CELLULAR IMMUNOLOGY 106,428-436 (1987)

The Effects of Cytokines and Adherent Cells on the lnterleukin 4-Mediated Induction of la Antigens on Resting B Cells’

KERRY OLIVER,* PETER H. KRAMMER,~ PHILIP W. TUCKER,*,* AND ELLEN S. VITETTA**$’

*Immunology Graduate Program, and $Department ofMicrobiology, University of Texas Health Science Center, Southwestern Medical School, Dallas, Texas 75235; and the f Institutefor Immunology

and Genetics, Heidelberg, Federal Republic of Germany

Received December 15.1986; accepted January 22,1987

In this report we have extended our previous studies on interleukin 4 (L-4) [previously termed B-cell stimulatory factor-l (BSF-l)]. Our results demonstrate that 8 hr of exposure to IL4 is sufficient to induce maximal expression of Ia antigens. This increase in expression of Ia antigens on resting B cells is due to the direct action of IL-4 on the B cells since adding or removing adherent cells or utilizing low density cultures of B cells at SO-lOO/culture had no effect on the L4mediated increase in Ia. Monoclonal anti-E-4 antibody completely abrogated the Ia- inducing activity of E-4. A variety of other purified lymphokines including interleukin 2 (IL 2), interleukin 1 (IL-l), and a source of either B-cell differentiation factor for IgM (BCDFp), or B-cell growth factor II (BCGF II), did not alter the expression of Ia antigens on resting B cells. However, interferon-r can partially inhibit the IL&mediated induction of Ia. 8 1987 Academic

Ress, Inc.

INTRODUCTION

Interleukin 4 (IG4),3 initially known as B-cell growth factor- 1 (BCGF- 1) and later as B-cell stimulatory factor- 1 (BSF- I), is a lymphokine which has multiple effects on resting and activated B cells. On resting B cells, IL-4 induces an increased expression of Ia antigens ( 1,2) and prepares the cells to enter the S phase of the cell cycle follow- ing stimulation with anti-Ig antibodies (3,4). In lipopolysaccharide (LPS) (5) or anti- Ig (6) activated B cells, IL4 induces the nuclear transcription (7), RNA modulation and increased half-life (8, 9), surface expression (7), and secretion (5-7, 10, 11) of IgGi . It also induces the secretion of IgE ( 12). The mechanisms by which this lympho- kine exerts multiple effects on resting and activated B cells are unclear. However, the

’ This work is supported by NIH Grants AI- 1185 1, AI-2 1229, AI- 180 16, NATO Grant 3 13-86, and by the Texas Department of the Ladies Auxiliary, Veterans of Foreign Wars.

* To whom reprint requests should be addressed: Department of Microbiology, University of Texas Health Science Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX 75235.

’ Abbreviations used: BCDFr(, B-cell diI%erentiation factor for IgM; BCGF II, B-cell growth factor II; BSF-I, B-cell stimulatory factor-l; FACS, fluorescence activated cell sorter; IFN-7, interferon--r; L-4, interleukin 4, H-3, interleukin 3; IG2, interleukin 2; IGl, interleukin 1; MFI, median fluorescence inten- sity; MV, median volume; PI, propidium iodide; SI, stimulation index.

OOOS-8749187 $3.00 Copyright Q 1987 by Academic I’ms, Inc. All rights of reproduction in any form reserved.

INTERLEUIUN 4 429

induction of Ia antigens on resting B cells indicates that receptors for IL-4 are present on these cells.

To gain further insight into the mechanism of action of IL-4 on resting B cells, we have carried out additional experiments on its Ia-inducing properties. Our results demonstrate that IL-4 acts directly on the B cell in the absence of either T cells or adherent cells. Cells must be cultured with IL-4 for approximately 8 hr before they will show a maximal increase in the expression of Ia antigens. Recombinant murine IFN-y partially inhibits and monoclonal anti-IL-4 completely inhibits the Ia-induc- ing properties of IL-4.

MATERIALS AND METHODS

Animals. Female (C57BL/6 X DBA/2)F, (BDFi) mice (from the University of Texas Health Science Center breeding colony), 8-12 weeks of age were used for all experiments, unless otherwise specified.

Preparation of B cells. Spleen cells were stained with biotin-conjugated, mono- clonal anti-Thy- 1.2 (HO- 13.4) ( 13) and fluoresceinated avidin (Vector Labs, Burlin- game, CA). Small B cells were enriched based on their negative surface fluorescence and low forward light scatter on a fluorescence activated cell sorter III (FACS III) (Becton-Dickinson, CA). The sorted cells were always greater than 99% surface-Thy- 1 .Znegative (sThy-I .2-) and represented a homogeneous population of small cells which were 90-95% surface Ig-positive (sIg+).

