ACTH receptor-mediated induction of leukocyte cyclic AMP

Vol. 157, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICALRESEARCH COMMUNICATIONS Pages 1205-1211

December 30, 1988

ACTH RECEPTOR-MEDIATED INDUCTION OF LEUKOCYTE CYCLIC AMP .

.

o

+

Eve W. Johnson , J.E. Blalock , and Eric M. Smith ' Departments of Microbiology and +Psychiatry and Behavioral Sciences University of Texas Medical Branch Galveston, Texas 77550 o

Department of Physiology and Biophysics University of Alabama at Birmingham Birmingham, Alabama 35294 Received October 28,

1988

SUMMARY. Studies were conducted to determine whether lymphocyte ACTH receptors behave as t h e i r s t r u c t u r a l l y similar adrenal cell counterparts, in terms of adenylate cyclase activation and cyclic AMP (cAMP) production in th~ presen~ of ACTH. Treatment of mouse mononuclear splenocytes with ACTH (lOto lO-~VM) induced a consistent rise in cAMP. ACTH treatment of more homogenous cell populations, represented by Molt 4 T lymphoblast and $49A T cell lymphoma lines, yielded a dramatic, dose-related increase in cAMP levels for $49A cells but not for Molt 4 c e l l s . Immunofluorescence assays, employing an antiserum to the adrenal cell ACTH receptor, indicated that 45% of splenocytes, 69% of $49A c e l l s , and < 1% of Molt 4 c e l l s possess ACTH receptors. Radioligand binding studies confirmed that Molt 4 c e l l s possess many fewer receptors than $49A c e l l s , and probably f a i l to respond to ACTH because they lack the appropriate receptor. This is the f i r s t report of ACTH induction of leukocyte cAMP, evidence important to understanding the mechanisms by which t h i s neuroendocrine hormone influences immune responses. © 1988 A c a d e m i c

Press,

Inc.

There is now considerable evidence which describes b i - d i r e c t i o n a l communication between cells of the neuroendocrine and immune systems (for reviews, see 1,2).

One putative network of interregulation

is characterized

by a set of ligands (hormones, lymphokines) and t h e i r receptors which are shared by cells of both systems.

Adrenocorticotropin

(ACTH) is one such

peptide hormone which is produced by anterior p i t u i t a r y cells as well as by lymphocytes (3,4).

In addition to i t s classic neuroendocrine effects, ACTH

has demonstrated a variety of immunoregulatory properties ranging from suppression of interferon x (IFNY) production (5) and in v i t r o antibody responses (6) to enhancement of B cell growth and d i f f e r e n t i a t i o n

(7) and

memory cytotoxic a c t i v i t y (8). 0006-291XJ88 $1.50 1205

Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

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Relevant to the effects of ACTH on immune function is the detection of specific ACTH receptors on c e l l s of the immune system.

Both human peripheral

blood mononuclear cells (9) and mouse spleen cells (6) have been shown to

express ACTH receptors by radioligand binding.

The affinity constants and

number of binding sites for both cell types closely correlated with those published for adrenal ACTH receptors.

A recent report describes ACTH

receptors on a murine B cell l i n e , BCLI, demonstrated by radioligand binding and by immunofluorescence receptor (10,11).

assay using an antiserum to a mouse adrenal ACTH

Thus, i t is logical

to presume that immunological effects

produced by ACTH are mediated via specific leukocyte receptors. In adrenal c e l l s , neuronal c e l l s , and adipocytes, ACTH mediates i t s intracellular

effects in part via activation of adenylate cyclase and

subsequent rise in cyclic AMP (cAMP) levels.

