Target cells for an immunosuppressive cytokine, glycosylation-inhibiting factor

Katsuji Sugie1,2, Takafumi Tomura3, Kenji Takakura4, Tetsu Kawano5, Masaru Taniguchi5, Howard M. Grey2 and Kimishige Ishizaka1

1 Division of Immunobiology and
2 Division of Immunochemistry, La Jolla Institute for Allergy and Immunology, San Diego, CA 92121, USA
3 Kirin Pharmaceutical Laboratory, Takasaki 370-12, Japan
4 Department of Obstetrics and Gynecology, Shiga University of Medical Science, Shiga 520-21, Japan
5 Division of Molecular Immunology, Center for Biomedical Science, Chiba University School of Medicine, Chiba 260, Japan

Correspondence to: K. Ishizaka


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Receptors for bioactive glycosylation-inhibiting factor (GIF) were demonstrated using a bioactive mutant of recombinant human (rh) GIF, which is comparable to the suppressor T (Ts) cell-derived bioactive GIF in its affinity for the receptors on helper T (Th) hybridoma cells. Both naive T and B cells in normal mouse spleen lacked GIF receptors. However, presentation of specific antigen to naive T cells resulted in the expression of the receptors on activated T cells. Furthermore, activation of small resting B cells with F(ab')2 fragments of anti-mouse IgM plus IL-4, lipopolysaccharide (LPS) plus IL-4 or LPS plus dextran sulfate induced the expression of the receptors within 48 h of B cell stimulation. It was also found that NK T cells freshly isolated from mouse spleen, but not conventional NK cells, expressed receptors for GIF. CD4+ and CD4 subpopulations of NK T cells showed a similar binding capability. Mature dendritic cells derived from bone marrow did not bear the receptors. The dissociation constant (Kd) of the interaction between the bioactive rhGIF mutant and the high-affinity receptors was 10–100 pM, whereas inactive wild-type rhGIF failed to bind to the receptors. A bioactive derivative of rhGIF suppressed both IgG1 and IgE synthesis by purified B cells activated by LPS and IL-4, indicating that the binding of bioactive GIF to its receptors on activated B cells results in suppression of their differentiation.

Keywords: cytokine receptor, Ig


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In the course of previous studies on regulation of the IgE antibody response, we described an immunosuppressive cytokine, glycosylation-inhibiting factor (GIF) (1,2). This cytokine is a 13 kDa protein secreted from antigen-specific suppressor T (Ts) cells (3,4) and appears to be a subunit of antigen-specific Ts cell factor (5,6). The protein secreted from Ts cells is bioactive, whereas that contained in Ts cytosol is inactive (7). Bioactive and inactive forms of GIF had an identical amino acid sequence (8), and no difference between the two forms was detected by SDS–PAGE analysis (7). These findings suggested to us the possibility that bioactive GIF is formed by post-translational modification of the inactive peptide in Ts cells and that heterogeneity of GIF in bioactivity is due to conformational transition of the same peptide. This idea was supported by subsequent findings that recombinant human (rh) GIF expressed in Escherichia coli was inactive, but it was converted to bioactive derivatives by chemical modification of a cysteine residue at position 60 (Cys60) with a sulfhydryl reagent, such as iodoacetate or 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) (9). In addition, the replacement of Cys57 in the inactive rhGIF with Ala by site-directed mutagenesis resulted in the generation of bioactivity (10).

In a separate experiment, we demonstrated high-affinity binding of Ts-derived bioactive GIF and bioactive derivatives of rhGIF to Th hybridoma cells which had been employed for the detection of GIF bioactivity (10). In contrast, inactive cytosolic GIF and E. coli-derived wild-type rhGIF failed to bind to the same cells. The capacity to bind to the cells was generated by replacement of Cys57 in rhGIF with Ala (C57A) or of Asn106 with Ser (N106S), or modification of Cys60 with DTNB, and these independent chemical changes in the molecule synergystically increased the affinity for GIF receptors. The dissociation constant (Kd) between the high-affinity receptors on the target cells and C57A/N106S, i.e. a mutated rhGIF which has both C57A and N106S replacements, or C57A-DTNB, i.e. a derivative of rhGIF in which Cys57 is replaced with Ala and Cys60 is modified with DTNB, was 10–100 pM, which was comparable to that between Ts-derived bioactive GIF and the receptors. Therefore the GIF receptors appear to recognize conformational structures essential for the bioactivity of GIF. The results of the binding assay also demonstrated the presence of high-affinity receptors on NK1.1+ cells, but not on naive T and B cells isolated from normal spleen. The receptors were detected on both Th1 and Th2 clones, but not on macrophage/monocyte lines. The present experiments were undertaken to extend these findings to determine whether GIF receptors are expressed on lymphocytes following their activation. The results show that the receptors are induced on both T and B cells activated through their antigen receptors, and indicate that the binding of bioactive GIF derivative to the receptors on activated B cells results in suppression of their capacity to synthesize and secrete IgG1 and IgE.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Animals
The AD10 mice, transgenic on a B10.A genetic background for a TCR {alpha}ß that recognizes moth and pigeon cytochrome c (PCC) (11), were provided by S. M. Hedrick (University of California, San Diego). B10.A/SgSnJ, BALB/cByJ and C57BL/6J mice were purchased from the Jackson Laboratory (Bar Harbor, ME). J{alpha}281-deficient mice do not have V{alpha}14 NKT cells (12). V{alpha}14 NKT mice are transgenic for V{alpha}14 and Vß8.2 on a recombination activating gene (RAG)-deficient background (13). These mice do not have T, B or NK cells.