Preparation ofperitoneal adherent cells. The peritoneal cavities of BDF, mice were flushed with balanced salt solution (BSS) plus 5% fetal calf serum (FCS) using a sy- ringe and 18-gauge needle. The cells were then washed, counted, and plated in com- plete media at different concentrations. After 24 hr, the nonadherent cells were re- moved by rinsing each well. Fresh medium containing the sorted B cells was then added to each well.

Cell culture conditions. Sorted B cells were cultured as described previously ( 14). Lymphokines. Five sources of lyrnphokines were used (a) the supematant (SN) of

concanavalin A (Con A)-pulsed PK 7.1 cells ( 15) which contains IL-4, B-cell differ- entiation factor for IgM (BCDFp) (I 5), and B-cell growth factor II (BCGF II) (Vitetta, unpublished observations) but lacks interferon-y (IFN-y) and interleukin 2 (IG2) (15); (b) a preparation of IL4 purified from the SN of phorbol my&ate acetate (PMA)-stimulated EL-4 or concanavalin A (Con A)-pulsed PK 7.1 cells by high per- formance liquid chromatography (HPLC) according to Ohara et al. ( 16); (c) recombi- nant human interleukin 1 (IGl) (Cistron, Pine Brook, NJ); (d) recombinant mouse interleukin 2 (B-2) (Amgen, Thousand Oaks, CA); and (e) recombinant mouse inter- feron-y (rIFN-y) (Genentech).

Analysis of cell surjbze antigen expression. Cultured B cells were stained with bioti- nylated monoclonal anti&Ad (clone MKD6 obtained from ATCC) and fluorescein- ated (FITC)-avidin (Vector Labs). Propidium iodide (PI) was added to all samples at a final concentration of 5 &ml to exclude dead cells during the analysis on the fluorescence activated cell sorter (FACS). Negative controls contained less than 5% positive-staining cells.

RESULTS Kinetics of Maximal Ia Induction

Previous kinetic studies have shown that after 8 hr of exposure of B cells to IL-4, an increase in Ia expression is detectable ( 1). Noelle et al. ( 17) have also demonstrated

430 OLIVER ET AL.

TABLE 1

Kinetics of IL&Mediated Ia Induction”

Time of exposure to IL-4”

(W Expt 1

Ia expression MFI (SI)*

Expt 2

No add 0 1 3 6 8

12 24

42.6 43.8 108.0 (2.5) 106.0 (2.4) 106.4 (2.5) 106.0 (2.4) 83.2 (2.0) -

65.6<1.5) 80.2(1.8) 67.2(1.5)

53.6 (1.3) 64.2(1.5) 52.2(1.2) 61.8(1.4)

LI After the exposure to puritkd IL-4 (5 units/ml), the celk were washed three times and recukured for the remainder of the 24-hr culture period in fresh medium lacking K-4.

* MFI = median fluorescence intensity; SI = stimulation index.

that by radioiodination and Northern blotting that increased Ia expression and in- creased levels of Ia-specific mRNA expression are detectable 5 hr after exposure of the B cells to IL4. To determine the time of exposure to IL-4 necessary to obtain maximal induction of surface-Ia antigens, further kinetic analysis has been done. This has involved varying the exposure time to IL-4 followed by culturing the cells in medium alone for the remainder of the 24-hr culture phase and then staining the cells with anti-Ia antibodies. Table 1 shows that B cells exposed to IL-4 for 8- 12 hr undergo an increase in expression of Ia antigens that is similar to that observed in B cells exposed to IL4 for 24 hr.

Adherent Cells Do Not Affect the Ability of IL-4 to Induce Increased Levels of Ia on B Cells

The possible role of macrophages in the IG4-mediated increase of Ia antigens on B cells was studied by altering the proportion of adherent cells present in the B-cell population. This was accomplished in two ways: (a) Adherent cells were removed by passage of spleen cells over two G- 10 Sephadex columns ( 18) followed by removing sThy-l+ cells on the FACS. These cells contained less than 1% monoesterase-positive cells when compared to normal splenocytes. The removal of G- 10 adherent cells had no effect on the Ia inducing properties of IL4 on B cells (data not shown). (b) Perito- neal adherent cells were added to cultures of B cells containing K-4. After overnight incubation of peritoneal cells in the wells of microtiter plates, the nonadherent cells were removed and the purified B cells were added. The data in Table 2 demonstrate that the addition of these cells had no effect on the la-inducing properties of E-4.