In the present study, i t was

hypothesized that ACTH would act s i m i l a r l y to increase levels of cAMP in leukocytes which express ACTH receptors. MATERIALS AND METHODS

Mice. C57 BI/6 female mice, 6-8 weeks of age, were obtained from the Jackson Laboratory (Bar Harbour, ME). Reagents. Highly purified porcine ACTH (40 u n i t s / v i a l ) was obtained from Armour Pharmaceuticals (Kankakee, IL). Both i s o b u t y l m e ~ I x a n t h i n e (IBMX) and forskolin were purchased from Sigma (St. Louis, MO). [ ~ I ] A C T H was obtained from New England Nuclear (DuPont Biotechnology Systems, Wilmington, DE) and had a specific a c t i v i t y of 170 uCi/~g. Cells. Mouse spleen cells were prepared from dissociated spleens and mononuclear leukocytes were separated for cAMP stimulation by gradient centrifugation on Lympholyte M (Cedarlane Laboratories, Hornby, Ontario). Molt 4 T lymphoblast cells were purchased from American Type Culture Collection (ATCC); the origin of the cells has been described (12). S49A lymphoma cells were derived from ATCC S49 c e l l s by Dr. Bahiru Gametchu; these were kindly provided by him. Both cell lines were maintained in RPMI 1640 containing 5% FCS and p e n i c i l l i n / s t r e p t o m y c i n . cAMP stimulation. Spleen mononuclear c e l l s , $49A cells or Molt 4 cells were washed 2-3 times in RPMI 1640 and resuspended in the same medium containing 0.5 mM IBMX; the c e l l s were then allowed to rest for 20-30_minutes. After a f i n a l wash, cells were added in 0.25 ml amounts (at 2 x lOl=cell/ml) to 0.25 ml of RPMI 1640, ACTH in various concentrations, or 2 x i0 "~ M forskolin in 16 x i00 mM glass tissue culture tubes. Cells were incubated at 37°C for 5 minutes or 25 minutes, then tubes were cooled on ice and centrifuged at 4°C. Supernatants and cell pellets were both assayed for cAMP content as described below. cAMP measurement, cAMP content of each s~mple was determined as a function of competition between unlabelled cAMP and [~H]cAMP (ICN Biochemicals, I r v i n e , CA) for binding to 3'5' cAMP dependent protein kinase (Sigma), as described (13). cAMP measurements for the cell pellet and supernate for each condition were determined separately and added together to yield total cAMP produced (pmole/lO U c e l l s ) . 1206

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Immunofluorescent staining. Staining of the various cell types were accomplished by the indirect method described by Mishell and Shiigi (14). Rabbit antiserum to the Y-1 adrenal ACTH receptor (11) was used as primary antibody, with normal rabbit serum used as a control for background fluorescence. After staining with secondary antibody (fluorescein-conjugated goat anti-rabbit IgG, Organon Teknika Corp., West Chester, PA), cells were viewed on an epifluorescence microscope. The percentage of positive cells was determined as the number of fluorescent cells counted out of at least 200 c@~s examined per sample. [~L~I] ACTHbinding. Cultured Molt 4 or $49A cells were washed 2-3 times=and resuspended in cold RPMI 1640 containing 0.2% BSA and 0.02% NaN3. 5 x 10U c ~ s / O . 5 ml were placed in individual siliconized glass tubes.- Binding of [~:~I]ACTH (Amersham, Arlington Heights, IL) was allowed to proceed for 30 minutes at 4"C. Cells were harvested on glass fiber f i l t e r s (Gelman Sciences, Ann Arbor, MI), then washed with 10 ml of media. Filters were counted in a gamma counter. Nonspecific binding was determined by preincubating cells with a lO00-fold excess of unlabelled ACTH (lOOIU/ml, Sigma); radiolabelled ACTH bound under these conditions was determined to be nonspecific.

RESULTS

Because spleen c e l l s are known to express ACTH receptors (6), experiments tested the a b i l i t y illustrated

initial

of ACTH to augment cAMP production.

As

in Figure I , ACTH is capable of inducing an increase in cAMP

accumulation by mouse spleen c e l l s .