Antibodies, recombinant cytokines and antigenic peptide
Biotinylated mAb specific for mouse CD8 (53-6.7), CD4 (RM4-5), CD5 (53-7.3), I-Ek (14-4-4S), CD19 (1D3), CD11b (M1/70), Thy1.2 (30-H12), NK1.1 (PK136), IgM (R6-60.2), IgG1 (A85-1) and IgE (R35-92), FITC-conjugated mAb specific for mouse IgM (R6-60.2), IgG1 (A85-1), I-Ad (AMS-32.1), I-Ek (14-4-4S), CD14 (rmC5-3) and CD62L (MEL-14), phycoerythrin (PE)-conjugated mAb specific for mouse TCRß (H57-597), CD23 (B3B4), CD45R/B220 (RA3-6B2), B7-2 (GL1), CD4 and NK1.1, mAb specific for mouse CD16/32 (2.4G2), IgM (II/41), IgG1 (A85-3) and IgE (R35-72), and monoclonal mouse IgM (G155-228) and IgG1 (MOPC-21) were purchased from PharMingen (San Diego, CA). The F(ab')2 fragments of goat anti-mouse IgM were purchased from ICN (Costa Mesa, CA). A monoclonal mouse IgE specific for the DNP hapten (14) was prepared as described previously (15). Recombinant murine granulocyte macrophage colony-stimulating factor (rmGM-CSF) was supplied by Dr H. Tsumura (Kirin Pharmaceutical, Takasaki, Japan). Recombinant murine tumor necrosis factor (TNF)-{alpha} and IL-4 were purchased from R & D (Minneapolis, MN) and Genzyme (Cambridge, MA) respectively. The wild-type rhGIF and its mutants, C57A and C57A/N106S, were expressed in E. coli and purified, as described (810). C57A was treated with DTNB, as documented previously (10). PCC peptide 88–104 (PCC 88–104) was synthesized, as described (16).

Purification of naive T cells, B cells and NK1.1+ cells
Naive T cells were obtained from erythrocyte-free splenocytes of the AD10 transgenic mice. Cells non-adherent to nylon wool were incubated with a cocktail of biotinylated anti-CD8, anti-I-Ek, anti-CD19, anti-NK1.1 and anti-CD11b antibodies. The antibody-treated cells were incubated with streptavidin–microbeads (Miltenyi Biotech, Auburn, CA) and CD4+ cells were purified by negative selection using a magnetic cell sorting system (MiniMACS; Miltenyi Biotech). Small high-density T cells in the CD4+ cells were obtained from the 62/80% interface by discontinuous Percoll density gradient centrifugation. Between 90 and 95% of the cells in the final cell preparation were CD4+CD62Lhigh, which is consistent with the naive T cell phenotype. To purify B cells, splenocytes of BALB/c mice were treated with a mixture of biotinylated anti-Thy1.2, anti-CD4, anti-CD8 and anti-CD11b antibodies, and incubated with streptavidin–microbeads for negative selection. Small resting B cells in the preparation were obtained from the 62/80% interface on a Percoll gradient. More than 90% of the cells in the final preparation were B220+. For purification of NK1.1+ cells, spleen cells from B10.A mice were first incubated with biotinylated anti-CD19 and streptavidin–microbeads to deplete B cells. The effluent was then treated with biotinylated anti-NK1.1 antibody and streptavidin–microbeads for positive selection. To deplete NK1.1+ T cells in the NK1.1+ cell preparation, immunomagnetically purified NK1.1 cells were stained with PE–anti-TCRß followed by sorting on a FACStar (Becton Dickinson). To purify V{alpha}14 NK1.1+ T cells, V{alpha}14 NKT mice were used. In these mice, all TCR{alpha}ß+ cells are NK1.1+ (13). Their spleen cells were stained with PE–anti-TCRß and FITC–anti-CD4, and sorted on FACStar. More than 98% of the sorted cells were TCRß+ cells.

