Regulation I. Effects of Cyclic

of Immune


and Cyclic

Responses GMP on Immune


Agents that raise intracellular CAMI’ lcvcls (tlihutyrpl cyclic :\A1 I’. nminoph~tlinc. atlenosine and butyric acid) increase the magnituttc of an irr 7ifYo priinarv humc~ral imniuncresponse when added al 10.” :\I during the first 12 hr of a 108 hr culture. Under the same conditions. cC;SlP has no ttirecl cffrct hut inhibits c.451 I’-metliatctt slimutation. I)txAhlP (lo-” A9 or 10.’ dll, prwznt from (1 to 12 hr, also incrcasrs the numhcr of cytotosic lymphocytes in CBA/J (H-2”) spleen cell cultures stimutatcd in a one-way mixed lymphocyte reaction with t)B:I/2J (EI-2’) spleen cells. The (thc.4,\1 F’ effect is antigen-depcndetlt in both humoral al~tt cell-mctliatctt immunity an(l antigen-specific in Ihe caw of humorat rcsp~m~es.

I STl~OT)UC’I’TOS Several

studies have suggestetl that CA AT 1’ n l~hys ;I role in immune responses. of the ATTC resl)oiise t(J SRIIC ]Jy ]JO]y i\. I. i?I ‘i’i7’0 ( l-3) :ulti iJl. vitro (2$ 4, 5 ) \vas potentiatetl 1)~ the(J]Jh~]]ille (2-A)) an inliihitor of cAAIE’ ]J]l(JSlhodiesterase. Ei-aun and coworkers (Z-3 j suggested that Lily A. r (6, 7) acts s\-ncrgisticall\bvith antigen to increase intrnccllular c.AiVI’. ant1 that ele\-ation of an activation signal for the immunc~ intracellular CAN P 1)~ antigen constitutes rcsI)onse. Studies by I’lescia cl 01. (8) slio\vetl that injected SKUC c~usetl a tkvoto illree-fold increaw iii mouse sI)lenic c:\ R’IP within 2 min. folIo\vctl 1,~ a tlrol) 10 next- normnl levels. Autologous (non-itlltnuliogcliic j ervtlirocytes _ or carhm Imticlcs were without effect. Similar results were repcn3etl 1)~ Y;lm;umoto mtl ~tilll~l]~~~iO~l







to 1~1 specifically





title to :Iriti~eti-stimul:ltetl


I{ cells.

CA\ A] 1'

since the




:r Ahhreviations used in this t,aprr : c.-\.\t I’, atlenosinc .i’.5’-cyclic Inollophost)hatc : cthcr\kl P. SC, O”‘-dihutyryladcnt,sitle 3’, 5’-cyclic inonophosI)hatr ; 5’.:IRI P, atlenosint* S’mot~ol~host~hatc ; d;Mt?, guanosinc 3’, j’kyctic monot)hosphate ; tthc(~ll P, N’, O”‘-tlihut, rylguanosinc 3’, 5’-cyctil. motlot)host)hate ; X-Br-cGhl I’, 8-hromaguatlosill~~ 3’. S’-cyclic rnonol,hos))hate ; :\ P, aminophylline ; cnrt~achot, carl~amylcholine rhtoridc ; pal) A .II, tlout)lc stranttvd hyhritl 0i 1,olyril,oatlcnylatc :md 1,~~lyrih(luritlylate ; XFC, antil)ott?-fornlillg cell(s) : PFC. plaque-forming cell(s) ; SRTSC, Act) crythrocytw ; I’OI+ Hagcllar t~r~~tc’in from .S~rlrIr~~rt~~lld urfcluid~~ ; C‘hl I, cell-mediatctt immunit b

less than 0.01% of the spleen cells. More likely, the cAh2P accumulation resulted from a general stimulation of the adenylate cyclase of spleen cells by an unknofiq~ mechanism. On the other hand, Watson et al. showed that the presence of dbcAMP in mouse spleen cultures reduced the number of AFC to SRBC (IO). These observations were interpreted by a “two-signal” model (1 l), in which CAMP is part of a paralytic signal delivered to B cells by antigen, and an inductive signal (normally delivered by T cells) involves cGMP. In that work, as in the experiments to be presented here, presence of CAMP-elevating agents only during the early phase of cultures stimulated the immune response. Further experimentation is therefore required to resolve this controversy regarding the role of cyclic nucleotides, especially CAMP, in the initiation of an immune response. We reported that dbcAMP or aminophylline are immunoenhancing when given at early times (0 to 24 hr) of a S-day in z&o AFC responseto SRBC, but that addition of these agents at later times (> 24 hr) inhibited the response (12). In this paper, we show that elevated intracellular CAMP levels during the first 12 hr of primary humoral or cell-mediated immune responsesincreases their magnitude. Under the same conditions, cGMP has no direct effect but inhibits CAMP-mediated stimulation of the AFC responseto SRBC. The effects of CAMP in the humoral responsesare antigen-depentleiit and antigen-specific. MATERIALS


Mice. Mice of the CBA/J (H-2’ 0.1).

