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Augmentation of phorbol ester-induced T cell proliferation by agents which raise intracellular cyclic adenosine monophosphate
CELLULAR
IMMUNOLOGY
145,240-253
(1992)
Augmentation of Phorbol Ester-Induced T Cell Proliferation by Agents Which Raise Intracellular Cyclic Adenosine Monophosphate’ HRISHEKESHR. CHAKKALATH* AND LAWRENCEK. L. JUNG? *Department of Tropical Health, Harvard School of Public Health, 665 Huntington Avenue, Boston, Massachusetts 02115; and fDivision of Pediatric Immunology, Department of Pediatrics, University of MassachusettsMedical Center, 55 Lake Avenue North, Worcester,Massachusetts 01655 Received February 12, 1992; acceptedSeptember 6. 1992 Although raising intracellular cyclic adenosine monophosphate (CAMP) levels is generally considered to be inhibitory on the mitogen-induced T cell proliferation, in this study we have shown that the addition of either dbcAMP (50 PM) or cholera toxin (1 rig/ml) resulted in an increase in [3H]thymidine uptake in PBMC cultures stimulated with phorbol ester, 12-tetradecanoylphorbol 13-acetate(TPA), or with a combination of TPA plus anti-CD3 mAb (mAb 235). In contrast, under similar culture conditions, the phytohemagglutininP (PHA-P) responsewas inhibited by these agents as has been reported. The augmentative effect of dbcAMP in PBMC cultures was due to an increase in IL-2 production and not to increased in IL-2R-a chain expression. The enhancing effect of dbcAMP and CT observed with PBMC was monocyte dependent and not seen with purified T cell preparations. The addition of monocytes reconstituted the ability of intracellular CAMP elevating agents to augment the T cell response to TPA with and without mAb to CD3. The monocytes mediate their action via soluble factor(s) with molecular weight (m.w.) of more than 10 kDa. Neither rIL-1, rIL-6, nor rTNF+ have any augmentative effect as contrast with the supernatant from treated monocytes. Taken together, our results indicate that CAMP can play a positive regulatory role in T cell proliferation due to factor(s) secreted by dbcAMP-treated monocytes resulting in increased IL-2 synthesis in T cells. o 1992 Academic Press.Inc.
INTRODUCTION Many investigators have established that cyclic adenosine monophosphate (CAMP) analogues and agents that raise intracellular CAMP levels inhibit or downregulate T lymphocyte proliferation (1-8, reviewed in 9). This conclusion is derived from experiments wherein the inhibitory effect of CAMP elevating agents or analogues was detected chiefly on the lectin-induced T cell proliferation. The stimulation of T cells by mitogens leads to early activation events which include phosphatidylinositol turnover (lo), elevation in cytoplasmic free calcium (11) and induction of endogenous IL2 production and IL-2 receptor expression (12, 13). Each of these events has been reported as targets for CAMP-mediated inhibition of T cell proliferation ( 14- 18). Such findings reaffirmed the earlier conclusion that CAMP mediated a general, antiproliferative signal (19). However, adenylate cyclase activators like cholera toxin and forskolin could be mitogenic in systemssuch as dog thyrocytes (20). Similar evidence although lesscom’ This work was supported in part by NIH Grant AI-25704. 240
0008-8749192$5.00 Copyright 0 1992 by Academic Press,Inc. All rights of reproduction in any form reserved
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plete is available for many epithelial and nonepithelial differentiated cells (2 1). Thus, a positive functional role for CAMP in mitogenic stimuli has also been documented. Indeed, earlier studies by Webb et al. (22-24) had shown that agents which raised intracellular CAMP could enhance DNA and RNA synthesisof lymphocytes stimulated at supraoptimal dosesof PHA. More recent studies supported Webb’s observation of the positive effect of CAMP on T cell proliferation, although the later authors did not focus on this aspect in their studies (25, 26). In this study, we have investigated the effect of intracellular CAMP elevating agents on the in vitro response of peripheral T cells to various stimuli. We confirm that dbcAMP2 and cholera toxin do have an inhibitory effect on T cell proliferation stimulated by optimal dose of PHA-P. Additionally, these reagents also augment T cell proliferation induced by TPA or TPA plus anti-CD3 mAb by upregulating IL-2 synthesis but not IL-2R-a! chain expression. We further show that the augmentative effect by CAMP is mediated by factor(s) elaborated by monocytes upon exposure to dbcAMP. MATERIALS AND METHODS Reagentsand antibodies. The complete medium was made of RPM1 1640 (GIBCO, Grand Island, NY) supplemented with penicillin/streptomycin (GIBCO), glutamine (GIBCO), and 10%heat-inactivated fetal bovine serum (FBS, HyClone Lab Inc., Logan, UT). N6-2’-O-dibutyrladenosine 3’:5’-cyclic monophosphate (dbcAMP) (Sigma, St. Louis, MO) was dissolved in sterile phosphate-buffered saline, aliquotted, and stored at -70°C until use. TPA (Sigma) was dissolved in dimethyl sulfoxide (DMSO, Sigma) and stored at -70°C. Prior to use in culture, TPA was diluted to the appropriate concentration in complete culture medium. Cholera toxin was obtained from ICN Biochemicals, (Cleveland, OH). Phytohemagglutinin-P was obtained from Difco Lab (Detroit, MI). Human rIL- 1, rIL-6, and rTNI+ were obtained from Genzyme (Boston, MA), aliquotted, and stored at -70°C until use. Monoclonal antibody against CD3, designated 235 (IgM) (27) was a generous gift of Dr. Shu Man Fu (University of Virginia, Charlottesville, VA). T cell preparation. PBMC were isolated by Ficoll-Hypaque (Pharmacia, Piscataway, NJ) density centrifugation from peripheral blood of normal volunteers collected in heparin-containing syringes.T cells from PBMC preparations were obtained by Percoll discontinuous density gradient (28). The cells were then plated onto plastic Falcon petri dishes (Becton-Dickinson labware, Lincoln Park, NJ) and incubated for 1 hr 15 min in a 5% CO2 incubator. The nonadherent cells were removed and the same procedure was repeated three times. Cells thus obtained were then depleted of B cells and monocytes by preincubating for 30 min at 4°C in a mixture of mAb’s to these cells (29) and removed using goat anti-mouse immunoglobulin-coated magnetic beads (Dynal A.S., Oslo, Norway). T cell preparations thus obtained were found to have less than 1% monocytes and B cells, as determined by flow cytometry. Enumeration of monocytes in T cell preparations by nonspecific esterasestaining (30) gave comparable results as with immunofluorescence and such cell preparations were used in assays. All the experiments in the present study were carried out with ~98% viable cells, as observed by trypan blue dye exclusion technique. Monocyte and supernatant preparation. Monocytes from PBMC were isolated by Percoll discontinuous density gradient asmentioned above. Plastic adherenceprocedure ’ Abbreviations used: CT, cholera toxin; dbcAMP, dibutyrl cyclic adenosine monophosphate; TPA, 12tetradecanoylphorbol 13-acetate;MCF, mean channel fluorescence.
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was repeated three times with the cells, each for 1 hr 15 min. The adherent cells were detached from the plates by using a rubber policeman in the presence of cold PBS containing neither Mg*+ nor Ca2+. The adherent cells thus obtained were 98% esterase positive and 95% viable as determined by esterasestaining and trypan blue dye exclusion technique, respectively. In experiments requiring treated or untreated monocytes, 5 X 1O5adherent cells were plated on a 6-well plate and were treated for various periods with reagentsor media. The adherent cells were removed from the wells using the rubber policeman, washed twice, and checked for viability before adding to T cell cultures. Treated monocytes used in the study were essentially 92-95% viable. The culture supernatants from treated monocytes (5 X lo5 to 10 X 1O6cells) were collected at different time points after treatment with reagents or media and subjected to fractionation and concentration using a centrifugal ultrafilter (Centricell, Polysciences Inc., Wanington, PA) with a molecular weight cutoff at 10,000 Da. The concentrates thus obtained were diluted in double-distilled water, sterilized by filtration through 0.22 pm filters, and stored at -70°C until use. Proliferation assays.PBMC or T cells (1 X 105)in 200 ~1of complete medium were incubated in triplicate in flat-bottom 96-well plates at 37’C for 3 days in a 5% CO2 atmosphere. During the last 16 hr cells were pulsed with 2.0 PCi of [3H]thymidine (6.7 Ci/mmol; NEN DuPont, Boston, MA) and the incorporated radioactivity was determined using a 1205 Betaplate counter (Pharmacia LKB Nuclear, Gaithersburg, MD). The percentage of control responsewas calculated by the following formula: % Control response = [counts per minute (cpm) in CAMP-treated experimental cultures/cpm in untreated control cultures] X 100. IL-2 determination. The 24-hr culture supernatants from stimulated PBMC were collected and analyzed for IL-2 levels using an Intertest- enzyme-linked immunosorbent assay(ELISA) kit (Genzyme). The assaywas performed as per the manufacturer’s instructions. Briefly, a monoclonal antibody to human IL-2 was coated on polystyrene microtiter plates, incubated, washed, and test samples were added. After overnight incubation and washing, purified polyclonal rabbit anti-human IL-2 was added and then reacted with alkaline phosphatase-conjugated goat-anti-rabbit immunoglobulin. The OD at 405 nm was determined on an ELISA reader. Results were calculated by comparison to IL-2 standards ranging between 20 and 1200 pg/ml. Flow cytometry. PBMC were incubated with media alone, PHA (10 pg/ml), TPA ( 10 rig/ml) or TPA plus anti-CD3 (1 pg/ml) in the presence or absence of dbcAMP (50 palm)for 24 hr, washed, and stained with a fluorescenated isotype control or antiIL-2R-(Y mAb (Ortho Diagnostics, NJ) for 30 min at 4°C. Five thousand gated cells were analyzed on a FACScan flow cytometer (Becton-Dickinson, CA). The data obtained were analyzed on FACScan Research software. Statistical analysis. Statistical analysis was done by Student’s t test or by analysis of variance. RESULTS Dose and time kinetics of dbcAMP eflect on mitogen-induced PBMC proliferation. The effect of dbcAMP on the mitogen-induced responsesof PBMC were studied using the following culture systems. Cells were stimulated with either PHA-P, TPA or a combination of TPA plus soluble anti-CD3 mAb, 235. The responses,monitored by [3H]thymidine incorporation by stimulated cells were expressedas counts per minute. In preliminary experiments, the proliferation of PBMC stimulated with anti-CD3 plus
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TPA (82,188 ~fi4040; mean cpm f SEM) was comparable to that in PHA-P cultures (88,044 + 5493 cpm) and significantly higher than that in TPA-treated cultures ( 13,237 + 1400 cpm). The responseof T cells to mAb 235 alone was similar to PBMC control cultures with media (19 1 + 66 cpm vs 256 + 86 cpm). Although [3H]thymidine uptake varied in PBMC cultures from different individuals, the same pattern of responsewas observed. For comparison, the results were normalized and expressedas percentage of control response computed from the formula mentioned under Materials and Methods. The dose-dependent effect of dbcAMP on PBMC proliferation to mitogens is shown in Fig. 1. dbcAMP concentrations ranging from 10 to 50 PM were found to enhance (P < 0.025) TPA and anti-CD3 plus TPA-induced proliferative response in PBMC cultures. The optimal dose of dbcAMP on both the responseswas 50 pIM. In contrast, under similar culture conditions, dbcAMP significantly (P < 0.005) inhibited the PHAP-induced proliferation. Thus, dbcAMP augmented phorbol ester-induced proliferation but inhibited PHA-P-induced proliferation. One possible explanation for our observation is that the addition of dbcAMP led to a shift in the kinetics of T cell proliferation. As shown in Fig. 2, the augmentative effect induced by dbcAMP in activated cells representsa real increase and not a kinetic shift in cell proliferation. Another possibility is that the increased thymidine uptake represented a shift in nucleotide pool metabolism and not to a real increase in cell proliferation. To address this possibility, lo5 PBMC were cultured for 3 days with media, TPA, or TPA plus anti-CD3 with or without the presence of 50 PM dbcAMP. The mean viable cell counts from triplicate cell cultures were determined by trypan blue dye exclusion and the means of four independent experiments are representedin Fig. 3. Thus, the addition
0 0
10
Cont.