To further demonstrate that IL-4 acts directly on B cells, cells were plated at either 50 cells/well (data not shown) or 100 cells/well. IL-4 was added and the cells were cultured for 18 hr. The cells were then pooled, stained with anti-h% and the median fluorescence intensity (MFI) was determined. As shown in Fig. 1, increases in the levels of Ia were similar whether the B cells were plated at 100 cells/well or were cultured in the usual manner in bulk culture.

INTERLEUKIN 4 431

TABLE 2

Effect of the Addition of Peritoneal Adherent Cells on the IL4Mediated Induction of Ia Antigens

Number of peritoneal cells added”

x lo-’ Addition of

IL-4 (k)

Ia induction MPI (SI)b

0 - 38.4 0 + 114.2 (3.0)

20 + 98.6 (2.6) 10 + 102.8 (2.7) 5 + 99.0 (2.6) 2 + 98.6 (2.6)

’ Peritoneal cells were prepared and cultured as described under Materials and Methods. After 24 hr the nonadherent cells were removed and B cells were cultured with the adherent cells in the presence of purified IL4 or medium. ARer 18 hr, the cells were stained with anti-Ia and analyzed by flow cytometry.

’ See legend to Table 1.

Hyper-la Induction Is Due to K-4 and Not to a Contaminating Cytokine

The presence of other cytokines (e.g., IL- 1, IG2, IFN-7, BCDFp, BCGF II) in SN of stimulated T cells or macrophages led to the possibility that in vivo, other factors may contribute to or inhibit the hyper-Ia expression induced by IL-4. To investigate this possibility, a preparation of crude SN from the T-cell line, PK 7.1, was passed over a monoclonal anti-IL-4 a5nity column ( 16). The lymphokines contained in the “fall-through” included BCDFr( (9) and BCGF II (Vitetta, unpublished observa- tions), however, PK 7.1 SN does not contain IL2 or IFN-7 ( 14). As shown in Table 3, the addition of crude PK 7.1 SN or aflinity-purified IL-4 resulted in a comparable increase of Ia expression on resting B cells. However, the effluent from the anti-IL-4 affinity column (which contained BCGF-II and BCDFp) did not induce increased expression of Ia antigens. The effluent also lacked both the anti& costimulatory ac- tivity (5) and IgGr-inducing properties of IL4 (5). Furthermore, when monoclonal

100

90 - MFI(S.1) p eo- - MEDIUM 24.0

._._._ IL-4 66.0(3~3

'.. 'v.,1

..?..... ..- .

0 20 40 60 80 100 120 140 160- 180 200

FLUORESCENCE INTENSITY (IA)

PK. 1. Effect of IL-4 on resting B cells cultured at low density. B cells were cultured at 100 cclls/Terasaki well in the presence of purified IL-4 (5 units/ml) or medium. After 18 hr, the cells were pooled, stained with anti-Ia, and analyzed by flow cytometry.

432 OLIVER ET AL.

TABLE 3

Effects of Soluble Mediators on the Induction of Ia Antigens on Resting B Cells

Cytokines Ia expression added a MFI (SI)e

SI of costimulation

=.=Y

- 28.8 - PK 7.1 (207)b 92.4 (3.3) 58.0 R-4’ 86.4 (3. I) 60.0 PK 7.1 (207abQd 28.4(1.0) 2.5

4 Costimulation of resting B cells with Sepharoseanti-6. ’ Unfractionated PK 7.1 SN (batch 207). ’ Affinity-purified IL4 (5 units/ml), adherent PK 7.1 SN (batch 207) from a monoclonal anti-IL-4 a&r-

ity column ( 14). d Nonadherent PK 7.1 SN (batch 207) from a monoclonal anti-IL-4 aIhnity column (14). This material

contained BCDF# and BCGF II but not IL-4 [as defined by lack of anti-d-mediated costimulating ability and BCDF? (8) activity].

’ See legend to Table 1.

anti-IL-4 was added to IG4-containing cultures, it abrogated the ability of IL4 to induce increased expression of Ia antigens (Fig. 2). An isotype matched rat hybridoma antibody had no effect on the IL-4-mediated hyper Ia-expression (data not shown).

