Selective Suppression of L ymphokine Production by Human Hybridoma Suppressor Factor (HSF)

Immunobiol., vol. 181, pp. 64-83 (1990)

Department of Immunology, Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio, U.S.A.

Selective Suppression of L ymphokine Production by Human Hybridoma Suppressor Factor (HSF)=:'

YASUYUKI TOMITA, BELINDA Y. LIEBERMAN, and MARTHA K. CATHCART

Received December 12, 1989· Accepted May 2, 1990

Abstract

We have made a human thymus cell hybridoma that secretes an immunosuppressive monoclonal lymphokine, referred to as hybridoma suppressor factor (HSF). This factor modulates the function of CD4+ cells suppressing their IL-2 production and suppressing PWM-induced B cell differentiation into Ig producing cells. Here we have examined the effect of HSF on the generation of T cell-derived lymphokines that regulate B cell growth and differentiation as well as the expression of other proteins involved in the control of T cell growth i.e., the p55 chain of the IL-2R and the transferrin receptor (TFR). HSF suppressed IFN-y activity produced by mitogen-stimulated PBMC without affecting the generation of lymphokines responsible for BCGF and BCDF activities. Additionally, HSF did not inhibit the expression of either IL-2R (p55) or TFR by activated T cells in spite of causing the suppression of IL-2 production. This evidence was further supported by experiments in which HSF selectively suppressed the accumulation of IL-2 mRNA without affecting IL-2R (p55) mRNA expression in mitogen-stimulated PBMC. The selective action of HSF may help to clarify the regulatory mechanisms involved in lymphokine gene expression as well as provide a way by which immune responses involved in autoimmunity and transplant rejection may be interrupted.

Introduction

A number of antigen nonspecific T cell-derived lymphokines that regulate B cell growth and differentiation have been identified. Among them, B cell growth factor (BCGF, now known as IL-5) (1, 2), B cell stimulatory factor-1 (BSF -1, now known as IL-4) (3), B cell differentiation factor (BCDF) or B cell stimulatory factor-2 (BSF-2, now known as IL-6) (4-6),

"·Portions of this work were published in abstract form in the Fed. Proc. 46:1216,1987. This work was supported by grants from the NIH (AI 26121), Seragen Inc., Boston, MA, U.S.A., and the Mudge Foundation.

Abbreviations: LSN=activated lymphocyte supernatants, BCGF=B cell growth factor; BSF = B cell stimulatory factor; BCDF = B cell differentiation factor; HSF = hybridoma suppressor factor; CVH = common variable hypogammaglobulinemia; TFR = transferrin receptor; IL-2R (p55) = Interleukin 2 receptor chain 55 kd; SAC= staphylococcus aureus Cowan 1; CsA=Cyclosporin A; PKC=protein kinase C.

HSF Selectively Suppresses Lymphokine Production . 65

IL-2 (7, 8), and IFN-y (9, 10) have been functionally and biochemically isolated from activated lymphocyte supernatants. Furthermore, recent advances in molecular biology have allowed the cloning of the cDNA of several of these factors (3, 6, 8, 10).

B cell growth factors such as IL-4 and IL-5 are clearly involved in inducing activation of resting B cells and proliferation of B cells activated with mitogen or anti-Id antibody, whereas B cell differentiation factor (IL-6) acts to promote B cell differentiation into Ig producing cells (11). IL-2, originally described as a T cell growth factor, has been shown to promote growth and differentiation of both human and murine B cells (12, 13) and IFN-y has been reported to serve as a differentiation factor for B cells (14-16). Thus, different B cell subsets at different stages of growth and differentiation may respond to different T cell-derived lymphokines.

PWM is widely used to study B cell growth and differentiation because of its ability to induce polyclonal B cell differentiation in vitro. It has been shown that Ig production in this system is both macrophage- and T celldependent (17). T cells, upon stimulation with PWM release soluble factors. These factors can replace the helper function of T cells for promoting B cell proliferation and differentiation to Ig secreting cells (18).

We have previously established a human thymus cell hybridoma producing a monoclonal lymphokine that is immunosuppressive (19, 20). This monoclonal lymphokine, referred to as hybridoma suppressor factor (HSF), caused significant suppression of polyclonal Ig production in PWMstimulated PBMC. It also significantly suppressed IL-2 production in both PHA- and PWM-stimulated PBMC without affecting IL-1 production or IL-1 or IL-2 interaction with their receptors (21). Similar suppression of Ig and IL-2 production was observed in reconstituted cultures of CD4+ cells and non T cells. Moreover, a recent study demonstrated that HSF suppressed PWM-induced IL-2 production by total T cells or CD4+ T cells but not by CD8+ cells and its suppressive activity was totally absorbed by CD4+ but not by CD8+ cells (22). In addition, we have demonstrated that reconstitution of normal culture levels of IL-2 by addition of exogenous rIL-2 completely abrogated the HSF-mediated suppression of PWMinduced IgM production (21). These results lead us to hypothesize that HSF modulates the function of CD4+ cells and inhibits their IL-2 production thus ultimately suppressing Ig production in PWM-stimulated PBMC. Here we examine whether HSF modulates the generation of other T cellderived lymphokines by examining the effect of HSF on the production of lymphokines with IL-4, IL-5, IL-6 and IFN-y activity. We also monitored the effect of HSF on the expression of two nonlymphokine proteins known to be produced by CD4+ cells that are directly involved in control of T cell growth and known to be affected by other immunosuppressive agents: the IL-2R (p55) (23) and transferrin receptor (TFR) (24).

