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Corticosterone-releasing activity of immune mediators
Life Sciences, Vo]. 40, pp. Printed in the U.S.A.
929-934
Pergamon
Journals
CORTICOSTERONE-RELEASING ACTIVITY OF IMMUNE MEDIATORS I. Torres-A]em&n, M.T. Rejas~" I. Barasoai~," J. Borre]] and C. Guaza Department of Psychobiology, Caja] Institute of Neurobiology and ~)epartment of Immunology, Biological Research Center, C.S.I.C. 28006 MADRID, Spain (Receiw~d
in final
form November
24,
]986)
Summary Produ('ts d,~rJved from the activated immune syste;m have been reported to modulate neuroendocrine function. In addition, a direct ('onnection between neuroendocrJne and immune responses to stress has recently been proposed. We now provide evidence that heterogenous ]ymphokine-containing supernatants from m i t o g e n - s t i m u ] a t e d rat spleen cells can stimulate both basal and c o r t i c o t r o p i n - i n d u c e d corticosterone secretion from rat adr',;na] cells in an in vitro perifusion system. Moreover, thymosin¢~l , a 28-aminoacid residue peptide found both in thymus and lymphocyte-derived supernatants was also able to sy:]ergistJ(:,-'~]]y stimu]at,e c o r t i c o t r o p i n - s t i m u ted corticosteron~, re]c~as(~, without affecting basal cor'ticosteron~ output, in this sam(; in vitro adrenal cell perifusion system. These resu]t,s reinforce the suggestion about the existence of bidirectional interactions between the immune and n e u r o e n d o c r i n e systems. They a]so indicate that this communication may occur direct,]y at the adrenal gland level, a fTajor effector site o f the body's respons~to stress. A bidirectional communication between the irmnune and n e u r o e n d o c r i n e systems has ree(:nt]y been proposed (I). There is ample evidence that num<~rous neuropeptides and hormones can influence immunoeompetence (2). On th(~' other hand, plasma eor'ticosteron(~ lev<,]s are increased during the course of the immune response (3,4). This steroidogenic effect of the activated immune system has been ascribed to different kinds of immune mediators. Thus, an a d r e n o e o r t i c o t r o p ] n - ] i k e peptide secrete(] by ]ymphocytes in response to a parti(:u]ar set. of stimuli has been d~scribed (5). Alternately, a "glucocorticoid increasing factor" secreted by immuno-competent eel Is has been shown to stimulate pituitary-adrenocortica] activity (2). Finally, although not yet Identified, a ]ymphokine-monokine peptide seems to be able to activate the p]tuirar'y-adr<~na] axis via the hypotha]amus (6). Thus, although a corticosteror1(:-re]eas]ng action of unknown immuno-re]at(~d products seems to be apparent, possible sites ~nvolved Jn this effect rt.ma~n unclear. We have now studied the effect of both ]ymphokine-containing supernatants and thymosin ¢~1 ( T ~ l) on adrenal corticoster'oid(,genesis, using an in vitro perifused rat isolated adrenal cells system, which is known to be a most sensitive bioassay for corl.i costerone-re] eas ing b ioac tiv i ty.
I.T.A. "was supported by a postd~ctoral Reincorporaci6n (M.E.C.).
Copyright
fe]l~Jwship
from
0024-3205/87 $3.00 + .00 (c) 1987 Pergamon Journals
the Urograma
Ltd.
de
930
l.ymphokines and C o r t i c o s t e r o i d o g e n e s i s
Materials
Vo].
40, No.
10,
1987
and M e t h o d s
A n i m a l s . - Adult male Wistar rats were kept under temperature and light-control dark schedule and 24°(]). Food and waler wet(: s a c r i f i c e d by d e c a p i t a t i o n and their adrenal below. Ten rats were used for each p ~ r i f u s i o n were set up per experiment.
(200-250 g) were" used. Animals led c o n d i t i o n s (]2 h I [ght-12 h a v a i l a b l e ad ]ibitum. Rats w~re~ glands processed a.~ de'scrib~.d column. Two ~xp~,r[mcntal celumns
Test. m a t e r i a l s . - H e t e r o g e n o u s ] y m p h o k i n e - c o n t a i n i n ~ .~up~Hu~tants (LCS) were ob~aa{n-e-d-~yy i ~ c u b a t i n g Lewis rat spleen ce] !s (SxlO ~ c<~i Is el) for 24 hours in the presence of concanaw-i] in A (con A, 5 C a ] b i o c h ~ m USA) in RPMI 1640 medium c o n t a i n i n g 2 mM g]utamin~.~, ]0 mM ]iepes, 7x]O-SM 2 - m e r c a p t h o ethanol and 5% fetal calf serum. This LCS is active in a thymoeyte co-stimulation assay (7), and has 340 i n t e r ] e u k i n - 2 T.U./m] as measured by the i n t e r ] e u k i n - 2 dependent p r o l i f e r a t i o n of a T cytotoxi(- T-ceil l in~ (CTLL-2) (8). In some e x p e r i m e n t s LCS was incubated with chick(~n red b]ood c{~]]s (CRBC) at a ratio of ]0/1 (supernatant v o ] u m e / C R B C volume), a[ 4-~C for ! hour' in order to remove con A (9). No s i g n i f i c a n t v a r i a t i o n in inter]eukin-2 activity was o b s e r v e d after this procedure. T h y m o s i n ~ l , a 2 8 - a m i n o a c i d r'esidu(.~ p(,ptid(, d e r i v e d from thymosin frac'.ion b (]0) was purchased from Sigma (Hi. I,oui.~{,USA).