100
of dbcAMP
1000
(@VI)
FIG. 1. Effect of dbcAMP on mitogen-induced PBMC proliferation. PBMC were stimulated with PHAP (10 pglml), TPA (10 rig/ml) alone, or TPA plus anti-CD3 mAb 235 (1 &ml) in the presenceof various concentrations of dbcAMP as indicated. The resultsare expressedaspercentagesof control mitogen response calculated by using the formula given under Materials and Methods. Values are given as the means f SE of three separate experiments. Cell viability tested by trypan blue dye exclusion technique, at all dosesof dbcAMP tested in PBMC treated with media or anti-CD3 was in the range of 90-95%. At dbcAMP doses below 100 &I, cell viability in PBMC cultures stimulated with PHA, TPA, and TPA plus anti-CD3 was 90-95s as against 70-80s viability in stimulated cultures treated with above 100 & concentration of dbcAMP. Mean cpm + SE in cultures treated with medium alone was 191 + 66; anti-CD3, 256 f 86; antiCD3 + TPA, 82,188 f 4040; TPA, 13,237 +- 1400; PHA-P, 88,044 + 5493.
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&I
15
10
5 A-A
TPA
A-A
TPA + dbcAMP
0 1
3
5
7
DAYS OF CULTURE FIG. 2. dbcAMP-induced enhancement of proliferation is not due to a shift in kinetics of thymidine uptake. The 1 X lo5 PBMC were stimulated with media or TPA (10 &ml) with and without dbcAMP (50 PM). [3H]Thymidine-pulsed cultures were harvested on Days 2, 3, 5, and 7. Results are expressedas mean cpm f SE of three independent experiments. Mean cpm f SE in cultures treated with medium alone was 290 f 63, and 245 + 55,646 -C90, 865 + 123,and 1090 f 234 for 2-, 3-, 5-, and 7-day cultures, respectively.
of dbcAMP led to the increase in total cell numbers in both cell cultures with TPA or TPA and anti-CD3 (P < 0.05). These experiments show that the effect of dbcAMP is to increase DNA synthesis and cell proliferation. dbcAMP augmented TPA and anti-CD3/TPA induced cell responseseven after 2 days of culture initiation (Fig. 4). Furthermore, the significant (P < 0.005) inhibitory
2.5
-2
2.0 x z $
1.5
B 3 u
1.0
0.5
: -+ media
TPA
dbcAMP anti-CD3 + TPA
FIG. 3. dbcAMP enhanced thymidine uptake by inducing proliferation of T lymphocytes. The 1 X lo5 PBMC were stimulated with media, TPA (10 rig/ml), or TPA + mAb (1 pg/ml) with and without dbcAMP (50 p&f). The mean viable cell counts from triplicate cell cultures were determined by trypan blue dye exclusion technique. Four independent experiments were carried out and the means of the experiments are depicted. Analysis by Student’s t test and by analysis of variance showed that dbcAMP enhanced T cell proliferation (P < 0.05).
CAMP AUGMENTS TPA-INDUCED . . . ..-. q
- - -0
245
T CELL PROLIFERATION PHA-P ad-CD3
+ TPA
o!
I 0
10
20
30
Time of dbcAMP
40 addition
50
60
(hrs)
FIG. 4. dbcAMP effectin PBMC mitogenic responses:Time kinetics. PBMC were stimulated with mitogens as described in the legend for Fig. 1 and dbcAMP (50 p,W) was added at the indicated time periods. The results are expressed as percentages of control response. Values are given as the means + SEM of two independent experiments. Mean cpm f SE in cultures treated with medium alone was 2 14 + 34; anti-CD3 + TPA, 39,091 + 701; TPA, 5566 + 555; and PHA-P, 92,904 f 3493.
effect on PHA-P response could be demonstrated only when dbcAMP was added within 2-4 hr after mitogen addition to cultures. Thus, we show that the effects of dbcAMP on PBMC mitogenesis varied depending on the kind of stimulus and state of activation of the cells. Cholera toxin mimics the efect of dbcAMP in mitogen-induced PBMCprolifeation. Cholera toxin catalyzes the ADP-ribosylation of the CYsubunit of G, protein rendering it unable to hydrolyze its bound GTP. This results in continual activation of adenylate cyclase and prolonged elevation of intracellular CAMP. To demonstrate that CAMP augments T cell proliferation, cholera toxin was used to induce a rise in intracellular CAMP. Figure 5A shows that cholera toxin at an optimal concentration of 1 rig/ml can significantly (P < 0.005) augment the responseof PBMC to TPA as well as to the combination of mAb 235 plus TPA. Again, the samedoseinhibited the PHA-P response (P < 0.025). A dose of 100 rig/ml of cholera toxin was found to be cytotoxic and thus was suppressive in proliferating cultures (Fig. 5A). The time course of cholera toxin addition to stimulated PBMC cultures (Fig. 5B) had a similar pattern as seen with dbcAMP in Fig. 3. Essentially, cholera toxin inhibited (P < 0.005) PHA-P response when added at the initiation of culture or within 2 hr. At later time points of addition, cholera toxin did not influence (P > 0.05) the lectin-induced proliferation. These results also indicated that inhibition by cholera toxin of PHA-P cultures at a concentration of 1 rig/ml was not due to cytotoxicity. It was also of interest to note in Fig. 5B that cholera toxin had a synergistic effect in TPA and in the mAb 235 plus TPA cultures even when added after 24 hr after culture initiation. Thus, these experiments confirmed the positive role of CAMP on mitogen-induced PBMC responses. Augmentative effect of dbcAMP is associated with an increase in IL-2 production and not due to IL-2R-a chain expression in activated T cells. To understand the mechanism of the dbcAMP-mediated augmentative effect, we measured IL-2 production in phorbol ester-activated PBMC using the Interest-2 ELISA kit. The presence of
246
CHAKKALATH
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PHA-P anti-CD3
+TPA
04 10
1
100
Cont. of CT (rig/ml)
0 z
* 0’: 0-l 0
. ....-. PHA
10
20
0. - -0
anti-CD3
A--r)r
TPA
30
time of CT addition
40
50
+ TPA
60
(hours)
FIG. 5. (A) Cholera toxin mimics the effectsof dbcAMP in mitogen-induced PBMC proliferation. PBMC were stimulated with PHA-P (10 pg/ml), TPA (10 q/ml) alone, or TPA plus anti-CD3 (I &ml) in the presenceof various concentrations of cholera toxin as indicated. The results are expressedas percentagesof control mitogen responsecalculated by using the formula given under Materials and Methods. Values are given as the means f SEM of three separate experiments. Cell viability at all dosesof cholera toxin tested in PBMC treated with media or anti-CD3 was in the range of 90-95s. With cholera toxin dosesbelow 10 rig/ml, cell viability tested by trypan blue dye exclusion in PBMC cultures stimulated with PHA, TPA, and TPA plus anti-CD3 was 90-95%. A 60-80s viability was observed in stimulated cultures treated with 100 rig/ml of cholera toxin. Mean cpm f SE in cultures treated with medium alone was 162 * 25; anti-CD3 + TPA, 32,397 ? 2729; TPA, 7166 + 502; and PHA-P, 64,549 + 2786. (B) Cholera toxin effect in PBMC mitogenic responses:Time kinetics. A dose of 1 rig/ml of cholera toxin was added at different time points to PBMC cultures stimulated with mitogens as described in the legend for Fig. 4. The results are expressed as percentagesof control response.Values are given as the means + SEM of two separateexperiments. Mean cpm + SE in cultures treated with medium alone was 214 + 34; anti-CD3 + TPA, 87,725 + 3831; TPA, 5294 & 356; PHA-P, 92,904 + 3493.