In this paper, we report that HSF suppresses T cell-derived lymphokine production in a selective manner without affecting the expression of IL-2R

66 . YASUYUKI TOMITA, BELINDA Y. LIEBERMAN, and MARTHA K. CATHCART

(p55) and TFR by mitogen-activated T cells. We also show that the HSFmediated suppression of IL-2 production is at least in part due to selective inhibition of the accumulation of IL-2 mRNA. Studies on the mode of action of HSF may provide useful information for understanding not only the regulation of Ig production, but also the selective regulatory mechanisms which affect T cell-derived lymphokine production.

Materials and Methods

Preparation of HSF

A human thymus cell hybridoma was established utilizing a hypoxanthine guanine phosphoribosyl transferase deficient human T cell line, CEM-CM3 and thymus cells from a patient with common variable hypogammaglobulinemia (CVH) (19, 20). To obtain HSF, 4 x 106/ml viable cloned hybridoma cells (from clone 8E24) were cultured in RPMI 1640 medium supplemented with 2 % FCS for 48 h in a humidified atmosphere of 10 % CO2 at 3rc and then culture supernatants were collected and stored at -20°C until use. The culture supernatants of CEM-CM3 were collected by a similar procedure and used as control supernatants.

Cell separation

PBMC were isolated from heparinized blood of healthy volunteers by Ficoll-Hypaque density gradient centrifugation. After three washes in PBS, cells were resuspended in 2 ml RPMI medium and overlaid onto 8 ml FCS in 15 ml centrifuge tubes and then subjected to centrifugation at 150 x g for 5 min to remove platelets. This procedure was repeated twice and then the final cell pellets were resuspended in RPMI 1640 medium supplemented with 2 mM L-glutamine, 100 IU/ml penicillin, 100 Ilg/ml streptomycin, 10 mM HEPES buffer solution, and 10 % FCS (10 % FCS-RPMI). In some experiments PBMC were further separated by a spontaneous rosetting procedure with SRBC as reported previously (25). SRBC rosettepositive cells were used as «T cells», and rosette-negative cells were used to as <<llon-T cells». The «T cells» were < 3 % Ig +, < 6 % OKMI + and> 90 % OKT3+.

B cell separation

Purified B cells were isolated from the non-T cell fraction by the following procedures. Adherent cells were removed by two incubations on plastic culture flasks (Corning 25120, Corning, NY, USA), each for 1 h at 37°C, and then nonadherent cells were incubated with a 1 % solution of neuraminidase-treated SRBC at a 1: 1 ratio for 2 h at 4°C according to the method described by HIRANO et al. (18). The cell suspension was then layered on a FicollHypaque density gradient and non-rosetting cells were recovered at the interface of the gradient following centrifugation (300 x g, 30 min). The residual T cells in the non-rosetting cell fraction were removed by the panning method utilizing the mAb OKT3 (20). B cells (nonadherent cells following OKT3 panning) contained less than 1 % CD3+ cells and more than 70 % sIg+ cells. These cells did not respond to PHA (16 Ilg/ml; Miles Scientific, Naperville, IL, USA) with significant cell proliferation or to PWM (1/200 dilution; Gibco, Grand Island, NY, USA) with cell differentiation into Ig producing cells.

Assay for HSF-mediated suppression

PBMC (2 or 3 x 105/200 Ill) were cultured with PWM (1/200 dilution) in the presence or absence of HSF (or the control CEM-CM3 supernatant) for 7 days at 3rC in 10 % FCSRPMI in round bottomed microtiter plates (Costar 3799, Costar, Cambridge, MA, USA). The amount of IgM in the culture supernatants was measured by an ELISA as previously described (26).


LSN production

PBMC or T cells (lor Z x 106/ml) were incubated with PHA (16 [.Ig/ml) or PWM (1/Z00 dilution) in 10 % FCS-RPMI in the presence or absence of HSF (1/6 dilution) or control CEM-CM3 supernatants (1/6 dilution) for the indicated times at 37°C and then supernatants were collected and filtered through 0.45 micron filters. These supernatants were used as LSN (18) and contained IL-Z, IFN-y, B cell growth and differentiation factor activities. In some experiments, LSN were incubated twice with the IL-Z-dependent cell line, CTLL-ZO (1 x 107

/

ml) for Z h at 4°C for removal of IL-Z activity. These absorbed LSN contained no detectable IL-Z activity.

B cell proliferation assay

Purified B cells (Z x 1051200 [.II/well) were cultured with Staphylococcus aureus Cowan I (SAC; BRL, Gaithersburg, MD, USA: a final dilution of 0.01 %) in the presence or absence of HSF (1/6 dilution) in 10 % FCS-RPMI medium for 3 days. The cultures were then pulsed with eH]-TdR (0.5 [.ICi/well; 6.7 Ci/mmol, New England Nuclear, Boston, MA, USA) during the final6h of incubation. Incorporation of eH]-TdR was measured by standard liquid scintillation counting techniques after harvesting the cells using a Skatron cell harvester (Skatron Inc., Sterling, VA, USA).

Assay for lymphokines mediating B cell growth factor activity

B cell growth factor activity, believed to be due to IL-4 and IL-5, was measured by monitoring the proliferative response of B cells (Z x lOS/ZOO [.II/well) in the presence of SAC (a final dilution of 0.01 %) and LSN (a final dilution of 1:4) for 3 to 5 days as reported by others (1). Proliferative responses were measured by pulsing with [3H]-TdR (0.5 [.ICi/well) during the final 6 h of incubation.