}~g/ml,
Experimental procedure.- Corticosterone-r<:]easin~ activity of both LCS and T~]was tested using an in vitro p e r i f u s e d d i s p e r s e d adr~.'nal cells system d e s c r i b e d e l s e w h e r e (il). Briefly, adrenal _F.,]unds from real(: :'at donors were dispersed with c o l l a g e n a s e (3 mg/m] in p~.~r~fus~on medium, Boehringer, Wv..~t Germany) and the r e s u l t i n g dispers~.'d c<~l Is placed onto a jacketed column which served as the p e r i f u s i o n chamber. C(,]Is received a continuous flow of perifusion medium (Hank's medium 199 with 0.2b% bovine albumin, 4ram liC03Na , and 5 mr/el gentamicin, pH 7.35) in which th<• test s u b s t a n c e s w~-,re dissoiv,,d immediately befor(~ use. I,CS was di],ited in the, perifhlsJon medJt;r:, to ~iv( ~~ .~Jna] c o n c e n t r a t i o n of 1/20 (5%), I/]0 (i0%) and !/b (20%). Cortico:.{~,r~n{, r~sponses to pulses of graded do.~;es ,')f ]y¢:phokin{~ c o n t a i n i n g sup~:rn~:nts, T~I, ACTH or ACTH plus either LCS or" T~(lwer~ (.~va]uated by f]uorimezry. [)ata wet< a n a l y z e d by one way analysis of variance followed by Student-N~-wman-Kuuls t~,st when appropriate. ~. R(~su]ts and D i s c u s s i o n
F i g u r e 1, p a n e l A, s h o w s t h a t c o r t . i c o s t ~ r o n t : r~]ea.~;cd by p,~r.ifu.~;~,d rat adrenal ceils was significantly increased by c o n A s t t m u l a t ~ . ~ d l y n p h o c y t e derived supernatants (F(15,32)=4.14, p~O.OOl; and F(15,32) 3 . ' / 9 , p&O.(),Ol); f o r d o s e s o f l y m p h o k i n e s o f 10% a n d 20%, r e . ~ p ~ c t i w . d y ) . This enhanced cort.icost~.~on~: s e c r e t i o n was dose-dependent., sugar.sting a specific effect of ]ymphokin~:s on adrenal gland corticost~r'oidogen~sis (r=0.99(5, p
Vol.
40, No.
I0,
1987
Lymphokines
and C o r t i c o s t e r o i d o g e n e s i s
A
931
B
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OOSE OF L Y M P H O K I N E
Figure
1
Effect of heterogenous ]ymphokine-containing supernatants (LCS) on in vitro adrenal corticosteroidogenesis. A: Corticosterone release from in vitro p e r i f u s e d dispersed rat adrenal cells in response to graded doses of LCS given as 15 min pulses. B: A significant direct linear correlation (r=O.996;p(O.O05) between total corticosterone output and increasing doses of LCS was found. The zero dose corresponds to control RPMI 1840 medium at i/5 dilution in perifusion medium. Results shown are the mean of 3 representative experiments. Standard errors are not shown for clarity in this and following figures, but they were always lower than 5% of the mean. TABLE
i
Effect of control media and heterogenous lymphokine-containing supernatants on corticosterone release by perifused rat adrenal cells. Media and supernatants were dissolved in perifusion medium to give a final concentration of 20%, and were given as 15 min pulses.
Corticosterone Treatment Fresh
RPMI
Exhausted
(ng/m])
Basal 1 1640 medium RPMI
1640 medium
11.6+O.1 4.2
~ 0.6
Post-pulse 2
14.1+O.5 5.7
+ 0.3
Lymphokine-containing supernatants
7 . 3 + 0 . 3
32.4+5.3
C R B C - t r e a t e d ]ymphokinecontaining supernatants
9 . 4 + 0 . 6
29.8+4.1
i. Mean basal corticosterone secretion rate during 12 min prior pulse. 2. Mean corticosterone secretion rate during 30 min in response test pulse.
to the test to a 15 min
Since under physiological conditions the adrenal gland receives a tonic c o r t i c o t r o p i n input, we studied in a subsequent experiment a possible interaction between ACTH and LCS in releasing corticosterone from the perifused adrenal cells system. Figure 2, panel A shows that when a 20% dose of LCS was
932
Lymphokines
and C o r t i c o s t e r o i d o g e n e s i s
Vol.
