The cellular basis of cortisone-induced immunosuppression of the antibody response studied by its reversal in Vitro

CELLULARIMMUNOLOGY17, 405-417 (1975)

The Cellular Basis of Cortisone-Induced Immunosuppression of the Antibody Response Studied by its Reversal in V i t r o 1 KWO:K-CHoY LEE, RODNEY ]~. LANG~AN, VERNER H. PAETKAU, AND ERWIN DIENER MRC Transplantation Group and Departments of Biochemistry and Immunology, University of Alberta, Edmonton, Alberta, Canada Received October 15, 1974 Spleen cells from mice pretreated with cortisone were impaired in their ability to support an immune response in vitro to two antigens: sheep erythrocytes (SRBC) and polymeric flagellar protein o~ Salmonella adeIaide (POL). We conclude that this imrnunosuppression is consistent with cortisone causing a dysfunction in accessory cells (macrophages?) and thymus-derixed (T) helper cells without seriously affecting antibody forming cell precursors. Evidence for this comes from reconstitution of the response with additions of peritoneal exudate cells (PEC, irradiated, and anti-0treated), activated T cells, and 2-mercaptoethanol (2-Me) in culture. Additions of PEC or 2-Me restored the T cell-independent anti-POL response not only in cortisone-treated spleen cell cultures, but, contrary to previous reports, also in cultures of normal spleen cells depleted of adherent (accessory) ceils by c~rbonyl iron treatment. The anti-SRBC response (which is dependent on T cells and accessory cells) in cortisone-treated spleen ceil cultures was fully restored only by a combination of aetixated T cells and PEC or activated T cells and 2-Me. However, with lower doses of cortisone pretreatment, activated T cells or 2-Me alone was effective. INTRODUCTION Glucorticosteroids are used extensively for immunosuppression in transplant patients and for treatment of inflammation and leukemia, but their mode of action at the cellular level is poorly understood. Suppression of humoral and cell-mediated immune responses in different mammalian species has been reported ( 1 ) , but there is conflicting evidence as to the identity of the steroid-sensitive cells. T h u s Claman and others reported that in mice, corticosteroids selectively destroy thymus leukemia ( T L ) positive cells in the thymus cortex, but do not affect others such as cells responsible for graft-versus-host reactivity (2), cells in bone m a r r o w (3), and thymus-derived ( T ) helper cells for humoral responses (4, 5). Cohen and Claman (5) concluded that bone marrow-derived precursors of antibody-forming cells were hydrocortisone sensitive since spleen cells from hydrocortisone treated mice could not fully reconstitute an a n t i - S R B C (sheep erythrocyte) response in irradiated recipients unless bone m a r r o w cells were added. O n the other hand, Segal et al. 1 This work was supported by the Medical Research Council and the National Cancer Institute of Canada. 405 Copyright© 1975by AcademicPress, Inc. All rights of reproductionin any formreserved.