Assay for lymphokines mediating B cell differentiation factor activity

The BCDF activity, presumably due to IL-6, was detected by measuring the amount of IgM produced by the BCDF-reactive Epstein Barr virus-(EBV) transformed cell line, SKW 6.4, in the presence or absence of LSN (Z7). Briefly, SKW 6.4 cells (1041200 [.II/well) were cultured in the presence or absence of LSN in 10 % FCS-RPMI medium for 4 days at 37°C. The amount of IgM in the culture supernatants was measured by an ELISA.

Assay for IL-2 activity

IL-Z activity was determined by measuring the IL-Z dependent proliferation of CTLL-ZO cells as described previously (Z8). One unit of activity was defined as the amount of sample inducing half maximal proliferation of CTLL-ZO cells as determined by probit analysis (Z8, Z9). Recombinant IL-Z was obtained from Amgen Biologicals (Thousand Oaks, CA, USA).

Assay for IFN-y activity

IFN-y activity in LSN was determined by measuring the inhibition of the cytopathic effect of Encephalomyocarditis (EMC) virus on human lung carcinoma cell, A549 (30). The A549 cells, seeded Z4 h earlier at a cell concentration of 3 x 104/well, were incubated with serial dilutions of each IFN-containing sample in Dulbecco's modified eagle medium containing 5 % FCS for Z4 h at 3rc. The monolayer cultures were then washed three times and EMC virus was added (100 [.II of a 1/1000 dilution of 165 x lOS PFU/ml)c After a 40-44 h incubation, the culture supernatants were removed, the monolayer was washed three times and the remaining cells were stained with 0.05 % crystal violet. Following elution of the stain with 95 % methanol, the absorbance was measured using a multichannel ELISA reader at 595 nM to evaluate the cytopathic effect of EMC virus. A unit of IFN-y activity was defined as the inverse of the sample dilution inducing the half maximal cytopathic effect of EMC virus on the A549 cells. Positive controls included an IFN-y laboratory reference, calibrated with NIH


HuIFN-a (Go23-901-527) and NIH HuIFN-y (Gg23-901-530) standard. Recombinant IFNy was obtained form Biogen (Cambridge, MA, USA). Under similar culture conditions we have never observed detectable levels of IFN-a as determined by using antisera specific for IFN-a or IFN-y. In such studies, anti-IFN-y completely inhibited the protection of A 549 cells and anti-IFN-a had no effect.

Analysis of IL-2R (p55) and TFR by flow cytometry

IL-2R (p 55) and TFR were detected using specific mAbs. For direct immunofluorescence, PBMC were cultured with PHA (16 ftg/ml) in the presence or absence of HSF (Yo dilution) for 3 days and then the cells were incubated with a Yo dilution of either fluorescein-coupled mAb OKTll (Ortho Diagnostic Systen Inc.) and phycoerythrin-coupled anti-IL-2R (p 55) antibody (Becton-Dickinson) or fluorescein-coupled anti-TFR antibody (Becton-Dickinson) and phycerythrin-coupled mAb OKT11 for 30 min at 4 0c. After three washes with PBS, cells were resuspended for FACS analysis. The percentage of stained cells was determined from computed histograms using the Becton-Dickinson Consort-40 computer program. Mouse monoclonal antibodies (Becton-Dickinson) of isotypic classes either IgGI (clone X40) or IgG2a (clone X39) coupled to phycoerythrin or FITC were used as fluorescence controls. Greater than 99.5 % of cells remained unstained when treated with these control antibodies.

Preparation of DNA probes

The IL-2 cDNA was obtained from Oncor (Gaithersburg, MD, USA) (8) and E. coli containg the plasmid IL-2R (p 55) DNA (pIL2R2) or the cardiac actin cDNA (pHRL83-IVS4) were obtained from American Type Culture (Rockville, MD, USA) (31). The method for plasmid DNA preparations was as described in MANIATIS et al. (32). One microgram of plasmid DNA or cDNA was radiolabeled by nick translation with 32p-dCTP. The resultant specific activity was approximately 108 cpm/ftg which was used at 7x106 cpm per blot.

Preparation of RNA and northern blot hybridization

After the indicated of culture with PHA in the presence or absence of HSF, PBMC were collected, washed with PBS and total cellular RNA was extracted by the guanidine isothiocyanate-cesium chloride method (33). A total of 20 ftg of denatured RNA per lane was sizefractionated by electrophoresis in 1 % agarose-formaldehyde gels and then transferred to nitrocellulose paper (Schleicher and SchueII, Keene, NH, USA) in a Northern capillary transfer (34). The blots were prehybridized 6 h at 42°C in 50 % formamide, 1 % SDS, 5x SSC, Ix Denhardt's solution, 0.25 mg/ml denatured herring testes DNA, and 50 mM sodium phosphate buffer. Hybridization was carried out at 42°C for 16 h with 7x 106 cpm of denatured probe (35). After hybridization, the blots were washed 4x with 0.1 % SDS in 2x SSC at room temperature and subsequently in 0.1 % SDS in O.5x SSC at 55°C for 20-30 min. Blots were then dried and exposed to XAR-5 X-ray film (Eastman Kodak Co., Rochester, NY, USA) with intensifying screens at -70°C. Blots were reutilized by stripping and rehybridizing with a different probe (32). Such rehybridization established the selective expression of individual messages. Hybridization with the actin probe verified that equal amounts of RNA were loaded onto the agarose gels.

