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Desensitization of delayed-type hypersensitivity in mice: Possible involvement of interleukin 2-dependent regulatory mechanisms in desensitized mice
CLINICAL
IMMUNOLOGY
AND
55,438-452 (IWO)
IMMUNOPATHOLOGY
Desensitization of Delayed-Type Hypersensitivity Possible involvement of lnterleukin 2-Dependent Mechanisms in Desensitized Mice KAZUO
KOBAYASHI,**’
STANLEY
COHEN,?
AND TAKESHI
in Mice: Regulatory
YOSHIDA~:
*The First Department of Internal Medicine, Showu University School oj’Medicine. f-3-b Hatanodai, Shina,gawa-ku, Tokyo 142, Japan; tDepartment of Pathology and Laborator> Medicine, Hahnemann University, Philudelphia, Pennsylvunia 19102; and STokyo Institute for Immunopharmacology. Tokyo, Japun
The systemic injection of high doses of antigen into a previously immunized animal results in a state of transient anergy with respect to cell-mediated immune responses. This phenomenon is known as desensitization. The results presented here demonstrated that serum interleukin 2 (IL-2) activity was found transiently in desensitized mice at early stage (3 hr after the challenge). Subsequently, these mice could not develop in Go (footpad swelling) and in vitro (lymphocyte proliferation) manifestations of cell-mediated immune responses 1 day after the challenge. Antigen-nonspecific and specific suppression of IL-2 production was observed in desensitized mice. The serum from 3 hrdesensitized mice containing endogenous IL-2 activity showed a marked suppressive effect on IL-2 production. Exposure of lymph node cells to IL-2 was capable of inhibiting IL-2 production in vitro. Additionally, in viva administration of exogenous IL-2 into preimmunized mice led to the failure of development of footpad responses to antigen. These results suggest that IL-2-dependent regulatory mechanisms of T cell-mediated immune responses play an important role in the immunosuppression of desensitized mice. & 19% Academx Press. Inc INTRODUCTION
Desensitization is defined as the suppression of a preexisting delayed-type hypersensitivity (DTH) state by the systemic administration of a large dose of homologous antigen. The unresponsive state may be specific for the desensitizing antigen (1, 2) or may be nonspecific (3-5). In the latter situation, if a doubly immunized animal is given a desensitizing dose of one antigen, it is also transiently unresponsive (anergic) to the other. In this case the specific phase of desensitization usually persists for a longer period of time than the nonspecific phase (3.5). In the guinea pig model, we have demonstrated that desensitization is a multistage process (5, 6), involving an initial overpopulation of lymphokines such as migration inhibition factor (MIF), followed by inhibition of lymphokine production by suppressor macrophages, and finally by nonspecific and specific suppressor T cells. Interleukin 2 (IL-2), an immunoregulatory T cell-derived molecule, has been shown to trigger the clonal expansion of antigen or mitogen-activated T cells including cytolytic T cells (7, 8), helper T cells (9-1 l), DTH effector T cells (1 I), and suppressor T cells (12, 13). Although compelling evidence supports IL-2 t To whom correspondence should be addressed 438 0090-1229190 $1.50 Copyright 0 1990 by Academic Press. inc. AU rights of reproduction in any form reserved.
SERUM
IL-2
ACTIVITY
IN
DESENSITIZED
MICE
439
modulation of in vitro immune reactivity, little is known concerning the role of IL-2 in vivo (14-19). Many of these studies dealt with the ability of exogenously administered IL-2 to induce T cell growth (15-17) or to restore suppressed T cell-mediated immune responses in Trypanosorna cruzi or Mycobacterium bovis infection (18, 19). Although desensitization involves immunologic responses that are mediated by effector lymphokines, afferent mediations such as IL-2 are likely to play important modulatory roles. Accordingly, we investigated IL-2 production during desensitization for this purpose and have utilized a murine model of desensitization. Evidence presented suggesting that IL-Zdependent regulatory mechanisms of T cell-mediated immune responses play an important role in the immunosuppression of desensitized mice. MATERIALS
AND METHODS
Mice Female BALB/c mice were purchased from West Seneca Laboratory NY). Mice 6 to 9 weeks of age were used in all experiments.
(Buffalo,
ImmunizationlDesensitization Methylated bovine serum albumin (MBSA) and egg albumin (EA) were obtained from Sigma (St. Louis, MO). Keyhole limpet hemocyanin (KLH) was purchased from Calbiochem (San Diego, CA). Purified protein derivative (PPD) was purchased from Connaught Laboratories, Willowdale (Ontario, Canada). BALBlc mice were immunized with 125 pg MBSA emulsified in complete Freund’s adjuvant (Difco, Detroit, MI) containing 2 mg/ml of heat-killed Mycobacterium tuberculosis (2&22). Ten days after immunization, mice were desensitized by injection intravenously and intraperitoneally with a total of 1.5 mg MBSA (0.5 mg iv and 1 mg ip) as described (23). Induction
of Cutaneous
DTH Reaction
At various intervals after desensitization, mice were challenged by 40 pg antigen (MBSA or PPD) into the hind footpad. The difference of footpad thickness at 24 hr after and just before challenge injection was measured. Measurements were made in triplicate for each mouse tested by using an engineer’s micrometer (Starret, Athol, MA), and mean swelling + SEM were calculated as described (21,22). Medium Cells were cultured in RPM1 1640 (GIBCO, Grand Island, NY) supplemented with 10% fetal calf serum, 2 mM glutamine, 100 U/ml penicillin, 100 l&ml streptomycin, and 2.5 x lop5 M 2-mercaptoethanol (referred to below as complete medium). Lymphocyte
Proliferative
Response
As described previously (21, 22), lymph node cell suspensions (2 x lo5 cells/O.2 ml/well) in complete medium were dispensed into 96-well, flat-bottomed culture
440
KOBAYASHI,
COHEN,
AND
YOSHIDA
plates (Falcon 3072, Becton-Dickinson, Oxnard, CA). Optimal antigen and Con A concentration in culture, selected by dose-response studies, was 50 p,g MBSA/ml, 100 kg PPD/ml, and 5 kg Con A/ml (21,22). Culture plates were incubated at 37°C in a humid, 5% CO, atmosphere for 4 days, and 0.5 l.~Ci = 18.5 kBq of [3H]thymidine ([3H]TdR; sp act, 5 Ci/mmol = 185 GBq/mmol, Amersham, Arlington Heights, IL) was added to each well 16 hr before harvesting. Cultured lymphocytes were collected by glass fiber filters and the E3H]TdR incorporation was measured by liquid scintillation counting. Antigen or Con A-induced proliferative responses were expressed as the difference in cpm (A cpm) t SEM between the mean cpm of stimulated cultures and the mean cpm of unstimulated cultures. Cell-Free Cultare Supernatants
and Standard
IL-2
Lymph node cell suspensions (5 X IO6 cells/ml) were cultured in the presence of MBSA (50 pg/ml) or Con A (5 kg/ml) at 37°C for 24 hr. These doses of MBSA and Con A and incubation period were previously shown to result in optimal lL-2 production from lymph node cells (21, 22). To inactivate residual Con A in Con A-containing supernatants, methyl-a-n-mannopyranoside (Sigma) was added to a final concentration of 10 mM at the termination of culture. All supernatants were clarified by centrifugation at 1800 g for 15 min, then Millipore filtered (pore size. 0.45 km), and stored at -70°C until use. The cultured cells showed E&90%, viability by trypan blue dye exclusion at the termination of incubation. Recombinant human IL-2 (sp act: 1 X 10’ U/mg) was purchased from Genzyme (Boston. MA). Assay for IL-2lIL-4
Activity
IL-2/IL-4 activity was determined by the method of Gillis et al. (24). The activity was evaluated using the IL-2 and IL-4 responsive cell line CTLL (kindly supplied from Dr. K. A. Smith, Dartmouth Medical School, Hanover, NH). In certain experiments in which antibodies were added directly to the proliferation assays, monoclonal anti-murine IL-2 receptor (clone AMT 13) from BoehringerMannheim Biochemica (Indianapolis, IN) and monoclonal anti-murine IL-4 antibodies (TexStar, Dallas, TX) were added at the initiation of the assay as described (25). Cell suspensions (5 X lo3 cells/O. lml/well) were cultured with 100 ~1 of serial twofold dilutions of sample to be assayed. After 20 hr incubation, 0.5 &i = 18.5 kBq of [3H]TdR was added to each well and the incubation was resumed for an additional 4 hr. Cells were then harvested and counted in a liquid scintillation counter, and the activity was determined and expressed as the mean half-maximal U/ml t SEM of triplicate cultures at various sample dilutions. Effects of Cells or Sera on IL-2 Production Lymph node cells (5 x lo6 cells/ml) from IO-day MBSA-immunized mice were stimulated with MBSA or Con A in the presence of cells or sera from desensitized mice. After 24 hr incubation, supernatants were collected and were then assayed for IL-2 activity. The cultured cells showed EO-90% viability by trypan blue dye
SERUM
IL-2
ACTIVITY
IN
DESENSITIZED
exclusion at the termination of incubation. Percentage duction was calculated by using the formula:
MICE
441
suppression of IL-2 pro-