406

nEE ET AL

(6), using a period of hydrocortisone pretreatment of much longer duration than that used by Cohen and Claman (5), found that the humoral response to dinitrophenylated rabbit serum albumin ( D N P - R S A ) was inhibited whereas the response to the T cell-independent antigen DNP-poly L-lysine was unaffected. Furthermore, Vann (7) found that the depressed response of spleen cells from hydrocortisonetreated mice against SRBC in vitro could be restored by activated T cells or soluble factors from cells stimulated with allogeneic antigens. These two latter results suggested that T helper cells were sensitive to eorticosteroids if the pretreatment period was sufficiently long, whereas bone marrow-derived (B) cells were resistant. However, Dracott (8) recently reported no suppression of the anti-SRBC response of spleen cells from hydrocortisone-treated mice in vitro. This is at variance with the other reports discussed above and may reflect differences in experimental conditions. The sensitivity of mononuclear phagocytes or macrophages to corticosteroids has been used to explain susceptibility to a number of infections (reviewed in 9). There is agreement that small doses of cortisone stimulate phagocytic activity in the reticuloendothelial system of rats, mice, and guinea pigs whereas large doses induce blockade (9, 10). Wiener et al. (11) showed by electron microscopy that Kupffer cells in cortisone-treated rats failed to phagocytose carbon particles. Using morphological criteria, Thompson and van Furth (12) found that injection of hydrocortisone into mice resulted in rapid sequestration of blood mononuclear phagocytes to unknown sites and halted their transit to the peritoneal cavity but did not cause a disappearance of resident macrophages. Hydrocortisone did not affect the production of monocytes in the bone marrow, but prevented their release into the blood (13). These observations could provide an explanation for the antiinflammatory and immunosuppressive activities of corticosteroids since macrophages have been shown to be required for the generation of cytotoxic lymphocytes to alloantigens (14), activation of T cells by antigen (15-17) and for mediation of T and B cell collaboration in humoral immune responses (18, 19). In order to elucidate the cellular basis of cortisone-induced suppression of the antibody response, we have examined the immune response of spleen cells from cortisone-treated mice against two antigens with known cellular requirements and attempted to reverse the immunosuppression by addition of defined cell populations. The antigens were: SRBC, which is T cell and accessory cell dependent (18, 2023), and polymeric flagellar protein of Salmonella adelaide ( P O L ) which is T cellindependent (20, 21). It was found that cortisone suppressed the response to both antigens. The anti-POL response was restorable by peritoneal exudate cells (PEC) which had been irradiated and treated with anti-0 ascitic fluid and complement, but the anti-SRBC response required the presence of both PEC and activated T cells for full restoration. These observations are consistent with the notion that cortisone induces functional defects in accessory and T helper cells and may therefore account for the immunosuppressive property of corticosteroids. M A T E R I A L S AND M E T H O D S Animals. Mice of the AKR/J, CBA/J, and CBA/CaJ strains were purchased from Jackson Laboratories, Bar Harbor, Maine. Only CBA/CaJ male mice (3-5 mo old weighing about 30 g) were used for cortisone experiments.

R E V E R S A L OF C O R T I S O N E - I N D U C E D

IMMUNOSUPPRESSION

407

Antigens. Sheep red blood cells (SRBC) were collected aseptically each week in Alsever's solution and washed three times in normal saline before use. Polymeric flagellar protein ( P O L ) was prepared from Salmonella adelaide (strain SW1338; H antigen, fg; 0 antigen, 35) according to the method of Ada et al. (24). The concentration of P O L was defined operationally by the extinction at 215 nm in 0.05 M HC1. Bovine serum albumin (Armour Pharmaceutical Co., Chicago) was used as standard. Cortisone. Cortisone acetate suspension was purchased from Merck Sharp and Dohme Ltd., Kirkland, Quebec. Mice were injected subcutaneously with various doses of this preparation 24 hr before being killed for experiments. Tissue Culture. A modification of the method of Diener and Armstrong (25) was used. Spleens were removed aseptically and placed in Puck's saline A. They were minced with scissors and teased through a stainless steel mesh. The cell suspension was centrifuged at 750g for 7.5 rain, the pellet was suspended in Puck's saline and clumps of cells removed by gentle centrifugation. The concentration of viable spleen cells was determined by counting in a hemocytometer using 0.1% (w/v) Trypan blue to stain dead cells. Aliquots of 60 × 106 viable cells were taken and washed once in Eagle's minimal essential medium (Gibco, Grand Island Biological Company, Santa Clara, California) supplemented with 10% (v/v) heat inactivated fetal calf serum (termed MEM-FCS medium). The 60 × 106 cells were suspended in 4 ml of MEM-FCS and SRBC (final concentration 0.01% v/v) and SW1338 P O L (various doses) were added. From this suspension of spleen cells and antigen, 1 ml aliquots were placed in 1.1 Cln diam glass tubes with ends covered by dialysis tubing. The tubes were then placed in conical flasks containing 50 ml of MEM-FCS medium and incubated at 37°C in a 10% CO2 in air gas mixture for 4 days. When required, 2-mereaptoethanol was added to the medium on both sides of the dialysis membrane at a final concentration of 5 × 10-5 M. Cells were harvested in Mishell-Dutton's balanced salt solution (26) and washed twice before assay. Assays. Anti-SRBC antibody forming cells (AFC) were assayed by the method of Cunningham and Szenberg (27). Anti-POL AFC were enumerated by the bacterial adherence method of Diener (28) with the modification that the pH of bacterial suspensions was brought to neutrality with 1 M NaOH before use and that the cells were incubated at 20°C for 15 min before being plated out in agar. This method yielded larger colonies without affecting the number. The results were expressed as the arithmetic mean --- standard error of the mean (SEM)I All calculations including Student's t test were done by a Warg 500 calculator. Anti-O Ascitic Fluid. A K R / J male mice were immunized with ten weekly intraperitoneal injections of 108 CBA/J thymocytes. The first injection was accompanied by l0 s killed Bacillus pertussis organisms (Connaught Medical Research Laboratories, Toronto). Three days after the last dose of thymocytes, the mice received intraperitoneal injections of Sarcoma 180 cells (0.2 ml of ascitic fluid from a tumorbearing mouse) (29). The asci,tic fluid was harvested 10 days after the final thymoeyte iniection. For anti-0 treatment, 20 × 106 cells were incubated for 40 min at 37°C in 1 ml of anti-0 ascitic fluid. After washing, the cells were incubated again for 4 (diluted 3 times in MEM-FCS) rain at 37°C in 2 ml of agarose-absorbed (30) guinea pig complement (c', diluted 6 times in MEM-FCS).