dbcAMP increased IL-2 production in PBL cultures stimulated with TPA and antiCD3 plus TPA (Table 1). However, there was no increase in IL-2R-LUchain expression in these stimulated cultures (Fig. 6) with no significant change in the mean channel of fluorescence (MCF) for activation by TPA (26 vs 24) or by TPA plus anti-CD3 (38 vs 35) in the absenceor presence of dbcAMP. In contrast, the MCF with PHA stim-
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TABLE 1 Effect of dbcAMP on Interleukin-2 Production from Stimulated PBMC” Culture additivesb
Mean pgjml & SEMc
Medium PHA-P TPA TPA + dbcAMP Anti-CD3 + TPA Anti-CD3 + TPA + dbcAMP
<20 666 k 43 <20 433 + 34 216 k 22 650 k 29
0 24-hr culture supematants were tested using the Intertest- ELISA kit (seeMaterials and Methods). bThe concentration of agents used to stimulate PBMC was as follows: TPA (10 rig/ml), dbcAMP (50 &f), anti-CD3 (1 pg/ml), and PHA-P (10 &ml). ‘The resultsare representedas mean pgjml f SEM obtained from three experiments with different donors.
ulation was 62 without and 46 with dbcAMP. Together, these results implied that dbcAMP increases IL-2 synthesis but not IL-2R-a chain expression. dbcAMP and cholera toxin have no efect on purified T cell cultures. The experiments done thus far had evaluated the effect of dbcAMP and cholera toxin in unfractionated
¶nOUSC IgGI-FITC I d& + dbcAMP
TPA
TPA
+ anti-CD3
+ dkAMP
Log Immunofluorescence FIG. 6. Augmentative effect of dbcAMP is not associatedwith an increase in IL-ZR-(Uexpression. The 1 lo6 PBMC were cultured in 6-well flat-bottom plates for 24 hr with media, PHA (10 pg/ml), TPA (10 rig/ml), or anti-CD3 (1 &ml) in the presence or absenceof dbcAMP (50 FM). The cells were washed and stained with fluorescenatedisotype control mAb or anti-IL-ZR-cYmAb. Fluorescenceintensity was displayed on a log scale. The mean channel fluorescenceintensity (MCF) for the histograms were as described in the text. X
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PBMC. To further characterizethe augmentative effectof CAMP on T cell proliferation, T cells were purified from PBMC as mentioned under Materials and Methods. The purity of these cell preparations were confirmed by the lack of response to PHA-P, a monocyte-dependent T cell mitogen (Table 2). These cell preparations were used to monitor the effects of dbcAMP and cholera toxin on T cell stimulation in response to anti-CD3 (mAb 235) and/or TPA. Table 2 demonstrated that unlike in PBMC cultures, no significant (P > 0.05) augmentative effect of dbcAMP (50 +V) was observed in TPA- or mAb 235 plus TPA-stimulated T cell cultures. These data strongly suggestedthat accessory cells participated in facilitating the effect of dbcAMP and cholera toxin as seenin stimulated PBMC cultures (Fig. 1). The possibility that an inadequate dose of the reagents was employed was ruled out since up to 80-100 PM of dbcAMP and 10 rig/ml of cholera toxin had no significant effect on T cell proliferation (data not shown). Augmentative e&t by dbcAMP is mediated via monocytes. To study the role of monocytes, monocytes were separatedfrom PBMC by discontinuous Percoll gradient centrifugation and plastic adherence. Table 3 shows that addition of 5% autologous monocytes restored the activity (P < 0.05) of dbcAMP to augment T cell proliferation in response to the mitogenic combination of TPA plus anti-CD3 mAb 235. Similar results were obtained in TPA-treated cultures. Thus, dbcAMP mediated its effect on stimulated T cells through monocytes. However, it was not clear from theseexperiments whether the phenomenon was the result of a direct T cell-monocyte interaction in presence of dbcAMP or via soluble factor(s). Supernatants from dbcAMP-treated monocytes have augmentative activity. To determine whether monokines mediate the augmentative activity, we concentrated culture supernatants from dbcAMP-treated monocytes using a microconcentrator with a molecular weight cutoff at 10 kDa (Centricell, Polysciences Inc.). Two fractions were collected; one below 10 kDa and another above 10 kDa. Dilutions of these fractions were made in sterile water before addition to cultures. Figure 7 depicts the results obtained from experiments wherein T cells were stimulated with TPA plus mAb 235 in the presenceof fractionated supernatant with a MW of > 10 kDa from control and dbcAMP-treated monocytes. As previously shown, dbcAMP alone (closed bar) had no effect on T cell proliferation; this excludes the possibility that retained dbcAMP is the active factor in the fractionated samples that acted on the activated T cells. In
TABLE 2 T Cell Responseto dbcAMP Treatment in Mitogen Culture [3H]Thymidine incorporation (cpm X 10e3)+ SEM” for T cells treated with b Experiment
dbcAMP (50 ww
I
-
2
+ +
Media 110 + 1122 179 + 190 s
12 11 17 21
Anti-CD3 21Ok 214 f 114+ 119*
11 14 9 11
PHA 846 f 805 f 2215 f 1914 +
TPA 41 39 120 274
6061 + 6293 k 782 f 695 f
Anti-CD3 + TPA 215 154 85 54
51650 f 51290 + 61978 f 62310 +
1254 1452 1225 1985
LIResults are expressedas means + SEM from triplicates of each sample. b 1 X lo5 T cells were cultured for 72 hr with medium alone or supplemented with the different activator PHA (10 &ml), anti-CD3 (1 pg/ml), or TPA (10 rig/ml), and the thymidine incorporation was measured.
CAMP AUGMENTS TPA-INDUCED
249
T CELL PROLIFERATION
TABLE 3 Role of Monocytes in T Cell Mitogenesis [‘H]Thymidine uptake (cpm X IO-‘) f SEM’ for T cells stimulated with b Expt. 2
Expt. 1 5% Monocytes
dbcAMP (50 ww
-
-
-
+ -
+ +
mAb 235 + TPA
TPA 9,954 f 564 10,110 _+448
107,191 + 1145 106,522 + 1589 112,134+1158 210,478 k2345
9,598 k651 15,678 2789
+
TPA
mAb 235 + TPA
12,589&423 12,998 f 548 11,558+328 18,971 f 554
213,907 + 1148 212,llOk 1584 225,108 + 1587 319,317 + 1897
a Results are presented as mean cpm t- SEM. Data are from two independent experiments. Mean cpm + SEM in cultures with medium alone was 245 + 14 and 119 k 15 in Experiments 1 and 2, respectively. bT cells were stimulated with TPA (10 rig/ml) alone or TPA plus mAb (1 rig/ml) in the presence or absenceof autologous monocytes as well as with or without dbcAMP (50 PM).
contrast, the supernatant from monocytes treated with dbcAMP for 24 hr and longer significantly (P < 0.005) enhanced T cell mitogenesis although the enhancement was already seen at 8 hr. Thus, factor(s) with a molecular weight above 10,000 Da were synthesized and secretedby monocytes in the presenceof dbcAMP. It is of interest to note that the fractionated supernatant with MW less than 10 kDa from the treated monocytes exerted significant inhibitory effect on T cell stimulation (data not shown). rIL-I, r-IL-6 and rTNF-CYdo not duplicate the enhancing efects of supernatant from treated monocytes. It is known that monocytes/macrophages secrete monokines that
500 .-s
ii
400 b a g ;r)^ .E 0 300
I 0
dbcAMP sup from ontrl
CD; 2 f% d" 7
200 100
3 0 cntrl
2 time
8
of supernatant
24 collection
48
72
(hours)
FIG. 7. Supernatants from dbcAMP-treated monocytes augment T cell mitogenesis. Supematants were collected at various time points from cultures containing 5 X IO’- 10 X IO6monocytes treated with complete medium (open bar) or dbcAMP (50 m, crosshatch bar). The fraction with greater than 10 kDa obtained from the supematant (seeMaterials and Methods) was added to T cells (1 X 105/well) stimulated with TPA (10 q/ml) plus anti-CD3 mAb 235 (1 pg/ml). As control, T cells were cultured in media (right diagonal bar) and dbcAMP (50 pw closed bar). Results are presentedas cpm. Values are given as the means + SEM of three separateexperiments done with supematant collected from monocytes of three different individuals.
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may influence T cell proliferation. Among these are IL-l, IL-6, and TNF-a (3 1, 32). Therefore, to determine the factor(s) responsible for the augmentative phenomenon described thus far, T cells were stimulated with mAb 235 plus TPA and varying concentrations of recombinant IL-l, IL-6, or TNF-a were added. The results of the experiments are shown in Figs. 8A and 8B. The addition of IL-1 and IL-6 in concentrations ranging from 0.1 to 10 and 1.0 to 100 U/ml was found not to have any influence on the proliferation of T cells in responseto TPA plus mAb 235. However, rIL- 1 enhanced the proliferation of cells at suboptimal concentrations of TPA (0.1 rig/ml) in the presence and absence of anti-CD3 mAb (data not shown) but not at optimal concentration of TPA as described above. In contrast, the addition of recombinant TNF-a at all concentrations tested exerted a suppressive effect (P < 0.005) in stimulated T cell cultures. Additionally, a combination of the recombinant factors again did not augment T cell proliferation (data not shown). Taken together, these results suggestedthat the augmentative factors with a MW of > 10 kDa in the super-
1
300
rTNF-a 200
100
ii e i
0 L--l
FIG. 8. (A) IL-I and IL-6 do not mimic the augmentative effect of supernatant from dbcAMP-treated monocytes. T cells were stimulated with TPA plus anti-CD3 and recombinant human IL-1 and IL-6 was added at the concentrations indicated. As control, supematant from monocytes treated with media and dbcAMP (50 pkf) were used. The results are expressedas mean cpm of 2 individual experiments. (B) Effect of rTNF-ol addition to mitogen-induced T cell cultures. T cells were stimulated as in A and recombinant human TNF-a~was added at the indicated concentrations. As control, supematant from monocytes treated with media and dbcAMP (50 PLM)were used. The results are expressed as mean cpm of 2 individual experiments. Cell viability in cultures treated with rTNF-a was in the range of 90-9X as determined by trypan blue exclusion. * representsP < 0.05.