IL-2 production in the presence of sample x 100. IL-2 production in control > Treatment of Lymph Node Cells with Exogenous IL-2 Lymph node cells (5 x IO6 cells/ml) from MBSA-immunized mice were incubated with recombinant IL-2 or MBSA-IL-2 at 37°C for 3 hr. As controls, lymph node cells were incubated with IL-2-depleted supernatants that were absorbed by CTLL cells (2 x IO6 cells/ml) at 37°C for 24 hr according to the method described previously (26, 27). After the incubation, lymph node cells were washed extensively and were resuspended at a density of 5 x lo6 cells/ml in complete medium. Cells were then stimulated with either MBSA or Con A for 24 hr. Culture supernatants were assayed for IL-2. In Vivo Administration
of Exogenous IL-2 into Immunized
Mice
Immunized mice were injected intravenously and intraperitoneally with recombinant IL-2 or MBSA-IL-2. Footpads of these mice were challenged with 40 pg of MBSA or PPD within 1 hr following the injection and the 24-hr footpad swelling was then determined with an engineer’s micrometer (Start-et, Athol, MA) just before and 24 hr after footpad challenge. The difference between both measurements was calculated and expressed as mean swelling (mm) + SEM. Gel Filtration
Chromatography
Gel filtration chromatography was performed at 4°C on a Sephacryl S200 (Pharmacia Fine Chemicals, Piscataway, NJ) column (85 x 2.2 cm). The column was calibrated with the MW standards: blue dextran, ferritin (MW 440 kDa), catalase (MW 232 kDa), human IgG (MW 150 kDa), EA (MW 43 kDa), chymotrypsinogen A (MW 25 kDa), and cytochrome c (MW 12 kDa). Fractions (4 ml) were collected and were lyophilized and reconstituted to the fourfold concentrated volume of the fractionated sample (1 ml). Statistical
Analyses
Data were analyzed by analysis of variance (ANOVA) and by the Student’s t test; P values less than 0.05 were considered significant. All statistical analyses were performed using Statview 512+ (Brainpower, Calabasas, CA) run on a Macintosh II PC (Apple, Cupertino, CA). RESULTS
Lack of Footpad Responses to Antigens in Desensitized
Mice
As shown in Table 1, footpad responses to either MBSA or PPD were significantly suppressed (83-91% inhibition) in sensitized mice with 1 day after desensitization when compared to those observed in immunized mice. Diminution of MBSA-induced footpad response (43-71% inhibition) was observed in desensi-
442
KOBAYASHI,
COHEN, AND YOSHIDA TABLE
LACK OF FOOTPAD
Days after I
Desensitization + .-
3 7 14
+ +
-+ -
I
RESPONSE TO ANTIGEN
MBSA 0.10 0.61 0.20 0.69 0.29 0.52 0.33 0.36
IN DESENSITIZED
MICE”
Footpad responseh (mean mm t SEM) ~~~~ ~~~~~~ PPD
t + It rt I 2 i t
0.02 0.07 0.01 0.05 0.03 0.08 0.05 0.02
(83%)"," (71%)" (43%)" (9%)
0.02 0.29 0.28 0.30 O.2Y 0.34 0.28 0.30
t " If: t_
0.01 (9f%i" 0.04 0.05 (X%,) 0.06
+- 0.02 (15%) -t- 0.04 2 0.02 (6%) t- 0.02
a BALB/c mice were immunized with MBSA emulsified in complete Freund’s adjuvant. Ten days after immunization, mice were desensitized with MBSA (1.5 mg/mouse). Control immunized mice were injected with saline. * The difference of footpad thickness at 24 hr after and just before challenge injection was measured. Data represent the mean swelling (mm) + SEM from three different experiments per four mice each condition. ’ Numbers in parentheses indicate percentage suppression of footpad swelling when compared with control immunized mice. d Significant difference when compared to controls (f’ < 0.02).
tized mice from Day 3 through Day 7. In contrast, mild inhibition of the PPDinduced response (815% inhibition) was seen in this stage. On Day 14, we could not find suppression of either MBSA or PPD-elicited footpad response. Thus, cutaneous unresponsiveness to both desensitizing (MBSA) and immunizing (PPD) antigens was observed 1 day (the first stage) after desensitization. In the second stage of desensitization, occurring between 3 and 7 days after the antigenic challenge, the footpad reaction to the desensitizing antigen remained to be suppressed, while the other skin reactivity to the immunizing antigen returned to normal. These results showed that there is both a specific and a nonspecific component to desensitization. Similar results were observed when PPD was employed as a desensitizing antigen. Failure of Lymphocyte Proliferative Responses in Desensitized Mice
As can be seen from Table 2, nonspecific inhibition of lymphocyte proliferation induced by MBSA (85% inhibition), PPD (38% inhibition), and Con A (36% inhibition) was observed in Day l-desensitized mice. In a subsequent step, suppression of the lymphocyte proliferative response to the desensitizing antigen (MBSA: 57-62% inhibition) was observed from 3 to 7 days after desensitization. Alternatively, very mild inhibition of the responses to PPD (12-22% suppression) and Con A (5-6% suppression) was seen at this stage when compared with those observed in immunized mice. On Day 14, negligible inhibition of lymphocyte proliferation induced by either antigens or Con A was observed in desensitized mice. The data show that the desensitized state is demonstrated by both in vivo (inhibition of footpad swelling) and in vitro (inhibition of antigen-derived cell proliferation) manifestations.
SERUM IL-2 ACTIVITY
IN DESENSITIZED
TABLE FAILURE
OF ANTIGEN-STIMULATED
LYMPH
2 CELL
PROLIFERATION
IN DESENSITIZED
MICE”
Lymph node cells stimulated with
Days
after
NODE
443
MICE
Desensitization
MBSA
+ + + + -
2,346 15,940 15,195 35,108 6,%1 18,351 4,088 5,260
1 3 7 14
PPD 2 -c + + f 2 2 f
536 (85%)b 1705 1366 (57%)b 1161 932 (62%)b 1255 327 (22%) 849
13,302 21,271 18,521 23,670 24,699 28,055 29,129 26,009
Con A 2 2 2 2 2 2 k ”
2476 (38%)’ 279 2056 (22%) 1935 2019 (12%) 865 1352 (- 11.9%) 1141
22,066 34,746 35,751 37,743 32,945 35,007 34,993 36,234
+- 1254 (36%)” 2 I349 k 2605 (5%) k 2790 ? 2313 (6%) 2 4801 t 784 (3%) 2 2641
y Lymph node cells (2 x lo5 cells/O.2 ml/well) from desensitized or control immunized mice were cultured in the presence of antigen or Con A for 4 days. Data represent the mean A cpm k SEM from six different experiments. Unstimulated cultures showed 30004000 cpm. Values in parentheses indicate percentage suppression. b Significant difference when compared to controls (P < 0.02). c Significant difference when compared to controls (P < 0.05).
Impaired
IL-2 Production
in Desensitized
Mice
As shown in Table 3, significantly impaired IL-2 production stimulated with MBSA (45-72% inhibition) in desensitized mice was observed 1 to 7 days after desensitization when compared with control-immunized mice. Con A-induced IL-2 production was suppressed 1 day after desensitization (34% inhibition), but showed almost normal levels of the production from Day 3- to Day 14desensitized mice. MBSA-induced IL-2 production in desensitized mice returned to control levels on Day 14. These results are consistent with in vivo (footpad responses; Table 1) and in vitro (lymphocyte proliferation; Table 2) manifestations of cell-mediated immunity observed in desensitized mice. To elucidate the nature of impaired IL-2 production observed in desensitized mice, we employed an IL-2 generating coculture system that was composed of lymph node cells from controlimmunized mice and those from desensitized mice. As can be seen in Table 3, lymph node cells from Day l-desensitized mice showed no suppressive effects (t&2% inhibition) on MBSA or Con A-induced IL-2 production. On the other hand, lymph node cells from 3- to 7-day-desensitized mice showed suppressive activity (33-39% inhibition) on MBSA-elicited IL-2 production but not on Con A-stimulated IL-2 generation. Detection of Serum IL-2 Activity in Desensitized Mice To determine circulating IL-2 activity in vivo, we examined IL-2 activity in the serum from desensitized mice. As shown in Table 4, we demonstrated the transient appearance of serum IL-2 activity in 3-hr desensitized mice. As shown in Table 5, chromatography of sera from 3-hr desensitized mice on Sephacryl S200 revealed the presence of at least two major peaks of IL-2 activity with molecular weights in the range of 12-25 kDa and 25-43 kDa. The activity obtained by gel filtration was totally destroyed by trypsin and by heat treatment (60°C 20 min).
444
KOBAYASHI,
COHEN, AND YOSHIDA TABLE
IMPAIRED
Days after 1
IL-2
PRODUCTION
BY LYMPH
NODE
3 CELLS
Lymph node cells from Immunized
Desensitized
FROM
DESENSITIZED
MICE”
IL-2 activity (U/ml f~ SEM) MBSA
Con A
1.5 ml 0 1.5 ml 1.5 ml 0 1.5 ml 1.5 ml 0 1.5 ml 1.5 ml 0 1.5 ml
0 64-t7 263 i- I7 1.5 ml 35 + 6 (45%)h 174 f 21 (34%)h 0.5 ml 62 r 9 (2%) 265 2 23 ( 1%) 3 0 71 -t IO 248 t- 22 1.5 ml 26 -+ 7 (63%)’ 229 +- 14 (X%i 0.5 ml 48 -c 9 (33%)h 136 It- 2s (4%) 7 0 101 -+ 12 1C8 i- 2: 1.5 ml 28 :t 4 (72%)’ 274-+24( 6%) 0.5 ml 62 -t IO (39%)’ 269 t 31 ( --4To 14 0 26 -c 4 258 t 29 1.5 ml 21 -+ 3 (19%) 271 -?I 32 ( --5%) 0.5 ml 24 t 5 (8%) 262 T!T2x ( - ‘o/r) .~ * Lymph node cells were stimulated with either MBSA (50 &ml) or Con A (5 &ml) for 24 hr. Culture supematants were assayed for IL-2 activity. Data indicate the mean U/ml t SEM from four separate experiments. Unstimulated culture supematants were reconstituted MBSA or Con A at the termination of incubation. Numbers in parentheses represent percentage suppression. ’ Significant difference when compared to controls (P < 0.05). ” Significant difference when compared to controls (P c 0.02).