408

LEE

ET AL.

Peritoneal Exudate Cells. A peritoneal exudate was induced by injecting each CBA/CaJ mouse with 3 ml sterile thioglycollate medium (BBL, Cockeysville, Maryland). Three days later, the mice were given 1000 fads whole body radiation from a laTCs source (Gamma cell 40, Atomic Energy of Canada Ltd.), killed, and the peritoneal cells harvested by washing out the peritoneal cavity with Puck's saline. The cells (80-90% macrophages by neutral red staining) were treated with anti-0 ascitic fluid and complement before use in all experiments and will be referred to as PEC. Activated T Cells. Activated T cells were obtained from the spleens and mesenteric lymph nodes of mice 7 days after they had been irradiated (950 rads) and injected with thymocytes (108/mouse) intravenously and antigen (0.2 ml of 10% v/v SRBC or 20/~g P O L ) intraperitoneally. Depletion of Adherent Cells from Spleen Cell Suspension with Carbonyl Iron Powder. A suspension of spleen cells in Puck's saline was treated to remove dead cells, capsular, and fibrous material as described previously (31). 80 X 106 Cells in 8 ml MEM-FCS were incubated at 37°C for 45 min with various amounts of a coarse grade carbonyl iron powder (batch GQ4-135, General Aniline and Film Corp., Linden, New Jersey) in a flat bottomed siliconized glass vessel. The mixture was shaken every 5 min. The pH was maintained at about 7.5 by gassing the vessel with a gas mixture of 5% carbon dioxide, 5% oxygen, and 90% nitrogen. After the incubation, without allowing the mixture to cool down, the carbonyl iron powder was removed by placing a powerful magnet below the vessel and decanting the supernatant fluid. Control ceils were treated in the same way with omission of the carbonyl iron powder. RESULTS

Suppression of the Immune Capacity of Spleen Cells in Vitro by Cortisone Treatment of Mice and its Reversal by 2-Mercaptoethanol. Spleen cells from mice which received various doses of cortisone 24 hr previously were immunized in vitro with SRBC and P O L for 4 days. Following the observation of others that hydrocortisone administration in vivo resulted in monocytopenia (12) and that 2mercaptoethanol (2-Me) would substitute for macrophage function in tissue culTABLE 1 CORTISONE-INDUCED SUPPRESSION OF THE ANTI-SRBC RESPONSE AND ITS REVERSAL BY 2-MERCAPTOETHANOLa Mean AFC/culture 4- SEM

Cortisone per mouse

Yield of cells per spleen

(mg)

(millions)

No additions

None 1.5 2.5 5.0 10.0

80 33 31 30 20

44194-569 (4.4) b 10004-471 3174-219 8 7 4 - 27 (6.4) 8 7 4 - 41

+5

X 10 - S M 2 - M e

36874-160 (4.4) 3158=t=275 3875 4-725 29834-239 (7.3) 3754-109

a Spleen cells from mice which received various doses of cortisone acetate subcutaneously 24 hr previously were cultured for 4 days (15 X l0 s viable cells/culture) in the presence of SRBC

(0.01% v/v). b Millions of viable cells recovered per culture after 4 days.

REVERSAL OF CORTISONE-INDUCED I?¢IMUNOSUPPRESSION ]04

409

(b)

10 a

w o

3 <

10 ?