Cell cultures
Cells were cultured in RPMI 1640 medium (Irvine Scientific, Santa Ana, CA) supplemented with 2 mM L-glutamine, 50 µM 2-mercaptoethanol, non-essential amino acids (Gibco/BRL, Gaithersburg, MD), 1 mM sodium pyruvate, 10% FCS (Harlan Bioproducts for Science, Indianapolis, IN) and antibiotics. Transgenic T cells were stimulated as described previously (17). Briefly, naive T cells from AD10 transgenic mice (2.5x105 cells/well) were plated at 2 ml/well in 24-well plates (Falcon) and stimulated with 0.5 µM PCC 88–104 presented by B10.A spleen cells (5x106 cells/well) which had been irradiated at 2600 rad. After 72 h of culture, activated T cells were collected by Ficoll density gradient centrifugation.

To obtain activated B cells, resting B cells from BALB/c mice were cultured with or without 30 µg/ml anti-mouse IgM F(ab')2 or 20 µg/ml of lipopolysaccharide (LPS) from Salmonella typhosa (Sigma, St Louis, MO) in the presence of 10 ng/ml rmIL-4 for 48 h Alternatively, resting B cells were stimulated by the procedures described by Snapper and Paul (18). The cells were cultured with or without 10 ng/ml rmIL-4 for 48 h and then cultured with 25 µg/ml LPS for 72 h. Resting B cells were also stimulated with 25 µg/ml LPS and 20 µg/ml dextran sulfate (mol. wt ~500,000; Pharmacia, Uppsala, Sweden) for 48 h.

To determine Ig formation, B cells were stimulated in vitro, as described (19). Briefly, small resting B cells from BALB/c spleen were cultured in 96-well flat-bottomed plates (Falcon) in 200 µl/well at 1x105 cells/ml in triplicate. LPS (20 µg/ml) and increasing concentrations of rmIL-4 were added on day 0. After 6 days of culture, supernatants were collected for determination of Ig by ELISA.

Dendritic cells were grown in vitro, as described previously (20,21). Briefly, bone marrow cells from B10.A mice were incubated with biotinylated anti-CD4, anti-CD8, anti-CD19 and anti-I-Ek, and treated with streptavidin–microbeads to remove mature T and B cells. Cells were cultured with 10 ng/ml rmGM-CSF for 6 days and the cells were subjected to panning on Petri dish (Falcon). Non-adherent cells were further cultured in the presence of 10 ng/ml rmGM-CSF and 25 ng/ml rmTNF-{alpha} for 48 h. To determine the capacity to present antigen, increasing concentrations of dendritic cells which had been treated with 50 µg/ml mitomycin C (Sigma) for 1 h were distributed into 96-well flat-bottomed plates (Falcon). CD4+ T cells were obtained from spleen cells of AD10 transgenic mice, which had been stimulated with 0.5 µM PCC 88–104 for 7 days, and were plated at 5x104 cells/well. PCC 88–104 was added to the cells at 5 nM. After 48 h of culture, [3H]thymidine was added. Cells were harvested 16 h later and radioactivity was counted by liquid scintillation. The B lymphoma line CH27 (provided by G. Houghton, University of North Carolina) was used as a control antigen-presenting cell.

GIF binding assay
Samples of 5 µg of either wild-type rhGIF or C57A/N106S were radiolabeled using 0.25 mCi of [125I]Bolton Hunter reagent (NEN, Boston, MA). The GIF binding assay was carried out as described previously (10). Briefly, viable cells isolated on Ficoll were suspended in culture medium at a concentration of 1x107/ml. Serially diluted 125I-labeled GIF was added to an equal volume of the cell suspension. Non-specific binding was determined by measuring the binding of radiolabeled GIF in the presence of a 300-fold excess of unlabeled GIF. After incubation for 20 min at 37°C, the mixtures were layered on 20% olive oil/80% di-n-butyl phthalate (Sigma) and centrifuged at 3500 g for 90 s. The number of binding sites per cell and the equilibrium dissociation constant (Kd) were calculated by Scatchard analysis using the `Ligand' computer program (22). This program fits biphasic Scatchard plots to curves which it subsequently approximates to two linear components according to the `Gauss–Newton approximation'.

The ability of unlabeled rhGIF or C57A/N106S to inhibit the binding of radiolabeled C57A/N106S was determined by the method previously described (10). Briefly, serially diluted rhGIF or C57A/N106S was mixed with an equal volume of 4 nM 125I-labeled C57A/N106S and each mixture was added to an equal volume of a cell suspension (1x106 cells/sample). After incubation for 20 min at 37°C, cell-bound radioactivity was determined. Non-specifically bound radioactivity was subtracted from cell-bound c.p.m. and the capacity of unlabeled rhGIF or C57A/N106S to inhibit the binding of radiolabeled C57A/N106S was determined by the ratio: (specifically bound c.p.m. in the presence of a sample)/(specifically bound c.p.m. in the absence of the sample).