40, No.
i0,
1987
given as a 15 rain pulse ]mined]at.ely after a 2 rain pulse of an in<-ff~ctiwt dose of ACTH (IO-12M) the release of corticosterone was sliF,htly increased ,,'to+.al corticosterone output: Basal release: ]25 ±i4 n~/30 rain; ACTH: 112 _+44nF/SOmin; LCS: 204 +24 ng/30 rain; and LCS plus ACT}{: 26"7 _+%7 n~/30 rain), indical.in~ a potentiation of the steroidogenic action of ACTH by LCS. Moreover, when an effective close regime of ACTH (6.6x]O-13M, 15 rain) was used, co-addition of 20% LCS (15 rain) results in a greater peak of corlicosterone release (t.t:la] cortJeosterone output: Basal release: 198 ±4 n[{/50 ,nin; ACTII: 956 ±125 ng/50 rain; ACTH plus LCS: 1803 +_66 nFJ50 rain; [,CS: 347 £58 nFJ50 mirl; p£O.Ol for all between-groups comparisons; Fiy urc 2, pane] ~). These data tndicat~ a syner~isti(" interaction between ACTII and LCS in stimu]atin Z adrenal corticosleroido~,enesis. As previously mcntLom:d, ]ymphocyt.es have been reported to produce an ACTH-]ike p~ptide in response io certain stimuli (5). In order Io as:{~s if the c o r t i c o s t e r o n e - r e l e a s i n g activity found could be ascribed io th( pr~,s,~nce of ACTH in the con A-stimulated supernatants, we assayed
A __
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0
PO
30
B
I
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TIME
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(mln)
FLy,uru 2 Effect of" combined ACTH plus [,CS puls<,s on corr, icosterone release by the in vitro perifusion system. A: Triangles, ACTII (]O-12M) ~ziv(~n as a 2 rain pulse (ineffective reff,imc.,). Ope,n c]r'c]cs, [,CS (20% in perifusion medium) giv(~.n as a 15 rain pulse. Closed cirelr~s, a 2 rain ACTH (IO-12M) pulse followed by a 15 rain LCS (20%) pu]s~. B. Triangles, ACTH (6.6xlO-13M) given as a 15 rain pulse (effect]v(: regime). Open circles, I,CS (20%) given as a 15 min pulse. Closed circ]es, a 15 min pulse of ACTH (6.6xlO-13M) plus LCS (20%).
Vol.
40, No.
i0,
1987
Lymphoklnes
and C o r t i c o s t e r o i d o g e n e s i s
933
and porcine ACTI{ (100% cross-reactivity) we did not find any specific ACTH-Iike immunoactivity. Since the w o r k i n g sensitivity (B/Bo~80%) of this radioimmunoassay is 8.4 pmol tube, we can reasonably rule out the possibility that an ACTH -like peptide present in the supernatants would be responsible for the corticosteroidogenic effect observed. [n addition, other authors have shown that cell mitogenesis is not an appropriate stimulus for lymphocytes to produce an ACTH-]ike peptide (13,14). H o w e v e r , o K - i n t e r f e r o n may be coproduced with -interferon by ]ymphocytes in response to con A. This lymphokine has been shown to share both biological and structural properties with ACTH (13,14). Moreover,~-interferon has been previously shown to directly stimulate corticosterone release by tumoral adrenal cells (]5). T h u s , ~ - i n t e r f e r o n might be the factor present in LCS which could account for the c o r t i c o s t e r o n e - r e ] e a s i n g effect of the supernatants. Figure 3 shows the effect of T ~ 1 in combination with ACTH on cortieosterone release by the in vitro perifused adrenal cells system. The single presence of T ~ l does not exert any effect on c o r t J c o s t e r o i d o g e n e s i s at. doses ranging from ]O-12M to lO-6M (data not shown). However, when a lo-lOM dose of this pepttde was eo-perifused during i0 min with a 6.6xlO-ISM dose of ACTH, a significantly greater corticosterone release was observed than when ACTH was given alone (total corticosterone output: ACTH: 1004 ±148 ng/70 min; To(] plus ACTH: 2756 ±6li ng/70 min; pKO.O01; Figure 3). Therefore, a potentiating effect of To41 upon the c o r t i e o s t e r o n e - r e l e a s i n g ability of ACTII is apparent. Since the
E v
'\
o
8 ,oo" Inr
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o
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Figur~ Effect of perifused circles, pulse of
.
.
.
.
,~o
(rain)
3
a c o m b i n e d ACTH p l u s To( 1 p u l s e on e o r t i c o s t e r o n e release by t h e adrenal cells. Triangles, To( I ( 1 0 - l O M ) g i v e n a s a 10 min p u l s e . Open ACTH ( 6 . 6 x l O - 1 3 M ) g i v e n a s a 10 min p u l s e . C l o s e d c i r c l e s , a 10 min ACTH ( 6 . 6 x l O - 1 3 H ) p l u s To( l ( l o - l O H ) .
dose of To( l u s e d in the p r e s e n t : study is within the reported physiological range (I0), a functional relation between T ~ l a n d ACTH on the modulation of adrenal steroidogenesis, can be suggested.
