Effects of streptozotocin-induced diabetes on lymphocyte POMC and growth hormone gene expression in the rat

E L S E V I E R Journal of Neuroimmunology 49 (1994) 35-44

Journal of Neuro immuno logy

Effects of streptozotocin-induced diabetes on lymphocyte POMC and growth hormone gene expression in the rat

Vincent Law, Linda C. Payne, Douglas A. Weigent *

University of Alabama at Birmingham, Department of Physiology and Biophysics, 1918 Unwersity Boulevard, BHSB894, Birmingham, AL 35294-0005, USA

(Received 26 April 1993) (Revision received 29 July 1993)

(Accepted 29 July 1993)

Abstract

Diabetes in the rat is associated with a change in the profiles of several neuroendocrine hormones resulting in poor growth and decreased immune function. Since lymphocytes can also serve as a source of neuroendocrine hormones, we have examined whether the change in hormone profiles are accompanied by an impairment of lymphocyte GH and POMC gene expression in the immune system. Diabetes was induced by the administration of streptozotocin (STZ; 10 mg/100 g body weight) and 3 days later GH and ACTH protein and mRNA were determined. The results show a modest diminution of GH RNA in the spleen of diabetic animals whereas the expression of POMC mRNA and ACTH by the thymus was enhanced. The expression of POMC in the spleen appeared unaltered while the increase of POMC RNA in the thymus was evident after the first day of STZ treatment. STZ had no direct effect on GH or POMC expression in the spleen or thymus cells in vitro. Insulin does not appear to be involved in the expression of lymphocyte GH or POMC. The administration of insulin to the diabetic animals had no significant effect on the expression of GH or POMC by the immune cells. In addition, lymphocytes do not appear to serve as a source of insulin or are the expression of genes for lymphocyte GH or ACTH altered by insulin in vitro. Taken together, the findings are the first to report on the expression of neuroendocrine genes in lymphocytes during diabetes. The mechanism for the inhibition of GH and stimulation of POMC expression by iymphocytes in diabetic animals is unknown, but it is tempting to speculate an important role in the development of the autoimmunity that characterizes this complex disease.

Key words: Diabetes; Growth hormone; POMC; Lymphocytes; Rat

I . Introduct ion

In the rat, streptozotocin (STZ), a potent diabetogenic substance which has cytotoxic effects upon pan- creatic B cells, decreases insulin content and secretion in the pancreas of rats and has been suggested as a suitable model for the study of artificial diabetes (Campbell et al., 1950; Junod et al., 1967; Like and Rossini, 1976). In this model, plasma GH levels are suppressed with a loss of high amplitude secretory pulses and reduced pituitary GH content (Gonzalez and Jolin, 1985; Tannenbaum, 1981). The diminution in GH may result in part from altered hypothalamic GH-releasing hormone (GHRH) or changes in the

* Corresponding author. Phone (205) 934 4227, Fax (205) 934 1446.

0165-5728/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0165-5728(93)E0123-Q

G H R H receptor number or affinity (Olchovsky et al., 1990; Simard et al., 1987). The effects of chronic stimulation of the hypothalamic-pituitary-adrenocortical axis by a diabetes induced through streptozotocin (STZ) treatment includes increased corticotrope metabolic activity and increased adrenal weight (DeNicola et al., 1977; DeNicola et al., 1976). Also, rats exposed to STZ-induced diabetes have been shown to exhibit decreased sensitivity to glucocorticoid-negative feedback compared to control rats (Young et al., 1990). A1- ti~ough STZ-treated rats exhibit consistent increases in adrenal weight, samples of basal plasma ACTH are not always significantly different between vehicle and STZ-treated rats (Scribner et al., 1991). In addition to alterations in the neuroendocrine system, STZ-induced diabetes is also known to induce a depression in immune reactivity (Gaulton et al., 1985). More recently, it

36 1/.. Law et al. /Journal of Neuroimmunology 49 (1994) 35-44

has been shown that some parts of the immune system may even be stimulated after STZ treatment. It has been suggested that increased systemic IFN-y levels (Cockfield et al., 1989) after multiple low doses of STZ may stimulate non-specific natural cytotoxicity (Kant- werk-Funke et al., 1991).

Recent work by ourselves (Smith et al., 1986; Weigent et al., 1988) and others (Buzzetti et al., 1989; Kao et al., 1989; Hattori et al., 1990) suggests that lymphocytes may also serve as a source of GH and ACTH. A complete understanding, however, of how the expression of these genes is controlled in lymphocytes is not yet clear. The mechanisms regulating lymphocyte GH synthesis and secretion appear in some respects to be both similar to and different from pituitary GH regulation (Weigent and Blalock, 1990a). It is known that lymphocyte GH can be elevated by GHRH (Guarcello et al., 1991) and inhibited by insulin-like growth factor-I (Baxter et al., 1991a,b). In fact, lymphocytes have been shown to produce GHRH (Weigent and Blalock, 1990b) and contain receptors for this hypothalamic releasing hormone (Guarcello et al., 1991). Much evidence has also accumulated to show that the lymphocyte synthesis and secretion of peptide hormones derived from pro-opiomelanocortin (POMC) and their regulation is similar to the pituitary (Blalock, 1989). The authenticity of ACTH was verified by showing identity in the nucleotide and amino acid sequence between lymphocyte and pituitary ACTH (Smith et al., 1990).

