Molecular and functional studies of recombinant soluble Fcγ receptors

0161-5890/90$3.00+ 0.00 Pergamon Press plc

MolecularImmunology,Vol. 27, No. 12, pp. 1201-1207,1990 Printed in Great Britain.

MOLECULAR AND FUNCTIONAL STUDIES OF RECOMBINANT SOLUBLE Fey RECEPTORS C. SAU’I%S,*N. VARIN, P. M. HoGARTH,? J. C. UNKELESS,~ C. TEILLALJD,J.EVEN, A. LYNCH and W. H. FRIDMAN INSERM U.255, Institut Curie, 26 rue d’Ulm, 75251 Paris, Ctdex 05, France; tuniversity of Melbourne, Parkville, Victoria 3001, Australia; and $Mount Sinai’ Medical Center, New York, U.S.A. (First received 21 February 1990; accepted 21 March 1990)

INTRODUCTION Immunoglobulin-Binding Factors (IBF) are molecules involved in the regulation of isotype production. IBF specific for each Ig isotype have been successively described: first IgG-Binding Factors (IgG-BF), then IgE-BF, IgA-BF and IgD-BF. IgGBF inhibits IgG production during secondary in vitro antibody responses and IgM production during primary responses in vitro (for review see Fridman and Sautks, 1990). Soon after their discovery, it became clear that, in the mouse, IBF are produced by T cells which express receptors for the Fc portion of Ig (FcR), and that T cells equipped with FcR for a given isotype produce IBF for the same isotype (Fridman et al., 1981). Moreover, it was shown, by several groups, that FcR disappear from the cell surface during IBF production, suggesting that IBF may be soluble forms of FcR (Fridman et al., 1981). This hypothesis was reinforced by the observations that IBF share antigenic determinants with low affinity FcR (Da&on et al., 1985). Besides these in vitro findings, evidence progressively emerged that IBF and FcR positive T cells may have an important role in viuo. Lynch and co-workers described that patients or mice with Ig secreting tumors have higher frequencies of T cells expressing receptors for the same isotype than the paraprotein (Hoover et al., 1981). An increase of FCCXand FccR positive T cells was also found in patients with IgA nephropathy (Adachi et al., 1983) and hyper IgE syndrome, respectively (Young et al., 1984). More recently, the use of monoclonal antibodies allowed the detection of IBF in serum in the mouse (Khayat et al., 1984, 1986; Pure et al., 1984) and in the human (Khayat et al., 1987; Sarfati et al., 1988; Fridman et al., 1988). In human lymphoproliferative disorders, it was shown that IgE-BF (Sarfati et al., 1988) and IgG-BF (Teillaud et al., 1990) circulating levels are significantly modified, suggesting that IBF may play a role in tumor progression. Circulating IgG-BF may be produced by different subsets of cells since it has *Author to whom correspondence

should be addressed.

been shown that, in the mouse, in addition to T cells (Ntauport-Sautes et al., 1975), macrophages (Loube and Dorrington, 1980) and B cells (PurC et al., 1984) shed their membrane Fey R. In the latter case, soluble Fey R had an A4, of 45,000. In the mouse, IgG-BF produced by T cells were identified as glycoproteins with size and charge heterogeneity. Several IgG-BF species of IV, 74-78,000, 38-40,000 and 19-23,000 and of p1 5.3, 6.3, 7.7 and 8.4 were described (Blank et al., 1986). The p1 5.3 and M, 38-40,000 component was found to react with a rat anti-mouse low affinity FcyR (Fey RII), mAb 2.462, and the 74-78 kDa and 19-23 kDa species were found to be aggregates and degradation products of that component respectively (Blank et al., 1989). Low affinity receptors for IgG (Fey RII) are transmembrane glycoproteins of M, 50-70,000 on SDS-PAGE (for review see Anderson et al., 1989; Ravetch and Anderson, 1990). Two genes, c( and p, encoding murine Fey RI1 recognized by mAb 2.462 have been described (Hibbs et al., 1986; Ravetch et al., 1986). The proteins display two nearly identical ligand-binding domains of 180 amino acids long coupled to divergent transmembrane and intracytoplasmic domains. The a gene is expressed on macrophages, NK and mast cells (Benhamou et al., 1990). Two fi gene transcripts, /3 1 and 82 have been identified. The 2.462 positive T and B cells contain /3 1 transcripts whereas 82 is expressed in macrophages and mast cells (Benhamou et al., 1990). The precise relationship between Fey RI1 and IgGBF is still not precisely understood, nor the cellular events which generate IgG-BF production. For instance, IgG-BF could be released by cleavage of membrane Fey RI1 or produced by alternative splicing of mRNA coding for Fey RII. The availability of j3 1Fey RI1 cDNA and of genetic engineering techniques recently gave us the opportunity to directly address this question. We show that (1) recombinant soluble Fey RI1 containing the two extracellular domains of the j 1 molecule have the same functional properties as IgG-BF and (2) IgG-BF can be formed by proteolytic cleavage of membrane Fey RI1 1201

c.