Other cytokines produced by activated T cells and macrophages were also tested. Neither recombinant IL- 1 nor recombinant IL2 at any dose alone, or in combination with IL-4, had any effect on the Ia expression on resting B cells (Fig. 3).

IFN-7 Partially Inhibits IL-4-Mediated Hyper-Ia Expression

Although IFN-y induces class II expression on many other cell types (macro- phages, mast cells, melanocytes, B-cell blasts, etc.) (19-22), Noelle et al. (1) have shown that EN-7 has no Ia inducing properties on resting B cells. However, EN-7

100 90

g 00 -I 70 d 60 v ? 50

40 5 30 d 20 Oz 40

0

MFI (SI) -MEDIUM 34.0 -IL-4 102.6(3.01 ----IL-4 + ANTI-IL-4 49.8(1.5)

20 40 60 80 100 120140 ?60 1802 lo

FLUORESCENCE INTENSITY (IA)

FIG. 2. Anti-IL4 inhibits the IL-&mediated induction of Ia. B cells were cultured with purified IL-4 (5 units/ml), IL-4 plus anti-Ic4 [ 11 B 1 1 ( 1411, or medium alone. After 18 hr, the cells were stained with anti- Ia and analyzed by flow cytometry.

INTERLEUIUN 4 433

r

0 MEDIUM l IL-4 0 IL-2 0 IL-4 + IL-2 0 IL-I l IL-4+IL-1

-2 -1 0 1 2 Log (units /ml)

FIG. 3. Effects of IL-1 and IL2 on IL4mediated induction of Ia. Cells were cultured for 24 hr with various amounts of either IGI, IL-2, or in combination with purified IL-4 (1 unit/ml). After 18 hr, the cells were stained with anti-Ia and analyzed by flow cytometry.

has recently been shown to inhibit the anti-Ig costimulatory activity (23,24) and the IgG,- and IgFkducing properties of IL4 ( 12).

We, therefore, addressed the question of whether IFN-7 could inhibit the IG4- mediated induction of Ia antigens. Cells were incubated in the presence of purified IL-4, IFN-y, or a combination for 24 hr, then analyzed by flow cytometry for the level of expression of Ia antigens. Figure 4 illustrates that by using 10 units/ml of IFN-7, 50% inhibition of the maximal IGCmediated hyper-Ia expression was ob- served. This inhibition occurs in a dose-dependent manner (Fig. 5), and inhibition is still observed with as little as 0.05 units/ml IFN-7. At doses of up to 500 units/ml of IFN-y, total inhibition of IL-4-mediated hyper-Ia expression was never observed. Inhibition was also dependent upon the dose of IL-4 used. When IL-4 was added

100

90

p 00 - - MEDIUM IL-4 106.6(3.4) J 70 ---- IL-4 + y-IFN 67.6 (2.1)

ii 60

; 50

-> 40

!ii 30

d 20

a 10

0 20 40 60 80 100 120 140 160 100 i

FLUORESCENCE INTENSITY

(IA)

FIG. 4. Effect of IFN-y on the IL4-mediated induction of Ia. Cells were cultured for 24 hr with 10 units/ ml rIFN-7, purified IL-4 (5 unit/ml), or a combination. At the end of the 24-hr incubation period, rIFN-7 was added to the control. Samples were washed twice, stained with monoclonal anti-Ia, and analyzed by tlow cytometry.

434 OLIVER ET AL.

40 -

. 35- a-0

Y 30 -

;; =25- P \ -/A. - I

20 -0 c@ cW+#_oP~o _

15

t

0 MEDIUM m IL-4 o Y-IFN

4n 0 IL-4 + Y-IFN

-3 -2 -1 0 1 2 Log (units/ml)

PIG. 5. Dose response of the IPN-y-mediated inhibition of IG4. Cells were cultured for 24 hr with different amounts of rIFN-7, alone, or in combination with IL-4 (1 unit/ml). At the end of the 24-hr incubation period, rIFN-y was added to the control. Samples were washed twice, stained with monoclonal anti-Ia, and analyzed by flow cytometry. Percentage inhibition is calculated as the ratio of Ia increase of r1IW-y + IL-4 to IL-4 alone.

at a 50% maximal dose (ECSO; 1 unit/ml), IFN-y-mediated inhibition of hyper-Ia expression was 85% (Fig. 5). Kinetic studies indicated that preincubation of B cells with IFN-y never caused complete inhibition of IL-4 activity (Fig. 6). However, IFN- y was still inhibitory when added up to 8- 10 hr after K-4.