Given the pivotal role that ACTH plays in activation of the HPA-axis and GH in the activation of cell-mediated immunity, we sought to determine whether the lymphocyte production of these hormones were altered in STZ-induced diabetes. We have measured ACTH and GH production in vivo and in vitro from lymphocytes of normal and STZ-diabetic rats. We have also tested the response of lymphocytes to the natural sec- retagogue GHRH and measured the GHRH receptor. The results suggest that lymphocytes of diabetic rats have reduced abilities to produce GH RNA and that GH made by lymphocytes does not appear to be regu- lated by the levels of glucose or insulin in vivo or in vitro. On the other hand, elevated levels of POMC were detected in the thymus of diabetic rats. Taken together, the data indicate that neuroendocrine hormone production by cells of the immune system is altered during drug-induced diabetes.

serum is highly specific and shows less than 0.0001% cross-reactivity to other anterior pituitary hormones according to the suppliers. The rat GH we used was from the Pituitary Hormones and Antisera Center (Torrance, CA; Dr. A.F. Parlow) for RIA and judged to be contaminated with less than 0.1% of the anterior pituitary hormones as determined by RIAs. Additional reagents of the highest purity available were purchased from Gibco (Grand Island, NY) and Sigma (St. Louis, MO) unless otherwise stated.

2.2. Cell preparations Adult male Sprague-Dawley rats (150-200 g) used

for the experiments were purchased from Harlan (Prattville, AL). Spleens and thymi were aseptically removed and teased apart in RPMI 1640 medium supplemented with penicillin, streptomycin, and myco- statin (100 U/ml) . Cell viability was monitored by Trypan blue exclusion (Boyum, 1968). Only cell preparations with a 95% viability or greater were used. Before use in any assay, the cells were washed in serum-free RPMI 1640 and antibiotics. After isolation, cells were usually either immediately frozen in microfuge tubes for RNA isolation or washed and spotted onto glass slides for immunofluorescent studies.

2.3. Animal preparation Adult male Sprague-Dawley rats weighing 150-200

g were housed under constant temperature (22°C) and a 12-h light, 12-h dark cycle, and provided free access to rat chow and tap water. Animals were adapted for at least 4 days to the animal facility before any proce- dures were performed.

Diabetes mellitus was induced by a single i.p. injection of streptozotocin (STZ; Sigma cat. no. S0130), 65 mg/kg freshly dissolved in 0.5 ml citrate buffer (pH 4.5). Control rats received an equal volume of citrate buffer vehicle. Diabetes was defined by the appearance of gross glycosuria and polyuria. All rats were killed by decapitation and the tissues immediately frozen or placed in culture for overnight incubation prior to RNA preparation. Blood was collected in tubes and plasma glucose concentrations were determined by keto-Diastix analysis (Miles, Inc., Diagnostics Division, Elkhart, IN 46515). All manipulations of animals were conducted according to the guidelines and require- ments of the University of Alabama at Birmingham Animal Welfare Committee.

2. Materials and methods

2.1. Materials Monkey anti-rat GH antiserum was generously do-

nated by Dr. S. Raiti of The National Hormone and Pituitary Program (NIDDK, Bethesda, MD). The anti-

2.4. Binding o f 125I-labeled GHRH Binding assays were carried out in the presence of

125I-labeled hGHRH (0.1-20 nM) in a total volume of 0.3 ml. The incubations were carried out in microfuge tubes treated with Sigmacote in RPMI 1640 containing 25 mM Hepes (pH 7.4) and 0.1% BSA at 4°C for 60 min. At the end of the incubations, the cells were

v. Law et al. /Journal of Neuroirnmunology 49 (1994) 35-44 37

centrifuged for 3 min in a Beckman 12 microfuge. The pellets were washed three times in ice-cold buffer, and the tip of the tube was cut off and counted in a gamma-counter. Radioactivity removed in the presence of an excess of unlabeled hGHRH (1-44 or 1-29) at 10 /zM was considered non-specific. Non-specific binding was 25-30% of total cell binding.

2. 5. Proliferation studies Cell suspensions (1 x 106/ml) of fractionated lym-

phoid cells were prepared in RPMI 1640 supplemented with 10% fetal calf serum containing 100 U of penicillin and streptomycin (Gibco, Grand Island, NY) per ml. These suspensions were incubated in the presence of [3H]thymidine ([3H]TdR; 24 h; 10 /zC/ml, New England Nuclear, Boston, MA) in plastic tissue culture dishes with and without various concentrations of STZ or GHRH, as indicated. The cultures were harvested using a Whittaker (Watertown, MA) Mini-Mash Har- vester, and the glass fiber filters were counted in a Tracor Analytic Model 6892 liquid scintillation system (Elk Village, IL).

2.6. R/A GH was measured by RIA using [125I]GH (NIDDK-

rGH-I-6) as radioligand and a polyclonal monkey anti- rat GH antibody. This antiserum (NIDDK-anti-rGH-S- 5) was a generous gift from the Hormone Distribution Program of the NIADDK and provided by Dr. S. Raiti. The specifications on this antibody show it to be highly specific, with low cross-reactivity with other highly purified rat pituitary hormones (rat PRL, < 0.0001%; rat FSH, 0.0009%; rat TSH, 0.008%) and has a sensitivity of 500 pg/ml. The reagents, including buffer, cold standard or unknown, [lZSI]GH and antiserum, were incubated at room temperature for 24 h, before the addition of insoluble protein-A (8 mg/tube) as a second agent. The immune complexes were pelleted by centrifugation and counted in a gamma counter. Rat ACTH was measured by RIA using a kit obtained from Incstar Corp. (Cat. No. 24065). The procedure is an equilibrium RIA using rabbit anti-ACTH serum fol- lowed by a second antibody complex of goat anti-rabbit serum and polyethylene glycol. The minimum de- tectable amount of ACTH is 15 pg /ml with less than 1% crossreactivity on a number of peptides including endorphin, enkephalin, calcitonin, growth hormone, vasopressin, and substance P. The intra- and interassay coefficients of variation for the assays used in these studies were 8.1% and 6.0%, respectively.