1202

SAUtiS

occurring near amino acids 175-180. Moreover, we present evidence that the circulating levels of IgG-BF are highly increased in mice during the growth of IgG-secreting tumors, suggesting a role for IgG-BF in the immune defense against B cell tumors.

RECOMBINANT SOLUBLE FcyRII HAS THE FUNCTIONAL PROPERTIES OF I$-BF

We first addressed the question of whether a recombinant soluble Fey RI1 constructed by genetic engineering has the suppressive activity of IgG-BF. For this purpose, a cDNA encoding /I 1FcyRII (Hibbs et al., 1986) was mutated by the creation of a stop codon at the Lys”’ codon. Mouse L cells were transfected with this cDNA inserted into an expression vector. A cell line CulB3 which secretes recombinant soluble Fey RI1 reacting with mAb 2.462 was selected. This truncated Fey RI1 had the following characteristics: (1) it is a glycoprotein with two molecular species of M, 44,000 and 34-38,000 and of p1 4.5 and 6.3; (2) it expresses the epitope reacting with mAb 2.462 and binds murine IgGl, IgG2a, IgG2b but not IgG3 or F(ab’)2 fragments of IgG; and (3) after deglycosylation by N-glycosidase F, it gives a single polypeptide of M, 19,000. The material was purified to homogeneity by ion exchange chromatography and affinity chromatog-

et ul

raphy on murine IgG2a. More recently, the second purification step was replaced by hydrophobic interaction chromatography on phenyl sepharose which led to the obtention of pure (more than 98%) truncated Fey RI1 (Fig. 1A). The biological activity of this recombinant molecule was tested on secondary in vitro IgG antibody responses against SRBC. As shown in Fig. 1, l-10 ng/ml of sFcyRI1 were able to inhibit 25573% of the production of IgG anti-SRBC as measured by indirect plaque formation. Similar results were obtained by using recombinant factor purified on IgG2aasepharose (Varin et al., 1989) and it was shown that: (1) 100 ng of sFcyRI1 were required for a complete inhibition of the response; (2) the suppressive activity of sFcy RI1 specifically bound to-and could be eluted from-insolubilized IgG2a or mAb 2.462, but not control F(ab’)2 fragments; and (3) the recombinant sFcyRI1 also inhibited the primary IgM response, although to a lower extent (Varin et al., 1989). In the same assays, control material secreted by non transfected L cells failed to exert any effect. Independently, Unkeless et al. have produced a cell line, D 1959, which secretes a 176 amino acid-long truncated Fey RI1 containing the 172 N-terminal amino acids of Fey RI1 and four accessory amino acids (GLIN) (Qu et al., 1988). This material was purified by ion-exchange chromatography and

A

60 -

0.02 ;g

SDS-PAGE Fig.

1A

s

Fc;R

10

Recombinant and soluble Fey receptors

1:10000

1:5000~

1203

l:loool

1:500

DILUTION

Fig. 1B Fig. I. Recombinant sFcyRI1 inhibits antibody production in vitro. Purified sFcy RIX secreted by the CuiB3 cell line (A) and by D1959 cells (8) was analysed by SDS-PAGE after labeling with [‘zsI]and 0.1 ml were added together with SRBC, to 1ml spleen cell cultures of mice sensitized with SRBC. Five days later, indirect IgG PFC were measured. Results are expressed in % inhibition of PFC as compared to control cultures: A = 1188 & 127 PFC. B = 1974 PFC + 129.

affinity chromatography on murine IgG2a. Material eluted at pH4 was analysed by SDS-PAGE after labeling with [“‘I]-Na (Fig. 1B) and tested for its suppressive activity on secondary in vitro IgG antiSRBC responses. As shown in Fig. 1B this sFcy RI1 also inhibited IgG anti-SRBC production, in a dosedependent manner. These results demonstrated that two distinct recombinant sFcyRI1 containing the two extracytoplasmic domains of the molecule, expressing the epitope recognized by mAb 2.462 and having respectively h4, of 44,000 and 34-38,000 (Varin et al., 1989) and M, of 31-33,000 (Qu et al., 1988) are able to suppress primary and secondary anti-SRBC responses in z&o. These molecules are thus biologically similar to the T cell produced-IgG-BF of Mr 38-40.000 which reacts also with mAb 2.462 (Blank er al., 1989). I&&BF