Results

HSP does not affect the production of lymphokines mediating BeGP activity

We first examined whether HSF suppressed the generation of lymphokines mediating BCGF activity in supernatants of PHA-stimulated T cells

HSF Selectively Suppresses Lymphokine Production' 69

Table 1. HSF does not inhibit production by T cells or PBMC of lymphokines mediating BCGF activityl

LSN preparation2 BCGF assay

Factor Absorption Lymphocytes Addition of LSN3 B Cells4 SAC Expt. I Expt. II

eHJ-TdR uptake (cpm±SEM)

+ 1149 ± 190 1042 ± 52 + + 2326± 405 3926 ± 151

+ + + 6336± 453 9981 ± 569

+ CEM + + 7640 ± 1064 N.T.5 + HSF + + 6093 ± 789 11870 ± 398

+ + + + N.T. 6356 ± 538 + HSF + + + N.T. 6131 ± 125

1 BCGF activity was determined by measuring the proliferation of B cells (2 x 105/well) with SAC plus LSN at a final dilution of 1:4 for 5 days. eHJ-thymidine (0.5 !!Ci/well) was added during the final 6 h of culture.

2 LSN were collected from PHA-activated T cells (Expt. I) and PBMC (Expt. II) in the presence or absence of factors (HSF or CEM-CM3 control supernatants).

3 For absorption, LSN were twice incubated with CTLL-20 cells (Ix 107 cells/ml) for 2 h at 4°C. Unabsorbed LSN contained 115 U/ml in the absence of HSF and 50 U/ml of IL-2 activity in the presence of HSF (Expt. II). CTLL-20-absorbed LSN contained less than 1 U of IL-2 activity.

4 B cells were obtained as described in Materials and Methods. PHA-stimulated B cells and resting B cells yielded the same level of [3HJ-thymidine incorporation at day 5.

5 N.T. = not tested.

or PBMC (Table 1). Results of representative experiments with these cell populations are presented in Table 1. T cells (Expt. 1) or PBMC (Expt. 2) were cultured with PHA in the presence or absence of HSF (or supernatants of CEM-CM3) for 48 h, and the culture supernatants were collected as LSN. LSN were then added to cultures of SAC-activated B cells at the initiation of a 5 day culture to monitor BCGF activity (1). Addition of LSN caused significant enhancement in the incorporation of eH} T dR into SAC-activated B cells. HSF (or control factor) had no effect on this enhancement of B cell proliferation in spite of causing significant suppression of IL-2 production in the LSN by HSF. This result suggests that HSF does not suppress the generation of lymphokines like IL-4 and IL-S that are known to contribute to the B cell growth factor activity generated by T cells. Yet, the possibility that the presence of some IL-2 in LSN might override the effect of the lymphokines causing BCGF activity in this assay system was not ruled out since IL-2 is known to enhance the proliferation of SAC-activated B cells (14). Therefore, in order to eliminate this possibility, LSN were absorbed with CTLL-20 cells, a step in which IL-2 activity was completely removed, prior to adding them to B cells. As shown in


Experiment 2, CTLL-20-absorbed LSN significantly enhanced the growth of SAC-activated B cells, and as before, HSF did not inhibit this enhancement of B cell proliferation. Similar results were obtained from these assays at 3 days (data not shown).

We next tested whether HSF had any direct effect on the proliferative response of SAC-activated PBMC or B cells. Results of representative experiments of several performed are shown in Table 2. No suppression was observed in either PBMC or B cells. Taken together, these results suggest that in spite of causing significant suppression of IL-2 production, HSF does not suppress the generation of lymphokines that contribute to the BCGF activity in supernatants of PHA-stimulated T cells. It is also evident that HSF does not directly interfere with B cell replication.

HSP does not affect the production of lymphokines mediating BCDP activity

To test whether HSF modulated the generation of IL-6-like BCDF in PWM-stimulated PBMC, LSN were prepared in the presence or absence of HSF and BCDF activity in these supernatants was examined. BCDF activity was monitored in LSN using the BCDF-reactive, EBV transformed cell line, SKW6.4 (27). This cell line, in response to B cell differentiation factors such as IL-6, will differentiate into an IgM secreting cell. Table 3 displays the results of two representative experiments. LSN were obtained from cultures of PWM-stimulated PBMC in the presence or absence of two different batches of HSF for 24 or 48 h. LSN, in the presence of HSF, induced IgM production in cultures of SKW6.4 cells to similar levels as those produced in the absence of HSF. This was evident when LSN were collected at 24 or 48 or 62 h (data not shown). In contrast to BCDF activity,

Table 2. HSF does not alter proliferation of SAC-stimulated PBMC or B cells

Uptake of [3H]-TdR (mean cpm ± SEM)1

Expt. Cells SAC PHA Medium control CEM HSF

PBMC2 1292 ± 404 + 6620 ±243 7075 ± 230 7285 ± 578

II B cells) 550 ± 65 + 344 ± 13

+ 6924 ± 595 5133±418 6571 ± 302

1 eH]-thymidine (0.5 JlCi/well) was added for the final 6 h of culture. Data are expressed as the mean ± SEM of triplicate samples.

2 PBMC (2 x 105/well) were incubated with SAC (0.01 %) in the presence or absence of supernatants of CEM-CM3 and HSF at a 1:6 dilution for 3 days.

) B cells (2 x 105/well) were incubated with SAC or PHA (16 Jlg/ml) in the presence or absence of supernatants of CEM-CM3 and HSF at a 1:6 dilution for 3 days.