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natant from dbcAMP-treated monocytes were not among the variety of known monokines tested above. DISCUSSION The results described here show that dbcAMP and CT could enhance phorbol esterinduced T cell proliferation. Under similar culture conditions, we confirmed that dbcAMP and CT could be inhibitory to PHA-induced T cell proliferation as reported by others (2-8). Although the augmentation of T cell proliferation by dbcAMP and CT was modest, it was reproducible with over 20 separate experiments and was statistically significant. This augmentative effect of CAMP-inducing agents has also been observed by other investigators. Although using different mitogens, our results are comparable to those reported by Webb et al. (22-24). More recently, Ledbetter et al. had shown that 100 PM dbcAMP moderately augmented TPA-induced T cell proliferation while the samedosesignificantly inhibited anti-CD3-induced T cell proliferation (25). Bismuth et al. reported that the CAMP-inducing agent PGE2 reproducibly augmented the proliferation of P28D cell clone induced by TPA/ionomycin (26). Nel et al. also reported that TPA-induced T cell proliferation, in contrast to PHA, was not affected by dbcAMP or CT (2). These observations led us to investigate the mechanism(s) whereby dbcAMP or CT could augment T cell proliferation. We observed a dose-dependent effect of CAMP agents in TPA-stimulated cell cultures. 1OO-1000 &4 of dbcAMP and lo- 100 rig/ml of CT were inhibitory (and this was partly due to toxic effects of the drugs), whereas at comparatively lower doses these agents tend to augment T cell responses.The augmentative effect of dbcAMP or CT is observable even when added 24 hr after T cell stimulation. This would suggest that the augmentative effect is inducible even at late phases of cell proliferation and an existing response is enhanced. The enhancement of response measured by [3H]thymidine uptake could be correlated with an increasein cell numbers in dbcAMP and phorbol ester-treated cultures versus the control cultures (P < 0.05, Fig. 3). Furthermore, the enhancement in response was not a consequence of a shift in kinetics of [3H]thymidine uptake by the proliferating cells. The augmentative effect of dbcAMP and CT is modest and this may be due in part to the dampening effectsof suppressive factors elaborated by monocytes and the direct suppressiveeffect of CAMP on T cells. In sharp contrast to the above results, when added within 4 hr of culture initiation, both dbcAMP and CT inhibited proliferation of PHA-stimulated cultures. However, addition of CAMP agents 4-8 hr after initiation of culture did not result in inhibition of the lectin-induced proliferation. This would support the hypothesis that once T cells acquire the ability to proliferate they become resistant to inhibition by CAMP agents. Similar data arguing for the early phase of T cell activation as the target for suppression by CAMP have been reported by others (I, 15, 18, 31, 32). Like most studies reported, monocytes were present in the cell preparations used in our initial experiments. To evaluate the role of monocytes, the cell preparations were extensively depleted of monocytes. The purity of the T cell preparations were found to be comparable when tested either by immunofluorescence method or by nonspecific esterase staining. Furthermore, the resulting population became unresponsive to PHA, a monocyte-dependent mitogen. When the purified T cells were stimulated with TPA or TPA with anti-CD3, addition of dbcAMP did not augment the responsesuggestinga role for accessorycells in this system. The need for accessory cells was confirmed when the augmentative effect of dbcAMP was reconstituted with monocytes.
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It should be emphasized that our study did not determine what additional direct effect CAMP may have on T cells, Imboden et al. (14) have shown that CT inhibited TcR- and CD3-mediated calcium flux and IP3 turnover in Jurkat cells. They concluded that CT affects events that occur subsequent to the binding of antigen to its receptor but prior to the activation of phospholipase C. Our results do not exclude the possibility that dbcAMP and CT could influence the early biochemical events as shown in cell lines by Imboden and co-workers. We have further demonstrated that monokine(s) mediated the effect of the monocytes.The identity of the monokine(s) has not been clarified but appearedto be different from IL- 1, IL-6, and TNF-(Y. A large number of cytokines have recently been identified and efforts are underway to correlate the activity with those described herein. Among these factors include IFN-ol, TGF-P, IL-7 and the less characterized ones such as the ADF/thioredixin as described by Yodoi et al. (33, 34). The precise mechanism(s) of action of the monokine(s) is yet to be determined. Since TPA activates PKC directly, it appears likely that the monokines act on the events subsequent to PKC activation. This is in agreement with our time kinetic studies. The likely site of action includes the production of IL-2 (Table 2) but not the expression of IL-2R-(r chain (Fig. 6). Our data indicate that in TPA-activated T cells, IL-2R-a! chain is expressed.However, little or small amounts of IL-2 are produced by those T cells. The addition of dbcAMP to mediate IL-2 releasecan therefore help to stimulate activated T cells to proliferate. Alternatively, dbcAMP-induced monokine(s) may potentiate the expression of the high affinity IL-2R-fi chain expression and thus enhance the proliferation of activated T cells. These alternate mechanisms are being studied in our laboratory. Our finding that CAMP analogues and CAMP elevating agents can modulate T cell function via monocytes may help to shed light on some of the controversial aspects discussedearlier since most studies reported to date have residual monocytes in their cell preparation. We have identified both helper and suppressive factors from the dbcAMP-treated monocyte supernatants. The kinetics of secretion of these factors may depend on the stimulus used. The net effect will thus depend on the relative proportion of these factors. Furthermore, the numbers of monocytes in the preparation and the stimulus used will contribute to the ability of the target T cells to proliferate. In conclusion, CAMP may positively regulate the proliferation of T cells as it has been appreciated in other systems (20). The effect appears to be a result of factor(s) produced by monocytes. The identity of the factor(s) and the mechanism(s) with which they regulate the augmentative activity remains to be elucidated. REFERENCES 1. Averill, L. E., Stein, R. L., and Kammer, G. M., Cell. Immunol. 115, 88, 1988. 2. Nel, A. E., Vandenplas, M., Wooten, M. M., Cooper, R., Vandenplas, S., Rheeder, A., and Daniels, J., Biochem. J. 256, 383, 1988. 3. Papadoggiannakis, N., Johnsen, S. A., Rosberg, S., Enderson, R. G., and Olding, L. B., Immunology 68, 378, 1989. 4. Daniel, A., Kowalski, D. J., Nodzenski, E., Micek, M., and Pearl, W., Biochem. Biophy. Res. Commun. 167,383, 1990.
5. 6. 7. 8.
Lingk, D. S., Chan, M. A., and Gelfand, E. W., J. Immunol. 145, 449, 1990. Murray, A. W., Froscio, M., and Kemp, B. E., Biochem. .I. 129,995, 1972. Kemp, B. E., Froscio, M., Rogers, A., and Murray, A. W., Biochem. J. 145,241, 1975. Chaplin, D. D., Wedner, H. J., and Parker, C. W., In “Biologic Basis of Immunodeficiency” (E. W. Gelfand and H. W. Dosch, Eds.), p. 269. Raven Press,New York, 1980.
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9. Kammer, G. M., Immunol. Today9,223, 1988. 10. Taylor, M. V., Metcalfe, J. C., Hesketh, T. R., Smith, G. A., and Moore, J. P., Nature 312,462, 1984. 11. Tsein, R. Y., Pozzan, T., and Rink, T. J., Nature 295,68, 1982. 12. Mills, G. B., Lee, J. W. W., Cheung, R. K., and Gelfand, E. W., J. Immunol. 135, 3087, 1985. 13. Kumagai, N., Benedict, S. H., Mills, G. B., and Gelfand, E. W., J. Zmmunol. 141, 3747, 1988. 14. Imboden, J. B., Shoback, D. M., Pattison, G., and Stobo, J. D., Proc. Natl. Acad. Sci. USA 83, 5673, 1986.
15. Goto, Y., Takeshita, T., and Sugumura, K., FEBS Lett. 239, 165, 1988. 16. Johnson, K. W., Davis, B. H., and Smith, K. A., Proc. Natl. Acad. Sci. USA 85, 6072, 1988. 17. Lemer, A., Jacobson, B., and Miller, R. A., J. Zmmunol. 140, 936, 1988. 18. Iwaz, J., Lafont, S., Cordier, G., and Revillard, J. P., J. Clin. Lab. Immunol. 29, 85, 1989. 19. Bourne, H. R., Lichtenstein, L. M., Melmon, K. L., Henney, C. S., Weinstein, Y., and Shearer,G. M., Science 184, 19, 1974. 20. Dumont, J. P., Jauniaux, J. C., and Roger, P. P., Trends Biochem. Sci. 14, 67, 1989. 21. Boynton, A. L., and Whitefield, J. F., Adv. Cyclic Nucleotide Res. 15, 193, 1983. 22. Webb, D. R., Stites, D. P., Perlman, J. D., Luong, D., and Fudenberg, H. H., Biochem. Biophys. Res. Commun. 53, 1002, 1973. 23. Webb, D. R., Stites, D. P., Perlman, J. D., Austin, K. E., and Fudenberg, H. H., Clin. Immunol. Immunopath. 2, 322, 1974. 24. Webb, D. R., Belohradsky, B., Hanes, D., Stites, D. P., Perlman, J. D., and Fudenberg, H. H., C/in. Immunol. Immunopath. 4, 226, 1975. 25. Ledbetter, J. A., Parsons, M., Martin, P. J., Hansen, J. A., Rabinovitch, P. S., and June, C. H., J. Immunol. 137,3299, 1986. 26. Bismuth, G., Theodurou, I., Gouy, H., Gouvello, S. L., Bernard, A., and Debre, P., Eur. J. Immunol. 18, 1351, 1988.