IS PRODUCED BY CLEAVAGE MEMBRANE FeyRII

OF

The structural relationship between FqRII and IgG-BF was then investigated by using eukaryotic cells which express recombinant membrane Fey RII,

and antibodies which react specifically either with the extracellular domain (mAb 2.462) or with the intracytoplasmic (IC#I) domain (Anti-ICj antibodies) of the #I1FqRII. The Anti-ICP antibodies were prepared by immunizing rabbits with a KLH-coupled peptide corresponding to the I5 COOH terminal amino acids of the fll molecule. Mouse L cells were transfected with a p lFcy RI1 cDNA inserted into a eukaryotic expression vector and transfectants were screened for Fey R expression as previously described (Da&on et af., 1989). A cell line, DIBl which expresses high levels of Fey RII, as measured by a direct binding assay using (‘251]-mAb 2.4G2, was selected. The DIBI cells bound 4.106 mAb 2.462 molecules per cell and formed rosettes with erythrocytes coated with IgGI, IgG2a, IgG2b but not IgG-3 (Da&on et al., 1989). We first investigated whether these cells release IgG-BF by solubilization of their membrane FqRII. For this purpose, cells were labeled with [‘*‘I]-Na using lactoperoxydase and incubated for 3 hr at 37°C in culture medium (DMEM 5% FCS). Supernatants and detergent-soluble membrane extracts from

1204

C. Smtis

MEMBRANE

55

et a/.

Fc y RII

SHED Fc y RII

I

200

I

92

m

69

I

46

kDa

-

.

30

.

14

39 kDa

Fig. 2. Membrane Fey RI1 is shed under a soluble form. membrane extract and su~matant from a 3 hr incubation at 37°C of surface labeled DlBl cells were incubated with sepharose coupled to mAb 2.462 or to F(ab’)Z anti IQ antipeptide antibodies. Bound material was eluted, run under reducing conditions on 12.5% acrylamide gels in the presence of SDS and submitted to autoradiography.

radiolabeled cells were then immunoprecipitated either with mAb 24G2-sepharose or with sepharose coupled to F(ab’)2 fragments of IgG from antiICP peptide antiserum. As shown in Fig. 2, immunoprecipitates from cell membranes contained 51-69 kDa molecules which react with 2.462 and correspond to recombinant Fey RII. Anti-IQ antibodies immunoprecipitated molecules of similar iw, showing that these antibodies react with native Fey RII. By contrast, DlBl supernatants contained a radioactive component of smaller M, (36-45,000), which bound to mAb 2.4G2-sepharose but not to anti-ICP-sepharose (Fig. 2). Further experiments

showed that this component binds with low affinity to mouse IgG2a (Sautes et al., unpublished data) but not to the corresponding F(ab’)2 fragments. We thus concluded that DlBl cells release a fragment of Fey RI1 which lacks the COOH terminal part of the molecule but has kept the epitope reacting with mAb 2.462 and the ability to bind the Fc portion of IgG. The domain of Fey RI1 that is cleaved to generate soluble Fey RI1 was then investigated more precisely by estimating the M, of the deglycosylated polypeptide, and was compared to the 175 amino acid-long truncated Fey RI1 described above which corresponds to the two extracellular domains of

Recombinant and soluble Fey receptors

1205

sFc yRII FROM CELLS TRANSFECTED

61 Fc yRll cDNA

WITH

mutated cDNA

92 69

46

19kDa

17kDa

14

+

-

Endo F

+

-

Endo F

Fig. 3. Comparison by SDS-PAGE and autoradi~graphy of soluble Fey RI1 shed by DiBl cells (left) and secreted by the CuIB3 cell line (right) before (-) and after (+) treatment by N-endoglycosydase F.

Fey RII. After endo F treatment,

both polypeptides had M, of 19,000 and 17,000 respectively (Fig. 3) whereas de~ycosylated Fey RI1 had the expected M, of 38,000 (data not shown). In a second set of experiments, we investigated whether soluble Fey RI1 was spontaneously secreted by DlBl cells during culture. The 2.462 soluble Fey RI1 present in supematants from five days culture of DlBl cells was purified by ion-exchange chromatography and affmity chromatography on mAb 2.4G2-sepharose. SDS-PAGE analysis of the material eluted at acid pH showed that culture supernatants contain 28-43 kDa soluble Fey RI1 which MIMM

