitometric tracing of the autoradiographies demonstrated that the intensity of the two heavier bands (3.5 and 2.9 kb), which encode t,, decreased 1.5-3-fold in LPS-stimulated cells, while the E,-specific 1.9-kb mRNA increased approx. two-fold. These data correlated well with the changes observed at the protein level (cf. Figs 1 and 2) and suggested that the control of cm and L, biosynthesis operates mostly at the RNA level.

1293

Biosynthesis of rnurine ~-chains DISCUSSION

hours

Fig. 3. Glycosylation is required for membrane expression of IgE. I.29 6 +2 cells were cultured in the presence (closed symbols) or absence (open symbols) of Z.ug/ml Tm. In b cells were first treated with pronase. The results are expressed as percentage of cells stained by tluorescent antiidiotype over total viable cells at the times indicated on abscissa.

We have investigated the biosynthesis and certain biochemical properties of cm- and Es-chains in a murine B-cell line expressing mIgE and inducible by LPS to increase the rate of secretion of IgE molecules. The cell line used in this study (I.29 ~+2) arose spontaneously from a subline of the I.29 tumor expressing mIgM only. It has been previously shown that these cells (I.29 p+) can switch to IgA and, less frequently, to IgE. The frequency of switching can be increased in vitro by the addition of LPS and antiidiotype (Stavnezer et al., 1985). Restriction enzyme analyses (Stavnezer et al., 1982, 1985) and nucleotide sequencing (Klein and Stavnezer, in preparation) demonstrated that an identical VDJ complex is ex-

b

a

LPS

-

I b !-

Fig. 4. Characterization of the LmRNAs. Northern blot analysis of 20 pg total RNA extracted from I.29 6+2 cells cultured for 3 days with (lanes 2 and 4) or without (lanes I and 3) LPS. (a) E RNAs from the cells labeled with )‘S in Fig. 2. (b) t RNAs from cells labeled with % in Fig. I. The blot was hybridized with a nick-translated DNA probe, specific for C, (Ishida ef al., 1982). Markers were end-labeled I Hind III fragments. The lower part of the figure (a’ and b’) shows the densitometric scanning of the blots. L, mRNA increased in LPS-treated cells 1.4- (a’) and 2.1- (b’) fold, while k mRNA (peaks I + II) decreased 2% (a’) and 1.2- (b’) fold. In both experiments the ratio between 3.5 and 2.9-kb e,,, mRNAs was inverted in LPS-treated cells (0.4 + I. I in a’ and 0.8 + 2.2 in b’, respectively).

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ROBERTO SITIA

pressed by I.29-derived p +-, u+- and t +-cells. At the protein level the three isotypes share the same idiotypic determinants (Sitia et al., 1981; Tada et al., 1981). These findings, together with the observation that all three types of cells from I.29 may be induced by LPS to terminally differentiate into secreting cells, make I.29 an optimal model system to investigate the regulation of Eg biosynthesis. At the RNA level, the same regulatory elements are present at the S-end of the messengers for the m and s forms of the three isotypes. This is particularly interesting in view of the fact that, as it will be reported elsewhere (Sitia er al., in preparation), the regulation of IgM differs from that of IgA and IgE. In this study the notion that ,u-, a- and c-chains from I.29 lines share an identical Vu-region allowed us to characterize L,- and es-chains and to compare certain biochemical properties of these two molecules with p- and u-chains. &,-Chains could be identi~ed by the radioiodination technique, that selectively labels externalized molecules: they appear as a broad band of -93 kd (90-96). This heterogeneity is typical of heavily glycosylated molecules, and also characterized c,-chains. The latter are present in large quantities in the supernatants of biosynthetically labeled cells, and display an AMW of -86,000 (82,000-90,000). The lysates of methionine-labeled cells contain two major L bands: the lighter and more abundant intracellular e (ric) has an AMW of - 75,000 (72,00~78,0~) and most probably consists of partially gIycosylat~ precursors of es, and to a lesser extent oft, molecules. The second eicband has a slower mobility (-88 kd). Since it appears to be 2 kd heavier than mature E, (86 kd), this band is likely to consist of partially glycosylated precursors to t,. In this connection, the relative content of the 88 kd tic decreases in LPS-stimulated cells. Alternatively, this band may represent the direct intracellular precursor of secretory chains. As expected, the difference between cmand c, is not due to differential glycosylation: two unglycosylated t-chains of 61 and 56 kd could be isolated from Tm-treated I.29 ~+-cells. The heavier chain (61 kd) corresponds to E,, and the lighter (56 kd) to c*. This assumption comes from: (i) an analogy with other Ig isotypes, where the membrane form is always heavier than the s counterpart (Williams et al., 1978; Vassalli et al., 1979; Kehry et al., 1980, Oi et al., 1980; Singer and Williamson, 1980; McCune et al., 1981; Kikutani et al., 1981; Sitia et al., 1982); (ii) the nucleotide sequence of the murine C-gene (Ishida et al., 1982) predicting a hydrophobic m form 64 residues longer than the &,-chain; and (iii) the observation that the heavier band decreases, while the lighter increases in LPS-treated cells. The data obtained with Tm indicate that e-chains are heavily glycosylat~. This is in agreement with data on human and rat myelomas (Ishizaka, 1977; Bennich and Bahr Lindstrom, 1974; Hickman et al.,