27. Ham, T., and Fu, S. M., J. Exp. Med. 161, 641, 1985. 28. Gutierrez, C., Bemabe, R. R., Vega, J., and Krieisler, M., J. Immunol. Methods 29, 57, 1979. 29. Jung, L. K. L., Haynes, B. F., Nakamura, S., Pahwa, S., and Fu, S. M., Clin. Exp. Immunol. 81, 466, 1990. 30. Yam, L. T., Li, C. Y., and Crosby, W. H., Am. J. Clin. Pathol. 55,28, 1971. 31. Rincon, M., Tugores, A., Lopez-Rivas, A., Silva, A., Alonso, M., De Landazuri, M. O., and LopezBotet, M., Eur. J. Immunol. 18, 179 1, 1988. 32. Takayama, H., Trenn, G., and Sitkowsky, M. V., J. Biol. Chem. 263, 2330, 1988. 33. Yodoi, J., and Tursz, T., Adv. Cancer Res. 57, 381, 1991. 34. Yamauchi, A., Masutani, H., Tagaya, Y., Wakasugi, N., Mitsui, A., Nakamura, H., Inamoto, T., Ozawa, K., and Yodoi, J., Molec. Immunol. 29, 263, 1992.
IMMUNOLOGY
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Augmentation of Phorbol Ester-Induced T Cell Proliferation by Agents Which Raise Intracellular Cyclic Adenosine Monophosphate’ HRISHEKESHR. CHAKKALATH* AND LAWRENCEK. L. JUNG? *Department of Tropical Health, Harvard School of Public Health, 665 Huntington Avenue, Boston, Massachusetts 02115; and fDivision of Pediatric Immunology, Department of Pediatrics, University of MassachusettsMedical Center, 55 Lake Avenue North, Worcester,Massachusetts 01655 Received February 12, 1992; acceptedSeptember 6. 1992 Although raising intracellular cyclic adenosine monophosphate (CAMP) levels is generally considered to be inhibitory on the mitogen-induced T cell proliferation, in this study we have shown that the addition of either dbcAMP (50 PM) or cholera toxin (1 rig/ml) resulted in an increase in [3H]thymidine uptake in PBMC cultures stimulated with phorbol ester, 12-tetradecanoylphorbol 13-acetate(TPA), or with a combination of TPA plus anti-CD3 mAb (mAb 235). In contrast, under similar culture conditions, the phytohemagglutininP (PHA-P) responsewas inhibited by these agents as has been reported. The augmentative effect of dbcAMP in PBMC cultures was due to an increase in IL-2 production and not to increased in IL-2R-a chain expression. The enhancing effect of dbcAMP and CT observed with PBMC was monocyte dependent and not seen with purified T cell preparations. The addition of monocytes reconstituted the ability of intracellular CAMP elevating agents to augment the T cell response to TPA with and without mAb to CD3. The monocytes mediate their action via soluble factor(s) with molecular weight (m.w.) of more than 10 kDa. Neither rIL-1, rIL-6, nor rTNF+ have any augmentative effect as contrast with the supernatant from treated monocytes. Taken together, our results indicate that CAMP can play a positive regulatory role in T cell proliferation due to factor(s) secreted by dbcAMP-treated monocytes resulting in increased IL-2 synthesis in T cells. o 1992 Academic Press.Inc.
INTRODUCTION Many investigators have established that cyclic adenosine monophosphate (CAMP) analogues and agents that raise intracellular CAMP levels inhibit or downregulate T lymphocyte proliferation (1-8, reviewed in 9). This conclusion is derived from experiments wherein the inhibitory effect of CAMP elevating agents or analogues was detected chiefly on the lectin-induced T cell proliferation. The stimulation of T cells by mitogens leads to early activation events which include phosphatidylinositol turnover (lo), elevation in cytoplasmic free calcium (11) and induction of endogenous IL2 production and IL-2 receptor expression (12, 13). Each of these events has been reported as targets for CAMP-mediated inhibition of T cell proliferation ( 14- 18). Such findings reaffirmed the earlier conclusion that CAMP mediated a general, antiproliferative signal (19). However, adenylate cyclase activators like cholera toxin and forskolin could be mitogenic in systemssuch as dog thyrocytes (20). Similar evidence although lesscom’ This work was supported in part by NIH Grant AI-25704. 240
0008-8749192$5.00 Copyright 0 1992 by Academic Press,Inc. All rights of reproduction in any form reserved
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plete is available for many epithelial and nonepithelial differentiated cells (2 1). Thus, a positive functional role for CAMP in mitogenic stimuli has also been documented. Indeed, earlier studies by Webb et al. (22-24) had shown that agents which raised intracellular CAMP could enhance DNA and RNA synthesisof lymphocytes stimulated at supraoptimal dosesof PHA. More recent studies supported Webb’s observation of the positive effect of CAMP on T cell proliferation, although the later authors did not focus on this aspect in their studies (25, 26). In this study, we have investigated the effect of intracellular CAMP elevating agents on the in vitro response of peripheral T cells to various stimuli. We confirm that dbcAMP2 and cholera toxin do have an inhibitory effect on T cell proliferation stimulated by optimal dose of PHA-P. Additionally, these reagents also augment T cell proliferation induced by TPA or TPA plus anti-CD3 mAb by upregulating IL-2 synthesis but not IL-2R-a! chain expression. We further show that the augmentative effect by CAMP is mediated by factor(s) elaborated by monocytes upon exposure to dbcAMP. MATERIALS AND METHODS Reagentsand antibodies. The complete medium was made of RPM1 1640 (GIBCO, Grand Island, NY) supplemented with penicillin/streptomycin (GIBCO), glutamine (GIBCO), and 10%heat-inactivated fetal bovine serum (FBS, HyClone Lab Inc., Logan, UT). N6-2’-O-dibutyrladenosine 3’:5’-cyclic monophosphate (dbcAMP) (Sigma, St. Louis, MO) was dissolved in sterile phosphate-buffered saline, aliquotted, and stored at -70°C until use. TPA (Sigma) was dissolved in dimethyl sulfoxide (DMSO, Sigma) and stored at -70°C. Prior to use in culture, TPA was diluted to the appropriate concentration in complete culture medium. Cholera toxin was obtained from ICN Biochemicals, (Cleveland, OH). Phytohemagglutinin-P was obtained from Difco Lab (Detroit, MI). Human rIL- 1, rIL-6, and rTNI+ were obtained from Genzyme (Boston, MA), aliquotted, and stored at -70°C until use. Monoclonal antibody against CD3, designated 235 (IgM) (27) was a generous gift of Dr. Shu Man Fu (University of Virginia, Charlottesville, VA). T cell preparation. PBMC were isolated by Ficoll-Hypaque (Pharmacia, Piscataway, NJ) density centrifugation from peripheral blood of normal volunteers collected in heparin-containing syringes.T cells from PBMC preparations were obtained by Percoll discontinuous density gradient (28). The cells were then plated onto plastic Falcon petri dishes (Becton-Dickinson labware, Lincoln Park, NJ) and incubated for 1 hr 15 min in a 5% CO2 incubator. The nonadherent cells were removed and the same procedure was repeated three times. Cells thus obtained were then depleted of B cells and monocytes by preincubating for 30 min at 4°C in a mixture of mAb’s to these cells (29) and removed using goat anti-mouse immunoglobulin-coated magnetic beads (Dynal A.S., Oslo, Norway). T cell preparations thus obtained were found to have less than 1% monocytes and B cells, as determined by flow cytometry. Enumeration of monocytes in T cell preparations by nonspecific esterasestaining (30) gave comparable results as with immunofluorescence and such cell preparations were used in assays. All the experiments in the present study were carried out with ~98% viable cells, as observed by trypan blue dye exclusion technique. Monocyte and supernatant preparation. Monocytes from PBMC were isolated by Percoll discontinuous density gradient asmentioned above. Plastic adherenceprocedure ’ Abbreviations used: CT, cholera toxin; dbcAMP, dibutyrl cyclic adenosine monophosphate; TPA, 12tetradecanoylphorbol 13-acetate;MCF, mean channel fluorescence.