Table 3. HSF inhibits production of IL-2 but not lymphokines mediating BCDF activity

Preparation of LSN1 IL-2 Activity2 BCDF Activity3

LSN PBMCPWM HSF4 Time (hr) (U/ml) IgM (ng/ml)

Expt. I 1 + 24 0 69± 2 2 + + 24 65 152 ± 4 3 + + + 24 25 152 ± 11 4 + 48 0 70 ± 4 5 + + 48 65 227± 4 6 + + + 48 25 217± 5

Expt. II 7 + 48 0 92± 7 8 + + 48 110 209± 8 9 + + + 48 55 199 ± 8

I PBMC (2 x 106 cells) were cultured with PWM in the presence or absence of HSF at a 1:6 dilution for 24 or 48 h and then the supernatants were collected as LSN.

2 IL-2 activity (U/ml) in LSN was measured using CTLL-20 cells. 3 BCDF activity was determined by measuring the amount of IgM production by SKW6.4

cells in the presence of LSN at a final dilution of 1:4 for 4 days. SKW6.4 cells did not secrete IgM in response to either rIL2 (up to 1000 U/ml) or medium alone (data not shown). Data are expressed as the mean ± SEM of triplicate samples.

4 Two different batches of HSF were utilized for preparation of LSN in Expts. I and II. These were prepared by the same procedure.

HSF significantly suppressed IL-2 production in LSN, which was consistent with our previous findings. We also tested the ability of rlL-2 to induce IgM production in SKW6,4 cells and found that rIL-2 up to 1000 Vlml did not induce any IgM production (data not shown). Therefore, SKW6,4 cells clearly responded to differentiation factors other than IL-2.

Since BCDF activity was monitored in the LSN at a 1:4 dilution (50 [tll 200 [tl) in these experiments, the possibility remained that greater or smaller amounts of these LSN, prepared in the presence or absence of HSF, might differentially modulate the IgM production in SKW6,4 cells. To examine this possibility, serial dilutions of LSN, between 1:2 and 1 :32 dilution, were added to the cultures of SKW6,4 cells. As shown in Figure 1, 100 [tllwell (1:2 dilution) of LSN, prepared in the absence of HSF, could induce 145 ± 15 nglml of IgM. Decreasing amounts of LSN induced proportionally decreased amounts of IgM in the SKW6,4 cells cultures. HSF did not influence the LSN-induced IgM production at any dilution tested. The results in Table 3 and Figure 1 suggest that HSF does not suppress the generation lymphokines that mediate B cell differentiation in this system in spite of causing significant suppression of IL-2 production.

HSF suppresses IFN-y activity in LSN

Since another T cell-derived lymphokine, IFN-y, has been demonstrated to be one of the differentiation factors for B cells (15,16), we tested whether


150

\ • -HSF o +HSF

~

E tOO ""'-OJ

~~ ..s

"" ~, OJ

50

~~ 0

-1 -2 -3 -4 -5 LOG 2 DILUTION

Figure 1. HSF does not suppress the production of lymphokines mediating BCDF activity in supernatants of PWM-stimulated PBMe. BCDF activity in LSN was detected by measuring IgM synthesis by the BCDF-reactive cell line SKW6.4. PBMC (2 x 106 cells) were cultured with PWM in the presence (open circles) or absence (closed circles) of HSF (1:6 dilution) for 48 h and the culture supernatants were collected and used as LSN. Serial dilutions of LSN were incubated with SKW6.4 cells (l04/well) for 4 days and the amount of IgM in these cultures was measured by ELISA. Data are expressed as the mean ± S.E.M. of triplicate samples.

HSF modulated the generation of IFN-y activity in mitogen-stimulated PBMC (LSN). LSN were obtained from cultures of either PHA- or PWMstimulated PBMC in the presence or absence of HSF as indicated and then antiviral activity in the LSN was determined. In contrast to our results detecting BCGF and BCDF activity, IFN-y activity in the LSN was significantly suppressed by HSF at 48 h after exposure to PHA or PWM

80

:z 0

60 ...... [J) [J) UJ a: a. a. 40 :::> [J)

I-:z UJ u 20 a: UJ a.

0

TIME OF INCUBATION (hrs)

Figure 2. HSF inhibits production of IFN-y activity in the supernatants of mitogen-stimulated PBMe. PBMC were cultured with PHA in the presence or absence of HSF (1:6 dilution) for the indicated times. Antiviral activity of the culture supernatants (IFN-y activity) was then evaluated. Data are expressed as the mean ± S.E.M. of % suppression of IFN-y activity from the indicated number of experiments. Control cultures produced 109 ± 27 Vlml (48 h), 92 ± 23 Vlml (72 h) and 138 ± 39 Vlml (96 h) of IFN-y activity, respectively. The asterisk denotes that the experimental data were significantly suppressed as compared to the matched control (p < 0.02, paired T test).


(Fig. 2 shows PHA data, PWM data not shown). HSF did not significantly suppress IFN-y activity at 72 or 96 h in the PHA-stimulated cultures (Fig. 2). Consistent with previous findings, the LSN prepared in the presence of HSF, contained diminished levels of IL-2 activity as compared to those prepared in the absence of HSF. Since it has been reported that endogenous IL-2 can enhance IFN-y mRNA and its protein synthesis (36, 37), the decreased level of IL-2 activity induced by mitogen-stimulated PBMC in the presence of HSF might contribute to the effect of HSF on IFN-y activity. To test this possibility, rIL-2 (50 D/ml) was added to the cultures of mitogen-stimulated PBMC in the presence or absence of HSF, and IFNy activity in these culture supernatants was measured at 48 or 72 h. HSF consistently suppressed IFN-y activity in cultures supplemented with rIL-2 (data not shown). HSF also did not inhibit anti-viral activity on the A549 cells by exogenous IFN-y (data not shown).