Other studies showed that the activity is stable to storage at 4°C for 24 hr and to freeze/thawing. Because CTLL could respond to IL-4 (28, 29) as well as IL-2 (26, 27), we used anti-IL-2 receptor (Boehringer-Mannheim Biochemica) and antimurine IL-4 monoclonal antibodies (TexStar) to test the possibility. As shown in Table 6, the CTLL proliferative activity found in sera from 3-hr desensitized mice was inhibited by anti-IL-2 receptor antibody but not by anti-IL-4 antibody. Similarly, the CTLL proliferation activity of fractionated samples was inhibited by anti-IL-2 receptor antibody but not by anti-IL-4 antibody. Thus, the results suggest that the activity was IL-2. As shown in Table 4, the activity were induced in MBSA-preimmunized mice challenged with MBSA but not with unrelated antigen (EA and KLH). Immunization procedures themselves could not induce the IL-2 activity since the serum from sensitized mice challenged with saline showed no IL-2 activity. Furthermore, MBSA itself had no effects on release of serum IL-2 activity because the serum from nonimmunized mice injected with MBSA did not contain IL-2 activity. Similar results were obtained by using mice immunized with other antigens (EA and KLH) and desensitized with the respective antigen. Thus, administration of a large amount of antigen into sensitized mice could induce serum IL-2 activity transiently and suppressed cell-mediated immunity. Suppression
of IL-2 Production
by Serum that Contained
Endogenous
iL-2
The next question we asked was whether the serum contains suppressive activity on IL-2 production. As can be seen from Table 7, remarkable suppression of IL-2 production was observed in only the serum obtained from 3-hr desensitized mice. The serum contained endogenous IL-2 activity (Table 4). The sup-
SERUM IL-2 ACTIVITY
IN DESENSITIZED
TABLE CTLL PROLIFERATION
ACTIVITY
4
IN SEFCA FROM
3-hr DESENSITIZED
Time after desensitization
Serum from
445
MICE
Desensitized with MBSA”
0 hr hr hr 12 hr 24 hr 3 days 7 days 14 days 3 6
Immunized mice injected with: EA’ KLH“ Nonimmunized mice injected with MBSA Normal mice
3 3 3
hr hr hr
MICE
IL-2 activity (mean U/ml -+ SEM) co. 1 (CO. ljb 57 2 7 (
co. co. co. co.
1 1 1 I
co.1 co.1 co. 1 co. 1
(1MBSA-immunized BALB/c mice were desensitized with MBSA. Sera were bioassayed for IL-2. Data represent IL-2 activity (mean U/ml 2 SEM) from three separate experiments of four mice per condition. b Numbers in parentheses express IL-2 activity in the sera from immunized mice injected with saline. c MBSA-immunized mice were injected intravenously and intraperitoneally with EA (1.5 mg/mouse) . d MBSA-immunized mice were challenged with KLH (1.5 ms/mouse).
pression was not caused by cytotoxic effects of the serum on cells since viability assessed by trypan blue was over 80% at the termination of culture. Moreover, the suppressive activity seen in 3-hr desensitized mouse serum was antigen nonspecific because both MBSA and Con A-induced IL-2 production was inhibited. The suppressive activity was found in only the 3-hr desensitized mouse serum that TABLE FRACTIONATION
Fraction I II III IV V VI VII VIII
OF
CTLL PROLIFERATION
5
ACTIVITY IN SERA FROM SEPHACRYL S200"
Range of MW &Da) >2000 2OOOA40 MO-232 232-150 150-43 43-25 25-12 <12
3-hr DESENSITIZED
MICE
ON
IL-2 activity (U/ml k SEM) co.2 co.2 co.2 co.2 0.7 2 0.3 23.5 2 4.2' 34.8 z!z 5.1b 2.2 k 0.4
u Sera from 3-hr desensitized mice were chromatographed on a Sephacryl S200 column. Each fraction was assayed for IL-2 activity. Data represent the mean U/ml k SEM from four to five different experiments. In each experiment, sera from 10 to 20 mice were pooled. ’ Statistically significant (P < 0.02).
446
KOBAYASHI,
COHEN, AND YOSHIDA TABLE
INHIBITION
OF
CTLL PROLIFERATION
BY ANTI-k-2
Dilution of antibody
Antibody added None Anti-IL-2
6
ACTIVITY
receptor
ANTIBODY
CTLL proliferation (U/ml) 54 6 2x 52 so
114 i/IO 114 l/IO
Anti-IL-4
RECEPTOR
2 8 z 3’ I T’ -i- 7 t 4
u Sera from 3-hr desensitized mice were added to CTLL assay in the presence or absence of antibodies. Sera were employed in the assay on adding at 10% concentration. Values represent the mean t SEM of three independent experiments. b Statistically significant (P < 0.01). ’ Statistically significant (P < 0.05).
contained endogenous IL-2 (Table 4). Thus, this suppressive activity of serum on IL-2 production was well correlated with serum IL-2 activity. Suppression of IL-2 Production by Exogenous IL-2
As shown in Table 8, IL-2 production by lymph node cells of immunized mice stimulated with either MBSA or Con A was inhibited by the pretreatment of cells with exogenous IL-2 (34-63% inhibition) when compared to controls. Culture supernatants of immunized lymph node cells stimulated with antigen contained multiple cytokine activities (30), therefore, we employed recombinant IL-2 and IL-Zdepleted preparations by CTLL absorption in order to explore the effect of IL-2 on its production. As shown in Table 8, the effects of recombinant IL-2 on TABLE EFFECTSOF
Time (hr) after desensitization
Serum (%I
0
1 5 I 5 I 5 I 5 1 5
3 6 12 24
SERUMON
7 IL-2 PRODUCTION" % Suppression of lL-2 production stimulated with MBSA 5.2 --7.3 57.5 88.9 16.1 9.5 6.1 -9.6 2.4 -4.5
2 t t + t t f r = z
4.7 6.3 8Sb 10.3” 4.9 3.3 2.1 2.2 I.1 3.9
Con A 4.3 i_ 3.7 7 .-? +.- 5.x
43.0 44.8 10.0 5.0 --6.3 9.4 4.3 4.4
? 7.4” It 6.Y” +_ 4.i t 2.3 i 2.5 f 3.7 c I.8 i 2.6
u Lymph node cells from IO-day immunized mice were stimulated with either MBSA (50 p&ml) or Con A (5 &ml) for 24 hr in the presence of desensitized mouse serum. Data represent the means t SEM from three independent experiments. b Significant difference (p < 0.02) when compared with suppression of lL-2 production in the presence of serum from immunized mice 3 hr after injection of saline. The effects of unimmunized mouse serum were similar to those of immunized mouse serum.
SERUM
IL-2
ACTIVITY
IN
TABLE SUPPRESSION
OF
IL-2 PRODUCTION
BY LYMPH
NODE
DESENSITIZED
447
MICE
8 CELLS PRETREATED
WITH
EXOGENOUS
IL-2
IL-2 production (U/ml k SEM) stimulated with’
Lymph node cells treated with”
Absorption with CTLLb
MBSA
Recombinant IL-2 Recombinant IL-2 MBSA-IL-2 MBSA-IL-2 MBSA aloneg Medium
+ + -
44 2 77 2 31 2 52 2 78 + 842
Con A 6 (48%)d,’ 8 (8%) 8 (63%)e 14 (38%)’ 13 (7%) 15
186 283 156 193 288 279
t 2 2 2 ? 2
26 (34%)’ 32(-l%) 21 (44%)’ 27 (31%)‘ 34 (-3%) 31
a Lymph node cells from MBSA-immunized mice were incubated with various samples for 3 hr at 37°C. After the incubation, cells were washed extensively, and then pretreated cells were stimulated with MBSA (50 &ml) or Con A (5 &ml) for 24 hr. Recombinant human IL-2 (100 U/ml) and MBSA-IL-2 (50 U/ml) were used. ’ To remove IL-2 activity, IL-2-containing preparations were incubated with CTLL (2 x IO6 cells/ml) at 37°C for 24 hr. After the absorption, residual IL-2 activity was <0.5 U/ml. c IL-2 activity of culture supematants was determined by CTLL proliferation. Data represent the mean U/ml 2 SEM from three separate experiments. d Numbers in parentheses indicate percentage suppression when compared to IL-2 production by lymph node cells pretreated with medium. e Significant difference (P < 0.02) when compared with IL-2 production pretreated with medium. f Significant difference (P < 0.05). * Cells were incubated with 50 t&ml of MBSA, which was equivalent to the content in MBSA-IL-2.
IL-2 production were diminished by absorption. However, IL-Zdepleted culture supernatants still showed a mild to moderate suppressive activity on IL-2 production (31-38% inhibition). Other cytokines besides IL-2 may have suppressive effects on IL-2 production. Again, the suppressive activity was not caused by cytotoxic effect of the supernatants on lymph node cells. Inhibition of Antigen-Induced Footpad Responses in Immunized Vivo Administration of Exogenous IL-2
Mice by In
We used the dose of IL-2 (250 U/mouse) which was almost equivalent to a total amount of in vivo release of IL-2 in a desensitized mouse estimated by contents of serum IL-2. As shown in Table 9, mice pretreated with crude or recombinant IL-2 exhibited antigen-nonspecific suppression of footpad responses induced by either MBSA or PPD (5581% inhibition). Suppression of DTH was observed when mice were injected with 2100 U of IL-2 per mouse but not with ~50 U. Next, preimmunized mice were injected with IL-2-depleted preparations. As shown in Table 9, marked diminution of suppression of antigen-induced footpad responses was observed in preimmunized mice injected with IL-2-depleted supematants (l-6% inhibition). IL-Zdepleted supematants, however, showed a mild to moderate inhibitory activity on the footpad response. The results of the in vivo study were similar to those of in vitro IL-2 production (Table 8). Thus, in vivo administration of a large amount of exogenous IL-2 into preimmunized mice could suppress antigen-induced footpad responses. A large amount of exogenous IL-2 suppressed
KOBAYASHI,
448
COHEN,
AND YOSHIDA
TABLE INHIBITION
OF ANTIGEN-INDUCED FOOTPAD ADMINISTRATION
Injected with” Recombinant IL-2 Recombinant IL-2 MBSA-IL-2 MBSA-IL-2 MBSA alone’ None
Absorption by CTLL + i -
9
RESPONSES IN PREIMMUNIZED OF EXOGENOUS IL-‘!