When f r e s h l y - i s o l a t e d mononuclear

splenocytes were incubated in the presence of increasing concentrations of ACTH, a consistent dose-related increase in cAMP levels was observed. Maximal stimulation was achieved at hormone concentrations of IO-8M to IO-5M r e s u l t i n g in a 2 . l - f o l d

increase at IO-8M ACTH. Responses shown were

manifested within 5 minutes.

Increases were less dramatic than expected and

were not augmented by extending the time of incubation to 25 minutes.

These

observations may r e s u l t from the fact that nonfractionated spleen c e l l s consist of a v a r i e t y of c e l l types ( p r i m a r i l y B c e l l s , T c e l l s and macrophages) each of which may respond d i f f e r e n t l y T cell

or not at a l l to ACTH. Two

lines were then examined as examples of more homogeneous c e l l

populations.

Results of treatment of Molt 4 lymphoblast and S49A lymphoma

c e l l s are shown in Figure 2.

$49A c e l l s incubated with increasing ACTH

concentrations demonstrated a dramatic increase in cAMP accumulation. of cAMP exceeded a 5 - f o l d increase over basal levels at IO-8M ACTH.

Levels In

contrast, Molt 4 c e l l s produced no s i g n i f i c a n t change in cAMP levels at any concentration of hormone even a f t e r 25 minutes of incubation. 1207

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

60-

.~. 120c

c

"~: 40-

E

, ~

• $49A 100-

.-T-

80.

"~ 3o¢)

i

-~. 20u

Q

10"

_I_

6040a. 2O

I~, O- ,_me, , =

1.

.

.

lO

0

6

- l o g [ACTH] (M)

O

Forskolin

0

,

(10-s M)

/

/

i

- l o g [ACTH] (M)

Forskolin 110-e M)

Figure 1. Effect of ACTH on cyclic AMP levels in mouse mononuclear spleen cells. Spleen cells (5 x 106/tube) were incubated in RPMI 1640 with the indicated concentrations of ACTH or forskolin (IO-~M), The reaction was stopped after 5 minutes and cAMP accumulation was measured as described in Methods. The data is a representative experiment. Each point is the mean of duplicate measurements ± standard deviation. Figure 2. Effect of ACTH on cyclic AMP levels in Molt 4 lymphoblast and $49A lymphoma cell lines. Cells (Sx 106/tube) were incubated in RPMI 16~0 with the indicated concentrations of ACTH or with forskolin (lO-UM). The reaction was stopped after 25 minutes and cAMP levels were measured as described in Methods. The data was representative of 4 experiments conducted for each cell type which yielded similar results. Each point is the mean of duplicate experiments ± standard deviation. I t is l o g i c a l to hypothesize that d i f f e r e n c e s in ACTH responsiveness between c e l l s may be explained as d i f f e r e n c e s in expression of ACTH receptor expression.

ACTH receptor expression by these c e l l s was t h e r e f o r e examined by

i n d i r e c t immunofluorescence a n a l y s i s and by [1251]ACTH binding. s t a i n i n g of spleen c e l l s ,

Molt 4 c e l l s and $49A c e l l s with an antiserum to the

Y-I adrenal ACTH receptor ( I I ) cells

Indirect

revealed the presence of receptor on spleen

(45%) and on $49A c e l l s (69%), w h i l e no ACTH receptors could be detected

on Molt 4 c e l l s

(Table I ) .

Figure 3 i l l u s t r a t e s

t h a t [1251]ACTH binding

experiments conducted f o r Molt 4 and $49A c e l l s revealed $49A c e l l s specifically cells,

bound as much as 5 times the amount of [1251]ACTH as did Molt 4

confirming t h a t $49A c e l l s probably possess many more ACTH receptors

than Molt 4 c e l l s ,

and t h a t Molt 4 c e l l s may comDletelv lack ACTH receotors.

DISCUSSION This r e p o r t describes an increase in cAMP production by leukocytes responding to ACTH treatment.