<20 [ 10'~

I

l

I

I

1

l

I

I

I

I

q--I

(d)

I

l

I

d m .L

10 3

.,<

<20

I

I

-i

E

~

l

l

l

l

l

l

125~125p~125~g125og125~g125,~g125,~12 5p9125~g125.g125o~125no125,~o125~g 1338 POL/Culture

Fla. 1. Effect of cortisone treatment on the antibody response to POE Cells from mice treated 24 hr previously with (a) 1.5 rag, (b) 2.5 rag, (c) 5.0 rag, and (d) 10.0 mg of cortisone acetate were cultured in the presence of various concentrations of POL for 4 days (15 × 106 viable cells/culture). (O) Control; ( • ) cortisone-treated. ture (32-34), 2-Me was added in an attempt to rescue the immune response. The results in Table 1 show that the anti-SRBC response was suppressed after cortisone treatment, and could be restored in the presence of 5 x 10-~ M 2-Me when cells were obtained from animals treated with low (1.5-5 mg) but not high (10 rag) doses of cortisone. These observations hold true with immunizing doses of SRBC between 0.0001 and 0 . 1 ~ ( v / v ) . The effect of cortisone on the anti-POL response is shown in Fig. 1. The degree of inhibition varied with the dose of cortisone administered and the concentration of P O L in culture. Cortisone-treated cells could respond to low but not high concentrations of P O E The response at tow P O L concentrations became progressively higher with increasing dose of cortisone when compared with the corresponding controls. This phenomenon could be a result of enrichment for a population of cortisone-resistant cells capable of responding at low antigen concentrations in culture since the yield of spleen cells decreased with increasing cortisone dose (Table 1), yet the same number of cells was cultured. In the presence of 2-Me, the cortisone-induced defect was largely corrected and the dose-response curve was shifted toward that of control cells (Fig. 2). In some experiments, 2-Me enhanced the response of cortisone-treated cells to levels greater than the control values. The ability of 2-Me to reverse cortisone-induced immunosuppression is consistent with a defect in accessory cell function. In order to probe further into this possibility, peritoneal exudate cells were used in attempts at counteracting the effect of cortisone.

Reversal of Cortisone-Induced Irnmunosuppression by Peritoneal Exudate Cells. Since any restorative effect with peritoneal exudate cells ( P E C ) may be due to small numbers of contaminating T or B cells, the exudate cells were irradiated then treated with anti-0 aseitic fluid and complement before use. The efficacy of these procedures was demonstrated by their effect on normal spleen cells (Table 2). Thus

410

L E E E T AL.

10'L

103. uJ uq +1

"-5 L)

102

<

<20. a I

I

I

I

I

I

-I

12.5pg 125pg 1'25ng 12'5rig 125ng 1"25¢ug 12'5~g 1338 POL/Culture 104 -

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D

2< 103

102

] [ I I I I 125pg 1"25ng 125ng 125ng l'25Hg 12'5,~g 1338 POL/Culture

FIG. 2. Effect of 2-mercaptoethanol on the antibody response to POL. Cells from mice treated 24 hr previously with (a) 5 mg and (b) 10 mg cortisone acetate were cultured in the presence of various concentrations of POL and 5 × 10-5 M 2-Me for 4 days (15 × 106 viable cells/ culture). (O) Control; ( • ) cortlsone-treated; ([B) control + 2-Me; ( • ) cortisone-treated + 2-Me. anti-0 treatment was effective in abrogating the T cell-dependent response to SRBC, but not the T celMndependent response to P O L , and irradiation eliminated the response to both antigens. Various numbers of P E C were added to cultures of spleen cells from cortisonetreated or normal mice and the immune responses to SRBC and P O L are shown in Fig. 3. In contrast to 2-Me, P E C were incapable of reversing the cortisonesuppressed response to S R B C ; however, they were effective in restoring the response to P O L . The minimum number of P E C for full restoration of the antiP O L response was 105 per culture when the spleen cell donors had received 2.5 mg cortisone whereas 106 per culture were needed after administration of 5 mg cortisone. In agreement with a previous report (35), the presence of P E C in normal spleen cell cultures was inhibitory when large numbers were added. Next, the effect of P E C on cortisone-treated spleen cells was studied at various P O L concentrations and the results are shown in Fig. 4. It is apparent that P E C

411

R E V E R S A L O F CORTISONE-INDUCED IM M U N O S U P P R E S S I O N TABLE

2

EFFECT OF q, RADIATIONAND ANTI-0 TREATMENT ON THE IMMUNE RESPONSE OF SPLEEN CELLS I n Vitro ~ Cells

Mean AFC/eulture ± SEM

Treatment

SRBC

POL

4217 -4- 613

6347 -4- 637

200 4- 88

6010 ± 810

1000 rads

<25

<20

1000 rads and anti-0 ascitic fluid + c'