934
Lymphokines
and C o r t i c o s t e r o i d o g e n e s i s
Vol.
40, No.
I[), 1987
The t:mergencu of p s y c h o n e u r o i m m u n o ] o g y as a part of" t h e behaviournI sciences is based on recent o b s e r v a t i o n s about interactions between the nervous and immune systems (1,2). These include both dii'e(:t effects of immune-der'ived products on the nervous system (6), as well as a varJ{~ty of actions of different n e u r o p e p t i d e s on the response of t.h(~ organism to infection, n~oplas[a, and genera] i m m u n o c o m p c t e n c e (2,16,]8). Our data lend left.her suppurt for a direct functiona] r e ] a t i o n s h i p between the a c t i w J t u d immune syst<{~1 and the, ad~'mna] gland, r e i n f o r c i n g the concept of bidirectional re]ation.~hips b~twet~n th~ immune and n e u r o e n d o c ~ i n e systems. On the or.her hand, our results furth(~r support. our previoHs studies s u g g e s t i n g t.h,: existence of mLi ltil~]c factors modtJ]sting basal and ACTH-st. imu]at,:d adrena] st,'~roidogenesis (19,2(]). Acknowlcdgements We
thank P. Baranda
and C. Bsi!Gn
for th~:ir <.xcel lent
t.~chnica]
assistant{,.
[~{?f't, r e r ] c ( : s
1. J . E . BI,ALOCK and E.M. SMITt!, Fed. Prc,c. ,44 108-~11 ( 1 9 8 5 ) . 11.0. BESEDOVSKL, A . E . DEI. kEY and K. SOEKLN, J. Tmmunol. 1:35 7 5 0 s - 7 5 4 s ( 1985). 3. H.O. BESEDOVSKI, E. SORKfK, M. KEI,I,ER and J . MULLER, P r e c . Soc. Exp. B i o l . Med. 150 4 6 6 - 4 7 0 ( 1 9 7 5 ) . 4. E.M. SMITH, W.J. MEYER and J . E . BLAI,OCK, S c i e n c e 218 1 3 1 1 - ] 3 1 2 f 1 9 8 2 ) . 5. J . E . BLALOCK and E.M. SMITtt, Human l , y m p h o k i n c s , p . 3 2 3 , Ac;~demic: Pr,~ss, New York (1982). 6. N.R. HALL, J . P . M ( ' G I L L ] S , !~.L. SPANGEI.O, D . L . ItEALEY and A.I.. GOI I)STEIN, Spring,{:rs Seminars in Immunopathol. 8 ]53-16a (1985). 7. A.I,. MAIZEI,, S . R . M3:THA, E.G. FORI', !,.B. LACHMAN, J. F:xpt. Med. 153 4 ' / 0 - 4 7 5 ( 198] ). 8. S. OlLLIS, M.M. FERM, W. OV and K.A. SMIT!I, d. Tmmur-ol. 120 2027-2032 (1978) 9. J. ALVAEEZ, J. RODRTGUEZ, M. I,OPF,Z-BOTET, A. SILVA and M.L',. DE LANDAZURI, J. Immune1. Meth. 40 289-296 (1981). ]D. J.P. McGILLIS, N.R. HAl,I,, G.V. VAHOUNY and A.I.. GOLDS'rEIN, ,;. Immunol. 134 3 9 5 2 - 3 9 5 5 (]985). ii. G.H. MULDER and P.G. SMEI,TK, E n d o c r i n o l o g y ]00 I143-]151 (19'77). 12. J.P. VAHOUNY, E. KYEYUNE-NYOMB1, J.P. McGILLIS, N.S. TAR[.:, K.Y. HUANG, R. TOMBES, A.L. GOLDSTI.:IN and N.R. HALL, J. Immune]. 130 791-794 (1983). 13. J.E. BLAI,OCK, J. immune]. ]32 1067-1070 (1984). 14, J.E. BLALOCK and J.M. SMITH, Pr'oc. Natn. Acad. Sci. USA 77 b972-59'/4 (1980). 15. J.P:. BLALOCK and C. MAEP, Arch. Viro]. 67 45-49 (1981). 16. V. REI.EY, Sci~.nce 212 1100-11(]9 (198]). 17. H.M. JOHNSON, E.M. SMITH, B.A. TORRES :-rod .I.E. BIALOCK, Prec. Natn. Acad. Sci. USA 79 417]-4L'/4 (1982). lB. I. TORRES-AI,EMAN, T.W. REDDING {Jnd A.V. %CHALLY, Pro(:. Na~n. Acaci. Sci. USA 82 1 2 [ ) 2 - 1 2 5 6 ( 1 9 8 5 ) . 19. ('. GUAZA and J. BOREEI.L, Pcpl.ides 5 895-897 (198a). Z]. C. GUAZA, M. ZUBIAUR and J. BORREL[, PeptJdes 7 P37-240 (1986). 2.