MICE
BY m Viw
Footpad swelling (mm I’ SEM)h induced by MBSA 0.23 0.52 0.12 0.34 0.57 0.63
i2 i t T ?-
PPD 0.06 0.08 0.04 0.03 0.05 0.08
(63%)’ I’ (17%) (81%)” (46%)’ (9%)
0.14 0.26 0.07 0.23 0.29 0.31
t 0.04 -t- 0.05 k 0.03 +- 0.04 s 0.05 + 0.06
(55%)d (16%) (77%i,)” (26%) 16%)
” Preimmunized mice were injected intravenously and intraperitoneally with IL-2 (250 liimouse). Mice were then tested for antigen-induced 24.hr footpad responses. ’ Data represent the mean swelling (mm) 5 SEM from three separate experiments of three mice per condition. ’ Values in parentheses indicate percentage suppression when compared to footpad swelling observed in preimmunized mice without any treatment. ” Significant difference @ < 0.02) when compared with footpad swelling observed in immunized mice without any treatment. ’ Significant difference tP < 0.05). ’ Preimmunized mice were injected with 125 pg of MBSA. which was equivalent to the content in MBSA-IL-2.
in vitro (IL-2 production; Table 8) and in vivo (footpad cell-mediated immune reactivity.
response: Table 9) ‘I’
DlSCUSSlON
Present studies on desensitization in mice have shown that serum lL-2 activity transiently appeared in the mice 3 hr after desensitization, and that, in a subsequent step, these mice were unable to produce IL-2 from lymph node cells and failed to express T cell-mediated immune responses in vivo (footpad responses) and in vitro (lymphocyte proliferation) simultaneously. Lymph node cells pretreated with exogenous IL-2-containing preparations could not produce IL-2 itI vitro stimulated with antigen or mitogen. Also, preimmunized mice injected with exogenous IL-2 were incapable of developing in vivo manifestations of T cellmediated immune responses. Thus, the IL-2-dependent regulatory circuit may play an important role in immunosuppression of desensitized mice. The mechanisms of desensitization of DTH are still lacking. There are several possibilities to interpret the mechanisms by induction of suppressor cells (31). antigen-mediated interference with effector cell function (32-34), and compartmentalization of effector cells (34). Our recent studies on desensitization in guinea pigs have demonstrated that the suppression is due to several lymphokinedependent mechanisms (4-6, 35). We and other investigators found that desensitizing doses of antigen, which elicited cutaneous anergy, induced the transient appearance of lymphokines such as MIF and interferon in the serum (4.6, 36,37). Subsequently, lymphocytes from desensitized guinea pigs showed the suppressed MIF production (5, 6). In addition, serum, which contained MIF from desensi-
SERUM
IL-2
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IN
DESENSITIZED
MICE
449
tized animals, was capable of passively transferring desensitization (38). The injection of exogenous lymphokine-containing supernatants could lead to a state of desensitization (39). The present study suggests that the IL-Zdependent regulatory mechanisms are presumably involved in desensitized mice and that such mechanisms are responsible for suppressed T cell-mediated immune responses in the mice. It has been reported that exposure of cloned helper T lymphocytes to IL-2 can induce the antigen-unresponsive state, and that unresponsive helper T lymphocytes are markedly impaired in their ability to produce IL-2 after antigenic restimulation (40). Similarly, it has been reported that the proliferative response (41) and IL-2 production (42) are inhibited when exogenous IL-2 is added. Taken together with our present study, these observations suggest that IL-2 is a part of the regulatory network in T cell-mediated immune responses in vivo and in vitro. We have not done the molecular analysis such as IL-2 mRNA and protein accumulation after antigen binding in vitro. We should examine this possibility. We and other investigators, however, have reported that in vitro and in vivo release of the migration inhibition factor, IL-2, and interferon-y was observed 3-6 hr after antigenic exposure (23, 30, 36, 37). The timing and appearance of these lymphokines early in desensitization suggests the possibility that they may play a role in the subsequent appearance of suppressor cells in later stages of desensitization. It should be noted that IL-2-depleted culture supernatants reduced the suppressive activity on IL-2 production in vitro (Table 8) and antigen-induced footpad responses in vivo (Table 9) when compared with the those of IL-Zcontaining supernatants. However, IL-Zdepleted preparations showed a moderate suppressive activity in both in vitro and in vivo systems when compared to controls. These results suggest that, besides IL-2, other cytokines (4347) may participate in this type of immune suppression. Because serum from desensitized mice (4,37, 38) and stimulated culture supernatants (5, 30) probably contain multiple cytokine activities such as MIF, macrophage-activating factors, and interferons, these cytokines may induce suppression of cell-mediated immunity via a feedback inhibition mechanism (5, 6). In addition, cytokineflymphokine-activated monocytes and/or macrophages release arachidonic acid metabolites, including prostaglandin E, and E,, which inhibit both the production and the activities of cytokines/ lymphokines (35, 48-51). Prostaglandin E, possesses an inhibitory effect on IL-2 production (49) and the ability to induce suppressor T cells (52,53). Lymphokines are also suggested as being capable of directly inducing suppressor cells for lymphokine production, thus exerting a feedback inhibition (5, 6, 35). Our present studies suggest the possibility of an IL-2-dependent regulatory circuit in T cell-mediated immune responses plays a role in immunosuppression in desensitized mice. Thus, IL-2 may be a down regulator of cell-mediated immune responses such as delayed-type hypersensitivity. ACKNOWLEDGMENTS We thank Dr. Kendall A. Smith for supplying an IL-2-dependent cytotoxic T lymphocyte line used in these studies. This work was supported by National Institutes of Health Grants HL-23982 and AI-16706. Dr. Kazuo Kobayashi was supported by a grant from The Ministry of Education, Science and Culture of Japan, The Showa Medical Foundation Research Grant, Tokyo, Japan, and a grant from The Mochida Memorial Foundation for Medical and Pharmaceutical Research, Tokyo, Japan.
450
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22. Kobayashi, K., Allred, C., Castriotta, R., and Yoshida, T., Strain variation of Bacillus Calmette Guerin-induced pulmonary granuloma formation is correlated with anergy and the local production of migration inhibition factor and interleukin-1. Amer. J. Pathol. 119, 223-235, 1985. 23. Cone, R. E., Gerardi, D. A., Davidoff, J., Petty, J., Kobayashi, K., and Cohen, S., Quantitation of T cell antigen-binding molecules (TABM) in the sera of nonimmunized, immunized, and desensitized mice. .Z. Zrnmunol. 138, 234-239, 1987. 24. Gillis, S., Ferm, M. M., Ou, W., and Smith, K. A., T cell growth factor: Parameters of production and quantitative microassay for activity. J. Zmmunol. 120, 2027-2032, 1978. 25. Wong, R. L., Ruddle, N. H., Padula, S. J., Lingenheld, E. G., Bergman, C. M., Rugen, R. V., Epstein, D. I., and Clark, R. B., Subtypes of helper cells: Non-inflammatory type 1 helper T cells. J. Zmmunol.
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26. Smith, K. A., Interleukin-2: Inception, impact, and implications. Science 240, 11691176, 1988. 27. Smith, K. A., and Ruscetti, F. W., T-cell growth factor and the culture of cloned functional T cells. Adv. Zmmunol. 31, 137-175, 1981. 28. Grabstein, K., Eiseman, J., Mochizuki, D., Shaneback, K., Conlon, P., Hopp, T., March, C., and Gillis, S., Purification to homogeneity of B cell stimulatory factor: A molecule that stimulates proliferation of multiple lymphokine dependent cell lines. J. Exp. Med. 163, 1405-1414, 1986. 29. Sideras, P., Noma, T., and Honjo, T., Structure and function of interleukin 4 and 5. Zmmunol. Rev. 102, 18%212, 1988. 30. Kobayashi, K., Cohen, S., and Yoshida, T., Antigen-mediated interleukin production by mononuclear cells from delayed hypersensitive mice. J. Exp. Pathol. 2, 191-209, 1985. 31. Swamy, P. K., Dwyer, J. M., and Kantor, F. S., Desensitization in mice: T cell requirement for nonspecific suppression of delayed-type hypersensitivity. Cell. Zmmunol. 60, 301313, 1981. 32. Polak, L., and Turk, J. L., Studies on the effect of systemic administration of sensitizer in guinea pigs with contact sensitivity to organic metal compounds. I. The induction of immunological unresponsiveness in already sensitized animals. Clin. Exp. Zmmunol. 3, 245-251, 1968. 33. Polak, L., and Gleick, H., Differing mechanisms of tolerance and desensitization to DNCB in guinea pigs. Eur. J. Zmmunol. 5, 94-99, 1975. 34. Schlossman, S. F., Levin, H. A., Rocklin, R. E., and David, J. R., The compartmentalization of antigen-reactive lymphocytes in desensitized guinea pigs. J. Exp. Med. 134, 741-750, 1971. 35. Suko. M., Yoshida, T., and Cohen, S., Desensitization. V. Suppression of MIF production by lymphokine-activated macrophages. Cell. Zmmunol. %, 49-60, 1985. 36. Yamamoto, K., and Takahashi, Y., Macrophage migration inhibition by serum from desensitized animals previously sensitized with tubercle bacilli. Nature (London) New Biol. 233, 261-263, 1971. 37. Salvin, S. B., Younger, J. S., and Lederer, W. H.. Migration inhibitory factor and interferon in the circulation of mice with delayed hypersensitivity. Infect. Zmmun. 7, 68-75, 1973. 38. Papermaster, V., Yoshida, T., and Cohen, S., Desensitization. II. Passive transfer of the desensitized state by serum from desensitized animals. Cell. Zmmunol. 35, 378-391, 1978. 39. Yoshida, T., and Cohen, S., Lymphokine activity in vivo in relation to circulating monocyte levels and delayed skin reactivity. J. Zmmunol. 112, 1540-1547, 1974. 40. Wilde, D. B., Prystowsky, M. B., Ely, J. M., Vogel, S. N., Dialynas, D. P., and Fitch, F. W., Antigen-reactive cloned helper T cells. II. Exposure of murine cloned helper T cells to IL 2containing supematant induces unresponsiveness to antigenic restimulation and inhibits lymphokine production after antigenic stimulation. J. Zmmunol. 133, 636-641, 1984. 41. Punnonen, J., and Viljanen, M. K., Recombinant interleukin 2 inhibits pokeweed mitogeninduced proliferation of human adult peripheral blood and cord blood mononuclear cells. Zmmuno/. Lett.