<25

<20

Normal A K R ascitic fluid + c' Spleen cells

Anti-0 ascitic fluid + c'

Peritoneal exudate cells

Spleen cells and peritoneal exudate cells from normal or irradiated (1000 rads) mice were treated with anti-O ascitic fluid and complement (c') and cultured for 4 days (15 X 106 viable cells/culture) in the presence of SRBC (0.01% v / v ) and POL (12.5 ng/ml).

restored the response to POL at all concentrations to levels near those of the control. The corresponding anti-SRBC (not shown) were not restored, The suppression of the T cell independent response to POL by cortisone and reconstitution of the response by PEC is consistent with defective accessory cell

104-

SRBC

i0~,

'+1

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,~ I041 0"4~

~u~q

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I I I I I 0 3x10'* 105 3x105 106 PECadded per Culture

FIG. 3. Effect of peritoneal exudate cells on the antibody response of cortisone-treated spleen cells. Cells from mice which received 2.5 or 5 nag cortisone acetate 24 hr previously were cultured for 4 days (15 × 106 viable cells/culture) in the presence of antigen (0.01% v / v SRBC and 12.5 ng P O L / m l ) and varying numbers of irradiated and anti-e-treated PEC. ( O ) Control; ( • ) 2.5 nag cortisone; ( • ) 5 mg cortisone,

412

L E E ET AL.

10 a.

uJ +1

10 3 .

u3 ,,u <

d

t0 2-

i

E <

<20

I l I I t I 125pg l'25ng 125ng 125ng ]-25,,ug ]2'5Asg 1338 POL/Culture

FIG. 4. Effect of peritoneal exudate cells on the antibody response of cortisone-treated spleen cells to various concentrations of POL. Cells from mice which received 2.5 mg cortisone acetate 24 hr previously were cultured for 4 days (15 × 106 viable cells/culture) with POL and irradiated and anti-0-treated PEC. ((2)) Control; (E]) control + 5 × 10~ PEC/culture; ( e ) cortisone-treated; (A) cortisone-treated + 10"~PEC/culture; ( • ) cortisone-treated + 5 × 10~ PEC/culture. function rather .than B cell function. Since the T cell dependent response to SRBC was not restored by PEC, the possibility of impaired T helper cell function was examined.

Reversal of Cortisone Immunosuppression with Activated T Cells and Peritoneal Exudate Cells. Thymocytes activated by SRBC in irradiated mice were used as a source of activated T cells (Ts~Be). As a control for antigen specificity, T cells activated by P O L (Tpo~) were used. Since the primary (4 days) IgM response against P O L in our tissue culture system (Table 2) and in vivo ( 3 6 ) is independent of T cells, the efficacy of T cell activation by P O L cannot be checked in vitro. However, the secondary IgG response against P O L in vivo is T cell-dependent (36), and hence it is likely that T cells can indeed be activated by POL. Spleen cells from mice which received 10 mg cortisone 24 hr earlier together with untreated controls were cultured with various additions, none of which alone would give rise to A F C in culture: Ts~Bo, TpoL, PEC, and 2-Me. At the concentration of P O L used for immunization in culture (50 ng/ml), good suppression by cortisone and restoration by 2-Me or P E C was observed. The results (Table 3) are in agreement with previous data (Table 1, Fig. 3), showing a depressed antiSRBC response in cultures of cortisone-treated cells which was restored partially by the addition of 2-Me but not at all by P E C (groups 1--4). P E C were again inhibitory in normal cell cultures at 5 × 105/culture (group 3). The addition of TsRBe enhanced the normal anti-SRBC response, but there was no reversal of cortisone suppression (group 5). However, TsBBc and 5 × 105 P E C when added together showed a synergistic effect and largely restored the response of cortisonetreated cells (group 7). The restoration was complete with the addition of TSRBO and 2-Me (group 8). Tt, oj~ were not effective in restoring the anti-SRBC (group 9), but produced a marginally significant effect in the presence of 2-Me (0.1 > P > 0.5 comparing groups 4 and 12). These observations suggest that cortisone treatment induced a dysfunction in both T helper and accessory cells.