Flow cytometry
For flow cytometric analysis, 5.0x105 cells were washed in Dulbecco's PBS containing 0.1% BSA and 0.1% sodium azide. Cells were incubated with monoclonal anti-CD16/32 at 50 µg/ml to block Fc receptors and then stained with PE- or FITC-conjugated mAb at 10 µg/ml. Flow cytometry was carried out on a FACScalibur (Becton Dickinson) using CellQuest software. An electronic gate for live cells was set through the window of the forward and side scatter profiles. To determine the levels of I-Ad, CD23, IgM and IgG1 expressed by B cells, cells were stained with PE–anti-B220, PE–anti-CD23 and either FITC-conjugated anti-I-Ad, anti-IgM or anti-IgG1. B220+ cells were gated for measurement of the latter markers. Cell size was analyzed using relative forward scatter (FSC) as a measure.

Measurement of Ig by ELISA
Monoclonal anti-mouse IgM, II/41 and R6-60.2, monoclonal anti-mouse IgG1, A85-3 and A85-1, and monoclonal anti-mouse IgE, R35-72 and R35-92 were used for capture and detection respectively. ELISA plates (96-well, MaxiSorp; Nunc, Roskilde, Denmark) were coated with 2 µg/ml capture antibody followed by blocking with BSA. As standards, G155-228 (mouse IgM), MOPC-21 (mouse IgG1) and anti-DNP mouse monoclonal IgE (14) were used. Samples serially diluted in culture medium were added to the plates and incubated overnight at 4°C. The plates were then washed and incubated with 2 µg/ml of biotinylated antibodies for detection. ELISA signals were developed by adding horseradish peroxidase-conjugated streptavidin (Zymed, South San Francisco, CA) and TMB substrate (Dako, Carpinteria, CA), and determined by absorption at 450 nm. Each assay system was specific and showed no cross-reactivity with the other isotypes being measured.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Expression of GIF receptors by activated T cells and NKT cells
Naive CD4+ T cells were purified from AD10 TCR {alpha}ß transgenic mice and stimulated with the peptide PCC 88–104 in the presence of antigen-presenting cells. After 72 h culture, T cells were recovered and tested for the binding of 125I-labeled C57A/N106S GIF. Scatchard plot analysis demonstrated both high-affinity (Kd = 10–20 pM) and low-affinity receptors (Kd = 300–500 nM) on the activated T cells (Fig. 1AGo). The number of high-affinity receptors was calculated to be 700–1000/cell. In contrast, naive unstimulated T cells did not express detectable numbers (>50 sites/cell) of the receptors. It was also found that 125I-labeled wild-type rhGIF, which lacks bioactivity (9), failed to bind to either activated or naive T cells. This finding was confirmed by the result that unlabeled C57A/N106S GIF inhibited the binding of 125I-labeled C57A/N106S GIF to the antigen-activated T cells in a dose-dependent manner, but even 100-fold excess of unlabeled wild-type rhGIF failed to do so. These results indicate that antigenic stimulation induced the expression of receptors for bioactive GIF on T cells.



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Fig. 1. Scatchard plot of the binding of 125I-labeled C57A/N106S to T, B and dendritic cells. The abscissa represents the number of GIF molecules bound per cell. The ordinate represents the number of GIF molecules bound to a cell divided by the concentration of unbound GIF. (A) Naive T cells were purified from AD10 transgenic mice. Activated T cells were obtained by stimulating naive T cells from AD10 with PCC 88–104 for 72 h. (B) Resting B cells were purified from BALB/c mice. B cells were stimulated with rmIL-4 alone or with rmIL-4 and F(ab')2 fragments of goat anti-mouse IgM for 48 h. (C) Purified B cells were cultured with or without rmIL-4 for 48 h. Cells were washed and cultured with LPS for 72 h. (D) Purified B cells were cultured in the presence of LPS and dextran sulfate for 48 h. Dendritic cells were induced in vitro from bone marrow cells of B10.A mice. Each experiment was reproduced at least 3 times.

 
Previous experiments demonstrated that radiolabeled C57A/N106S specifically bound to NK1.1+ cells isolated from normal mouse spleen (10). Since the NK1.1+ population consists of conventional NK cells and NK1.1+ T cells (NKT cells), we wished to determine which subpopulation expressed GIF receptors. In view of the fact that CD5 is expressed on NKT cells, but not on conventional NK cells (23), B10A splenic lymphocytes were treated with biotinylated anti-CD5 together with anti-CD19 to deplete CD5+ cells and B cells, prior to the positive selection of NK1.1+ cells (see Methods). As shown in Fig. 2Go(A), depletion of CD5+ cells from the NK1.1+ cell population resulted in almost complete loss of binding capacity of radiolabeled C57A/N106S. To further investigate the role of NKT cells in the binding of GIF, we depleted the TCR{alpha}ß+ population of NK1.1+ cells, since >85% of NKT cells bear TCR {alpha}ß (23). FACS sorting was used to remove the TCR{alpha}ß+ population from immunomagnetically purified NK1.1+ cells. It was found that removal of TCR{alpha}ß+ cells from NK1.1+ cells virtually abolished their capacity to bind radiolabeled C57A/N106S (Fig. 2BGo). NK1.1+ cells purified from J{alpha}281-deficient mice, which fail to generate NKT cells (12), did not express GIF receptors (data not shown). TCR{alpha}ß+ NK1.1+ cells express an invariant TCR {alpha} chain, V{alpha}14–J{alpha}281 (24). To examine directly whether GIF binds to V{alpha}14 NKT cells, we sorted TCR{alpha}ß+ cells from V{alpha}14 NKT mice by FACS because NK1.1 TCR{alpha}ß+ cells were not detectable in their spleen cells by flow cytometric analysis. Scatchard plot of the binding of 125I-labeled C57A/N106S showed that V{alpha}14 NKT cells expressed GIF receptors to a degree comparable to that of antigen-primed T cells (Fig. 2CGo). The number of high-affinity binding sites (Kd = 70 pM) was 1000/cell. Both CD4+ and CD4 fractions of V{alpha}14 NKT cells showed a similar binding of radiolabeled C57A/N106S (Fig. 2DGo).