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42. Kolitz, J. E., Welte, K., Wong, G. Y ., Holloway, K., Merluzzi, V. J., Engert, A., Konrad, M., Polivka, A., Gabrilove, J. L., Sykora, K. W., Miller, G. A., Fiedler, Oettgen, H. F., and Mertelsmann, R., Expansion of activated T-lymphocytes in with recombinant interleukin 2. J. Biol. Response Modif. 6, 412-429, 1987. 43. Asherson, G. L., and Zembala, M., Suppressor T cells in cell-mediated immunity. 32, 158-164,
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AND
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in mice. I. Suppression of contact sensitivity by soluble suppressor factor released in vttro by lymph node cell populations containing specific suppressor cells. J. Immunol. 119,315-321, 1977. Greene. M. I.. Pierres. A.. Dot-f, M. E.. and Benacerraf. B.. The I-J subregion codes for determinants on suppressor factor(s) which limit the contact sensitivity response to picryl chloride. J. Exp. Med. 146, 293-296. 1977. Ptak. W.. Zembala, M.. Heczakowska-Rewicka, M.. and Asherson. G, L.. Nonspecific macro phage suppressor factor: Its role in the inhibition of contact hypersensitivity to picryl chloride by specific T suppressor factor. Eur. J. Itnmccnol. 8, 645-649. 1978. Ptak. W.. Zembala. M.. Asherson, G. L., and MarcinkiewicL. J.. Inhibition of contact sensittvtty by macrophages: The separate phenomenon of the production of nonspecific macrophage suppressor factor by macrophages armed with specific T suppressor factor, and the nonspecific inhibition of passive transfer by high density of macrophages. Inr. Arch. Allergy Appi. Immunol 65, 121-128. 1981. Gordon, D.. Bray, M. A.. and Morley. J.. Control of lymphokine secretion by prostaglandrns. Nature (London) 262, 401402. 1976. Walker, C.. Kristensen. F., Bettens, F.. and de Week. A. l... Lymphokine regulation ofactivated (G,) lymphocytes. I. Prostaglandin &induced inhibition of interleukin 2 production. J. Immrrnol. 130, 177&1773. 1983. Kunkel, S. L.. Chensue. S. W., and Phan, S. H.. Prostaglandtna d\ endogenous medtators t>t interleukin 1 production. J. Immunol. 136, 186192. 1986. Kunkel. S. L.. Wiggins. R. C., Chensue. S. W.. and Larrick. J., Regulatmn ofmacrophage tumor necrosis factor production by prostaglandin EZ. Biochem. Biopi7yv. Res. Cornmrrn. 137. 404-410. 1986. Fulton. A. M.. and Levy. J. G., The induction of non\pecttX I’ \uppressor lymphocyte> by prostaglandin E,. Ceil. Immlrnol. 59, 54-60, 1981. Fisher. A.. Durandy. A.. and Griscelli. C., Role ofprostaglandtn t:, tn the mduction oi nonspecn’ic T lymphocyte suppressor activity. J. Imm~md. 126. 1452--1455. 1981
Received
September
35. 1989: accepted
with
revision
February
i. 1990
IMMUNOLOGY
AND
55,438-452 (IWO)
IMMUNOPATHOLOGY
Desensitization of Delayed-Type Hypersensitivity Possible involvement of lnterleukin 2-Dependent Mechanisms in Desensitized Mice KAZUO
KOBAYASHI,**’
STANLEY
COHEN,?
AND TAKESHI
in Mice: Regulatory
YOSHIDA~:
*The First Department of Internal Medicine, Showu University School oj’Medicine. f-3-b Hatanodai, Shina,gawa-ku, Tokyo 142, Japan; tDepartment of Pathology and Laborator> Medicine, Hahnemann University, Philudelphia, Pennsylvunia 19102; and STokyo Institute for Immunopharmacology. Tokyo, Japun
The systemic injection of high doses of antigen into a previously immunized animal results in a state of transient anergy with respect to cell-mediated immune responses. This phenomenon is known as desensitization. The results presented here demonstrated that serum interleukin 2 (IL-2) activity was found transiently in desensitized mice at early stage (3 hr after the challenge). Subsequently, these mice could not develop in Go (footpad swelling) and in vitro (lymphocyte proliferation) manifestations of cell-mediated immune responses 1 day after the challenge. Antigen-nonspecific and specific suppression of IL-2 production was observed in desensitized mice. The serum from 3 hrdesensitized mice containing endogenous IL-2 activity showed a marked suppressive effect on IL-2 production. Exposure of lymph node cells to IL-2 was capable of inhibiting IL-2 production in vitro. Additionally, in viva administration of exogenous IL-2 into preimmunized mice led to the failure of development of footpad responses to antigen. These results suggest that IL-2-dependent regulatory mechanisms of T cell-mediated immune responses play an important role in the immunosuppression of desensitized mice. & 19% Academx Press. Inc INTRODUCTION
Desensitization is defined as the suppression of a preexisting delayed-type hypersensitivity (DTH) state by the systemic administration of a large dose of homologous antigen. The unresponsive state may be specific for the desensitizing antigen (1, 2) or may be nonspecific (3-5). In the latter situation, if a doubly immunized animal is given a desensitizing dose of one antigen, it is also transiently unresponsive (anergic) to the other. In this case the specific phase of desensitization usually persists for a longer period of time than the nonspecific phase (3.5). In the guinea pig model, we have demonstrated that desensitization is a multistage process (5, 6), involving an initial overpopulation of lymphokines such as migration inhibition factor (MIF), followed by inhibition of lymphokine production by suppressor macrophages, and finally by nonspecific and specific suppressor T cells. Interleukin 2 (IL-2), an immunoregulatory T cell-derived molecule, has been shown to trigger the clonal expansion of antigen or mitogen-activated T cells including cytolytic T cells (7, 8), helper T cells (9-1 l), DTH effector T cells (1 I), and suppressor T cells (12, 13). Although compelling evidence supports IL-2 t To whom correspondence should be addressed 438 0090-1229190 $1.50 Copyright 0 1990 by Academic Press. inc. AU rights of reproduction in any form reserved.
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modulation of in vitro immune reactivity, little is known concerning the role of IL-2 in vivo (14-19). Many of these studies dealt with the ability of exogenously administered IL-2 to induce T cell growth (15-17) or to restore suppressed T cell-mediated immune responses in Trypanosorna cruzi or Mycobacterium bovis infection (18, 19). Although desensitization involves immunologic responses that are mediated by effector lymphokines, afferent mediations such as IL-2 are likely to play important modulatory roles. Accordingly, we investigated IL-2 production during desensitization for this purpose and have utilized a murine model of desensitization. Evidence presented suggesting that IL-Zdependent regulatory mechanisms of T cell-mediated immune responses play an important role in the immunosuppression of desensitized mice. MATERIALS
AND METHODS
Mice Female BALB/c mice were purchased from West Seneca Laboratory NY). Mice 6 to 9 weeks of age were used in all experiments.