Both spleen c e l l s 1208

(45% of which possess ACTH

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE i

Detection of ACTH Receptors on Leukocytes by Immunofluorescence Analysis Cell type

% fluorescent c e l l s

House spleen mononuclear

45%

Molt 4 lymphoblast

<1%

$49A lymphoma

69%

Cells were stained by the indirect immunofluorescence assay, using an antiserum to the ACTH receptor ( i i ) , as described in Methods. Cells were counted v i s u a l l y and percentages r e f l e c t the number of positive c e l l s of at least 200 c e l l s counted per sample. Nonspecific bindng, as assessed by use of NRS as primary antibody, was always <5%. The data represent the mean of two experiments for $49A c e l l s , and four experiments for the other cell types. The standard deviations were always less than 5%.

receptors)

and $49A c e l l s

(69% of which possess ACTH r e c e p t o r s )

yielded

increased cAMP l e v e l s when incubated w i t h ACTH. $49A c e l l s demonstrated a more dramatic r i s e in cAMP, ( > 5 - f o l d vs 2 . l - f o l d

f o r spleen c e l l s

at Io-SM

ACTH) probably because a g r e a t e r percentage of these c e l l s were capable of responding to ACTH v i a s p e c i f i c

receptors.

Molt 4 c e l l s demonstrate l i t t l e

no ACTH r e c e p t o r expression and a c c o r d i n g l y do not produce an increase in c y c l i c AMP f o l l o w i n g hormone t r e a t m e n t .

Thus, we present the f i r s t

t h a t lymphocyte ACTH r e c e p t o r s mimic those of adrenal c e l l s 300

in terms o f an

• $49Acells • Molt4 cells

--

evidence

240

/

/

180

/

0

20

40

60

80

100

120

Free (pM)

Figure 3. [1251]ACTH binding to Molt 4 and $49A c e l l s . Holt 4 and $49A c e l l s were washed and resuspended in ice-cold RPMI 1640 containing 0.2% BSA and 0.02% ~ . 5 x i0 ~ cells/tube were incubated with increasi~ ~ concentrations of [ ~I]ACTH for 15 minutes at 4°C. S p e c i f i c a l l y bound [ I]ACTH was determined as that blockable by a lO00-fold excess of cold ACTH.

1209

or

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induction of a common i n t r a c e l l u l a r mediator (cAMP) probably through activation of adenylate cyclase.

The question remains, however, whether the

immunomodulatory a c t i v i t i e s of ACTH are mediated through elevation of cAMP. In several instances, the effects of ACTH and cAMP on immune function, as described in the l i t e r a t u r e ,

can be compared.

For example, early studies

demonstrated an i n h i b i t o r y effect of dibutyryl cAMP on antibody production when the cyclic nucleotide analogue was added to nonfractionated spleen c e l l s (15).

S i m i l a r l y , ACTH has been found to i n h i b i t antibody responses to T c e l l -

dependent and -independent antigens when added to cultures of nonfractionated splenocytes (6).

However, when purified B cells are cultured with cAMP in the

presence of antigen and IL-1, a stimulation of antibody production is observed (16).

The same study reported that cAMP impairs helper T cell function,

inhibiting

synthesis of IL-2 and T c e l l - r e p l a c i n g factor.

a c t i v i t y of ACTH observed by Johnson et al.

Thus the i n h i b i t o r y

(6) in mixed spleen cell cultures

may be via a cAMP-mediated blockage of T cell help; indeed, these authors reported that T cell-dependent antibody responses were more sensitive to ACTH i n h i b i t i o n that T cell-independent responses.

ACTH has also been shown to

enhance immunoglobulin production when added with IL-2 and a B cell differentiation

factor to cultures of purified B cells (7).

In addition, both

dibutyryl cAMP (17) and ACTH (5) have been shown to i n h i b i t production of interferon by mitogen-stimulated spleen c e l l s .

We recognize that the

contributions of individual cell populations responding to ACTH and the involvement of other i n t r a c e l l u l a r mediators (Ca2÷, cGMP, phosphoinositides) have yet to be elucidated, and that these examples represent s l i g h t l y different

systems which can be reasonably compared in only a general sense.