2. 7. Direct imrnunofluorescence The cells used for immunofluorescence were washed

three times in 0.01 M PBS (1 X 107 cells/ml) and air-dried onto glass slides. After fixation with ice-cold 95% ethanol, the cells were rehydrated in PBS and

incubated with appropriate serum (1:200) for 1 h at 37°C. The cells were washed three times with PBS and then covered in a 1 : 50 dilution of fluorescein isothio- cyanate (FITC)-conjugated monkey-anti-rat GH or rabbit-anti-human ACTH prepared in our laboratory by standard techniques (Johnstone and Thorpe, 1987). The slides were allowed to incubate 1 h at 37°C fol- lowed by washing three times in PBS and rinsing in distilled water. To confirm the specificity of the conjugated antibodies, the labeled antibody and excess hormone were mixed and incubated at 4°C for 2 h before addition to cells. The cells were observed using an Olympus (Marietta, GA) vertical fluorescence illumina- tor model A-RFL. For some experiments, cells were analyzed by flow cytometry. Cells (1 x 105) were first incubated with 5% normal rat serum for 15 rain prior to staining to block Fc receptors. Cells were then directly stained without being permeabilized with a FITC-conjugated anti-rat GH or anti-human ACTH. Ceils without fluorochrome treatment were used to determine the proper window setting. Cells were washed extensively after each incubation, suspended in PBS containing 1% paraformaldehyde, and analyzed using an EPICS cell sorter (Coulter Electronics, Hialeah, FL) for unlabeled and for single and double positive populations. Data were collected on 5000 cells.

2.8. R N A isolation and blotting Total cytoplasmic RNA was isolated by the pro-

teinase K method (Maniatis et al., 1982). After ethanol precipitation, the RNA pellet was dried under vacuum and dissolved in sterile water. An aliquot was removed to determine the yield and purity by absorbance mea- surements at 260 and 280 nM. The RNA (10/zg) was blotted on nitrocellulose membranes with the Minifold II slot blotter (Schleicher and Schuell, Inc., Keene, NH) as described by the manufacturers. After hybridization with a GH-specific complementary (c) DNA probe as described below, the membranes were washed with 0.165 M NaCI, 14 mM sodium citrate, and 0.1% sodium dodecyl sulfate (SDS) at 100°C for 30 min to elute the cDNA probe (Maniatis et al., 1982). Densito- metric values of GH RNA were determined as a per- centage with 0.1/xg control pituitary RNA as the 100% reference point. Membranes were then reprobed with a synthetic oligodeoxynucleotide 18S ribosomal probe to detect differences in the amount of RNA bound to the membrane. Small corrections determined by pro- portion were applied to the densitometric cDNA data according to the amount of RNA bound to the nitrocellulose determined after probing with the 18S ribosomal probe.

2. 9. P O M C and G H c D N A s and hybridization Plasmids containing the rat (Seeburg et al., 1977)

GH cDNA were kindly provided by Dr. John Baxter

38 V. Law et al. /Journal of Neuroimmunology 49 (1994) 35-44

Table I Effects of STZ treatment on normal rats compared to vehicle- injected controls

Variable Vehicle-injected STZ-injected

Initial body weight (g) 185 ± 8 Final body weight (g) 208± 10 Plasma glucose (mg/dl) 250_+ 25 Spleen weight (rag/100 g BW) 650 5:50 Thymus weight (mg/100 g BW) 500± 100 Adrenal weight (mg/100 g BW) 41 ± 10 Corticosterone (ng/ml) < 20

170± 10 * 1150±287 * 450± 50 * 270± 30 * 71 J: 20

591 + 236 *

Initial body weight was measured at the time of vehicle or STZ injection. Final body weight was measured 3 days after injection. Glucose levels, spleen, thymus, and adrenal weights were measured on day 3 immediately after sacrifice of animals. Values are the mean +_ SEM from a typical experiment done three times. Data were analyzed by Student's unpaired t-test comparisons. * P < 0.05 vs. vehicle-injected.

and Dr. F r a n D e n o t o (Neu rochemis t ry Labora to r i e s , V A Med ica l Cen te r , Sepu lveda , CA). Escherichia col±- con ta in ing the P O M C p la smid were k indly p rov ided by F.S. Gal in . P la smid D N A was p r e p a r e d essent ia l ly as desc r ibed (Man ia t i s et al., 1982). Inser t s were pur i f i ed f rom the p l a smid (Mania t i s et al., 1982), and the cD- N A s were l abe l ed with [3zp]deoxycyt idine t r i p h o s p h a t e by nick t r ans l a t ion using a commerc ia l ly avai lable kit (BRL, Ga i the r sbu rg , M D ) to a specific activity of 1 - 2 × 108 c p m / / x g . T h e mouse 18S synthet ic o l igode- oxynuc leo t ide r ibosomal p r o b e was p r e p a r e d and pur i - f ied by our l abora tory . The p r o b e c o r r e s p o n d s to nu- c leo t ides 1056-1067 and was e n d - l a b e l e d with T4 po lynuc leo t ide k inase by s t a n d a r d p r o c e d u r e s (Man ia - tis et al., 1982). P rehybr id i za t ion was done for 4 h at 42°C, and hybr id iza t ion was done for 18 h at 42°C in SDS, d e n a t u r e d sa lmon s p e r m D N A D e n h a r d t ' s solu- t ion, f o rmamide , NaCI, N a H 2 P O 4 , H 2 0 , E D T A , and s t a n d a r d buf fe r con ta in ing 32p- labeled inser t (Mania t i s et al., 1982). A f t e r hybr id iza t ion , the m e m b r a n e s were extensively washed unt i l the radioac t iv i ty in the f inal wash was close to b a c k g r o u n d using 0.4 M NaCI, 0.04 M sod ium ci t ra te , and 0.1% SDS for 10 min, and 0.04 M NaCI, 0.004 M sod ium ci t ra te , and 0.1% SDS for a n o t h e r 10 min. Bo th washings were done at 25°C with gent le mixing. Two washes were done in 0.03 M NaC1,

0.003 M sod ium ci t ra te , and 0.1% SDS for 30 min at 55°C with gent le mixing. T h e p a p e r s were then r insed in 0.04 M NaCI and 0.004 M sod ium ci t rate . T h e n i t roce l lu lose pape r s were exposed to x-ray fi lm at - 7 0 ° C with D u P o n t (Wi lming ton , D E ) Cronex Light- n ing-Plus intensifying screens for 2 - 3 days. The au- t o r ad iog raphs were ana lyzed using a GS 300 dens±to- m e t e r (Hoe fe r Scient if ic Ins t ruments , San Franc isco , CA).