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Fig. 2. The binding of 125I-labeled C57A/N106S to NKT cells. (A) NK1.1+ cells were purified after removing B cells (solid line) or both B cells and CD5+ cells (dashed line) from spleen cells. Ninety percent of the purified cells in both preparations were NK1.1+, as determined by flow cytometry. Samples of 1.0x106 cells sample were incubated with 2 or 4 nM 125I-labeled C57A/N106S. Specific binding was calculated by subtracting non-specifically bound radioactivity in the presence of 300-fold excess of unlabeled C57A/N106S from cell-bound c.p.m. (B) NK1.1+ cells were stained with PE–anti-TCRß to remove TCR{alpha}ß+ cells by FACS. The proportion of TCR{alpha}ß+ cells in the original NK1.1+ cells was 20%, while >98% of sorted cells were NK1.1+ TCR{alpha}ß. Samples of 5x105 cells were incubated with 4 nM 125I-labeled C57A/N106S. Cell-bound c.p.m. were determined in duplicate samples. Specific binding, calculated by subtracting non-specifically bound radioactivity in the presence of 100-fold excess of unlabeled C57A/N106S from cell-bound c.p.m., is shown. (C) Scatchard plot of the binding of 125I-labeled C57A/N106S to V{alpha}14 NKT cells. NK1.1+ TCR{alpha}ß+ cells were purified by FACS from V{alpha}14 NKT mouse spleen. (D) Comparison of CD4+ and CD4 V{alpha}14 NKT cells for binding of C57A/N106S. Spleen cells obtained from V{alpha}14 NKT mouse were stained with FITC–anti-CD4 and PE–anti-TCRß, and sorted into CD4+ TCR{alpha}ß+ and CD4 TCR{alpha}ß+ fractions by FACS. 125I-labeled C57A/N106S (4 nM) was added to 5x104 cells. Cell-bound c.p.m. were determined in duplicate. Specific binding, calculated by subtracting non-specifically bound radioactivity in the presence of 100-fold excess of unlabeled C57A/N106S from cell-bound c.p.m., is shown. Each experiment was repeated 3 times.

 
Induction of GIF receptor expression on B cells
We next determined if B cells expressed GIF receptors upon activation. 125I-labeled C57A/N106S failed to bind to small resting B cells purified from BALB/c spleen cells (Fig. 1BGo). However, GIF receptors were induced on B cells 48 h after stimulation with the F(ab')2 fragments of anti-IgM in the presence of IL-4 (Fig. 1BGo). The high-affinity receptors (Kd = 10 pM) were induced at 430 sites/cell. As reported by previous investigators, culture of B cells for 48 h in the presence of 10 ng/ml IL-4 alone resulted in a marked increase in the expression of CD23 (25) and MHC class II (26), but IL-4 alone failed to induce the expression of GIF receptors. Approximately two-thirds of the cells recovered after stimulation with anti-IgM and IL-4 were enlarged (Table 1Go), and the yield of B cells after the stimulation was 6 times higher than that cultured with IL-4 alone. It was also found that stimulation of resting B cells with LPS together with 0.37–10 ng/ml IL-4 for 48 h or with 10 ng/ml IL-4 for 48 h followed by LPS alone for 72 h (Table 1Go and Fig. 1CGo) markedly induced GIF receptors. LPS alone was sufficient for activating B cells, judging from the enlargement of cells, but barely induced GIF receptors (Table 1Go and Fig. 1CGo). The combination of LPS and dextran sulfate also induced a high number of GIF receptors (Table 1Go and Fig. 1DGo). The receptors on activated B cells appear to be specific for bioactive GIF because radiolabeled wild-type rhGIF failed to bind to B cells activated with LPS and dextran sulfate, and even 100-fold excess of unlabeled wild-type rhGIF was unable to inhibit the binding of radiolabeled C57A/N106S to these activated B cells (results not shown). To characterize the activated B cells that expressed GIF receptors, cells were stained for surface IgM (sIgM) and IgG1 (sIgG1), and analyzed by flow cytometry. B cells cultured for 2 days with 10 ng/ml IL-4 followed by LPS for 72 h in the absence of IL-4, or those stimulated with LPS and dextran sulfate for 48 h expressed sIgM. The proportion of sIgG1+ cells was <2% as determined by FACS analysis. It was also found that B cells stimulated with IL-4 and anti-IgM did not contain sIgG1+ cells. Thus the activated B cells bearing GIF receptors were sIgM+ sIgG1.