However, there is an interesting agreement between those immunoregulatory effects mediated by cAMP and those described for ACTH. Taken together with the information present in t h i s report,

i t appears l i k e l y that at least some

of the ACTH-mediated i n t r a c e l l u l a r events, which lead to functional changes in lymphocyte behavior, involve activation of adenylate cyclase and increases in cAMP. 1210

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Further studies must explore the effect of ACTH on cAMP levels in purified, non-transformed

lymphocyte populations, and w i l l determine whether

ACTH treatment of lymphocytes w i l l result in activation of protein kinase A with subsequent protein phosphorylation.

Such information is v i t a l in

determining the physiologic role for ACTH in immune regulation. these data convey s t i l l

Additionally,

another common denominator between cellular a c t i v i t i e s

in the immune and neuroendocrine systems in terms of shared ligands, shared receptors, and shared intracellular mechanisms of action. ACKNOWLEDGEMENTS

E.W.J. was supported by a James W. McLaughlin Predoctoral Fellowship. This work was also funded in part by the Office of Naval Research (NOOO-14-84K-0486). The authors wish to thank Dr. Kenneth L. Bost for furnishing the antiserum to the ACTH receptor.

We also would like thank Ms. Karen Goodwin

for typing the manuscript. REFERENCES

1.

Smith, E.M. and Blalock, J.E. (1988) Intern. J. Neuroscience. 38, 455464. 2. Johnson, E.W. and Smith, E.M. (1987) In Immunopharmacology of Infectious Diseases: Vaccine Adjuvants and Modulators of Nonspecific Resistance (J.E. Majde, ed.), pp. 61-73, Alan R. Liss, Inc. 3. Smith, E.M. and Blalock, J.E. (1981) Proc. Natl. Acad. Sci. USA. 78, 7530-7534 4. Woloski, B.M.R.N.J., Smith, E.M., Meyer, W.J., Fuller, G.M. and Blalock, J.E. (1985) Science, 230, 1035-1037. 5. Johnson, H.M., Torres, B.A., Smith, E.M., Dion, D.L. and Blalock, J.E. (1984) J. Immunol. 132, 246-250. 6. Johnson, H.M., Smith, E.M., Torres, B.A. and Blalock, J.E. (1982) Proc. Natl. Acad. Sci. USA, 79, 4171-4174. 7. Alvarez-Mon, M., Kehrl, J.H. and Fauci, A.S. (1985) J. Immunol. 135, 3823-3826. 8. Johnson, E.W., Smith, E.M. and Klimpel, G.R. (1987) Fed. Proc. 46, 1221. 9. Smith, E.M., Brosnan, P., Meyer, W.J. and Blalock, J.E. (1987) N. Eng. J. Med. 317, 1266-1269. I0. Bost, K.L., Smith, E.M. and Blalock, J.E. (1987) J. Biol. Reg. and Hom. Agents, I , 23-27. I I . Bost, K.L. and Blalock, J.E. (1986) Mol. Cell. Endocrinol. 44, 1-9. 12. Minowada, d., Ohnuma, T., and Moore, G.E. (1972) J. Nat. Cancer Inst. 49, 891-895. 13. Peterson, J.W., Molina, N.C., Houston, C.W. and Fader, R.C. (1983) Toxicon. 21, 761-776. 14. Mishell, B.B. and S h i i g i , S.M. (1980) In Selected Methods in Cellular Immunology, pp. 298-299. W.H. Freeman and Co., San Francisco. 15. Melmon, K.L., Bourne, H.R., Weinstein, Y., Shearer, G.M., Kram, J. and Bauminger, S. (1974) J. Clin. Invest. 53, 13-21. 16. Gilbert, K.M. and Hoffmann, M.K. (1985) J. Immunol. 135, 2084-2089. 17. Johnson, H.M. (1977) Nature 265, 154-155. 1211