IFN-y addition does not ablate HSF suppression of PWM induced antibody production

Since we demonstrated that HSF suppressed the generation of IFN-y activity in mitogen-stimulated PBMC, we next examined whether the addition of exogenous rIFN-y could have any effect on the HSF-mediated suppression of PWM-induced IgM production. PBMC were cultured with PWM and HSF in the presence or absence of rIFN-y for 7 days and then the

Table 4. HSF-induced suppression of antibody production is not reversed by addition of rIFN-y

Culture IgM production (ng/culture)! Components Recombinant IFN-y (Units/ml)

0 50 100

Expt. I PBMC alone 116 ± 6 154 ± 14 91 ± 12 +PWM 358 ± 40 329 ± 34 417 ± 6 +PWM + HSF 184 ± 16 (72i,3 188 ± 13 (81)3 208 ± 10 (64t

Expt. II PBMC alone 10 ± 2 15 ± 5 9± 2 +PWM 166 ± 11 188 ± 16 181 ± 16 +PWM + HSF 65 ± 3 (65t 77± 9 (64? 80 ± 4 (59)4

! PBMC (Jx lOs/culture) were cultured with PWM and HSF (1/6 dilution) in the presence of rIFN-y at the indicated concentrations. After 7 days of culture, the amount of IgM was measured by ELISA. Data are expressed as the mean ± SEM of quadruplicate samples.

2 Percent suppression as compared to the PWM control is given in parentheses. Percent suppression was calculated according to the following equation: [l-(value with HSF -background value)/(value with PWM - background value)) x 100.

3 Significantly different from the PWM control (p < 0.01). 4 Significantly different from the PWM control (P < 0.001).


HSF-mediated suppression of Ig production in these cultures was assessed. As shown in Table 4, exogenous IFN-y up to 100 Vlml did not have any effect on the HSF-mediated suppression. The control level of IFN-y production in the PWM-stimulated cultures in the absence of HSF was in the range of 32--64 Vlml (data not shown). Therefore, the inhibition of IFN-y activity by HSF in the PWM-stimulated PBMC is not responsible for the suppression of IgM production.

HSF does not affect the expression of IL-2R (p55) or TFR by PHAactivated T cells

We also evaluated the effect of HSF on the expression of T cell activation products such as IL-2R (p55) or TFR by PHA-activated T cells (23, 24).

A B

' • . : t 1.1 161--'-,....,,-:.:....:'..:+=---'------ t-~. :...,:.:~Il.,.,.s.-rJ-, .-----

: ::~:~ ~! IV

16 32 48 16 Jl

GREEN FLUORESCENCE Figure 3. IL-2R and TFR are equally expressed by mitogen activated T cells despite the presence of HSF. PBMC (1 x 106/ml) were incubated with PHA (16 f.lg/ml) for 48 h (IL-2 assay) or 72h (FACS analysis) in the absence (quadrants A and C) or presence (quadrants B and D) of HSF. The cells were then stained with monoclonal antibodies conjugated with FITC (green fluorescence) or phycoerythrin (red fluorescence). PBMC were stained with FITCconjugated OKT11 and phycoerythrin-conjugated anti-IL-2R (quadrants A and B) or phycoerythrin-conjugated OKT11 and FITC-conjugated anti-TFR (quadrants C and D) as described in Methods. IL-2 activity was measured using the CTLL-20 cell assay and IL-2 levels in the culture supernatants of cells stained for FACS analysis were 115 U/ml in cultures without HSF and 50 U/ml in the cultures with HSF. 95.0 % and 95.8 % of the T cells stained with the IL-2R antibody (quadrants A and B) and 69.6 % and 66.6 % of the T cells stained with anti-TFR (quadrants C and D).


PBMC were cultured with PHA in the presence or absence of HSF for 48 h (IL-2 assay) or 72 h (FACS analysis). The cells were then doubly stained with mAbs OKTll and either anti-IL-2R (p55) or anti-TFR and the percentage of receptor-positive cells in the CD2+ T cell population was obtained by F ACS analysis. Results of a representative experiment of three performed are shown in Figure 3. HSF did not prevent or alter the expression of either IL-2R (p55) or TFR by PHA-activated T cells. Moreover, the kinetics of appearance of IL-2R (p55) positive T cells in HSF-treated cultures was very similar to that observed in untreated cultures, suggesting that HSF does not alter the kinetics of the expression of IL-2R (p55) by activated T cells (data not shown).

A. IL-2 123

B. IL-2R

~1.0kb

~3.5kb

~1.5kb

Figure 4. HSF suppresses expression of lL-2 and but not IL-2R (p55) genes in PHAstimulated PBMe. PBMC were cultured alone or with PHA in the presence or absence of HSF (1:6 dilution) for 5 h. Cells were then collected and washed with PBS. Total RNA was extracted, subjected to electrophoresis in formaldehyde-agarose gels, and transferred to

nitrocellulose membranes. Lane 1 contains RNA from cells incubated in the absence of PHA. Lane 2 contains RNA from cells incubated with PHA. Lane 3 contains RNA from cells incubated with PHA and HSF. The blots were sequentially hybridized to 32P-labeled probes for IL-2 and the IL-2R (p55). Blots were stripped and reutilized for rehybridization. IL-2 activity for each of the culture supernatants incubated for 48 h was as follows: PBMC alone (0 Vlml), PBMC + PHA (70 Vlml) and PBMC + PHA + HSF (35 Ulml).