929-934
Pergamon
Journals
CORTICOSTERONE-RELEASING ACTIVITY OF IMMUNE MEDIATORS I. Torres-A]em&n, M.T. Rejas~" I. Barasoai~," J. Borre]] and C. Guaza Department of Psychobiology, Caja] Institute of Neurobiology and ~)epartment of Immunology, Biological Research Center, C.S.I.C. 28006 MADRID, Spain (Receiw~d
in final
form November
24,
]986)
Summary Produ('ts d,~rJved from the activated immune syste;m have been reported to modulate neuroendocrine function. In addition, a direct ('onnection between neuroendocrJne and immune responses to stress has recently been proposed. We now provide evidence that heterogenous ]ymphokine-containing supernatants from m i t o g e n - s t i m u ] a t e d rat spleen cells can stimulate both basal and c o r t i c o t r o p i n - i n d u c e d corticosterone secretion from rat adr',;na] cells in an in vitro perifusion system. Moreover, thymosin¢~l , a 28-aminoacid residue peptide found both in thymus and lymphocyte-derived supernatants was also able to sy:]ergistJ(:,-'~]]y stimu]at,e c o r t i c o t r o p i n - s t i m u ted corticosteron~, re]c~as(~, without affecting basal cor'ticosteron~ output, in this sam(; in vitro adrenal cell perifusion system. These resu]t,s reinforce the suggestion about the existence of bidirectional interactions between the immune and n e u r o e n d o c r i n e systems. They a]so indicate that this communication may occur direct,]y at the adrenal gland level, a fTajor effector site o f the body's respons~to stress. A bidirectional communication between the irmnune and n e u r o e n d o c r i n e systems has ree(:nt]y been proposed (I). There is ample evidence that num<~rous neuropeptides and hormones can influence immunoeompetence (2). On th(~' other hand, plasma eor'ticosteron(~ lev<,]s are increased during the course of the immune response (3,4). This steroidogenic effect of the activated immune system has been ascribed to different kinds of immune mediators. Thus, an a d r e n o e o r t i c o t r o p ] n - ] i k e peptide secrete(] by ]ymphocytes in response to a parti(:u]ar set. of stimuli has been d~scribed (5). Alternately, a "glucocorticoid increasing factor" secreted by immuno-competent eel Is has been shown to stimulate pituitary-adrenocortica] activity (2). Finally, although not yet Identified, a ]ymphokine-monokine peptide seems to be able to activate the p]tuirar'y-adr<~na] axis via the hypotha]amus (6). Thus, although a corticosteror1(:-re]eas]ng action of unknown immuno-re]at(~d products seems to be apparent, possible sites ~nvolved Jn this effect rt.ma~n unclear. We have now studied the effect of both ]ymphokine-containing supernatants and thymosin ¢~1 ( T ~ l) on adrenal corticoster'oid(,genesis, using an in vitro perifused rat isolated adrenal cells system, which is known to be a most sensitive bioassay for corl.i costerone-re] eas ing b ioac tiv i ty.
I.T.A. "was supported by a postd~ctoral Reincorporaci6n (M.E.C.).
Copyright
fe]l~Jwship
from
0024-3205/87 $3.00 + .00 (c) 1987 Pergamon Journals
the Urograma
Ltd.
de
930
l.ymphokines and C o r t i c o s t e r o i d o g e n e s i s
Materials
Vo].
40, No.
10,
1987
and M e t h o d s
A n i m a l s . - Adult male Wistar rats were kept under temperature and light-control dark schedule and 24°(]). Food and waler wet(: s a c r i f i c e d by d e c a p i t a t i o n and their adrenal below. Ten rats were used for each p ~ r i f u s i o n were set up per experiment.
(200-250 g) were" used. Animals led c o n d i t i o n s (]2 h I [ght-12 h a v a i l a b l e ad ]ibitum. Rats w~re~ glands processed a.~ de'scrib~.d column. Two ~xp~,r[mcntal celumns
Test. m a t e r i a l s . - H e t e r o g e n o u s ] y m p h o k i n e - c o n t a i n i n ~ .~up~Hu~tants (LCS) were ob~aa{n-e-d-~yy i ~ c u b a t i n g Lewis rat spleen ce] !s (SxlO ~ c<~i Is el) for 24 hours in the presence of concanaw-i] in A (con A, 5 C a ] b i o c h ~ m USA) in RPMI 1640 medium c o n t a i n i n g 2 mM g]utamin~.~, ]0 mM ]iepes, 7x]O-SM 2 - m e r c a p t h o ethanol and 5% fetal calf serum. This LCS is active in a thymoeyte co-stimulation assay (7), and has 340 i n t e r ] e u k i n - 2 T.U./m] as measured by the i n t e r ] e u k i n - 2 dependent p r o l i f e r a t i o n of a T cytotoxi(- T-ceil l in~ (CTLL-2) (8). In some e x p e r i m e n t s LCS was incubated with chick(~n red b]ood c{~]]s (CRBC) at a ratio of ]0/1 (supernatant v o ] u m e / C R B C volume), a[ 4-~C for ! hour' in order to remove con A (9). No s i g n i f i c a n t v a r i a t i o n in inter]eukin-2 activity was o b s e r v e d after this procedure. T h y m o s i n ~ l , a 2 8 - a m i n o a c i d r'esidu(.~ p(,ptid(, d e r i v e d from thymosin frac'.ion b (]0) was purchased from Sigma (Hi. I,oui.~{,USA).