2.10. Data analysis Each da t a po in t f rom the expe r imen t s r e p r e s e n t

r ep l i ca te samples f rom th ree to five rats. Each exper i - m e n t was r ep l i ca t ed at leas t t h r ee t imes. D a t a a re p r e s e n t e d as m e a n _+ SEM. R a t p i tu i t a ry R N A (0.1 / ~ g / s l o t ) was used as a s t a n d a r d in each p rob ing experiment to no rma l i ze the va lues in expe r imen t - to -expe r i - m e n t var ia t ion . Di f fe rences b e t w e e n groups were de- t e r m i n e d by S tuden t ' s t - tes t and M a n n - W h i t n e y test w h e r e d i f fe rences are ind ica t ed as be ing signif icant , P is less than 0.05.

3. Results

3.1. Characteristics of rats with STZ-induced diabetes S T Z - t r e a t e d ra ts d e m o n s t r a t e d m a r k e d hyper -

g lycemia c o m p a r e d to veh ic le - in jec ted controls . Fu r - t he rmore , the animals exhib i ted an overal l lower body weight as well as spleen, and thymus weights than cont ro ls (Table 1). By contras t , a d r e na l weight was only modes t ly inc reased in S T Z d iabe t ic c o m p a r e d to con- t rol rats. Cons i s ten t wi th all these f indings, the levels of co r t i cos te rone in the s e rum were also s ignif icant ly ele- va ted . A d m i n i s t r a t i o n of S T Z to ra ts r e su l t ed in po lyur ia and po lyd ips ia as ear ly as 24 h af ter inject ion.

3.2. Comparison of GH and A C T H RNA and protein levels from control and diabetic animals

T h e levels of G H and A C T H R N A and p ro t e in were d e t e r m i n e d by do t blot hybr id iza t ion of homolo- gous c D N A to to ta l cy top lasmic R N A and flow cytometry, respect ively, f rom sp leen and thymus cells ob-

Table 2 Lymphocyte GH RNA and protein levels from control and diabetic rats

Treatment Plasma glucose Serum GH Source of GH RNA (relative GH protein percent group a (mg/dl) (ng/ml) tissue absorbance by densitometry positive by immunofluorescence

Control 235 ± 31 140 ± 40 Spleen 16.5 +_ 2.6 1.3 ± 0.4 Diabetic 1610 ± 168 * 29 ± 8 * Spleen 5.0 ± 2.0 * 0.9 ± 0.5 Control - - Thymus 10.0 ± 2.9 1.2 ± 0.2 Diabetic - - Thymus 7.0 ± 1.4 1.0 ± 0.4

a 3 days after treatment with vehicle or STZ rats were sacrificed and spleen and thymus removed, teased, and part immediately frozen for RNA isolation and probing while the remainder was analyzed for GH production by immunofluorescence analysis. Values are the mean ± SEM from a typical experiment done three times. Data were analyzed by Student's unpaired t-test comparisons. * P < 0.05 vs. vehicle-injected.

V. Law et al. /Journal of Neuroimmunology 49 (1994) 35-44 39

Table 3 Lymphocyte POMC RNA and ACTH levels from control and diabetic rats

Treatment Plasma glucose Serum ACTH Source of tissue POMC RNA (relative ACTH protein (Percent group a (mg/dl) (pg/ml) tissue absorbance by densitometry positive by immunofluorescence

Control 100 + 25 369 + 69 Spleen 5.25 _+ 0.37 4.35 ± 0.35 Diabetic 942 ± 207* 606 ± 91" Spleen 4.69 ± 0.45 4.57 ± 0.79 Control - - Thymus 16.25 + 1.9 1.85 ± 0.24 Diabetic - - Thymus 22.75 ± 1.5 * 3.025 ± 0.35 *

a 3 days after treatment with vehicle or STZ rats were killed and spleen and thymus removed, teased, and part immediately frozen for RNA isolation and probing for POMC RNA while the remainder was analyzed for ACTH production by immunofluorescence analysis. Values are the mean + SEM from a typical experiment done three times. Data were analyzed by Student's unpaired t-test comparisons. * P < 0.05 vs. vehicle-injected.

t a ined f rom animals 3 days fol lowing S T Z t r e a t m e n t (Tab les 2 and 3). Analys i s by scanning d e n s i t o m e t r y r evea led the re were s ignif icant d i f fe rences be twe e n the d iabe t ic and con t ro l g roup for G H R N A and A C T H R N A . A l t h o u g h no d i f fe rences in G H - and A C T H - p r o - ducing ceils in con t ro l and d iabe t ic rats were a p p a r e n t in the h i s tog ram prof i les a f te r flow cy tomet ry (da t a not shown), a small inc rease in the p e r c e n t a g e o f cells p roduc ing A C T H was de t ec t ab l e in the thymus of d iabe t ic animals . In a previous work, we showed tha t t r ansc r ip t ion of the lymphocy te G H gene begins imme- d ia te ly af te r r emova l o f t issues f rom an imals and peaks af te r 6 h of in vi t ro cul ture . T h e levels of G H in the sp leen and thymus in d iabe t i c an imals were found to be lower at the t ime of sacrif ice of t r e a t e d animals . These d a t a suggest tha t d iabe t i c an imals have a re- d u c e d abi l i ty to express G H R N A c o m p a r e d to no rma l an imals once t issues are r emoved f rom the animal . No s ignif icant G H p ro t e in was d e t e c t e d in e i the r the sp leen o r thymus cells (Tab le 2). Conf i rma t ion of the iden t i ty of the GH-hybr id i z ing sequences has b e e n o b t a i n e d previous ly by N o r t h e r n blot hybr id iza t ion (We±gent et al., 1988). T h e resul ts o f expe r imen t s p rob ing for A C T H express ion dur ing S T Z - i n d u c e d d i abe t e s suggest in the thymus tha t this gene is expressed (Tab le 3). T h e d a t a show tha t an imals a f te r 3 days were hyperg lycemic with s ignif icant ly h igher levels o f A C T H in the serum. In addi t ion , h igher levels (1.5-fold) of bo th the R N A and p r o t e i n for A C T H were obse rved in the thymus but not in the spleen.