HSF selectively suppresses the expression of JL-2 mRNA

To determine the level at which HSF interrupts IL-2 synthesis, we examined its effect on IL-2 and IL-2R (p55) mRNA levels in PHAstimulated PBMC using Northern blot hybridization. In preliminary experiments, we found that the accumulation of IL-2 mRNA in PHAstimulated PBMC peaked at 5-6 h, and declined to undetectable levels at 24 h after stimulation. In contrast, IL-2R (p55) mRNA accumulation was detectable at 4-5 h, peaked at 24 h, and declined but was still detectable at 48 h after stimulation (Fig. 5) consistent with previous published observations (37, 38). Therefore, total RNA was collected from PBMC stimulated with PHA for 5 h in the presence or absence of HSF and transferred to a nitrocellulose membrane after size-fractionation. This blot was hybridized with an IL-2 cDNA probe and then rehybridized with an IL-2R (p55)

A.24hr

B.48hr

IL-2R

......:3.5kb

......:1.5kb

~.5kb

..... 1.5kb

Figure 5. HSF doe< not suppress the IL-2R (p55) message levels at 24 or 48 h. Experiments

similar to those described in Figure 3 were performed but lymphocytes were harvested at 24 or 48 h. The peak message levels for IL-2R (p55) are observed at 24 h. Cells were incubated with control medium (lane 1), PHA (lane 2) or PHA + HSF (lane 3).

HSF Selectively Suppresses Lymphokine Production· 77

plasmid DNA probe. As shown in Figure 4, HSF suppressed the accumulation of IL-2 mRNA without altering that of IL-2R (p55) mRNA in PBMC. HSF also did not alter the accumulation of IL-2R (p55) mRNA at either 24 or 48 h after stimulation in PBMC (Fig. 5). This evidence is consistent with the results obtained with detection of functional IL-2 and the presence of the IL-2R (p55) in cultures including HSF (Fig. 3) and suggests that HSFmediated suppression of IL-2 production is at least in part due to a selective effect on pretranslational events. Although we also attempted to analyze the effect of HSF on IL-4 (i.e. BSF1) mRNA levels in PHA-stimulated PBMC, we did not obtain sufficient amounts of IL-4 mRNA to assess the HSF effect.

Discussion

Among T cell-derived lymphokines, BCGF (IL-4/5) and BCDF (IL-6) have been determined to be important in the process of B cell activation and differentiation into Ig producing cells (11). Human BCGF (or BSF-1), which supports growth of anti-Ig antibody- or SAC-activated B cells, has been dissected into two major distinct groups, low m.w. BCGF (12-25K) (1,39) and high m.w. BCGF (50-60K) (2, 40). The cDNA sequence coding for a human BSF-1 has been recently cloned and the deduced m.w. has been determined as 15,000 daltons (3). Although rBSF-1 (rIL-4) shows BCGF activity, the relationship of this lymphokine to other low or high m.w. BCGF is unknown at the present time. Different human BCGFs may respond to different human B cell subsets at different stages of activation and differentiation, and/or the large variation in the observed m.w. of BCGFs may be attributed to differences in the cell source used for BCGF production.

Human BCDF (BSF-2 or IL-6) which acts on B cells in the final differentiation step into Ig producing cells has been functionally separated from supernatants of mitogen- or alloantigen-stimulated PBMC (4) and recently purified to homogeneity (5). Its cDNA has also recently been cloned (6). Purified BCDF induces Ig production in SAC-activated B cells and EBV -transformed B cell lines, but does not contain any IL-2 or IFN-y activity. Based on this evidence, we tested whether HSF interfered with the production of lymphokines responsible for the BCGF or BCDF activity in supernatants of mitogen-stimulated PBMC or T cells. We demonstrated the following: 1) Supernatants of PHA-stimulated PBMC or T cells (LSN) could enhance proliferation of SAC-activated B cells as long as 5 days in culture. This BCGF activity was not altered by addition of HSF to PHA-stimulated cultures (Table 1); 2) CTLL-20-absorbed LSN, which did not contain any IL-2 activity, also enhanced B cell growth and again HSF did not alter the production of lymphokines mediating BCGF activity in these LSN (Table 1); 3) Supernatants of PWM-stimulated PBMC could


induce Ig production in SKW6.4 cells, and HSF did not alter the production of lymphokines mediating BCDF activity in these culture supernatants (Table 3).

In contrast to the results for lymphokines mediating BCGF and BCDF activities, HSF suppressed IFN-y activity generated by both PHA- and PWM-induced PBMC at 48 h after initiation of culture in addition to suppressing mitogen-induced IL-2 production. Several investigators have demonstrated that the expression of IL-2 and IFN-y genes may be controlled by a common regulatory mechanism (41). Our present results may support these reports in that HSF suppressed both IL-2 and IFN-y activity produced by T cells, however, it should be noted that HSF did not significantly suppress IFN-y activity at later time points. The reason for this is not known at the present time. Since we observed that HSF suppressed IFN-y activity generated by the mitogen-stimulated PBMC in either the presence or absence of exogenous IL-2, the possibility that the decreased level of IL-2 production indirectly caused the decrease in IFN-y activity appears to be ruled out. The direct effect of HSF on the production of IFNy activity is presently being investigated using an IFN-y cDNA probe.