These data suggest two possible mechanisms of IFN-y-mediated regulation of IL- 4 activity. Different subpopulations of B cells may respond differently to IF%y and

0 IL-4+Y-IFN

-4 0 4 6 12 16 20 24

Time of Addition of y-IFN (hours)

FIG. 6. Kinetics of the IPN--r-mediated inhibition of IL-4. Cells were cultured with IPN-7 ( 10 units/ml) alone or in combination with IL-4 (1 unit/ml). 1FN-y was added at various times after the initiation of culture. The zero time point indicates the time point that IL-4 was added. After 24 hr, the cells were stained with anti-Ia and analyzed by flow cytometry.

INTERLEUKIN 435

IL-4. This seems unlikely since the inhibition of hyper-Ia expression by IFN-y results in a homogeneous expression of Ia antigens only at a decreased surface density (Fig. 4). If different subpopulations were involved then a bimodal distribution of Ia anti- gens would be expected. The other possibility is that the IL-Cmediated hyper-Ia in- duction may be a result of a two step process. The initial step would consist of a IFN- y-insensitive step that results in a low level increase in Ia expression. The second step would be IFN-7 sensitive whereby the addition of IFN-7 would block any further increase in Ia expression.

Taken together, the above results suggest that IL-4 acts directly on the B cell to induce hyper-Ia expression. Furthermore, IFN-y can partially inhibit this activity. The mechanism by which this occurs is currently being investigated.

DISCUSSION

IL-4 is a T-cell-derived lymphokine that induces increased levels of Ia expression on resting, noncycling (Go) B cells (1,2). Studies by Noelle et al. (17) have demon- strated that the K-4-mediated induction of Ia requires protein synthesis but not DNA synthesis and is accompanied by increases in the steady-state levels of Ia-specific mRNA. IL-4 can also prepare B cells for entry into cycle following the subsequent addition of anti-Is (3, 4). It has been demonstrated that IL-4 can also induce IgG, expression and secretion in anti-Ig (6) or lipopolysaccharide (LPS)-stimulated B-cell blasts (5) and IgE secretion in LPS blasts ( 12). In this report, we have further extended our studies on the action of IL-4 on resting B cells. The major results to emerge from these studies follow: (a) IL-4, but not a variety of other lymphokines such as IL-2, IL 1, IFN--y, BCDFp, and BCGF II, induces increased expression of Ia antigens on rest- ing B cells. Expression can be blocked by the addition of monoclonal anti-IL-4 anti- body ( 15). (b) IL-4 acts directly on the B cell since cells cultured with IL-4 in wells of Terasaki plates at 50- 100 cells/well can be induced to express Ia as effectively as cells cultured at higher densities. (c) Adherent cells are not involved in the Ia-inducing activity of IL-4. The removal of adherent cells or their addition to small, resting B cells has no effect on the ability of IL-4 to induce increased expression of Ia antigens. (d) It takes approximately 8 hr of exposure of resting B cells to IL-4 to induce a maximal increase in the expression of Ia. (e) The IL-Cmediated hyper-Ia expression can be partially inhibited by the addition of small amounts of IFN-y.

Further studies will be required to determine the precise mechanisms of action of IL-4 and IFN-7 on resting B cells, but the ability of some antigen-activated T cells to produce antagonistic lymphokines is intriguing and demonstrates that T cells can both enhance and inhibit the activation of B cells. One possibility to explain the inhibitory vs stimulatory activity of T-cell-derived lymphokines is that some lympho- kines are secreted by different T cells at different times after their activation. In this regard, it is known that clones of T helper cells (Th) fall into two groups, those secret- ing 1FN-y and IG2, and those secreting IL4 ( 12,25). It is possible that IL4 is secreted by one Th subset early after activation with antigen, and IFN--y may be produced later by another T-cell subset with more stringent activation requirements. Alternatively, resting B cells may express receptors for IL-4 before they express receptors for IFN- y. Therefore, the positive activation signals (IL-4) would be exerted early and the negative signal (IFN-7) might prevent continued activation of B cells.

436 OLIVER ET AL.

ACKNOWLEDGMENTS

We thank Ms. L. Trahan, Ms. R. Baylis, Ms. S. Gorman, Ms. B. Smith, and Ms. A. Buser for technical assistance and Ms. G. A. Cheek and Ms. C. Base& for secretarial assistance. We thank Dr. J. Ohara and Dr. W. Paul, NIH, for their gift of monoclonal anti-IL-4.

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