To d e t e r m i n e the k inet ics of P O M C and G H R N A synthesis, we in jec ted an imals with S T Z on day 1 and ki l led an imals for R N A analysis at 18, 48, and 72 h af te r t r ea tmen t . T h e resul ts (Tab le 4) f rom the ra t thymus d e a r l y show tha t s ignif icant levels of P O M C R N A were de t ec t ab l e a f te r the 1st day of t r ea tmen t . This c o r r e s p o n d s to the same t ime the cl inical signs of d i abe t e s were evident . A s imi lar expe r imen t measu r ing G H R N A levels in the sp leen a f te r S T Z t r e a t m e n t showed a g radua l loss in G H R N A p r o d u c t i o n with s ignif icant d i f fe rences obse rved only on day 3 (da t a not shown).

Since we were ab le to m e a s u r e a smal l inc rease in P O M C R N A by specif ic p rob ing with a c D N A as well

as cells p roduc ing A C T H by immunof luo rescence , we inves t iga ted by R I A w h e t h e r the ac tua l levels of A C T H in the thymus f luid were e levated . Thus, f luids were o b t a i n e d f rom cont ro l and d iabe t ic an imals a f te r teas- ing lymphocytes in to suspens ions and used as the sam- p le to me a su re for A C T H . The da ta (Table 5) suggest a s ignif icant inc rease in A C T H in the f luids of the dia- be t ic thymus. The levels of A C T H in the sp leen of no rma l and d iabe t ic an imals r e m a i n e d u n c h a n g e d (da ta not shown), whe reas the se rum levels o f A C T H in d iabe t i c an imals were inc reased three- fo ld . These da t a should be v iewed with some cau t ion since they may ref lec t the inc reased abi l i ty of cells in the thymus of d iabe t i c an imals to concen t r a t e se rum A C T H ra the r than the add i t i ona l A C T H p r o d u c e d in the thymus.

3.3. Effect o f STZ-induced diabetes on lymphocyte function in viuo and in citro

In a p rev ious repor t , m o n o n u c l e a r sp leen cells f rom S T Z - t r e a t e d mice showed e n h a n c e d cytotoxic activity and m a c r o p h a g e activity and a r e d u c e d thymid ine in- co rpo ra t i on af te r t r e a t m e n t wi th C o n A (Kan twerk- F u n k e et al., 1991). In the p r e se n t repor t , we have

Table 4 Kinetics of production of POMC RNA in lymphocytes from control and diabetic rats a

Source Dia- POMC RNA (relative absorbance of tissue betic by densitometry)

18 h 48 h 72 h

Spleen - 9.5+0.5 N.D. 9.5±0.5 Spleen + 9.3 ± 1.8 N.D. 8.7 ± 0.3 Thymus - 23.0 ± 1.0 29.0 ± 1.0 16.0 ± 1.0 Thymus + 39.6±0.7* 41.3+5.9 * 38.3±2.0 * Bone marrow - 6.0 ± 1.0 N.D. 6.5 _+ 0.5 Bone marrow + 6.0 ± 1.7 N.D. 5.3 _+ 1.9

a Kinetics of induction of POMC-related RNA in rats treated with STZ. Groups of animals (five per group) were injected i.p. with STZ as described in Materials and methods. Animals were killed at the indicated time points and the tissues frozen as pellets at -26°C. After all the samples were collected, the RNA was isolated and slotted on nitrocellulose and probed as described in Materials and methods. Data points represent the percent of control of average densitometric scans ± SEM. * Significantly different from control at P < 0.05. N.D., not determined.

40 I~. Law et al. /Journal of Neuroimmunology 49 (1994) 35-44

Table 5 Effect of diabetes on serum and lymphoid content of ACTH a

Source Diabetic ACTH content

of fluid Serum Thymus

(pg/ml) (pg/g tissue)

Serum - 369 + 69 - + 6065:91 * -

Thymus - - 718 5:196 + - 21965:692 *

a Control and diabetic rats treated with STZ for 3 days were sacrificed and serum collected for ACTH RIA. Thymus tissues were teased into single-cell suspensions in 2 ml of RPMI without serum and centrifuged for 2 min at 10000x g in a microfuge. The resulting supernatant fluids were collected and the ACTH content determined by RIA. Data points represent the average values + SEM from a typical experiment done three times. * Significantly different from control at P < 0.08.

m e a s u r e d t h e ab i l i ty o f s p l e e n a n d t h y m u s ce i l s f r o m

a n i m a l s t r e a t e d w i t h S T Z t o p r o l i f e r a t e ( T a b l e 6) as

we l l as d e t e r m i n e d t h e d i r e c t e f f e c t s o f S T Z o n P O M C

a n d G H g e n e e x p r e s s i o n in v i t r o ( T a b l e 7). T h e p r e s -

e n c e a n d f u n c t i o n o f t h e G H R H r e c e p t o r w a s a l so

e x a m i n e d to d e t e r m i n e w h e t h e r t h i s r e c e p t o r h a d b e e n

a l t e r e d as a r e s u l t o f t h e d i a b e t e s a n d c o u l d a c c o u n t

f o r t h e r e d u c e d G H leve ls . T h e d a t a s h o w n in T a b l e 6

s u g g e s t t h a t d i a b e t i c a n i m a l s h a v e a d e p r e s s e d t h y m i -

d i n e i n c o r p o r a t i o n b a s e d o n t h e p r o l i f e r a t i o n assay.