413

REVERSAL OF CORTISONE-INDUCED IM M U N O S U P P R E S S I O i ~

TABLE 3 REVERSAL OF CORTISONE-INDUCED IMMUNOSUPPRESSION BY ACTIVATED T CELLS AND PERITONEAL EXUDATE CELLSa

Groups

Additions per culture

Mean AFC/culture 4- SEM SRBC Normal

1. 2. 3. 4.

None 106 PEC 5 X 106PEC 2-Me

5. 6. 7. 8.

106 TSRBO 106 TSRBO+105 PEC 106TsRBe+5 X 105PEC 106TsRBC+2-Me

9. 10. 11. 12.

106TPoL 106 TpOL+10 ~ PEC 10 6 T p o L + 5 X 105 PEC 106Tpo~+2-Me Controls 15 X l0 s TSl~Be,TpoL, or PEC cultured alone

7500-4- 656 8500=I: 372 4238+ 489 87255:743

POL Cortisone

588-4- 69 4884. 43 7134.177 26634.283

Normal

Cortisone

91604.692 4804. 9060-4-384 7804. 8440-4-166 71074. 125704.729 21450:k

156 191 557 273

126384.1435 10624.288 82404-297 1290-4- 216 13575-4-469 17634.4-354 80604-510 24304. 493 94884. 372 77384.860 76504.451 101004.1319 127254. 670 122004.786 118104.624 220104. 307 5113± 73254. 39504. 88634.

<50

331 322 408 365

5254. 78 7450-4-291 6004. 91 10754. 78 7860-4-749 2250-4- 410 1388:1:401 4880+514 6987~ 355 41634.668 123704.763 17450q- 710

<40

Spleen cells from mice which received 10 mg cortisone acetate 24 hr earlier and from normal mice were cultured for 4 days (15 X 106 viable cells/culture) in the presence of SRBC (0.01% v/v) and POL (50 ng/ml) with additions of PEC, TSRBC,TpoL, and 5 X 10-5 M 2-Me. The effect of cortisone on the anti-POL response was reversed by the addition of P E C (5 x 105/culture) or 2-Me as shown previously (Figs. 2 and 3). In this experiment (group 4, Table 3), 2-Me restored the response of cortisone-treated cells to levels in excess of normal controls as noted before (Fig. 2). Activated T cells did not alter the anti-POL response of normal or cortisone-treated cells (groups 5 and 9). However, a small restorative effect was obtained with either TsEBe or TpoL when 105 P E C were also present in the cultures (0.02 > P > 0.01 comparing groups 6 and 10 with 2). This nonspecific T cell effect may be contrasted with the specific effect seen in anti-SRBC responses, and is consistent with the T-independent character of the anti-POL response (Table 2). The requirement for accessory cells to reconstitute an anti-POL response in cortisone-treated spleen cell cultures is in apparent conflict with previous work (22, 23, 35). This discrepancy may be due to a more stringent requirement for accessory cells in the immune response to SRBC and more exhaustive depletion may reveal a similar requirement for the anti-POL response. To test this possibility, normal spleen cells were treated with large amounts of carbonyl iron and tested for their ability to support an anti-POL response.

Effect of Severe Depletion of Adherent Cells on the Immune response of Normal Spleen Cells. It is likely that adherence rather than phagocytosis is the basis for accessory cell depletion using carbonyl iron (37). Normal spleen cells were incu-

414

L E E E T AL.

TABLE

4

EFFECT OF CARBONYL IRON T R E A T M E N T ON TIIE IMMUNE RESPONSE

OF NORMAL MOUSE SPLEEN CELLS In

ng POL/ Further culture additions/ in add±- culture tion to SRBC

Mean AFC/culture 4- SEM SRBC

POL

Carbonyl iron (g/80 X 106 cells)

Carbonyl iron (g/80 X 108 cells)

0 (Control) 12.5 250

None l0 sPEC 2-Me

Vi[Yo a

0.6

1.0

2750±201 417± 58 308±178 3158± 98 2350±740 2600±643 2441±155 933=f=192 4424-144

0 (Control)

0.6

1.0

5040-4-417 4453±959 1290± 180 4547 ±260 5180±954 5240±1202 44634-401 5300± 64 3580± 174