}~g/ml,
Experimental procedure.- Corticosterone-r<:]easin~ activity of both LCS and T~]was tested using an in vitro p e r i f u s e d d i s p e r s e d adr~.'nal cells system d e s c r i b e d e l s e w h e r e (il). Briefly, adrenal _F.,]unds from real(: :'at donors were dispersed with c o l l a g e n a s e (3 mg/m] in p~.~r~fus~on medium, Boehringer, Wv..~t Germany) and the r e s u l t i n g dispers~.'d c<~l Is placed onto a jacketed column which served as the p e r i f u s i o n chamber. C(,]Is received a continuous flow of perifusion medium (Hank's medium 199 with 0.2b% bovine albumin, 4ram liC03Na , and 5 mr/el gentamicin, pH 7.35) in which th<• test s u b s t a n c e s w~-,re dissoiv,,d immediately befor(~ use. I,CS was di],ited in the, perifhlsJon medJt;r:, to ~iv( ~~ .~Jna] c o n c e n t r a t i o n of 1/20 (5%), I/]0 (i0%) and !/b (20%). Cortico:.{~,r~n{, r~sponses to pulses of graded do.~;es ,')f ]y¢:phokin{~ c o n t a i n i n g sup~:rn~:nts, T~I, ACTH or ACTH plus either LCS or" T~(lwer~ (.~va]uated by f]uorimezry. [)ata wet< a n a l y z e d by one way analysis of variance followed by Student-N~-wman-Kuuls t~,st when appropriate. ~. R(~su]ts and D i s c u s s i o n
F i g u r e 1, p a n e l A, s h o w s t h a t c o r t . i c o s t ~ r o n t : r~]ea.~;cd by p,~r.ifu.~;~,d rat adrenal ceils was significantly increased by c o n A s t t m u l a t ~ . ~ d l y n p h o c y t e derived supernatants (F(15,32)=4.14, p~O.OOl; and F(15,32) 3 . ' / 9 , p&O.(),Ol); f o r d o s e s o f l y m p h o k i n e s o f 10% a n d 20%, r e . ~ p ~ c t i w . d y ) . This enhanced cort.icost~.~on~: s e c r e t i o n was dose-dependent., sugar.sting a specific effect of ]ymphokin~:s on adrenal gland corticost~r'oidogen~sis (r=0.99(5, p
Vol.
40, No.
I0,
1987
Lymphokines
and C o r t i c o s t e r o i d o g e n e s i s
A
931
B
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OOSE OF L Y M P H O K I N E
Figure
1
Effect of heterogenous ]ymphokine-containing supernatants (LCS) on in vitro adrenal corticosteroidogenesis. A: Corticosterone release from in vitro p e r i f u s e d dispersed rat adrenal cells in response to graded doses of LCS given as 15 min pulses. B: A significant direct linear correlation (r=O.996;p(O.O05) between total corticosterone output and increasing doses of LCS was found. The zero dose corresponds to control RPMI 1840 medium at i/5 dilution in perifusion medium. Results shown are the mean of 3 representative experiments. Standard errors are not shown for clarity in this and following figures, but they were always lower than 5% of the mean. TABLE
i
Effect of control media and heterogenous lymphokine-containing supernatants on corticosterone release by perifused rat adrenal cells. Media and supernatants were dissolved in perifusion medium to give a final concentration of 20%, and were given as 15 min pulses.
Corticosterone Treatment Fresh
RPMI
Exhausted
(ng/m])
Basal 1 1640 medium RPMI
1640 medium
11.6+O.1 4.2
~ 0.6
Post-pulse 2
14.1+O.5 5.7
+ 0.3
Lymphokine-containing supernatants
7 . 3 + 0 . 3
32.4+5.3
C R B C - t r e a t e d ]ymphokinecontaining supernatants
9 . 4 + 0 . 6
29.8+4.1
i. Mean basal corticosterone secretion rate during 12 min prior pulse. 2. Mean corticosterone secretion rate during 30 min in response test pulse.
to the test to a 15 min
Since under physiological conditions the adrenal gland receives a tonic c o r t i c o t r o p i n input, we studied in a subsequent experiment a possible interaction between ACTH and LCS in releasing corticosterone from the perifused adrenal cells system. Figure 2, panel A shows that when a 20% dose of LCS was
932
Lymphokines
and C o r t i c o s t e r o i d o g e n e s i s
Vol.