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Table 1. Proportion of enlarged cells and numbers of high-affinity GIF receptors per B cella
 
We next wished to determine if the binding of bioactive GIF to activated B cells might affect the differentiation of B cells into Ig-secreting cells. To test this, we determined the effect of GIF on Ig synthesis by B cells stimulated with LPS and IL-4. Since C57A-DTNB has 10–20 times higher GIF bioactivity than C57A/N106S (10), we employed the former derivative of rhGIF. As preliminary experiments, small resting B cells were cultured for 6 days with 20 µg/ml LPS in the presence of varying concentrations of IL-4, and concentrations of IgM, IgG1 and IgE were measured. As shown in Fig. 3Go(A), IgM levels progressively declined with increasing IL-4 concentration. As reported by Snapper et al. (19), the dose response of IgG1 synthesis to IL-4 concentration was bimodal; IgG1 levels peaked at 0.37 ng/ml of IL-4, declined to a minimum at 3.3 ng/ml of IL-4 and then rose with a further increase in IL-4. Formation of IgE became detectable at 3.3 ng/ml of IL-4 and increased progressively at higher concentrations of IL-4 (results not shown). To determine the optimum time for treatment with GIF, C57A-DTNB was added at 500 ng/ml at 0, 24 or 48 h of the culture, and levels of IgM, IgG1 and IgE were determined at the end of day 6 in culture (Fig. 3AGo). If C57A-DTNB was added at 24 or 48 h, both the IgG1 and IgE synthesis, but not IgM, was suppressed. When C57A-DTNB was added to B cells at the initiation of the culture, it did not suppress IgG1 or IgE formation. Repeated experiments with different concentrations of C57A-DTNB showed that 100 ng/ml, but not 20 ng/ml, of the derivative significantly suppressed IgG1 and IgE formation if it was added 24 h after the initiation of the culture (Fig. 3BGo). As expected, 500 ng/ml of wild-type rhGIF failed to affect the Ig synthesis.



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Fig. 3. Effect of C57A-DTNB on Ig synthesis by B cells. Small resting B cells were stimulated with 20 µg/ml LPS and increasing concentrations of rmIL-4. After 6 days of culture, concentrations of IgM, IgG1 and IgE were determined by ELISA. The means ± SD of triplicate cultures are shown. (A) C57A-DTNB (500 ng/ml) was added at 0 (open circles), 24 (open squares) or 48 (open triangles) h after initiation of the culture. Closed circles represent the results of cultures without C57A-DTNB. Since substantial IgE formation was obtained only with 10 ng/ml of IL-4, the effect of C57A-DTNB on IgE formation was evaluated from the results obtained with 10 ng/ml IL-4. (B) At 24 h, C57A-DTNB was added at 20 (open squares) or 100 (open circles) ng/ml. Control cultures (closed circles) received no C57A-DTNB. Effect of the GIF derivative on IgE formation was evaluated with the cultures containing 10 ng/ml IL-4. The results shown in both (A) and (B) were reproduced in three independent experiments of the same design.

 
We also determined the possible effect of C57A-DTNB on the induction of CD23 and MHC class II (I-Ad) on B cells by LPS and IL-4. Small resting B cells were stimulated with 20 µg/ml LPS and various concentrations of IL-4. Figure 4Go shows that the expression of CD23 and I-Ad increased after B cells were cultured for 48 h with 0.37 ng/ml IL-4 and 20 µg/ml LPS, and addition of 500 ng/ml C57A-DTNB at 24 h of culture did not have any effect on the expression of these molecules. A further increase in IL-4 concentrations up to 10 ng/ml did not augment the expression of CD23 or I-Ad and addition of C57A-DTNB did not affect their expression at any concentration of IL-4.



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Fig. 4. Effect of C57A-DTNB on expression of CD23 and MHC class II on B cells. Small resting B cells were stimulated with 20 µg/ml LPS and 0.37 ng/ml rmIL-4, and 500 ng/ml C57A-DTNB was added at 24 h. At 48 h of culture, expression of CD23 and MHC class II was determined by FACS analysis. The ordinate and abscissa represent the cell number and the fluorescence intensity on a logarithmic scale respectively. The shaded histograms represent background fluorescence. The bold solid histograms represent immuno- fluorescence of resting B cells. The thin solid and thin dotted histograms represent the absence and presence of C57A-DTNB respectively. The results were reproduced in three independent experiments of the same design.