27112-D

react with mAb 2.462 and contain after deglycosylation, two polypeptides of M, 19,000 and 20,000 (Sautes et al., unpublished data). These data demonstrate that IgG-BF can be formed by enzymatic cleavage of membrane Fey RII, the proteolytic site(s) being probably located around amino acid 175. Interestingly, in this area, the amino acid sequence contains several sites for putative enzymatic attack near the transmembrane domain. In the human, Delespesse et al. (1989) have also shown that the 25 kDa IgE-BF is produced by proteolytic cleavage of the 45 kDa B cell Fcr RII. In that case, the enzymatic attack generates a 37 kDa component

1206

c. SAW!&

et al

A

which, by autoproteolysis, gives 25 kDa IgE-BF. Other mechanisms may also generate IBF from FcR. For instance, for human neutrophils (Scallon et al., 1989; Huizinga et al., 1989), Fey RI11 molecules are anchored to cell surface glycans via phosphatidylinositol linkage and can be solubilized by the action of PI-phospholipase C. Indeed, these observations do not exclude the possibility that some IBF may be secretory molecules produced by splicing mechanisms (Warderman et al., 1989).

I&-BF CIRCULATING LEVELS ARE INCREASED MICE BEARING IgC-SECRETING TUMORS

"NORMAL LICE-

IN

In order to approach the question of the role of IgG-BF in the host defense against tumors, the levels of circulating IgG-BF were measured in tumorbearing mice. by using a semi-quantitative immunodot assay on nitrocellulose with [i251]-labeled mAb 2.462 followed by autoradiography. In a first set of experiments, normal mice were inoculated with syngeneic IgG2a secreting tumors which grew either as ascites or as solid tumors, such as hybridoma B cells (UN2 which secrete IgG2a anti-SRBC), lymphoma cells (A.20.2J and II A1.6) or myeloma cells (HOPC 1). Mice were bled at various days after tumor injection and the amount of circulating IgG-BF was estimated by measuring the spot density area as compared to that observed at day 0. As illustrated in Fig. 4A. IgG-BF levels increased in tumor-bearing animals, reaching up to 19 times the normal level. The circulating IgF-BF most probably does not come from the tumor but from the host, since mice injected with IgG2a-secreting tumor cells (IIA1.6) in which the Fc;RII fl gene is deleted, had similar levels of circulating factor as mice injected with the Fey RI1 positive A20.2J cells (Fig. 4A). Moreover, in nude mice bearing IgG secreting-tumors (IIA1.6 and A20.2J secreting IgG2a, BIOT secreting IgGl or Ar 13.4.9 secreting IgG2b anti-PC), the amounts of serum IgG-BF were either not significantly modified or multiplied by a factor below 4 (Fig. 4B). Indeed, these results are reminiscent of the observations of higher frequencies of Fey R positive T cells in patients or mice which bear IgG-secreting tumors (Hoover et al., 1981) and of the possibility that an isotypic regulatory circuit involving FcR positive T cells, B cells, IBF and Ig may operate in vivo (Fridman and Da&on, 1986). To investigate the role of IgG in this phenomenon, normal mice were grafted with syngeneic non Ig-secreting tumors such as a B cell hybridoma (SP2/0), T lymphoma (BW5147) or a melanoma (B16). In these cases, the levels of circulating IgG-BF remained unchanged or were multiplied by less than 3 (Fig. 4A), suggesting that IgG act as an important inducer of IgG-BF secretion in vivo. The existence of sera which contain high levels of IgG-BF gave us the opportunity to isolate and characterize this molecule. Serum IgG-BF was purified by affinity chromatography with mAb 2.462.

B “NUDE

IgG SECRET.

MICE”

TUMORS

Fig. 4. IgG-BF serum levels in tumor-bearing mice. The IgC-BF content of 5 ~1 of sera of groups of 611 normal or nude mice before and during tumor growth were estimated by an immunodot assay on nitrocellulose with [rZ51]mAb 2.462. The means of the ratio of the spot areas given by sera taken at 20-30 days and at day 0 after injection of tumor cells are indicated on the vertical axis.

Analysis by SDS-PAGE showed that both in reducing and non-reducing conditions, IgG-BF had a M, between 41 and 48,000 and bound to mAb 2.462, and to mouse IgGl, IgG2a and IgG2b isotypes but not to F(ab’)2 fragments (Lynch et al., 1990, submitted for publication). Obviously, this circulating factor has a M, which is significantly higher than the factor which is produced upon incubation of T cells in vitro (3%40,000) or to that of the recombinant molecules

Recombinant described

herein

these

discrepancies

further

studies.

(34644,000). The may

be

of

significance

interest

and

and soluble of await

Acknowledgemenrs--We wish to acknowledged the expert technical assistance of Annie Galinha and Marie-Annick Marloie and the skilful secretarial help of Micheline Charm This work was performed with a grant from the Institut Scientifique Roussel Uclaf (83 013).

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