40, No.
i0,
1987
given as a 15 rain pulse ]mined]at.ely after a 2 rain pulse of an in<-ff~ctiwt dose of ACTH (IO-12M) the release of corticosterone was sliF,htly increased ,,'to+.al corticosterone output: Basal release: ]25 ±i4 n~/30 rain; ACTH: 112 _+44nF/SOmin; LCS: 204 +24 ng/30 rain; and LCS plus ACT}{: 26"7 _+%7 n~/30 rain), indical.in~ a potentiation of the steroidogenic action of ACTH by LCS. Moreover, when an effective close regime of ACTH (6.6x]O-13M, 15 rain) was used, co-addition of 20% LCS (15 rain) results in a greater peak of corlicosterone release (t.t:la] cortJeosterone output: Basal release: 198 ±4 n[{/50 ,nin; ACTII: 956 ±125 ng/50 rain; ACTH plus LCS: 1803 +_66 nFJ50 rain; [,CS: 347 £58 nFJ50 mirl; p£O.Ol for all between-groups comparisons; Fiy urc 2, pane] ~). These data tndicat~ a syner~isti(" interaction between ACTII and LCS in stimu]atin Z adrenal corticosleroido~,enesis. As previously mcntLom:d, ]ymphocyt.es have been reported to produce an ACTH-]ike p~ptide in response io certain stimuli (5). In order Io as:{~s if the c o r t i c o s t e r o n e - r e l e a s i n g activity found could be ascribed io th( pr~,s,~nce of ACTH in the con A-stimulated supernatants, we assayed
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FLy,uru 2 Effect of" combined ACTH plus [,CS puls<,s on corr, icosterone release by the in vitro perifusion system. A: Triangles, ACTII (]O-12M) ~ziv(~n as a 2 rain pulse (ineffective reff,imc.,). Ope,n c]r'c]cs, [,CS (20% in perifusion medium) giv(~.n as a 15 rain pulse. Closed cirelr~s, a 2 rain ACTH (IO-12M) pulse followed by a 15 rain LCS (20%) pu]s~. B. Triangles, ACTH (6.6xlO-13M) given as a 15 rain pulse (effect]v(: regime). Open circles, I,CS (20%) given as a 15 min pulse. Closed circ]es, a 15 min pulse of ACTH (6.6xlO-13M) plus LCS (20%).
Vol.
40, No.
i0,
1987
Lymphoklnes
and C o r t i c o s t e r o i d o g e n e s i s
933
and porcine ACTI{ (100% cross-reactivity) we did not find any specific ACTH-Iike immunoactivity. Since the w o r k i n g sensitivity (B/Bo~80%) of this radioimmunoassay is 8.4 pmol tube, we can reasonably rule out the possibility that an ACTH -like peptide present in the supernatants would be responsible for the corticosteroidogenic effect observed. [n addition, other authors have shown that cell mitogenesis is not an appropriate stimulus for lymphocytes to produce an ACTH-]ike peptide (13,14). H o w e v e r , o K - i n t e r f e r o n may be coproduced with -interferon by ]ymphocytes in response to con A. This lymphokine has been shown to share both biological and structural properties with ACTH (13,14). Moreover,~-interferon has been previously shown to directly stimulate corticosterone release by tumoral adrenal cells (]5). T h u s , ~ - i n t e r f e r o n might be the factor present in LCS which could account for the c o r t i c o s t e r o n e - r e ] e a s i n g effect of the supernatants. Figure 3 shows the effect of T ~ 1 in combination with ACTH on cortieosterone release by the in vitro perifused adrenal cells system. The single presence of T ~ l does not exert any effect on c o r t J c o s t e r o i d o g e n e s i s at. doses ranging from ]O-12M to lO-6M (data not shown). However, when a lo-lOM dose of this pepttde was eo-perifused during i0 min with a 6.6xlO-ISM dose of ACTH, a significantly greater corticosterone release was observed than when ACTH was given alone (total corticosterone output: ACTH: 1004 ±148 ng/70 min; To(] plus ACTH: 2756 ±6li ng/70 min; pKO.O01; Figure 3). Therefore, a potentiating effect of To41 upon the c o r t i e o s t e r o n e - r e l e a s i n g ability of ACTII is apparent. Since the
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.
.
.
.
,~o
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3
a c o m b i n e d ACTH p l u s To( 1 p u l s e on e o r t i c o s t e r o n e release by t h e adrenal cells. Triangles, To( I ( 1 0 - l O M ) g i v e n a s a 10 min p u l s e . Open ACTH ( 6 . 6 x l O - 1 3 M ) g i v e n a s a 10 min p u l s e . C l o s e d c i r c l e s , a 10 min ACTH ( 6 . 6 x l O - 1 3 H ) p l u s To( l ( l o - l O H ) .
dose of To( l u s e d in the p r e s e n t : study is within the reported physiological range (I0), a functional relation between T ~ l a n d ACTH on the modulation of adrenal steroidogenesis, can be suggested.