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was repeated three times with the cells, each for 1 hr 15 min. The adherent cells were detached from the plates by using a rubber policeman in the presence of cold PBS containing neither Mg*+ nor Ca2+. The adherent cells thus obtained were 98% esterase positive and 95% viable as determined by esterasestaining and trypan blue dye exclusion technique, respectively. In experiments requiring treated or untreated monocytes, 5 X 1O5adherent cells were plated on a 6-well plate and were treated for various periods with reagentsor media. The adherent cells were removed from the wells using the rubber policeman, washed twice, and checked for viability before adding to T cell cultures. Treated monocytes used in the study were essentially 92-95% viable. The culture supernatants from treated monocytes (5 X lo5 to 10 X 1O6cells) were collected at different time points after treatment with reagents or media and subjected to fractionation and concentration using a centrifugal ultrafilter (Centricell, Polysciences Inc., Wanington, PA) with a molecular weight cutoff at 10,000 Da. The concentrates thus obtained were diluted in double-distilled water, sterilized by filtration through 0.22 pm filters, and stored at -70°C until use. Proliferation assays.PBMC or T cells (1 X 105)in 200 ~1of complete medium were incubated in triplicate in flat-bottom 96-well plates at 37’C for 3 days in a 5% CO2 atmosphere. During the last 16 hr cells were pulsed with 2.0 PCi of [3H]thymidine (6.7 Ci/mmol; NEN DuPont, Boston, MA) and the incorporated radioactivity was determined using a 1205 Betaplate counter (Pharmacia LKB Nuclear, Gaithersburg, MD). The percentage of control responsewas calculated by the following formula: % Control response = [counts per minute (cpm) in CAMP-treated experimental cultures/cpm in untreated control cultures] X 100. IL-2 determination. The 24-hr culture supernatants from stimulated PBMC were collected and analyzed for IL-2 levels using an Intertest- enzyme-linked immunosorbent assay(ELISA) kit (Genzyme). The assaywas performed as per the manufacturer’s instructions. Briefly, a monoclonal antibody to human IL-2 was coated on polystyrene microtiter plates, incubated, washed, and test samples were added. After overnight incubation and washing, purified polyclonal rabbit anti-human IL-2 was added and then reacted with alkaline phosphatase-conjugated goat-anti-rabbit immunoglobulin. The OD at 405 nm was determined on an ELISA reader. Results were calculated by comparison to IL-2 standards ranging between 20 and 1200 pg/ml. Flow cytometry. PBMC were incubated with media alone, PHA (10 pg/ml), TPA ( 10 rig/ml) or TPA plus anti-CD3 (1 pg/ml) in the presence or absence of dbcAMP (50 palm)for 24 hr, washed, and stained with a fluorescenated isotype control or antiIL-2R-(Y mAb (Ortho Diagnostics, NJ) for 30 min at 4°C. Five thousand gated cells were analyzed on a FACScan flow cytometer (Becton-Dickinson, CA). The data obtained were analyzed on FACScan Research software. Statistical analysis. Statistical analysis was done by Student’s t test or by analysis of variance. RESULTS Dose and time kinetics of dbcAMP eflect on mitogen-induced PBMC proliferation. The effect of dbcAMP on the mitogen-induced responsesof PBMC were studied using the following culture systems. Cells were stimulated with either PHA-P, TPA or a combination of TPA plus soluble anti-CD3 mAb, 235. The responses,monitored by [3H]thymidine incorporation by stimulated cells were expressedas counts per minute. In preliminary experiments, the proliferation of PBMC stimulated with anti-CD3 plus
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TPA (82,188 ~fi4040; mean cpm f SEM) was comparable to that in PHA-P cultures (88,044 + 5493 cpm) and significantly higher than that in TPA-treated cultures ( 13,237 + 1400 cpm). The responseof T cells to mAb 235 alone was similar to PBMC control cultures with media (19 1 + 66 cpm vs 256 + 86 cpm). Although [3H]thymidine uptake varied in PBMC cultures from different individuals, the same pattern of responsewas observed. For comparison, the results were normalized and expressedas percentage of control response computed from the formula mentioned under Materials and Methods. The dose-dependent effect of dbcAMP on PBMC proliferation to mitogens is shown in Fig. 1. dbcAMP concentrations ranging from 10 to 50 PM were found to enhance (P < 0.025) TPA and anti-CD3 plus TPA-induced proliferative response in PBMC cultures. The optimal dose of dbcAMP on both the responseswas 50 pIM. In contrast, under similar culture conditions, dbcAMP significantly (P < 0.005) inhibited the PHAP-induced proliferation. Thus, dbcAMP augmented phorbol ester-induced proliferation but inhibited PHA-P-induced proliferation. One possible explanation for our observation is that the addition of dbcAMP led to a shift in the kinetics of T cell proliferation. As shown in Fig. 2, the augmentative effect induced by dbcAMP in activated cells representsa real increase and not a kinetic shift in cell proliferation. Another possibility is that the increased thymidine uptake represented a shift in nucleotide pool metabolism and not to a real increase in cell proliferation. To address this possibility, lo5 PBMC were cultured for 3 days with media, TPA, or TPA plus anti-CD3 with or without the presence of 50 PM dbcAMP. The mean viable cell counts from triplicate cell cultures were determined by trypan blue dye exclusion and the means of four independent experiments are representedin Fig. 3. Thus, the addition
0 0
10
Cont.
100
of dbcAMP
1000
(@VI)
FIG. 1. Effect of dbcAMP on mitogen-induced PBMC proliferation. PBMC were stimulated with PHAP (10 pglml), TPA (10 rig/ml) alone, or TPA plus anti-CD3 mAb 235 (1 &ml) in the presenceof various concentrations of dbcAMP as indicated. The resultsare expressedaspercentagesof control mitogen response calculated by using the formula given under Materials and Methods. Values are given as the means f SE of three separate experiments. Cell viability tested by trypan blue dye exclusion technique, at all dosesof dbcAMP tested in PBMC treated with media or anti-CD3 was in the range of 90-95%. At dbcAMP doses below 100 &I, cell viability in PBMC cultures stimulated with PHA, TPA, and TPA plus anti-CD3 was 90-95s as against 70-80s viability in stimulated cultures treated with above 100 & concentration of dbcAMP. Mean cpm + SE in cultures treated with medium alone was 191 + 66; anti-CD3, 256 f 86; antiCD3 + TPA, 82,188 f 4040; TPA, 13,237 +- 1400; PHA-P, 88,044 + 5493.
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DAYS OF CULTURE FIG. 2. dbcAMP-induced enhancement of proliferation is not due to a shift in kinetics of thymidine uptake. The 1 X lo5 PBMC were stimulated with media or TPA (10 &ml) with and without dbcAMP (50 PM). [3H]Thymidine-pulsed cultures were harvested on Days 2, 3, 5, and 7. Results are expressedas mean cpm f SE of three independent experiments. Mean cpm f SE in cultures treated with medium alone was 290 f 63, and 245 + 55,646 -C90, 865 + 123,and 1090 f 234 for 2-, 3-, 5-, and 7-day cultures, respectively.
of dbcAMP led to the increase in total cell numbers in both cell cultures with TPA or TPA and anti-CD3 (P < 0.05). These experiments show that the effect of dbcAMP is to increase DNA synthesis and cell proliferation. dbcAMP augmented TPA and anti-CD3/TPA induced cell responseseven after 2 days of culture initiation (Fig. 4). Furthermore, the significant (P < 0.005) inhibitory
2.5
-2
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dbcAMP anti-CD3 + TPA
FIG. 3. dbcAMP enhanced thymidine uptake by inducing proliferation of T lymphocytes. The 1 X lo5 PBMC were stimulated with media, TPA (10 rig/ml), or TPA + mAb (1 pg/ml) with and without dbcAMP (50 p&f). The mean viable cell counts from triplicate cell cultures were determined by trypan blue dye exclusion technique. Four independent experiments were carried out and the means of the experiments are depicted. Analysis by Student’s t test and by analysis of variance showed that dbcAMP enhanced T cell proliferation (P < 0.05).
CAMP AUGMENTS TPA-INDUCED . . . ..-. q
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+ TPA
o!
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40 addition
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FIG. 4. dbcAMP effectin PBMC mitogenic responses:Time kinetics. PBMC were stimulated with mitogens as described in the legend for Fig. 1 and dbcAMP (50 p,W) was added at the indicated time periods. The results are expressed as percentages of control response. Values are given as the means + SEM of two independent experiments. Mean cpm f SE in cultures treated with medium alone was 2 14 + 34; anti-CD3 + TPA, 39,091 + 701; TPA, 5566 + 555; and PHA-P, 92,904 f 3493.
effect on PHA-P response could be demonstrated only when dbcAMP was added within 2-4 hr after mitogen addition to cultures. Thus, we show that the effects of dbcAMP on PBMC mitogenesis varied depending on the kind of stimulus and state of activation of the cells. Cholera toxin mimics the efect of dbcAMP in mitogen-induced PBMCprolifeation. Cholera toxin catalyzes the ADP-ribosylation of the CYsubunit of G, protein rendering it unable to hydrolyze its bound GTP. This results in continual activation of adenylate cyclase and prolonged elevation of intracellular CAMP. To demonstrate that CAMP augments T cell proliferation, cholera toxin was used to induce a rise in intracellular CAMP. Figure 5A shows that cholera toxin at an optimal concentration of 1 rig/ml can significantly (P < 0.005) augment the responseof PBMC to TPA as well as to the combination of mAb 235 plus TPA. Again, the samedoseinhibited the PHA-P response (P < 0.025). A dose of 100 rig/ml of cholera toxin was found to be cytotoxic and thus was suppressive in proliferating cultures (Fig. 5A). The time course of cholera toxin addition to stimulated PBMC cultures (Fig. 5B) had a similar pattern as seen with dbcAMP in Fig. 3. Essentially, cholera toxin inhibited (P < 0.005) PHA-P response when added at the initiation of culture or within 2 hr. At later time points of addition, cholera toxin did not influence (P > 0.05) the lectin-induced proliferation. These results also indicated that inhibition by cholera toxin of PHA-P cultures at a concentration of 1 rig/ml was not due to cytotoxicity. It was also of interest to note in Fig. 5B that cholera toxin had a synergistic effect in TPA and in the mAb 235 plus TPA cultures even when added after 24 hr after culture initiation. Thus, these experiments confirmed the positive role of CAMP on mitogen-induced PBMC responses. Augmentative effect of dbcAMP is associated with an increase in IL-2 production and not due to IL-2R-a chain expression in activated T cells. To understand the mechanism of the dbcAMP-mediated augmentative effect, we measured IL-2 production in phorbol ester-activated PBMC using the Interest-2 ELISA kit. The presence of
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. ....-. PHA
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anti-CD3
A--r)r
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time of CT addition
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FIG. 5. (A) Cholera toxin mimics the effectsof dbcAMP in mitogen-induced PBMC proliferation. PBMC were stimulated with PHA-P (10 pg/ml), TPA (10 q/ml) alone, or TPA plus anti-CD3 (I &ml) in the presenceof various concentrations of cholera toxin as indicated. The results are expressedas percentagesof control mitogen responsecalculated by using the formula given under Materials and Methods. Values are given as the means f SEM of three separate experiments. Cell viability at all dosesof cholera toxin tested in PBMC treated with media or anti-CD3 was in the range of 90-95s. With cholera toxin dosesbelow 10 rig/ml, cell viability tested by trypan blue dye exclusion in PBMC cultures stimulated with PHA, TPA, and TPA plus anti-CD3 was 90-95%. A 60-80s viability was observed in stimulated cultures treated with 100 rig/ml of cholera toxin. Mean cpm f SE in cultures treated with medium alone was 162 * 25; anti-CD3 + TPA, 32,397 ? 2729; TPA, 7166 + 502; and PHA-P, 64,549 + 2786. (B) Cholera toxin effect in PBMC mitogenic responses:Time kinetics. A dose of 1 rig/ml of cholera toxin was added at different time points to PBMC cultures stimulated with mitogens as described in the legend for Fig. 4. The results are expressed as percentagesof control response.Values are given as the means + SEM of two separateexperiments. Mean cpm + SE in cultures treated with medium alone was 214 + 34; anti-CD3 + TPA, 87,725 + 3831; TPA, 5294 & 356; PHA-P, 92,904 + 3493.