 
Absence of GIF receptors on dendritic cells
Dendritic cells obtained by culturing bone marrow cells in the presence of GM-CSF and TNF-{alpha} were tested for their ability to bind 125I-labeled C57A/N106S. They were 71% MHC class II+, 72% B7-2+, but did not express CD14 as determined by FACS analysis. As shown in Fig. 1Go(D), these dendritic cells did not express GIF receptors. To confirm their capacity to present antigen, antigen-primed T cells obtained from AD10 TCR transgenic mice were cultured with 10–105 mitomycin C-treated dendritic cells or B lymphoma cell line CH27 cells in the presence of 5 nM PCC 88–104. Measurement of [3H]thymidine incorporation showed that 102–103 dendritic cells/well were sufficient to give maximum stimulation of T cells, while 103–104 CH27 cells/well were necessary to achieve a comparable proliferative response of T cells.


    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The present experiments, together with previous findings (10), demonstrated that GIF receptors are expressed on activated T and B cells and freshly isolated NK1.1+ T cells, but not on naive T and B cells, conventional NK cells, macrophages or dendritic cells. The receptors consist of two types having different affinities, i.e. one with a Kd of 10–100 pM and the other with that of 300–500 nM. When C57A/N106S, a derivative of rhGIF, was employed as a ligand, the Kds and receptor numbers per cell on activated T and B cells fell in the range previously observed in the binding of Ts-derived bioactive GIF to T cell clones (10). Inactive wild-type rhGIF did not bind to either activated T or B cells. Thus it appears that antigen-primed T cells, activated B cells and NKT cells bear the receptors for the natural ligand.

Previous experiments have shown that 125I-labeled C57A/N106S bound to NK1.1+ cells in normal spleen, but the number of high-affinity receptors on these cells was ~100/cell (10). The present experiments demonstrated that NKT cells, comprising 5–15% of NK1.1+ cells in the spleen (23), are responsible for the binding of GIF and that conventional NK cells do no bear the receptors. The number of the high-affinity receptors on NKT cells in V{alpha}14 NKT mice was of the order of 1000/cell, which was comparable to that on activated T cells and B cells. We suspect that `naive' NKT cells in normal spleen may have been primed by natural ligand(s) presented by CD1 expressed on Langerhans cells, dendritic cells, macrophages, T or B cells (23,27). This possibility may be supported by the fact that NKT cells stand out by their bearing several markers of an activated T cell phenotype including HSAlo, CD44hi and LECAM-1lo (23). It is known that the majority of NKT cells express an invariant TCR{alpha} chain encoded by V{alpha}14–J{alpha}281 gene segments (24), which were originally cloned from a keyhole limpet hemocyanin-specific Ts hybridoma (28). One may wonder that NKT cells might share the capacity of secreting bioactive GIF with Ts cells. However, the formation of bioactive GIF has no relationship with TCR genes utilized by T cells. A representative ovalbumin (OVA)-specific Ts hybridoma, 231F1 cells and an OVA-specific Th2 hybridoma, 12H5 cells utilize V{alpha}8.3 gene segment (6), and TCR on the two hybridomas recognize a common T cell epitope (29). It has been shown that the Ts hybridoma secretes bioactive GIF, while the Th hybridoma, which expresses GIF receptors (10), fails to do so (7).

The presence of GIF receptors on NKT cells suggests the possibility that GIF regulates the immune response through these cells. NKT cells can rapidly secrete large amounts of Th2 cytokines such as IL-4, IL-5 and IL-10, as well as Th1 cytokines such as IFN-{gamma} and TNF-ß (23). It is possible that GIF may regulate the profile of the cytokines produced by NKT cells and thereby affect the antibody response. Our preliminary experiments showed that the secretion of IL-4 and IFN-{gamma} from normal spleen cells upon stimulation with {alpha}-galactosylceramide, which is a specific ligand for V{alpha}14 mouse NKT cells (13), could be markedly inhibited by C57A-DTNB, suggesting that the bioactive GIF regulates the cytokine production of NKT cells. Induction of GIF receptors by antigen stimulation of T cells may also have biological significance. Previous experiments have shown that stimulation of spleen cells obtained from OVA-primed mice with homologous antigen and subsequent propagation of the activated T cells with IL-2 in the presence of GIF resulted in the generation of T cells which could produce OVA-specific GIF upon antigenic stimulation (30). The presence of GIF receptors on activated T cells suggests that selective binding of GIF to antigen-activated T cells induces their differentiation toward Ts cells.