(Buffalo,
ImmunizationlDesensitization Methylated bovine serum albumin (MBSA) and egg albumin (EA) were obtained from Sigma (St. Louis, MO). Keyhole limpet hemocyanin (KLH) was purchased from Calbiochem (San Diego, CA). Purified protein derivative (PPD) was purchased from Connaught Laboratories, Willowdale (Ontario, Canada). BALBlc mice were immunized with 125 pg MBSA emulsified in complete Freund’s adjuvant (Difco, Detroit, MI) containing 2 mg/ml of heat-killed Mycobacterium tuberculosis (2&22). Ten days after immunization, mice were desensitized by injection intravenously and intraperitoneally with a total of 1.5 mg MBSA (0.5 mg iv and 1 mg ip) as described (23). Induction
of Cutaneous
DTH Reaction
At various intervals after desensitization, mice were challenged by 40 pg antigen (MBSA or PPD) into the hind footpad. The difference of footpad thickness at 24 hr after and just before challenge injection was measured. Measurements were made in triplicate for each mouse tested by using an engineer’s micrometer (Starret, Athol, MA), and mean swelling + SEM were calculated as described (21,22). Medium Cells were cultured in RPM1 1640 (GIBCO, Grand Island, NY) supplemented with 10% fetal calf serum, 2 mM glutamine, 100 U/ml penicillin, 100 l&ml streptomycin, and 2.5 x lop5 M 2-mercaptoethanol (referred to below as complete medium). Lymphocyte
Proliferative
Response
As described previously (21, 22), lymph node cell suspensions (2 x lo5 cells/O.2 ml/well) in complete medium were dispensed into 96-well, flat-bottomed culture
440
KOBAYASHI,
COHEN,
AND
YOSHIDA
plates (Falcon 3072, Becton-Dickinson, Oxnard, CA). Optimal antigen and Con A concentration in culture, selected by dose-response studies, was 50 p,g MBSA/ml, 100 kg PPD/ml, and 5 kg Con A/ml (21,22). Culture plates were incubated at 37°C in a humid, 5% CO, atmosphere for 4 days, and 0.5 l.~Ci = 18.5 kBq of [3H]thymidine ([3H]TdR; sp act, 5 Ci/mmol = 185 GBq/mmol, Amersham, Arlington Heights, IL) was added to each well 16 hr before harvesting. Cultured lymphocytes were collected by glass fiber filters and the E3H]TdR incorporation was measured by liquid scintillation counting. Antigen or Con A-induced proliferative responses were expressed as the difference in cpm (A cpm) t SEM between the mean cpm of stimulated cultures and the mean cpm of unstimulated cultures. Cell-Free Cultare Supernatants
and Standard
IL-2
Lymph node cell suspensions (5 X IO6 cells/ml) were cultured in the presence of MBSA (50 pg/ml) or Con A (5 kg/ml) at 37°C for 24 hr. These doses of MBSA and Con A and incubation period were previously shown to result in optimal lL-2 production from lymph node cells (21, 22). To inactivate residual Con A in Con A-containing supernatants, methyl-a-n-mannopyranoside (Sigma) was added to a final concentration of 10 mM at the termination of culture. All supernatants were clarified by centrifugation at 1800 g for 15 min, then Millipore filtered (pore size. 0.45 km), and stored at -70°C until use. The cultured cells showed E&90%, viability by trypan blue dye exclusion at the termination of incubation. Recombinant human IL-2 (sp act: 1 X 10’ U/mg) was purchased from Genzyme (Boston. MA). Assay for IL-2lIL-4
Activity
IL-2/IL-4 activity was determined by the method of Gillis et al. (24). The activity was evaluated using the IL-2 and IL-4 responsive cell line CTLL (kindly supplied from Dr. K. A. Smith, Dartmouth Medical School, Hanover, NH). In certain experiments in which antibodies were added directly to the proliferation assays, monoclonal anti-murine IL-2 receptor (clone AMT 13) from BoehringerMannheim Biochemica (Indianapolis, IN) and monoclonal anti-murine IL-4 antibodies (TexStar, Dallas, TX) were added at the initiation of the assay as described (25). Cell suspensions (5 X lo3 cells/O. lml/well) were cultured with 100 ~1 of serial twofold dilutions of sample to be assayed. After 20 hr incubation, 0.5 &i = 18.5 kBq of [3H]TdR was added to each well and the incubation was resumed for an additional 4 hr. Cells were then harvested and counted in a liquid scintillation counter, and the activity was determined and expressed as the mean half-maximal U/ml t SEM of triplicate cultures at various sample dilutions. Effects of Cells or Sera on IL-2 Production Lymph node cells (5 x lo6 cells/ml) from IO-day MBSA-immunized mice were stimulated with MBSA or Con A in the presence of cells or sera from desensitized mice. After 24 hr incubation, supernatants were collected and were then assayed for IL-2 activity. The cultured cells showed EO-90% viability by trypan blue dye
SERUM
IL-2
ACTIVITY
IN
DESENSITIZED
exclusion at the termination of incubation. Percentage duction was calculated by using the formula:
MICE
441
suppression of IL-2 pro-
IL-2 production in the presence of sample x 100. IL-2 production in control > Treatment of Lymph Node Cells with Exogenous IL-2 Lymph node cells (5 x IO6 cells/ml) from MBSA-immunized mice were incubated with recombinant IL-2 or MBSA-IL-2 at 37°C for 3 hr. As controls, lymph node cells were incubated with IL-2-depleted supernatants that were absorbed by CTLL cells (2 x IO6 cells/ml) at 37°C for 24 hr according to the method described previously (26, 27). After the incubation, lymph node cells were washed extensively and were resuspended at a density of 5 x lo6 cells/ml in complete medium. Cells were then stimulated with either MBSA or Con A for 24 hr. Culture supernatants were assayed for IL-2. In Vivo Administration
of Exogenous IL-2 into Immunized
Mice
Immunized mice were injected intravenously and intraperitoneally with recombinant IL-2 or MBSA-IL-2. Footpads of these mice were challenged with 40 pg of MBSA or PPD within 1 hr following the injection and the 24-hr footpad swelling was then determined with an engineer’s micrometer (Start-et, Athol, MA) just before and 24 hr after footpad challenge. The difference between both measurements was calculated and expressed as mean swelling (mm) + SEM. Gel Filtration
Chromatography
Gel filtration chromatography was performed at 4°C on a Sephacryl S200 (Pharmacia Fine Chemicals, Piscataway, NJ) column (85 x 2.2 cm). The column was calibrated with the MW standards: blue dextran, ferritin (MW 440 kDa), catalase (MW 232 kDa), human IgG (MW 150 kDa), EA (MW 43 kDa), chymotrypsinogen A (MW 25 kDa), and cytochrome c (MW 12 kDa). Fractions (4 ml) were collected and were lyophilized and reconstituted to the fourfold concentrated volume of the fractionated sample (1 ml). Statistical
Analyses
Data were analyzed by analysis of variance (ANOVA) and by the Student’s t test; P values less than 0.05 were considered significant. All statistical analyses were performed using Statview 512+ (Brainpower, Calabasas, CA) run on a Macintosh II PC (Apple, Cupertino, CA). RESULTS
Lack of Footpad Responses to Antigens in Desensitized
Mice
As shown in Table 1, footpad responses to either MBSA or PPD were significantly suppressed (83-91% inhibition) in sensitized mice with 1 day after desensitization when compared to those observed in immunized mice. Diminution of MBSA-induced footpad response (43-71% inhibition) was observed in desensi-
442
KOBAYASHI,
COHEN, AND YOSHIDA TABLE
LACK OF FOOTPAD
Days after I
Desensitization + .-
3 7 14
+ +
-+ -
I
RESPONSE TO ANTIGEN
MBSA 0.10 0.61 0.20 0.69 0.29 0.52 0.33 0.36
IN DESENSITIZED
MICE”
Footpad responseh (mean mm t SEM) ~~~~ ~~~~~~ PPD
t + It rt I 2 i t
0.02 0.07 0.01 0.05 0.03 0.08 0.05 0.02
(83%)"," (71%)" (43%)" (9%)
0.02 0.29 0.28 0.30 O.2Y 0.34 0.28 0.30
t " If: t_
0.01 (9f%i" 0.04 0.05 (X%,) 0.06
+- 0.02 (15%) -t- 0.04 2 0.02 (6%) t- 0.02
a BALB/c mice were immunized with MBSA emulsified in complete Freund’s adjuvant. Ten days after immunization, mice were desensitized with MBSA (1.5 mg/mouse). Control immunized mice were injected with saline. * The difference of footpad thickness at 24 hr after and just before challenge injection was measured. Data represent the mean swelling (mm) + SEM from three different experiments per four mice each condition. ’ Numbers in parentheses indicate percentage suppression of footpad swelling when compared with control immunized mice. d Significant difference when compared to controls (f’ < 0.02).
tized mice from Day 3 through Day 7. In contrast, mild inhibition of the PPDinduced response (815% inhibition) was seen in this stage. On Day 14, we could not find suppression of either MBSA or PPD-elicited footpad response. Thus, cutaneous unresponsiveness to both desensitizing (MBSA) and immunizing (PPD) antigens was observed 1 day (the first stage) after desensitization. In the second stage of desensitization, occurring between 3 and 7 days after the antigenic challenge, the footpad reaction to the desensitizing antigen remained to be suppressed, while the other skin reactivity to the immunizing antigen returned to normal. These results showed that there is both a specific and a nonspecific component to desensitization. Similar results were observed when PPD was employed as a desensitizing antigen. Failure of Lymphocyte Proliferative Responses in Desensitized Mice
As can be seen from Table 2, nonspecific inhibition of lymphocyte proliferation induced by MBSA (85% inhibition), PPD (38% inhibition), and Con A (36% inhibition) was observed in Day l-desensitized mice. In a subsequent step, suppression of the lymphocyte proliferative response to the desensitizing antigen (MBSA: 57-62% inhibition) was observed from 3 to 7 days after desensitization. Alternatively, very mild inhibition of the responses to PPD (12-22% suppression) and Con A (5-6% suppression) was seen at this stage when compared with those observed in immunized mice. On Day 14, negligible inhibition of lymphocyte proliferation induced by either antigens or Con A was observed in desensitized mice. The data show that the desensitized state is demonstrated by both in vivo (inhibition of footpad swelling) and in vitro (inhibition of antigen-derived cell proliferation) manifestations.
SERUM IL-2 ACTIVITY
IN DESENSITIZED
TABLE FAILURE
OF ANTIGEN-STIMULATED
LYMPH
2 CELL
PROLIFERATION
IN DESENSITIZED
MICE”
Lymph node cells stimulated with
Days
after
NODE
443
MICE
Desensitization
MBSA
+ + + + -
2,346 15,940 15,195 35,108 6,%1 18,351 4,088 5,260
1 3 7 14
PPD 2 -c + + f 2 2 f
536 (85%)b 1705 1366 (57%)b 1161 932 (62%)b 1255 327 (22%) 849
13,302 21,271 18,521 23,670 24,699 28,055 29,129 26,009
Con A 2 2 2 2 2 2 k ”
2476 (38%)’ 279 2056 (22%) 1935 2019 (12%) 865 1352 (- 11.9%) 1141
22,066 34,746 35,751 37,743 32,945 35,007 34,993 36,234
+- 1254 (36%)” 2 I349 k 2605 (5%) k 2790 ? 2313 (6%) 2 4801 t 784 (3%) 2 2641
y Lymph node cells (2 x lo5 cells/O.2 ml/well) from desensitized or control immunized mice were cultured in the presence of antigen or Con A for 4 days. Data represent the mean A cpm k SEM from six different experiments. Unstimulated cultures showed 30004000 cpm. Values in parentheses indicate percentage suppression. b Significant difference when compared to controls (P < 0.02). c Significant difference when compared to controls (P < 0.05).