dbcAMP increased IL-2 production in PBL cultures stimulated with TPA and antiCD3 plus TPA (Table 1). However, there was no increase in IL-2R-LUchain expression in these stimulated cultures (Fig. 6) with no significant change in the mean channel of fluorescence (MCF) for activation by TPA (26 vs 24) or by TPA plus anti-CD3 (38 vs 35) in the absenceor presence of dbcAMP. In contrast, the MCF with PHA stim-
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TABLE 1 Effect of dbcAMP on Interleukin-2 Production from Stimulated PBMC” Culture additivesb
Mean pgjml & SEMc
Medium PHA-P TPA TPA + dbcAMP Anti-CD3 + TPA Anti-CD3 + TPA + dbcAMP
<20 666 k 43 <20 433 + 34 216 k 22 650 k 29
0 24-hr culture supematants were tested using the Intertest- ELISA kit (seeMaterials and Methods). bThe concentration of agents used to stimulate PBMC was as follows: TPA (10 rig/ml), dbcAMP (50 &f), anti-CD3 (1 pg/ml), and PHA-P (10 &ml). ‘The resultsare representedas mean pgjml f SEM obtained from three experiments with different donors.
ulation was 62 without and 46 with dbcAMP. Together, these results implied that dbcAMP increases IL-2 synthesis but not IL-2R-a chain expression. dbcAMP and cholera toxin have no efect on purified T cell cultures. The experiments done thus far had evaluated the effect of dbcAMP and cholera toxin in unfractionated
¶nOUSC IgGI-FITC I d& + dbcAMP
TPA
TPA
+ anti-CD3
+ dkAMP
Log Immunofluorescence FIG. 6. Augmentative effect of dbcAMP is not associatedwith an increase in IL-ZR-(Uexpression. The 1 lo6 PBMC were cultured in 6-well flat-bottom plates for 24 hr with media, PHA (10 pg/ml), TPA (10 rig/ml), or anti-CD3 (1 &ml) in the presence or absenceof dbcAMP (50 FM). The cells were washed and stained with fluorescenatedisotype control mAb or anti-IL-ZR-cYmAb. Fluorescenceintensity was displayed on a log scale. The mean channel fluorescenceintensity (MCF) for the histograms were as described in the text. X
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PBMC. To further characterizethe augmentative effectof CAMP on T cell proliferation, T cells were purified from PBMC as mentioned under Materials and Methods. The purity of these cell preparations were confirmed by the lack of response to PHA-P, a monocyte-dependent T cell mitogen (Table 2). These cell preparations were used to monitor the effects of dbcAMP and cholera toxin on T cell stimulation in response to anti-CD3 (mAb 235) and/or TPA. Table 2 demonstrated that unlike in PBMC cultures, no significant (P > 0.05) augmentative effect of dbcAMP (50 +V) was observed in TPA- or mAb 235 plus TPA-stimulated T cell cultures. These data strongly suggestedthat accessory cells participated in facilitating the effect of dbcAMP and cholera toxin as seenin stimulated PBMC cultures (Fig. 1). The possibility that an inadequate dose of the reagents was employed was ruled out since up to 80-100 PM of dbcAMP and 10 rig/ml of cholera toxin had no significant effect on T cell proliferation (data not shown). Augmentative e&t by dbcAMP is mediated via monocytes. To study the role of monocytes, monocytes were separatedfrom PBMC by discontinuous Percoll gradient centrifugation and plastic adherence. Table 3 shows that addition of 5% autologous monocytes restored the activity (P < 0.05) of dbcAMP to augment T cell proliferation in response to the mitogenic combination of TPA plus anti-CD3 mAb 235. Similar results were obtained in TPA-treated cultures. Thus, dbcAMP mediated its effect on stimulated T cells through monocytes. However, it was not clear from theseexperiments whether the phenomenon was the result of a direct T cell-monocyte interaction in presence of dbcAMP or via soluble factor(s). Supernatants from dbcAMP-treated monocytes have augmentative activity. To determine whether monokines mediate the augmentative activity, we concentrated culture supernatants from dbcAMP-treated monocytes using a microconcentrator with a molecular weight cutoff at 10 kDa (Centricell, Polysciences Inc.). Two fractions were collected; one below 10 kDa and another above 10 kDa. Dilutions of these fractions were made in sterile water before addition to cultures. Figure 7 depicts the results obtained from experiments wherein T cells were stimulated with TPA plus mAb 235 in the presenceof fractionated supernatant with a MW of > 10 kDa from control and dbcAMP-treated monocytes. As previously shown, dbcAMP alone (closed bar) had no effect on T cell proliferation; this excludes the possibility that retained dbcAMP is the active factor in the fractionated samples that acted on the activated T cells. In
TABLE 2 T Cell Responseto dbcAMP Treatment in Mitogen Culture [3H]Thymidine incorporation (cpm X 10e3)+ SEM” for T cells treated with b Experiment
dbcAMP (50 ww
I
-
2
+ +
Media 110 + 1122 179 + 190 s
12 11 17 21
Anti-CD3 21Ok 214 f 114+ 119*
11 14 9 11
PHA 846 f 805 f 2215 f 1914 +
TPA 41 39 120 274
6061 + 6293 k 782 f 695 f
Anti-CD3 + TPA 215 154 85 54
51650 f 51290 + 61978 f 62310 +
1254 1452 1225 1985
LIResults are expressedas means + SEM from triplicates of each sample. b 1 X lo5 T cells were cultured for 72 hr with medium alone or supplemented with the different activator PHA (10 &ml), anti-CD3 (1 pg/ml), or TPA (10 rig/ml), and the thymidine incorporation was measured.
CAMP AUGMENTS TPA-INDUCED
249
T CELL PROLIFERATION
TABLE 3 Role of Monocytes in T Cell Mitogenesis [‘H]Thymidine uptake (cpm X IO-‘) f SEM’ for T cells stimulated with b Expt. 2
Expt. 1 5% Monocytes
dbcAMP (50 ww
-
-
-
+ -
+ +
mAb 235 + TPA
TPA 9,954 f 564 10,110 _+448
107,191 + 1145 106,522 + 1589 112,134+1158 210,478 k2345
9,598 k651 15,678 2789
+
TPA
mAb 235 + TPA
12,589&423 12,998 f 548 11,558+328 18,971 f 554
213,907 + 1148 212,llOk 1584 225,108 + 1587 319,317 + 1897
a Results are presented as mean cpm t- SEM. Data are from two independent experiments. Mean cpm + SEM in cultures with medium alone was 245 + 14 and 119 k 15 in Experiments 1 and 2, respectively. bT cells were stimulated with TPA (10 rig/ml) alone or TPA plus mAb (1 rig/ml) in the presence or absenceof autologous monocytes as well as with or without dbcAMP (50 PM).
contrast, the supernatant from monocytes treated with dbcAMP for 24 hr and longer significantly (P < 0.005) enhanced T cell mitogenesis although the enhancement was already seen at 8 hr. Thus, factor(s) with a molecular weight above 10,000 Da were synthesized and secretedby monocytes in the presenceof dbcAMP. It is of interest to note that the fractionated supernatant with MW less than 10 kDa from the treated monocytes exerted significant inhibitory effect on T cell stimulation (data not shown). rIL-I, r-IL-6 and rTNF-CYdo not duplicate the enhancing efects of supernatant from treated monocytes. It is known that monocytes/macrophages secrete monokines that
500 .-s
ii
400 b a g ;r)^ .E 0 300
I 0
dbcAMP sup from ontrl
CD; 2 f% d" 7
200 100
3 0 cntrl
2 time
8
of supernatant
24 collection
48
72
(hours)
FIG. 7. Supernatants from dbcAMP-treated monocytes augment T cell mitogenesis. Supematants were collected at various time points from cultures containing 5 X IO’- 10 X IO6monocytes treated with complete medium (open bar) or dbcAMP (50 m, crosshatch bar). The fraction with greater than 10 kDa obtained from the supematant (seeMaterials and Methods) was added to T cells (1 X 105/well) stimulated with TPA (10 q/ml) plus anti-CD3 mAb 235 (1 pg/ml). As control, T cells were cultured in media (right diagonal bar) and dbcAMP (50 pw closed bar). Results are presentedas cpm. Values are given as the means + SEM of three separateexperiments done with supematant collected from monocytes of three different individuals.