I n t e r e s t i n g l y , h o w e v e r , d i a b e t i c a n i m a l s a p p e a r to

m a i n t a i n spec i f i c b i n d i n g o f G H R H a n d c a n b e s t i m u -

l a t e d to p r o l i f e r a t e b y G H R H ( P a w l i k o w s k i e t al.,

1988; G u a r c e l l o e t al., 1991) as we l l as c o n t r o l s ( T a b l e

6). T h e s e d a t a c o n f i r m a n e a r l i e r f i n d i n g t h a t t h e

e f f e c t s o f S T Z o n i m m u n e ce l l s m a y b e spec i f i c ( C o c k -

f i e ld e t al., 1989). A l t h o u g h in t h i s i n s t a n c e , G H R N A

leve l s w e r e n o t d i r e c t l y m e a s u r e d , t h e d a t a d o n o t

s u p p o r t t h e i d e a t h a t t h e r e d u c e d G H R N A p r o d u c -

t i o n in d i a b e t i c a n i m a l s is l i n k e d to a d e f e c t i v e l y m p h o -

cy te G H R H r e c e p t o r .

S i n c e S T Z h a d b e e n s h o w n to d a m a g e l y m p h o i d

o r g a n s ( K o l b e t al., 1987) as we l l as i s le t s w e inves t i -

g a t e d w h e t h e r t h e d r u g h a d a d i r e c t e f f e c t o n l y m p h o -

cy te G H a n d P O M C R N A s y n t h e s i s in v i t ro . W e cu l -

t u r e d s p l e e n a n d t h y m u s ce l l s in v i t r o w i t h v a r i o u s

c o n c e n t r a t i o n s o f S T Z fo r 18 h a n d m e a s u r e d ce l l

v iab i l i ty , p r o l i f e r a t i v e abi l i ty , a n d G H a n d P O M C R N A

s y n t h e s i s ( T a b l e 7). I n al l t r e a t m e n t g r o u p s , t h e l eve l o f

G H a n d P O M C R N A w a s u n a f f e c t e d a l t h o u g h a t t h e

h i g h e s t l eve l ( 3 0 0 / x g / m l ) t h e p r o l i f e r a t i v e c a p a c i t y o f

l y m p h o c y t e s w a s s i g n i f i c a n t l y i m p a i r e d . T h e s e r e s u l t s

s u g g e s t t h a t t h e S T Z t r e a t m e n t o f l y m p h o c y t e s in v i t r o

w i t h u p to 3 0 0 / z g / m l S T Z d o e s n o t d i r e c t l y i n h i b i t t h e

p r o d u c t i o n o f G H R N A o r s t i m u l a t e t h e s y n t h e s i s o f

P O M C R N A .

Table 6 Effect of STZ treatment on the lymphocyte GHRH receptor

Treatment Cell source Total specific 1251 hGHRH group bound (pM) b

3H-thymidine incorporation a

No GHRH control + GHRH

Percent above control

Normal Spleen 104.5 5:28.0 31672 5 : 9 9 5 44423 + 3558 140 * Thymus 161.75 5:21.3 54598 _+ 1030 87874 5 : 5 4 7 161 *

Diabetic Spleen 114.6 -t- 20.4 6144 5 : 4 1 0 11006 + 616 179 * Thymus 204.2 + 3.7 17468 + 1 217 25864 + 2768 148 *

a Rats were made diabetic as previously described. Lymphoid tissues were teased into single-cell suspensions and 1 × 106 cells each were treated with GHRH 1-29 (10/~g/ml) in 1% Nutridoma for 18 h in microtiter wells. Plates were mash harvested and [3H]thymidine counted as previously described. Data points represent the average values of six replicates 5: SEM. * Significantly different from control at P < 0.05. b Binding assays were carried out as described in Materials and methods. Each point represents the mean of three determinations, pM were calculated based on the number of specific counts per min bound to the cells and a specific activity of 2000 C i / m m for iodinated hGHRH. The differences were not statistically significant.

Table 7 Effect of STZ on lymphocyte functions in vitro

STZ treatment Percent viability (~g /ml )

[3H]thymidine incorporation

Spleen Thymus Spleen Thymus

Relative absorbance by densitometry

GH (spleen) cells POMC (thymus) cells

None 94.6 + 0.7 96.4 + 0.4 46 637 + 1589 136 343 + 12 529 67.3 + 2.1 8.0 + 1.08 1 95.6+0.3 94.3+0.2 42607+ 890 128171+ 5505 61.3+1.2 7.8+0.5 3 - - 44924 ± 1471 140149 5 : 7 2 4 8 - -

10 92.5+1.1 95.7+0.6 46878+1235 160968+ 6527 66.0+2.5 10.1 + 1.1 30 - - 47805 + 1640 155659 + 5936 71.0 + 2.3 7,5 + 1.3

100 94.5 + 1.0 95.7 + 0.5 46 840 J: 1682 135 659 + 9 806 70.3 ± 2.3 7.3 + 2.2 300 - - 22188 5 : 5 2 8 * 71416 + 6160 * 73.5 5:2.6 6.5 + 1.4

a Rats were killed and spleen and thymic tissues teased and placed in medium containing the indicated concentrations of STZ for 18 h. Cells (1 × 106/ml) were harvested and [3H]thymidine counted as previously described. Cells from selected cultures were removed for RNA isolation, slotting, and probing to measure the level of GH and POMC RNA as described earlier. Data points represent the percent of control of average values + SEM. * Significantly different from control at P < 0.05.