Impaired
IL-2 Production
in Desensitized
Mice
As shown in Table 3, significantly impaired IL-2 production stimulated with MBSA (45-72% inhibition) in desensitized mice was observed 1 to 7 days after desensitization when compared with control-immunized mice. Con A-induced IL-2 production was suppressed 1 day after desensitization (34% inhibition), but showed almost normal levels of the production from Day 3- to Day 14desensitized mice. MBSA-induced IL-2 production in desensitized mice returned to control levels on Day 14. These results are consistent with in vivo (footpad responses; Table 1) and in vitro (lymphocyte proliferation; Table 2) manifestations of cell-mediated immunity observed in desensitized mice. To elucidate the nature of impaired IL-2 production observed in desensitized mice, we employed an IL-2 generating coculture system that was composed of lymph node cells from controlimmunized mice and those from desensitized mice. As can be seen in Table 3, lymph node cells from Day l-desensitized mice showed no suppressive effects (t&2% inhibition) on MBSA or Con A-induced IL-2 production. On the other hand, lymph node cells from 3- to 7-day-desensitized mice showed suppressive activity (33-39% inhibition) on MBSA-elicited IL-2 production but not on Con A-stimulated IL-2 generation. Detection of Serum IL-2 Activity in Desensitized Mice To determine circulating IL-2 activity in vivo, we examined IL-2 activity in the serum from desensitized mice. As shown in Table 4, we demonstrated the transient appearance of serum IL-2 activity in 3-hr desensitized mice. As shown in Table 5, chromatography of sera from 3-hr desensitized mice on Sephacryl S200 revealed the presence of at least two major peaks of IL-2 activity with molecular weights in the range of 12-25 kDa and 25-43 kDa. The activity obtained by gel filtration was totally destroyed by trypsin and by heat treatment (60°C 20 min).
444
KOBAYASHI,
COHEN, AND YOSHIDA TABLE
IMPAIRED
Days after 1
IL-2
PRODUCTION
BY LYMPH
NODE
3 CELLS
Lymph node cells from Immunized
Desensitized
FROM
DESENSITIZED
MICE”
IL-2 activity (U/ml f~ SEM) MBSA
Con A
1.5 ml 0 1.5 ml 1.5 ml 0 1.5 ml 1.5 ml 0 1.5 ml 1.5 ml 0 1.5 ml
0 64-t7 263 i- I7 1.5 ml 35 + 6 (45%)h 174 f 21 (34%)h 0.5 ml 62 r 9 (2%) 265 2 23 ( 1%) 3 0 71 -t IO 248 t- 22 1.5 ml 26 -+ 7 (63%)’ 229 +- 14 (X%i 0.5 ml 48 -c 9 (33%)h 136 It- 2s (4%) 7 0 101 -+ 12 1C8 i- 2: 1.5 ml 28 :t 4 (72%)’ 274-+24( 6%) 0.5 ml 62 -t IO (39%)’ 269 t 31 ( --4To 14 0 26 -c 4 258 t 29 1.5 ml 21 -+ 3 (19%) 271 -?I 32 ( --5%) 0.5 ml 24 t 5 (8%) 262 T!T2x ( - ‘o/r) .~ * Lymph node cells were stimulated with either MBSA (50 &ml) or Con A (5 &ml) for 24 hr. Culture supematants were assayed for IL-2 activity. Data indicate the mean U/ml t SEM from four separate experiments. Unstimulated culture supematants were reconstituted MBSA or Con A at the termination of incubation. Numbers in parentheses represent percentage suppression. ’ Significant difference when compared to controls (P < 0.05). ” Significant difference when compared to controls (P c 0.02).
Other studies showed that the activity is stable to storage at 4°C for 24 hr and to freeze/thawing. Because CTLL could respond to IL-4 (28, 29) as well as IL-2 (26, 27), we used anti-IL-2 receptor (Boehringer-Mannheim Biochemica) and antimurine IL-4 monoclonal antibodies (TexStar) to test the possibility. As shown in Table 6, the CTLL proliferative activity found in sera from 3-hr desensitized mice was inhibited by anti-IL-2 receptor antibody but not by anti-IL-4 antibody. Similarly, the CTLL proliferation activity of fractionated samples was inhibited by anti-IL-2 receptor antibody but not by anti-IL-4 antibody. Thus, the results suggest that the activity was IL-2. As shown in Table 4, the activity were induced in MBSA-preimmunized mice challenged with MBSA but not with unrelated antigen (EA and KLH). Immunization procedures themselves could not induce the IL-2 activity since the serum from sensitized mice challenged with saline showed no IL-2 activity. Furthermore, MBSA itself had no effects on release of serum IL-2 activity because the serum from nonimmunized mice injected with MBSA did not contain IL-2 activity. Similar results were obtained by using mice immunized with other antigens (EA and KLH) and desensitized with the respective antigen. Thus, administration of a large amount of antigen into sensitized mice could induce serum IL-2 activity transiently and suppressed cell-mediated immunity. Suppression
of IL-2 Production
by Serum that Contained
Endogenous
iL-2
The next question we asked was whether the serum contains suppressive activity on IL-2 production. As can be seen from Table 7, remarkable suppression of IL-2 production was observed in only the serum obtained from 3-hr desensitized mice. The serum contained endogenous IL-2 activity (Table 4). The sup-
SERUM IL-2 ACTIVITY
IN DESENSITIZED
TABLE CTLL PROLIFERATION
ACTIVITY
4
IN SEFCA FROM
3-hr DESENSITIZED
Time after desensitization
Serum from
445
MICE
Desensitized with MBSA”
0 hr hr hr 12 hr 24 hr 3 days 7 days 14 days 3 6
Immunized mice injected with: EA’ KLH“ Nonimmunized mice injected with MBSA Normal mice
3 3 3
hr hr hr
MICE
IL-2 activity (mean U/ml -+ SEM) co. 1 (CO. ljb 57 2 7 (
co. co. co. co.
1 1 1 I
co.1 co.1 co. 1 co. 1
(1MBSA-immunized BALB/c mice were desensitized with MBSA. Sera were bioassayed for IL-2. Data represent IL-2 activity (mean U/ml 2 SEM) from three separate experiments of four mice per condition. b Numbers in parentheses express IL-2 activity in the sera from immunized mice injected with saline. c MBSA-immunized mice were injected intravenously and intraperitoneally with EA (1.5 mg/mouse) . d MBSA-immunized mice were challenged with KLH (1.5 ms/mouse).
pression was not caused by cytotoxic effects of the serum on cells since viability assessed by trypan blue was over 80% at the termination of culture. Moreover, the suppressive activity seen in 3-hr desensitized mouse serum was antigen nonspecific because both MBSA and Con A-induced IL-2 production was inhibited. The suppressive activity was found in only the 3-hr desensitized mouse serum that TABLE FRACTIONATION
Fraction I II III IV V VI VII VIII
OF
CTLL PROLIFERATION
5
ACTIVITY IN SERA FROM SEPHACRYL S200"
Range of MW &Da) >2000 2OOOA40 MO-232 232-150 150-43 43-25 25-12 <12
3-hr DESENSITIZED
MICE
ON
IL-2 activity (U/ml k SEM) co.2 co.2 co.2 co.2 0.7 2 0.3 23.5 2 4.2' 34.8 z!z 5.1b 2.2 k 0.4
u Sera from 3-hr desensitized mice were chromatographed on a Sephacryl S200 column. Each fraction was assayed for IL-2 activity. Data represent the mean U/ml k SEM from four to five different experiments. In each experiment, sera from 10 to 20 mice were pooled. ’ Statistically significant (P < 0.02).
446
KOBAYASHI,
COHEN, AND YOSHIDA TABLE
INHIBITION
OF
CTLL PROLIFERATION
BY ANTI-k-2
Dilution of antibody
Antibody added None Anti-IL-2
6
ACTIVITY
receptor
ANTIBODY
CTLL proliferation (U/ml) 54 6 2x 52 so
114 i/IO 114 l/IO
Anti-IL-4
RECEPTOR
2 8 z 3’ I T’ -i- 7 t 4
u Sera from 3-hr desensitized mice were added to CTLL assay in the presence or absence of antibodies. Sera were employed in the assay on adding at 10% concentration. Values represent the mean t SEM of three independent experiments. b Statistically significant (P < 0.01). ’ Statistically significant (P < 0.05).
contained endogenous IL-2 (Table 4). Thus, this suppressive activity of serum on IL-2 production was well correlated with serum IL-2 activity. Suppression of IL-2 Production by Exogenous IL-2
As shown in Table 8, IL-2 production by lymph node cells of immunized mice stimulated with either MBSA or Con A was inhibited by the pretreatment of cells with exogenous IL-2 (34-63% inhibition) when compared to controls. Culture supernatants of immunized lymph node cells stimulated with antigen contained multiple cytokine activities (30), therefore, we employed recombinant IL-2 and IL-Zdepleted preparations by CTLL absorption in order to explore the effect of IL-2 on its production. As shown in Table 8, the effects of recombinant IL-2 on TABLE EFFECTSOF
Time (hr) after desensitization
Serum (%I
0
1 5 I 5 I 5 I 5 1 5
3 6 12 24
SERUMON
7 IL-2 PRODUCTION" % Suppression of lL-2 production stimulated with MBSA 5.2 --7.3 57.5 88.9 16.1 9.5 6.1 -9.6 2.4 -4.5
2 t t + t t f r = z
4.7 6.3 8Sb 10.3” 4.9 3.3 2.1 2.2 I.1 3.9
Con A 4.3 i_ 3.7 7 .-? +.- 5.x
43.0 44.8 10.0 5.0 --6.3 9.4 4.3 4.4
? 7.4” It 6.Y” +_ 4.i t 2.3 i 2.5 f 3.7 c I.8 i 2.6
u Lymph node cells from IO-day immunized mice were stimulated with either MBSA (50 p&ml) or Con A (5 &ml) for 24 hr in the presence of desensitized mouse serum. Data represent the means t SEM from three independent experiments. b Significant difference (p < 0.02) when compared with suppression of lL-2 production in the presence of serum from immunized mice 3 hr after injection of saline. The effects of unimmunized mouse serum were similar to those of immunized mouse serum.