None 2521 4- 219 375±222 108 4- 58 2873±236 3187±301 207± 77 105 PEC 3600 ± 412 2217 ± 456 1617 ± 282 2593 ±591 3320.4- 155 2605 ± 223

a Normal spleen cells (80 X l0 Gcells in 8 ml) were treated with either 0.6 or 1.0 g of carbonyl iron powder for 45 min at 37°C. After removal of the carbonyl iron, the cells were cultured for 4 days (15 X 106 viable cells/culture) in the presence of SRBC (0.01% v/v) and additions of POL, PEC, and 5 X 10.5 M 2-Me. The data was pooled from two separate experiments. bated for 45 rain at 37°C with various amounts of carbonyl iron powder and the immune response of the treated cells to SRBC and P O L is shown in Table 4. Treatment of 80 × 106 cells with 0.6 g carbonyl iron powder resulted in the abrogation of the anti-SRBC response leaving the a n t i - P O L response intact whereas 1.0 g carbonyl iron powder was needed to produce significant depression of the response to P O L ( P < 0.01). Addition of P E C restored the response of treated cells to both antigens. 2-Me failed to restore the response to SRBC, but did restore the a n t i - P O L response. There was considerable nonspecific adherence to the carbonyl iron powder during the treatment, and the recovery of cells for spleen cell suspension treated with 0.6 and 1.0 g carbonyl iron were 50 and 3 3 ~ , respectively. Although immunocompetent ( T or B) cells could have been removed, the recovery of cells was similar to that of other workers (22), and the restoration of the responses with small numbers of irradiated and anti-0-treated P E C would argue against selective removal of T or B cells. DISCUSSION Spleen cells from cortisone-treated mice were suppressed in the generation of antibody-forming cells in vitro against S R B C and certain concentrations of P O L (Table 1, Fig. 1). The a n t i - P O L response was restorable by P E C or 2-Me, but the anti-SRBC response required both activated T cells (TsRBO) and P E C for full restoration. These findings not only suggested that cortisone induced functional defects in both accessory cells and T helper cells, but also pose questions on the identity of some of the cell types participating in the normal immune response. Our finding of cortisone sensitivity in T helper cell function is in agreement with the conclusions of Segal et al. (6) and Vann (7), but at variance with those of Cohen and Claman (5) and Andersson and Blomgren (4). The failure of Cohen and Claman (5) to demonstrate suppression of T cell activation to S R B C in vivo

REVERSAL

OF CORTISONE-INDUCED

IMMUNOSUPPRESSION

415

may be related to the short time used for cortisone pretreatment before adminis,tration of antigen. Andersson and Blomgren (4) found that T helper cells in the thymus capable of restoring the humeral response of thymectomized, irradiated and bone-marrow protected mice to SRBC and heterologous serum proteins were cortisone-resistant. However, the dose of cortisone used in this study was much lower than ours (c.1.5 mg/mouse) and in the environment of the new host, the T helper cells could recover their activity. The possibility of an accessory cell defect was not demonstrated in these studies (4--7). Vann (7) reported that the suppressive effect of hydrocortisone on the anti-SRBC response was reversed by Ts•Bc or soluble factors from cells activated by allogeneic antigens without any necessity for accessory cells. This is probably due to the presence of 2-Me which obviated the need for accessory cells. In our experiments, it was only when mice had been treated with 10 mg cortisone that the anti-SRBC response was not restorable by 2-Me and the dual requirement for TsRBo + 2-Me or Ts~Be + PEC could then be observed (Table 3). When spleen cells had been treated w~th a lower dose of cortisone (2.5-5 rag/mouse), the response was restored to normal levels by 2-Me (Table 1), or by TseBe without concomitant addition of PEC (unpublished) whereas PEC alone were ineffective (Fig. 3). Reconstitution of the response by 2-Me could be the result of direct stimulation of T or B cells for which some evidence exists in the literature (32, 34, 48, 49) whereas the restorative effect of TSRBC in the absence of PEC could be due to active accessory cells persisting in cortisone-treated spleens in numbers sufficient for collaboration with TSl~BC. Thus the failure of Dracott (8) to observe immunosuppression in 2-Me-containing cultures of cells from hydrocortisonetreated mice may be due to insufficient steroid used in this particular strain of mice to induce immunosuppression observable in the presence of 2-Me. Our findings could provide an explanation for the apparently contradictory reports of Vann (7) and Dracott (8) based on variation in the dose of steroid used and the use of 2-Me in culture. Since 2-Me is increasingly used in tissue culture, caution should be exercised in the interpretation of data related to the behaviour of immunocompetent and accessory cells in such cultures. It is consistent with the T-independent character of POL to find that TsaBc or TroL had marginal effects on the anti-POL response of normal or cortisone-treated cells, particularly in the case of TpoL which may not have been activated by the POL. Since POL has been reported to be macrophage or accessory cell independent (22, 23, 35), the reversal of cortisone inhibition by PEC or 2-Me was unexpected and raised two possibilities. Firstly, B cells' could be inactivated by cortisone and the inactivation relieved by PEC or 2-Me through unknown mechanisms. There is, however, no clear-cut evidence to support this notion. Secondly, the anti-POL response could be dependent on cortisone-sensitive accessory cells but far less so than SRBC and the methods employed by other workers for depletion of accessory cells were inadequate for depression of the anti-POL response. In support of this possibility, we found that the anti-POL response was depressed by exhaustive depletion of adherent cells with carbonyl iron powder, and the response was restored by PEC or 2-Me. We have no direct evidence for inactivation or removal of splenic accessory cells or macrophages after cortisone administration, but we have observed that the frequency of macrophages full of endocytosed erythroeytes was greatly reduced in cortisone-treated cultures. The most likely cell