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may influence T cell proliferation. Among these are IL-l, IL-6, and TNF-a (3 1, 32). Therefore, to determine the factor(s) responsible for the augmentative phenomenon described thus far, T cells were stimulated with mAb 235 plus TPA and varying concentrations of recombinant IL-l, IL-6, or TNF-a were added. The results of the experiments are shown in Figs. 8A and 8B. The addition of IL-1 and IL-6 in concentrations ranging from 0.1 to 10 and 1.0 to 100 U/ml was found not to have any influence on the proliferation of T cells in responseto TPA plus mAb 235. However, rIL- 1 enhanced the proliferation of cells at suboptimal concentrations of TPA (0.1 rig/ml) in the presence and absence of anti-CD3 mAb (data not shown) but not at optimal concentration of TPA as described above. In contrast, the addition of recombinant TNF-a at all concentrations tested exerted a suppressive effect (P < 0.005) in stimulated T cell cultures. Additionally, a combination of the recombinant factors again did not augment T cell proliferation (data not shown). Taken together, these results suggestedthat the augmentative factors with a MW of > 10 kDa in the super-
1
300
rTNF-a 200
100
ii e i
0 L--l
FIG. 8. (A) IL-I and IL-6 do not mimic the augmentative effect of supernatant from dbcAMP-treated monocytes. T cells were stimulated with TPA plus anti-CD3 and recombinant human IL-1 and IL-6 was added at the concentrations indicated. As control, supematant from monocytes treated with media and dbcAMP (50 pkf) were used. The results are expressedas mean cpm of 2 individual experiments. (B) Effect of rTNF-ol addition to mitogen-induced T cell cultures. T cells were stimulated as in A and recombinant human TNF-a~was added at the indicated concentrations. As control, supematant from monocytes treated with media and dbcAMP (50 PLM)were used. The results are expressed as mean cpm of 2 individual experiments. Cell viability in cultures treated with rTNF-a was in the range of 90-9X as determined by trypan blue exclusion. * representsP < 0.05.
CAMP AUGMENTS TPA-INDUCED
T CELL PROLIFERATION
251
natant from dbcAMP-treated monocytes were not among the variety of known monokines tested above. DISCUSSION The results described here show that dbcAMP and CT could enhance phorbol esterinduced T cell proliferation. Under similar culture conditions, we confirmed that dbcAMP and CT could be inhibitory to PHA-induced T cell proliferation as reported by others (2-8). Although the augmentation of T cell proliferation by dbcAMP and CT was modest, it was reproducible with over 20 separate experiments and was statistically significant. This augmentative effect of CAMP-inducing agents has also been observed by other investigators. Although using different mitogens, our results are comparable to those reported by Webb et al. (22-24). More recently, Ledbetter et al. had shown that 100 PM dbcAMP moderately augmented TPA-induced T cell proliferation while the samedosesignificantly inhibited anti-CD3-induced T cell proliferation (25). Bismuth et al. reported that the CAMP-inducing agent PGE2 reproducibly augmented the proliferation of P28D cell clone induced by TPA/ionomycin (26). Nel et al. also reported that TPA-induced T cell proliferation, in contrast to PHA, was not affected by dbcAMP or CT (2). These observations led us to investigate the mechanism(s) whereby dbcAMP or CT could augment T cell proliferation. We observed a dose-dependent effect of CAMP agents in TPA-stimulated cell cultures. 1OO-1000 &4 of dbcAMP and lo- 100 rig/ml of CT were inhibitory (and this was partly due to toxic effects of the drugs), whereas at comparatively lower doses these agents tend to augment T cell responses.The augmentative effect of dbcAMP or CT is observable even when added 24 hr after T cell stimulation. This would suggest that the augmentative effect is inducible even at late phases of cell proliferation and an existing response is enhanced. The enhancement of response measured by [3H]thymidine uptake could be correlated with an increasein cell numbers in dbcAMP and phorbol ester-treated cultures versus the control cultures (P < 0.05, Fig. 3). Furthermore, the enhancement in response was not a consequence of a shift in kinetics of [3H]thymidine uptake by the proliferating cells. The augmentative effect of dbcAMP and CT is modest and this may be due in part to the dampening effectsof suppressive factors elaborated by monocytes and the direct suppressiveeffect of CAMP on T cells. In sharp contrast to the above results, when added within 4 hr of culture initiation, both dbcAMP and CT inhibited proliferation of PHA-stimulated cultures. However, addition of CAMP agents 4-8 hr after initiation of culture did not result in inhibition of the lectin-induced proliferation. This would support the hypothesis that once T cells acquire the ability to proliferate they become resistant to inhibition by CAMP agents. Similar data arguing for the early phase of T cell activation as the target for suppression by CAMP have been reported by others (I, 15, 18, 31, 32). Like most studies reported, monocytes were present in the cell preparations used in our initial experiments. To evaluate the role of monocytes, the cell preparations were extensively depleted of monocytes. The purity of the T cell preparations were found to be comparable when tested either by immunofluorescence method or by nonspecific esterase staining. Furthermore, the resulting population became unresponsive to PHA, a monocyte-dependent mitogen. When the purified T cells were stimulated with TPA or TPA with anti-CD3, addition of dbcAMP did not augment the responsesuggestinga role for accessorycells in this system. The need for accessory cells was confirmed when the augmentative effect of dbcAMP was reconstituted with monocytes.
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It should be emphasized that our study did not determine what additional direct effect CAMP may have on T cells, Imboden et al. (14) have shown that CT inhibited TcR- and CD3-mediated calcium flux and IP3 turnover in Jurkat cells. They concluded that CT affects events that occur subsequent to the binding of antigen to its receptor but prior to the activation of phospholipase C. Our results do not exclude the possibility that dbcAMP and CT could influence the early biochemical events as shown in cell lines by Imboden and co-workers. We have further demonstrated that monokine(s) mediated the effect of the monocytes.The identity of the monokine(s) has not been clarified but appearedto be different from IL- 1, IL-6, and TNF-(Y. A large number of cytokines have recently been identified and efforts are underway to correlate the activity with those described herein. Among these factors include IFN-ol, TGF-P, IL-7 and the less characterized ones such as the ADF/thioredixin as described by Yodoi et al. (33, 34). The precise mechanism(s) of action of the monokine(s) is yet to be determined. Since TPA activates PKC directly, it appears likely that the monokines act on the events subsequent to PKC activation. This is in agreement with our time kinetic studies. The likely site of action includes the production of IL-2 (Table 2) but not the expression of IL-2R-(r chain (Fig. 6). Our data indicate that in TPA-activated T cells, IL-2R-a! chain is expressed.However, little or small amounts of IL-2 are produced by those T cells. The addition of dbcAMP to mediate IL-2 releasecan therefore help to stimulate activated T cells to proliferate. Alternatively, dbcAMP-induced monokine(s) may potentiate the expression of the high affinity IL-2R-fi chain expression and thus enhance the proliferation of activated T cells. These alternate mechanisms are being studied in our laboratory. Our finding that CAMP analogues and CAMP elevating agents can modulate T cell function via monocytes may help to shed light on some of the controversial aspects discussedearlier since most studies reported to date have residual monocytes in their cell preparation. We have identified both helper and suppressive factors from the dbcAMP-treated monocyte supernatants. The kinetics of secretion of these factors may depend on the stimulus used. The net effect will thus depend on the relative proportion of these factors. Furthermore, the numbers of monocytes in the preparation and the stimulus used will contribute to the ability of the target T cells to proliferate. In conclusion, CAMP may positively regulate the proliferation of T cells as it has been appreciated in other systems (20). The effect appears to be a result of factor(s) produced by monocytes. The identity of the factor(s) and the mechanism(s) with which they regulate the augmentative activity remains to be elucidated. REFERENCES 1. Averill, L. E., Stein, R. L., and Kammer, G. M., Cell. Immunol. 115, 88, 1988. 2. Nel, A. E., Vandenplas, M., Wooten, M. M., Cooper, R., Vandenplas, S., Rheeder, A., and Daniels, J., Biochem. J. 256, 383, 1988. 3. Papadoggiannakis, N., Johnsen, S. A., Rosberg, S., Enderson, R. G., and Olding, L. B., Immunology 68, 378, 1989. 4. Daniel, A., Kowalski, D. J., Nodzenski, E., Micek, M., and Pearl, W., Biochem. Biophy. Res. Commun. 167,383, 1990.
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9. Kammer, G. M., Immunol. Today9,223, 1988. 10. Taylor, M. V., Metcalfe, J. C., Hesketh, T. R., Smith, G. A., and Moore, J. P., Nature 312,462, 1984. 11. Tsein, R. Y., Pozzan, T., and Rink, T. J., Nature 295,68, 1982. 12. Mills, G. B., Lee, J. W. W., Cheung, R. K., and Gelfand, E. W., J. Immunol. 135, 3087, 1985. 13. Kumagai, N., Benedict, S. H., Mills, G. B., and Gelfand, E. W., J. Zmmunol. 141, 3747, 1988. 14. Imboden, J. B., Shoback, D. M., Pattison, G., and Stobo, J. D., Proc. Natl. Acad. Sci. USA 83, 5673, 1986.
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27. Ham, T., and Fu, S. M., J. Exp. Med. 161, 641, 1985. 28. Gutierrez, C., Bemabe, R. R., Vega, J., and Krieisler, M., J. Immunol. Methods 29, 57, 1979. 29. Jung, L. K. L., Haynes, B. F., Nakamura, S., Pahwa, S., and Fu, S. M., Clin. Exp. Immunol. 81, 466, 1990. 30. Yam, L. T., Li, C. Y., and Crosby, W. H., Am. J. Clin. Pathol. 55,28, 1971. 31. Rincon, M., Tugores, A., Lopez-Rivas, A., Silva, A., Alonso, M., De Landazuri, M. O., and LopezBotet, M., Eur. J. Immunol. 18, 179 1, 1988. 32. Takayama, H., Trenn, G., and Sitkowsky, M. V., J. Biol. Chem. 263, 2330, 1988. 33. Yodoi, J., and Tursz, T., Adv. Cancer Res. 57, 381, 1991. 34. Yamauchi, A., Masutani, H., Tagaya, Y., Wakasugi, N., Mitsui, A., Nakamura, H., Inamoto, T., Ozawa, K., and Yodoi, J., Molec. Immunol. 29, 263, 1992.