A number of studies concerning the importance of IFN -y in promoting B cell growth and differentiation into Ig producing cells have been reported. Several reports have demonstrated that IFN-y could be one of the differentiation factors for B cells (14-16). In contrast, JELINEK et al. (42) have demonstrated that rIFN-y does not support the generation of Ig producing cells from SAC-activated B cells. Moreover, NAKAGAWA et al. (43) have recently demonstrated that IFN-y is not required for terminal differentiation of B cells into Ig producing cells in the PWM system, whereas IL-2 plays the essential role in this process. Since we demonstrated that the reconstitution of culture levels of IFN-y (50 or 100 D/ml) did not abrogate the suppressive effect of HSF on PWM-induced IgM production (Table 4) in contrast to the effect of exogenous rIL-2 (21), the decrease of IFN-y activity may not be responsible for the HSF-mediated suppression of PWM -induced Ig production. Therefore, our data support the concept that IL-2 is essential in the PWM system for Ig production and that suppression of IL-2 levels by HSF decreases the amount of antibody produced.

We demonstrated that HSF acts selectively in suppressing the production of T cell-derived lymphokines. The mechanism of the selectivity of HSFmediated suppression remains unclear. SAIKI et al. (44) have found one CVH patient whose T cells release BCDF and BCGF activity without IL-2 production in response to mitogen-stimulation. Moreover, a human T cell hybridoma (45) and a human T cell leukemia virus-transformed T cell line (46) have been shown to secrete BCGF or BCDF but not IL-2. Further, Cyclosporin A (CsA) does not inhibit BCDF production by T cells at concentrations which completely inhibit IL-2 production (43). Thus, B cell stimulatory factors (BCGF, BCDF) and IL-2 and IFN-y may be produced by different subsets of CD4+ cells. In this regard, MOSMANN et al. have


demonstrated that mouse T cell clones, producing BSF-1 or IL-4 (TH2) are distinct from those producing IL-2 and IFN-y (TH1) (47). Thus, the selectivity of HSF action may be due its specific interaction with the subset of CD4 cells that makes IL-2 and IFN-y but not IL-4, IL-5 or IL-6. Alternatively these lymphokines may be produced by the same cell and their expression may be independently regulated by HSF. Discrimination between these alternative mechanisms of regulation of lymphokine production will aid in elucidating the mechanism of HSF action.

Recently, MOSMANN et al. have reported the production of a suppressive lymphokine, produced by TH2 cells that acts on TH1 cells. It inhibits the production of IL-2, IL-3, LT/TNF, IFN-y and GM-CSF (48). It thus closely resembles HSF in biologic function. The biochemical similarities between this cytokine termed CSIF and HSF remain to be determined.

Another selective effect of HSF was that HSF did not inhibit the appearance of IL-2R (p55) mRNA and the expression of the receptor in spite of causing significant suppression of the appearance of IL-2 mRNA and its product by mitogen-stimulated T cells (Figs. 3-5). A number of studies focussing on the biochemical and molecular events of T cell activation have demonstrated that stimulation with PMA induced IL-2R (p55) gene transcription and expression of the receptor on the surface of both activated T cells and T lymphocytic leukemic cells, however, this agent alone only minimally induced IL-2 production (49). In contrast, combination of PMA with either PHA or calcium ionophores allowed marked IL-2 production in normal or malignant T cells (41, 50). Since PMA appears to exert its effect by activating protein kinase C (PKC) and both PHA and calcium ionophores cause the increase of cytoplasmic free calcium by mobilizing calcium from intracellular stores in the endoplasmic reticulum (51), IL-2 production is likely dependent on both of these two transmembrane signaling events. In contrast, the activation of PKC appears to be sufficient to signal IL-2R (p55) expression. In addition, it has been shown that CsA blocks expression of IL-2 mRNA but not that of IL-2R (p55) mRNA by mitogen-stimulated T cells (52). Consistent with this finding, is the report that CsA does not inhibit the activation of PKC but does prevent the calcium signal either by directly affecting calmodulin, or by blocking calcium channels and inhibiting the increase of cytoplasmic-free calcium (53). Therefore, an especially tempting hypothesis regarding HSF action is that HSF may inhibit a step of transmembrane calcium flux without affecting activation of PKC. This area is currently being further explored.

We have previously shown that HSF does not exhibit IL-1 or IL-2-like activity in that it does not promote the proliferation of the CTLL20 or D10 lines, respectively (21). In the control cultures for the experiments included in this manuscript it is evident that HSF does not express IL-4, IL-5, IL-6 or IFN-y-like activity. HSF activity appears to reside in the < 10K MW fraction of the supernatants (20) thus further distinguishing it from other lymphokines. In this paper, we have demonstrated that HSF suppresses


both IL-2 and IFN-y production but not the production of lymphokines mediating BCGF or BCDF activity or the expression of IL-2R (p55) or TFR. Moreover, we have found that HSF selectively suppresses the appearance of the IL-2 transcript. These observations provide additional support for the unique nature of HSF. These studies may lead to further elucidation of regulatory mechanisms involved in lymphokine production, and may provide important information for future therapeutic application of HSF in autoimmunity and transplantation.

Acknowledgement

We thank Dr. T. HAMILTON and Dr. C. TANNENBAUM for invaluable advice on Northern blot hybridization; Dr. M. J. THOMASSEN for providing rIFN-y; Mrs. R. TURINIC and Mr. G. BURGESS for FACS analysis; and the Blood Bank of the Cleveland Clinic Foundation for their cooperation in collecting units of blood.

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Dr. MARTHA K. CATHCART, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, Ohio 44195, U.S.A.

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Selective Suppression of L ymphokine Production by Human Hybridoma Suppressor Factor (HSF)