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in the P E C preparation responsible for reversal of the cortisone effect is the macrophage, but other cells cannot be ruled out. Such cells will have to be 0 negative, radioresistant, and active at very low numbers. There is uncertainty in the literature on whether splenic accessory cells or indeed the active accessory cells in peritoneal exudates are identical with the classical macrophage (22, 40-45). Our finding of a cortisone-sensitive accessory cell function restorable by P E C and Thompson and van Furth's (12) observation that peritoneal macrophages were resistant to hydrocortisone suggest that the splenic accessory cells may be distinguished from classical macrophages on the basis of cortisone sensitivity. If accessory and T helper cells are the sites of cortisone sensitivity, it follows that B cells are resistant. Nevertheless, cortisone inducted a loss of up to 7 5 ~ of cells from the spleen (Table 1) and B cells which comprise 42 3 of spleen cells according to Basten et al. (46), are probably lost also. A proportion of .the cell loss could be a result of nonspecific cytolytic effect of cortisone. However, the shift of the anti-POL dose-response curve of cortisone-treated cells toward low P O L concentrations (Fig. 1) and the observation that 2-Me often boosted the response of cortisone-treated cells to levels higher than control values (Fig. 2, Table 3) suggest that there is a progressive enrichment of a small subpopulation of cortisoneresistant B cells responsive to low concentrations of POL. At a gross level, cortisone-treated and normal spleen cells had similar proportions of 0-positive cells and of immunoglobulin-bearing cells (stained by rabbit anti-mouse IgM and flourescein-conjugated goat anti-rabbit Fc). The altered dose-response to P O L based on other explanations such as the absence of accessory cells, enrichment of memory cells or a lower threshold of B cell triggering cannot be ruled out at present, and this phenomenon must await further study. In conclusion, four main points can be inferred as the most likely explanation for the data presented. Firstly, cortisone inactivated helper T cells possibly by interfering with their activation by antigen or expression of helper activity. Secondly, cortisone induces a dysfunction in accessory cells through inactivation, cell death, or migration from spleen. Thirdly, mature immunocompetent B cells are not selectively sensitive to cortisone. Finally, there is evidence that the response of B cells to P O L at high antigen concentrations (1.25-250 ng/ml) was dependent on cortisone-sensitive accessory cells whereas at low P O L concentrations, a B cell response occurred in the apparent absence of accessory cells. The mechanism of inactivation of T helper cells and accessory cells is unclear but it is expected that this experimental system will be useful for the further study of corticosteroid action as well as cellular events in immune responses. ACKNOWLEDGMENTS We wish to thank Drs. N. Kraft and T. Wegmann for their interest and criticism and Misses E. Keller, J. Cummins, and J. Berezowsky for technical assistance. We are indebted to Dr. C. Shiozawa for a gift of rabbit anti-mouse IgM and fluorescein-conjugated goat antirabbit Fc. Note added to proof. We found that the content of immunoglobulin-bearing cells in spleen cell suspensions fell from 52 to 47% after treatment with carbonyl iron (lg/80 × 106 cells). Hence the depressed anti-POL response of carbonyl iron-treated cells is probably not the result of removal of B cells.

REVERSAL OF CORTISONE-INDUCED I3£1ViUNOSUPPRESSION

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