934
Lymphokines
and C o r t i c o s t e r o i d o g e n e s i s
Vol.
40, No.
I[), 1987
The t:mergencu of p s y c h o n e u r o i m m u n o ] o g y as a part of" t h e behaviournI sciences is based on recent o b s e r v a t i o n s about interactions between the nervous and immune systems (1,2). These include both dii'e(:t effects of immune-der'ived products on the nervous system (6), as well as a varJ{~ty of actions of different n e u r o p e p t i d e s on the response of t.h(~ organism to infection, n~oplas[a, and genera] i m m u n o c o m p c t e n c e (2,16,]8). Our data lend left.her suppurt for a direct functiona] r e ] a t i o n s h i p between the a c t i w J t u d immune syst<{~1 and the, ad~'mna] gland, r e i n f o r c i n g the concept of bidirectional re]ation.~hips b~twet~n th~ immune and n e u r o e n d o c ~ i n e systems. On the or.her hand, our results furth(~r support. our previoHs studies s u g g e s t i n g t.h,: existence of mLi ltil~]c factors modtJ]sting basal and ACTH-st. imu]at,:d adrena] st,'~roidogenesis (19,2(]). Acknowlcdgements We
thank P. Baranda
and C. Bsi!Gn
for th~:ir <.xcel lent
t.~chnica]
assistant{,.
[~{?f't, r e r ] c ( : s
1. J . E . BI,ALOCK and E.M. SMITt!, Fed. Prc,c. ,44 108-~11 ( 1 9 8 5 ) . 11.0. BESEDOVSKL, A . E . DEI. kEY and K. SOEKLN, J. Tmmunol. 1:35 7 5 0 s - 7 5 4 s ( 1985). 3. H.O. BESEDOVSKI, E. SORKfK, M. KEI,I,ER and J . MULLER, P r e c . Soc. Exp. B i o l . Med. 150 4 6 6 - 4 7 0 ( 1 9 7 5 ) . 4. E.M. SMITH, W.J. MEYER and J . E . BLAI,OCK, S c i e n c e 218 1 3 1 1 - ] 3 1 2 f 1 9 8 2 ) . 5. J . E . BLALOCK and E.M. SMITtt, Human l , y m p h o k i n c s , p . 3 2 3 , Ac;~demic: Pr,~ss, New York (1982). 6. N.R. HALL, J . P . M ( ' G I L L ] S , !~.L. SPANGEI.O, D . L . ItEALEY and A.I.. GOI I)STEIN, Spring,{:rs Seminars in Immunopathol. 8 ]53-16a (1985). 7. A.I,. MAIZEI,, S . R . M3:THA, E.G. FORI', !,.B. LACHMAN, J. F:xpt. Med. 153 4 ' / 0 - 4 7 5 ( 198] ). 8. S. OlLLIS, M.M. FERM, W. OV and K.A. SMIT!I, d. Tmmur-ol. 120 2027-2032 (1978) 9. J. ALVAEEZ, J. RODRTGUEZ, M. I,OPF,Z-BOTET, A. SILVA and M.L',. DE LANDAZURI, J. Immune1. Meth. 40 289-296 (1981). ]D. J.P. McGILLIS, N.R. HAl,I,, G.V. VAHOUNY and A.I.. GOLDS'rEIN, ,;. Immunol. 134 3 9 5 2 - 3 9 5 5 (]985). ii. G.H. MULDER and P.G. SMEI,TK, E n d o c r i n o l o g y ]00 I143-]151 (19'77). 12. J.P. VAHOUNY, E. KYEYUNE-NYOMB1, J.P. McGILLIS, N.S. TAR[.:, K.Y. HUANG, R. TOMBES, A.L. GOLDSTI.:IN and N.R. HALL, J. Immune]. 130 791-794 (1983). 13. J.E. BLAI,OCK, J. immune]. ]32 1067-1070 (1984). 14, J.E. BLALOCK and J.M. SMITH, Pr'oc. Natn. Acad. Sci. USA 77 b972-59'/4 (1980). 15. J.P:. BLALOCK and C. MAEP, Arch. Viro]. 67 45-49 (1981). 16. V. REI.EY, Sci~.nce 212 1100-11(]9 (198]). 17. H.M. JOHNSON, E.M. SMITH, B.A. TORRES :-rod .I.E. BIALOCK, Prec. Natn. Acad. Sci. USA 79 417]-4L'/4 (1982). lB. I. TORRES-AI,EMAN, T.W. REDDING {Jnd A.V. %CHALLY, Pro(:. Na~n. Acaci. Sci. USA 82 1 2 [ ) 2 - 1 2 5 6 ( 1 9 8 5 ) . 19. ('. GUAZA and J. BOREEI.L, Pcpl.ides 5 895-897 (198a). Z]. C. GUAZA, M. ZUBIAUR and J. BORREL[, PeptJdes 7 P37-240 (1986). 2.