F. Law et al. /Journal of Neurotmrnunology 49 (1994) 35-44 41

Table 8 Effect of insulin treatment of rats expression

on lymphocyte GH and POMC

Treatment Glucose (concentration (mg/dl) of insulin)

RNA expression (Relative absorbance by densitometry)

GH RNA POMC RNA

None 300±100 6.0±0.6 6.7±1.5 1001U Negatwe* 8.6±2.2 7.0±1.2 30IU 50±25* 7.7±1.5 6.7±1.3 10IU 1~±50 8.0±0.6 9.0±0.6 3IU 250±1~ 5.3±2.4 7.3±1.7 I lU 350±100 6.3±2.7 8.3±2.3

a Rats (230-240 g) were injected daily with the designated amounts of insulin preparation for 3 days. A mixture of equal amounts of Ultralente (100 U/ml) and lente (100 U/ml) in zinc suspension were given subcutaneously. On the 4th day, animals were killed and the spleen removed and RNA isolated slotted, and probed as described in Materials and Methods. Data points represent the percent of control of average densitometric scans ± SEM. * Significantly different from control at P < 0.05.

3.4. Effects o f insulin on rat lymphocyte G H levels The importance of glucose and more recently in-

sulin in regulating somatotroph secretion of G H has been reported (Yamashita and Melmed, 1986). The possibility that glucose and insulin may be involved in the control of lymphocyte G H and POMC was there- fore more directly investigated both in vitro and in vivo. The effects of varying doses of insulin (0-1 /~M) and glucose (0-10%) were tested for their effect on lymphocyte G H and POMC expression in vitro at 4, 18, and 48 h of culture. The results showed that at all doses tested for each t imepoint that no significant inhibition or enhancement of G H R N A levels were observed. Another set of in vitro experiments were performed using mixtures of insulin and glucose at the concentrations described above and again no differences were observed in G H or POMC R N A levels in spleen or thymus cells (data not shown).

The effects of insulin on G H expression were also investigated in vivo after the t reatment of animals with different doses of insulin for 3 days (Table 8). The data show that t reatment of animals with the highest dose of insulin resulting in an undetectable blood glucose level had no significant effect on the level of G H or A C T H R N A expression in the spleen or thymus. Oth- ers have found by R I A the levels of insulin in the spleen to be much greater than those observed in serum (Rosenzweig et al., 1980). Lymphocytes, however, in our studies do not appear to be a source of insulin. We cultured lymphocytes alone and probed the cells for the presence of R N A to insulin as well as a t tempted to radiolabel and immunoaffinity-purify the insulin to measure endogenous de novo production of insulin. In both cases, the results were negative (data

not shown). Taken together, these findings suggest that insulin is not produced by cells of the immune system and that the levels of A C T H and G H produced and secreted by lymphocytes does not appear to be subject to regulatory influences by insulin. Thus although insulin modulates G H secretion at the level of the somatotroph (Yamshita and Melmed, 1986), it does not appear to play a role in modulating the levels of lymphocyte GH.

4. Discussion

The following studies suggest that neuroendocrine hormone production by cells of the immune system is altered during diabetes. In particular, we have observed a decrease in the expression of G H in the spleen and an increase in P O M C expression in the thymus. The data show that the increase in POMC R N A levels occurs within 24 h after t reatment with STZ. Although the mechanism(s) of altered gene regulation are unclear, the data shown here indicate that insulin does not appear to be involved in controlling the transcription or translation of the POMC or G H gene in lymphocytes. In vivo studies examining spleen and thymus cells from insulin-treated animals and in vitro studies testing insulin and glucose-free medium alone a n d / o r in combinations were ineffective in modulating the level of lymphocyte A C T H and G H expression. The lack of effect of insulin and glucose on gene expression do not appear to be time- or dose-related (data not shown). It has been reported that insulin immunoreactivity is present in all tissues in humans and rats as well as in cultured lymphocytes at concentrations that are 2-100 times those present in plasma or culture medium. The lymphocyte insulin was shown to be indistinguishable from genuine insulin and unaf- fected by culture conditions (Rosenzweig et al., 1980). Although the concentration of insulin in the pancreas is 10000 times the concentration in extrapancreatic tissues, the data suggest that extrapancreatic insulin may be synthesized by the tissues themselves. Our inability to detect the insulin message or protein suggests that lymphocytes are not a source of insulin. The difference in our findings and others may be that we tested for de novo synthesis rather than the presence of immunoreactive material.

STZ-treated rats showed all the characteristic fea- tures of diabetes: hyperglycemia, glycosuria, polyuria, polydipsia, and weight loss. In STZ-induced diabetic rats, the cellularity of lymphoid organs is diminished. T-cell responses (allograft reactivity and delayed T-cell hypersensitivity), B-cell responses (humoral antibodies) and phagocytic activities have all been reported to be impaired (Mahmoud et al., 1976; Ishibashi et al., 1980; Saiki et al., 1980; Gaulton et al., 1985; Rebuffat et al.,