An important finding in the present study was that GIF receptors are induced within 48 h after stimulation of resting B cells with F(ab')2 fragments of anti-IgM or LPS in the presence of IL-4. B cells expressing GIF receptors were enlarged, proliferated and expressed sIgM. However, expression of the GIF receptors appears to be limited to a certain differentiation stage of activated B cells because stimulation by LPS alone caused cell enlargement to a similar extent to that by LPS plus IL-4, but the number of GIF receptors on LPS-stimulated B cells was <10% of that induced by LPS plus IL-4. We found that addition of as little as 0.37 ng/ml IL-4 to B cells in the presence of 20 µg/ml LPS was capable of inducing ~400 receptors/cell within 48 h (results not shown). However, the number of receptors can be increased ~3-fold by increasing the amount of IL-4 to 10 ng/ml. Thus the concentration of IL-4 may be an important factor in determining the density of the receptors or the proportion of the B lymphoblasts that express GIF receptors. However, in view of the result that a high number of GIF receptors was induced by stimulating resting B cells with LPS and dextran sulfate (cf. Table 1Go), there also may be an IL-4-independent pathway for the induction of GIF receptors on B cells. Snapper et al. (18,19,31) have shown that stimulation of resting B cells with LPS plus 104 units (~50 ng/ml)/ml IL-4 resulted in the expression of sIgG1 and/or sIgE on the cells, and promoted IgG1 and IgE production. However, time course of the induction of sIgG1 expression in the cultures showed that after 2 days culture, the proportion of sIgG1+ cells was <1% (31). The present experiments also showed that after 48 h culture of resting B cells with 10 ng/ml IL-4 and LPS, sIgG1+ cells were not detectable. Since the activated B cells express GIF receptors, it would appear that the receptors are expressed on activated B cells prior to class switching.

The biochemical mechanisms involved in the induction of GIF receptors are unknown. Nevertheless, expression of the receptors on activated B cells may have biological significance. The present experiments have shown that C57A-DTNB, a bioactive derivative of rhGIF, suppressed both IgG1 and IgE synthesis induced by stimulation of resting B cells with LPS and IL-4. Since C57A-DTNB affected neither the induction of CD23 or MHC class II, nor IgM synthesis, one might speculate that the bioactive GIF derivative specifically suppressed the isotype switch toward IgG1 and IgE. In the culture system employed, major Ig formed in the culture are limited to IgM, IgG1 and IgE (32). Further studies are required to determine whether the bioactive rGIF derivative can also regulate the differentiation of B cells toward IgG2 formation. It should be noted that the derivative was ineffective if it was added at the initiation of cultures, but gave substantial suppression of Ig synthesis if added 24–48 h after the stimulation with LPS and IL-4 (cf. Fig. 3AGo). This finding is in agreement with the fact that the GIF receptors are induced within 24–48 h after initiation of activation and are lacking on small resting B cells. It is known that the disulfide bond formed between DTNB and Cys60 in the rhGIF derivative is relatively unstable (unpublished results). Thus, if added at the initiation of B cell stimulation, the bioactive derivative might undergo reduction or degradation before a sufficient number of GIF receptors are expressed on the cell surface. In any event, a direct suppressive effect on B cell synthesis of IgG1 and IgE might explain, at least in part, the suppressive effects of Ts-derived GIF on both IgE and IgG antibody responses to alum-adsorbed antigen (2).

The present findings may also provide a clue to resolve controversial issues on the mechanisms for the immunosuppressive effect of antigen-specific GIF or Ts cell factor, which suppresses the antibody responses in an antigen- or carrier-specific manner (29,30). Previous studies revealed that antigen-specific GIF is a post-translationally formed conjugate of TCR{alpha}ß and bioactive GIF (5,6,33), and that the antigen receptor of the T cells that produce antigen-specific GIF recognizes a processed antigen in the context of class II MHC molecules (34, 35). Considering that the Kd of interaction between bioactive GIF and its receptors is in the range of 10–10 to 10–11 M, we suspect that the GIF portion of the conjugate may play a major role in the binding to its target cells. If this is the case, the present experiments may indicate that possible target cells for antigen-specific GIF would be antigen-primed T cells, activated B cells or NKT cells, rather than macrophages or dendritic cells. Among these candidates, only antigen-activated B cells can present antigenic peptides associated with class II MHC on their surface. Although the affinity of the MHC–peptide complex for TCR is low (Kd = 10–4 to 10–7 M) (3638), we suspect that the interaction between the complex and the TCR portion of antigen-specific GIF contributes to selective association of the molecule with such B cells. One may speculate that dual interaction of antigen-specific GIF with antigen-activated B cells renders the immunosuppressive effect of GIF specific for the homologous antigen system.


    Acknowledgments
 
We express our appreciation to Karen Anderson and Dung Huynh for technical help. This paper is publication no. 245 from the La Jolla Institute for Allergy and Immunology. The work was supported partly by research grant AI-14784 from the US Department of Human and Health Services.


    Abbreviations
 
DTNB5,5'-dithiobis(2-nitrobenzoic acid)
GIFglycosylation-inhibiting factor
LPSlipopolysaccharide
PCCpigeon cytochrome c
PEphycoerythrin
GM-CSFgranulocyte macrophage colony stimulating factor
TNFtumor necrosis factor
OVAovalbumin

    Notes
 
Transmitting editor: T. Sasazuki

Received 28 December 1998, accepted 1 April 1999.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
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