0.017, v/v of the 12 hr SRBC only, All cultures



effective or ineffective. Adenosine or butyric acid gave about a two-fold enhancement of the response at 10e3 M. These agents increase CAMP levels in cytotoxic lymphocytes (26) and neuroblastoma cells (27). CAMP or 5’-AMP were completely ineffective in enhancing the anti-SRBC response. Similar results were obtained in the Diener-Armstrong and the Mishell-Dutton culture systems. On the other hand, cGMP, dbcGMP, S-Br-cGMP, or carbachol did not have any effect ‘on the anti-SRBC response under similar experimental conditions (Table 1). These studies indicate that CAMP, but not cGMP, is immunoenhancing during the early phase (O-12 hr) of immune responses. The length of the 10m3 L%! dbcAMP pulse required for maximal stimulating effects was next determined. It is clear from Table 2 that the best stimulation was obtained with a O-12 hr pulse. It should be noted that at the concentration used, dbcAMP was cytotoxic, as indicated by the smaller number of viable cells recovered at the end of the pulse compared to control cultures. Despite this, an increase in the number of AFC (uncorrected for lower recovery of cells) was obtained. This was true even when dbcAMP was present for up to 22 hr. Reversal of the stimulating #Qrts of dbcAMP. If elevated CAMP levels stimulate immune inductions, then prevention of this elevation should counteract the stimulation. Cyclic GMP and imidazole have been reported to stimulate the intracellular hydrolysis of ‘CAMP (28, 29). Consequently, these compounds were tested for their ability to reverse the effects of dbcAMP, as shown in Table 3. Cyclic

2 1.i






Stimulatory Effect of dbcAMP Incubation with cGMP,


3 Is Diminished by Simultaneous Imidazole, or Con A

Additives during the O-24 hr incubation


Additives during the 24120 hr incubation SRBC

+ + + + +


dbcAMP cGMP imidazole cGMP + dbcAMP imidazole + dbcAMP



the O-12


Anti-SRBC PFC/culture (mean f SEM)

3,766 15,000 7,541 6,300 9,225 7,441

f f f f f f

450 1,030 610 470 630 600

6,366 20,333 6,900 7,133

f f f f

168' 1,230 408 1,145

hr incubation

& dbcAMP + Con A + Con A & dbcAMP

The spleen cells were incubated with 0.01 7c v/v SRBC f test agent(s) in Petri dishes for O-24 hr or O-12 hr as indicated. At the end of the incubation the agents were removed and the cells recultured in the Diener-Armstrong system. Cultures in experiment I were assayed at 120 hr and cultures in experiment II were assaved at 108 hr. Concentrations of the agents used: dbcAMP, 10-S M; cGMP, lo-” M; imidazole, 1OW M; and Con A, 5 rg/ml.





FIG. 1. Effects of dbcAMP on the induction of CMI. (A) Cytotoxic lymphocytes to DBA/ 2J were generated with various concentrations of dbcAMP added for the first 12 hr only. The drug was removed by washing tthe cells twice with medium. The cultures were assayed for cytotoxic lymphocytes using 61Cr-Iabeled P81.5 mastocytes as targets. Background supernatant counts were 120 cpm and freeze/thaw supernatant counts (100% lysis) were 1.500 cpm. (O), CBA/J+DBA/2J; (n----n), CBA/J+lO-” M &CAMP; (n----n) CBA/J 4 DBA/ 2J + lo-” M dbcAMP; (n), CBA/J + DB.4/2J + IO-” M dbcAMP; (o), CBA/2J t- lo-’ nb dbcAMP. (5’) Conditions used were similar to (A) except that the dose of DBA/2J cells was 0.2 X 10” per culture.

SIIl3C Slil3C

2.665 .3,275

* xk

4X.5 716

1’01. 1’01. SI< I%(‘ SIC IiC

U.(l. 11.0. 2.701 *


SKBC SliBC 1’01~

+ +

1’01. POL


Regulation of immune responses. I. Effects of cyclic AMP and cyclic GMP on immune induction.

Regulation I. Effects of Cyclic of Immune AMP and Cyclic Responses GMP on Immune Induction Agents that raise intracellular CAMI’ lcvcls (tlihuty...
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