42 II.. Law et al. / Journal of Neuroirnmunology 49 (1994) 35-44

1988). Adrenals of diabetic rats are hypertrophic and hyperactive as indicated by increased adrenal weight and corticosterone levels (Spangelo et al., 1987). Our results confirm earlier findings by showing that GH secretion in the diabetic rats is decreased in the pe- riphery (Hayford et al., 1980; Patel et al., 1980; Tan- nenbaum et al., 1981; Locatelli et al., 1985; Rebuffat et al., 1988). Others have shown that this most likely is a result of reduction in the amount of GH released by somatotrophs in the pituitary (Hayford et al., 1980) since transcription of pituitary GH and hypothalamic GHRH and somatostatin appear normal in diabetic animals (Lee et al., 1991). A more recent report suggested that a combined defect involving increased hypothalamic somatostatin and decreased GHRH is responsible for decreased pituitary GH synthesis (Olchovsky et al., 1990). Although somatostatin secretion increases during diabetes, this is not thought to be a primary factor responsible for the production of GH secretion since the changes in the levels of somatostatin occur after the reduction of GH secretion (Joanny et al., 1992). Thymocytes of diabetic rats incorporated less thymidine into DNA than did thymocytes of healthy rats; however, responsiveness to ConA and GHRH was preserved suggesting the cells can respond to certain hormones and a T-cell mitogen and are functionally intact. Some authors have reported direct depression of the immune response by STZ (Cockfield et al., 1989; Kantwerk-Funke et al., 1991). Our finding that normal spleen cells or thymus cells treated with STZ in vitro did not inhibit GH synthesis or induce POMC RNA argues against direct STZ activity. The mechanism of POMC induction, although unknown, may be mediated by secretion of thymus-derived peptides or a result of chronic corticosterone oversecretion in vivo. It can be noted that since thymosins appear to exert a direct effect on pituitary function in vivo and in vitro, they may also participate in the neuroendocrine hormone production by thymocytes (Spangelo et al., 1987).

The regulation and physiological role of lymphocyte-derived GH and ACTH are under active investigation. The lymphocyte gene for GH appears to be under negative control in lymphoid tissue in vivo since removal of spleen or thymic tissues results in rapid spontaneous gene transcription (Weigent et al., 1988). On the other hand, the expression of GH is inducible since elevated levels of GH produced by lymphocytes can be detected after treatment of animals with lipopolysaccharide (Baxter et al., 1991a,b). In vitro, the levels of GH can be modulated by GHRH, GH, and IGF-I (Weigent and Blalock, 1990a). It is also well established that POMC products are made by lymphocytes both in vivo (Smith et al., 1982, 1986; Buzzetti et al., 1989; Kavelaars et al., 1990a,b) and in vitro (Kavelaars et al., 1990a,b; Galin et al., 1991). Through both DNA and protein sequencing techniques, the

lymphocyte POMC product has been shown to be identical to its neuroendocrine counterpart (Smith et al., 1990). Leukocytes are similar to corticotrophs with respect to control of the POMC gene by CRF and AVP and feedback inhibition by a synthetic glucocorticoid hormone. The actions of CRF and dexamethasone in the immune system are indirect, however, requiring the production of IL-1 by macrophages (Kavelaars et al., 1989). Recently, the detection of lymphocyte POMC has been reported in an adjuvant-arthritis rat model (Stephanou et al., 1992). The response of the pituitary and the spleen to produce POMC mRNA to adjuvant arthritis in the rat suggests that in this disease the activation of both the neuroendocrine and the immune systems and their interaction may play a role in arthritis (Stephanou et al., 1992).

Numerous reports have appeared showing that neuroendocrine polypeptide hormones including POMC products can regulate several important immune functions (Johnson et al., 1992). In vitro studies have described receptors for ACTH and endorphins on lymphocytes (Carr, 1991). The effects can be either positive or negative and all the major immune cell types appear to be influenced. ACTH has been found to be a potent inhibitor of antibody production (Johnson et al., 1982), suppress MHC class II expression by murine peritoneal macrophages (Zwelling et al., 1992), stimulate natural killer cell activity (McGlone et al., 1991), modulate the rise in intracellular-free calcium concentration after T cell activation (Kavelaars et al., 1988), and suppress the production of IFN-3, (Johnson et al., 1984). The endogenous opiates/3-, 3~- and a-endorphin are also contained in the polyprotein POMC and have also been shown to modulate the activity of cells in the immune system (for review, see Kavelaars et al., 1988). In another report, treatment of diabetic animals with an anti-IL-2 receptor monoclonal antibody could suppress STZ-induced diabetes in mice (Hatamori et al., 1990). These data suggest an important role for acti- vated T cells and /o r macrophages in the immunological mediated insulitis thought to follow the toxic injury by STZ (Rossini et al., 1985). The available data suggest that the small amounts of neuroendocrine hormones released by cells of the immune system (10-100 pg/107 cells) most likely function in a paracrine/ autocrine fashion (Weigent and Blalock, 1990a). The increase in the expression of POMC in the thymus and possible release of ACTH and endorphins may influence the development of T cells and subsequent au- toimmune disease since these two peptides have been shown to have profound effects on T lymphocyte activities (Blalock, 1989). Further investigations are neces- sary to understand the mechanisms controlling the synthesis of lymphocyte POMC in diabetes and whether or not this alteration affects the outcome of diabetic episodes.

F. Law et al. /Journal of Neuroirnmunology 49 (1994) 35-44 43

Acknowledgements

T h e r e s e a r c h was s u p p o r t e d by a g r a n t f r o m t h e

N a t i o n a l I n s t i t u t e o f N e u r o l o g y a n d C o m m u n i c a t i v e

D i s o r d e r s ( R O 1 NS24636) to D . A . W . ; a g r a n t f r o m t h e

N a t i o n a l I n s t i t u t e o f A r t h r i t i s , D i a b e t e s , D i g e s t i v e , a n d

K i d n e y D i s e a s e ( R O 1 D K 3 8 0 2 4 ) ; N I H t r a i n i n g g r a n t

NS07335 a n d C o m p r e h e n s i v e M i n o r i t y F a c u l t y D e v e l -

o p m e n t P r o g r a m to L .C.P . W e t h a n k D i a n e W e i g e n t

fo r h e r h e l p in t h e p r e p a r a t i o n o f t h e m a n u s c r i p t .

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Effects of streptozotocin-induced diabetes on lymphocyte POMC and growth hormone gene expression in the rat