1977; Zajdel-Blair et al., 1981>, and with Ishida et al. (1982) who predicted six sites of N-linked glycosylation from the nucleotide sequence of C,. We have previously shown that unglycosylated IgA molecules could be secreted, but not expressed on the cell surface by Tm-treated 1.29 xi-cells. On the contrary Tm inhibited completely both ~cretion and membrane expression of IgM (Sitia er al., 1984). The data shown here demonstrate that IgE molecules behave like IgM. The inhibition of IgM and IgE secretion by Tm, but not of IgA, is probably related to the different degrees of N-linked glycosylation of the three isotypes. In I.29 cells, sugar chains account for a mass of -30,000 daltons (- 35%) in E-, -20,000 (-25%) in p-, and -8000 (-12%) in a-chains. Similarly to ~1 +- and tl +-cells (Sitia et al., in press, in preparation) I.29 c:‘2 cells respond to LPS by losing membrane-bound IgE and increasing the rate of secretion of Ig molecules. The possibility that LPS selects ceils that lacked an c,-chain is unlikely in view of the fact that the Ioss of surface staining with anti-IgE antibodies occurs within 24 hr after the addition of the mitogen. (The cell cycle of I.29 c +-cells is approx. 15-20 hr.) The changes in protein biosynthesis, best followed in the Tm experiments, are well matched by changes in the mRNAs specific for em (3.5 and 2.9 kb) and es (1.9 kb). Like in the a-system (Sitia et al., in press) the decrease in the heavier E, mRNA (3.5 kb) in LPS-treated cells is more pronounced than that of the minor 2.9-kb em mRNA, suggesting that premature te~ination of transcription rather than preferential splicing of a common transcript is responsible for the increase in the 6,: t, mRNA ratio, although this issue is still unclear. Whatever the molecular mechanism involved, our data suggest that the regulation of t, and E, biosynthesis occurs, like in the cl-system, mostly at the pretranslational level. However, the radioiodination experiments show that I.29 t +2 cells no longer express membrane-associated IgE after 3 days of LPS stimulation. On the other hand LPSstimulated cells do synthesize f,-chains, although at a lower rate than unstimulated cells. Taken together, these findings imply a defect in the transport mechanisms of membrane IgE in LPS-treated celis. As, on the contrary, secretion of IgE increases in such cells, it appears that the intracellular transport systems of mIgE and sIgE are independent. The same conciusion was also reached for IgA molecules on the basis of the differential sensitivity of CI, and c(, to Tm (Sitia et al., 1984). I.29 c +2 cells secrete constitutively a large amount of Ig. We have previously observed a certain variability in the stage of differentiation of a+-clones, reflected in different ratios between a, and CL, (Sitia et al., 1982) but the rate of secretion in I.29 tit- and x+-clones is always at least lo-fold lower than in I.29 e +2 cells. We are presently investigating whether this is a peculiarity of this clone or if a high secretion rate

1295

Biosynthesis of murine f-chains is a property of the IgE+ cell. Perhaps it is worth noting that IgE-bearing human (Romagnani et al., personal communication) and murine (Radbruch, personal communization) normal Iymphocy~es also

secrete considerable

amounts of IgE.

Acknow~edgemenfs-I am indebted to Dr Janet Stavnezer for providing I.29 E+-cells, to Drs T Honjo and Z. Ovary for generous gifts of reagents, to Drs Manlio Ferrarini, Ulrich Hammerling, Anna Rubartelli, Janet Stavnezer and Giorgio Vidali for many stimulating discussions, to MS Susanna Caorile and MS Gabriella Tirelli for tvoina the manuscript, ;o Mr Giovanni Molinari for the phdtographic work, and to Drs Cristina Alberini, Sandro De Ambrosis and Sefania Guazzi for their invaluable help in many experiments. This work was supported by grants from Consiglio Nazionale Ricerche (I’. F. Ingegneria Genetica, 84/00912.51-83/01026.51) and NATO (RG 186jSO).

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NOTE ADDED

IN PROOF

Eight independent I.29 mIgE+ ively high levels of IgE.

clones

secrete

constitut-