SERUM
IL-2
ACTIVITY
IN
TABLE SUPPRESSION
OF
IL-2 PRODUCTION
BY LYMPH
NODE
DESENSITIZED
447
MICE
8 CELLS PRETREATED
WITH
EXOGENOUS
IL-2
IL-2 production (U/ml k SEM) stimulated with’
Lymph node cells treated with”
Absorption with CTLLb
MBSA
Recombinant IL-2 Recombinant IL-2 MBSA-IL-2 MBSA-IL-2 MBSA aloneg Medium
+ + -
44 2 77 2 31 2 52 2 78 + 842
Con A 6 (48%)d,’ 8 (8%) 8 (63%)e 14 (38%)’ 13 (7%) 15
186 283 156 193 288 279
t 2 2 2 ? 2
26 (34%)’ 32(-l%) 21 (44%)’ 27 (31%)‘ 34 (-3%) 31
a Lymph node cells from MBSA-immunized mice were incubated with various samples for 3 hr at 37°C. After the incubation, cells were washed extensively, and then pretreated cells were stimulated with MBSA (50 &ml) or Con A (5 &ml) for 24 hr. Recombinant human IL-2 (100 U/ml) and MBSA-IL-2 (50 U/ml) were used. ’ To remove IL-2 activity, IL-2-containing preparations were incubated with CTLL (2 x IO6 cells/ml) at 37°C for 24 hr. After the absorption, residual IL-2 activity was <0.5 U/ml. c IL-2 activity of culture supematants was determined by CTLL proliferation. Data represent the mean U/ml 2 SEM from three separate experiments. d Numbers in parentheses indicate percentage suppression when compared to IL-2 production by lymph node cells pretreated with medium. e Significant difference (P < 0.02) when compared with IL-2 production pretreated with medium. f Significant difference (P < 0.05). * Cells were incubated with 50 t&ml of MBSA, which was equivalent to the content in MBSA-IL-2.
IL-2 production were diminished by absorption. However, IL-Zdepleted culture supernatants still showed a mild to moderate suppressive activity on IL-2 production (31-38% inhibition). Other cytokines besides IL-2 may have suppressive effects on IL-2 production. Again, the suppressive activity was not caused by cytotoxic effect of the supernatants on lymph node cells. Inhibition of Antigen-Induced Footpad Responses in Immunized Vivo Administration of Exogenous IL-2
Mice by In
We used the dose of IL-2 (250 U/mouse) which was almost equivalent to a total amount of in vivo release of IL-2 in a desensitized mouse estimated by contents of serum IL-2. As shown in Table 9, mice pretreated with crude or recombinant IL-2 exhibited antigen-nonspecific suppression of footpad responses induced by either MBSA or PPD (5581% inhibition). Suppression of DTH was observed when mice were injected with 2100 U of IL-2 per mouse but not with ~50 U. Next, preimmunized mice were injected with IL-2-depleted preparations. As shown in Table 9, marked diminution of suppression of antigen-induced footpad responses was observed in preimmunized mice injected with IL-2-depleted supematants (l-6% inhibition). IL-Zdepleted supematants, however, showed a mild to moderate inhibitory activity on the footpad response. The results of the in vivo study were similar to those of in vitro IL-2 production (Table 8). Thus, in vivo administration of a large amount of exogenous IL-2 into preimmunized mice could suppress antigen-induced footpad responses. A large amount of exogenous IL-2 suppressed
KOBAYASHI,
448
COHEN,
AND YOSHIDA
TABLE INHIBITION
OF ANTIGEN-INDUCED FOOTPAD ADMINISTRATION
Injected with” Recombinant IL-2 Recombinant IL-2 MBSA-IL-2 MBSA-IL-2 MBSA alone’ None
Absorption by CTLL + i -
9
RESPONSES IN PREIMMUNIZED OF EXOGENOUS IL-‘!
MICE
BY m Viw
Footpad swelling (mm I’ SEM)h induced by MBSA 0.23 0.52 0.12 0.34 0.57 0.63
i2 i t T ?-
PPD 0.06 0.08 0.04 0.03 0.05 0.08
(63%)’ I’ (17%) (81%)” (46%)’ (9%)
0.14 0.26 0.07 0.23 0.29 0.31
t 0.04 -t- 0.05 k 0.03 +- 0.04 s 0.05 + 0.06
(55%)d (16%) (77%i,)” (26%) 16%)
” Preimmunized mice were injected intravenously and intraperitoneally with IL-2 (250 liimouse). Mice were then tested for antigen-induced 24.hr footpad responses. ’ Data represent the mean swelling (mm) 5 SEM from three separate experiments of three mice per condition. ’ Values in parentheses indicate percentage suppression when compared to footpad swelling observed in preimmunized mice without any treatment. ” Significant difference @ < 0.02) when compared with footpad swelling observed in immunized mice without any treatment. ’ Significant difference tP < 0.05). ’ Preimmunized mice were injected with 125 pg of MBSA. which was equivalent to the content in MBSA-IL-2.
in vitro (IL-2 production; Table 8) and in vivo (footpad cell-mediated immune reactivity.
response: Table 9) ‘I’
DlSCUSSlON
Present studies on desensitization in mice have shown that serum lL-2 activity transiently appeared in the mice 3 hr after desensitization, and that, in a subsequent step, these mice were unable to produce IL-2 from lymph node cells and failed to express T cell-mediated immune responses in vivo (footpad responses) and in vitro (lymphocyte proliferation) simultaneously. Lymph node cells pretreated with exogenous IL-2-containing preparations could not produce IL-2 itI vitro stimulated with antigen or mitogen. Also, preimmunized mice injected with exogenous IL-2 were incapable of developing in vivo manifestations of T cellmediated immune responses. Thus, the IL-2-dependent regulatory circuit may play an important role in immunosuppression of desensitized mice. The mechanisms of desensitization of DTH are still lacking. There are several possibilities to interpret the mechanisms by induction of suppressor cells (31). antigen-mediated interference with effector cell function (32-34), and compartmentalization of effector cells (34). Our recent studies on desensitization in guinea pigs have demonstrated that the suppression is due to several lymphokinedependent mechanisms (4-6, 35). We and other investigators found that desensitizing doses of antigen, which elicited cutaneous anergy, induced the transient appearance of lymphokines such as MIF and interferon in the serum (4.6, 36,37). Subsequently, lymphocytes from desensitized guinea pigs showed the suppressed MIF production (5, 6). In addition, serum, which contained MIF from desensi-
SERUM
IL-2
ACTIVITY
IN
DESENSITIZED
MICE
449
tized animals, was capable of passively transferring desensitization (38). The injection of exogenous lymphokine-containing supernatants could lead to a state of desensitization (39). The present study suggests that the IL-Zdependent regulatory mechanisms are presumably involved in desensitized mice and that such mechanisms are responsible for suppressed T cell-mediated immune responses in the mice. It has been reported that exposure of cloned helper T lymphocytes to IL-2 can induce the antigen-unresponsive state, and that unresponsive helper T lymphocytes are markedly impaired in their ability to produce IL-2 after antigenic restimulation (40). Similarly, it has been reported that the proliferative response (41) and IL-2 production (42) are inhibited when exogenous IL-2 is added. Taken together with our present study, these observations suggest that IL-2 is a part of the regulatory network in T cell-mediated immune responses in vivo and in vitro. We have not done the molecular analysis such as IL-2 mRNA and protein accumulation after antigen binding in vitro. We should examine this possibility. We and other investigators, however, have reported that in vitro and in vivo release of the migration inhibition factor, IL-2, and interferon-y was observed 3-6 hr after antigenic exposure (23, 30, 36, 37). The timing and appearance of these lymphokines early in desensitization suggests the possibility that they may play a role in the subsequent appearance of suppressor cells in later stages of desensitization. It should be noted that IL-2-depleted culture supernatants reduced the suppressive activity on IL-2 production in vitro (Table 8) and antigen-induced footpad responses in vivo (Table 9) when compared with the those of IL-Zcontaining supernatants. However, IL-Zdepleted preparations showed a moderate suppressive activity in both in vitro and in vivo systems when compared to controls. These results suggest that, besides IL-2, other cytokines (4347) may participate in this type of immune suppression. Because serum from desensitized mice (4,37, 38) and stimulated culture supernatants (5, 30) probably contain multiple cytokine activities such as MIF, macrophage-activating factors, and interferons, these cytokines may induce suppression of cell-mediated immunity via a feedback inhibition mechanism (5, 6). In addition, cytokineflymphokine-activated monocytes and/or macrophages release arachidonic acid metabolites, including prostaglandin E, and E,, which inhibit both the production and the activities of cytokines/ lymphokines (35, 48-51). Prostaglandin E, possesses an inhibitory effect on IL-2 production (49) and the ability to induce suppressor T cells (52,53). Lymphokines are also suggested as being capable of directly inducing suppressor cells for lymphokine production, thus exerting a feedback inhibition (5, 6, 35). Our present studies suggest the possibility of an IL-2-dependent regulatory circuit in T cell-mediated immune responses plays a role in immunosuppression in desensitized mice. Thus, IL-2 may be a down regulator of cell-mediated immune responses such as delayed-type hypersensitivity. ACKNOWLEDGMENTS We thank Dr. Kendall A. Smith for supplying an IL-2-dependent cytotoxic T lymphocyte line used in these studies. This work was supported by National Institutes of Health Grants HL-23982 and AI-16706. Dr. Kazuo Kobayashi was supported by a grant from The Ministry of Education, Science and Culture of Japan, The Showa Medical Foundation Research Grant, Tokyo, Japan, and a grant from The Mochida Memorial Foundation for Medical and Pharmaceutical Research, Tokyo, Japan.
450
KOBAYASHI,
COHEN, AND YOSHIDA
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IL-2
ACTIVITY
IN DESENSITIZED
451
MICE
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Received
September
35. 1989: accepted
with
revision
February
i. 1990