Journal of Neuroimmunology, 45 (1993) 127-132 127 © 1993 Elsevier Science Publishers B.V. All rights reserved 0165-5728/93/$06.00

JNI 02381

Changes in hypothalamic corticotrophin-releasing factor and anterior pituitary pro-opiomelanocortin mRNA

during the course of experimental allergic encephalomyelitis

M.S. Harbuz a, j.p. Leonard b, S.L. Lightman a and M.L. Cuzner b a University Department of Medicine, University of Bristol, Bristol Royal Infirmary, Bristol, UK, and b Multiple Sclerosis Society Laboratory,

Institute of Neurology, London, UK

(Received 9 November 1992) (Revision received 20 January 1993)

(Accepted 20 January 1993)

Key words: Hypothalamus; Corticotrophin-releasing factor; Pituitary pro-opiomelanocortin; Pituitary; Experimental allergic encephalomyelitis

Summary

The pituitary-adrenal axis is activated during the course of experimental allergic encephalomyelitis (EAE), a paralytic disease resulting from an immunological reaction against central nervous system myelin. The magnitude of the adrenal response not only correlates with the severity of disease, but also serves an important functional role in recovery. We have shown that in EAE there are short-term changes in anterior pituitary pro-opiomelanocortin (POMC) mRNA as well as plasma corticosterone which can be detected before the development of clinical disease. At peak clinical signs when corticosterone and POMC mRNA are maximal, hypothalamic corticotrophin-releasing factor (CRF) mRNA is suppressed, suggesting that pituitary-adrenal activation is not mediated by CRF. Following recovery all parameters return to normal.

Introduction

Experimental allergic encephalomyelitis (EAE) is a paralytic disease resulting from an immunological reac- tion against central nervous system (CNS) myelin, in- duced in the Lewis rat either by direct inoculation of antigen with adjuvant or by adoptive transfer of cul- tured spleen cells taken from animals with the disease (Hickey and Gonatas, 1984). This model has been used as an experimental model of the human demyelinating disease multiple sclerosis (MS). It is an acute, monophasic disease from which the animals recover with subsequent resistance to disease induction.

The hypothalamo-pituitary-adrenal (HPA) axis is ac- tivated during the course of EAE and the magnitude of the adrenal response not only correlates with the sever- ity of the disease, but also serves an important func-

Correspondence to: M. Harbuz, University Department of Medicine, University of Bristol, Bristol Royal Infirmary, Lower Maudlin Street, Bristol BS2 8HW, UK.

tional role in recovery (MacKenzie et al., 1989; MacPhee et al., 1989; Mason et al., 1990).

In previous studies on another model of immunolog- ically mediated activation of the HPA axis (adjuvant- induced arthritis) we have confirmed that there is increased corticosterone secretion (Harbuz et al., 1992a; Sarlis et al., 1992) and anterior pituitary POMC mRNA accumulation, but surprisingly found a fall both in corticotrophin-releasing factor (CRF) mRNA in the paraventricular nucleus (PVN) of the hypothalamus and in CRF-41 content of hypophyseal-portal blood (Harbuz et al., 1992a).

It is clearly very important to know whether this very important discordance between the changes in CRF and POMC mRNAs is a peculiarity of the arthritic model or a more common response to endogenous immune activation of the HPA axis. In the present study we have investigated changes in CRF and POMC mRNAs together with plasma corticosterone during both the development of, and also the recovery from, adoptively transferred EAE.

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Materials and Methods

Immunization protocol Female Lewis rats (Bantin and Kingman) were im-

munized in each hind foot with 50 /zl of complete Freund's adjuvant (CFA) containing 10 mg ml-1 M y -

cobacterium butyricum and 0.1% of crude myelin basic protein (MBP) prepared from guinea pig spinal cord. 11 days after imunization the animals were killed and the spleens removed, chopped and sieved and splenic lymphocytes cultured at a concentration of 2 x 106 m1-1 for 3 days in Earle's modified basal medium (BME, Gibco) containing 10% foetal calf serum (Flow Lab., UK), 100 U ml-1 penicillin, 100/zg ml-1 strepto- mycin, 1 tzg ml - t indomethacin and 20 /~M 2- mercaptoethanol (Leonard et al., 1990). MBP was added at a concentation of 1 tzg ml - t for the duration of the culture period as the stimulating antigen. After the incubation period the cells were spun down, washed twice with fresh medium and then 40 x 106 cells were injected i.p. into naive recipients. Animals were decap- itated on days 3 (preclinical), 7 (clinical) and 11 (re- covery) after the transfer of MBP-sensitized spleno- cytes. The experiment was repeated to confirm the data. Trunk blood was collected for determination of serum corticosterone (tzSI-labelled corticosterone ra- dioimmunoassay kit, Immunodiagnostics) and the pitu- itary and brain rapidly removed and frozen on dry ice for subsequent determination of POMC and CRF mRNAs by in situ hybridization.

In previous studies the splenic noradrenergic and adrenocortical response 3 days after transfer with PPD-primed cells (CFA control) mirrored that with MBP-primed cells (Leonard et al., 1990; Leonard et al., 1991) and in the absence of any subsequent clinical signs there was no further response in these controls. Hence they were not included in the present work.

Analysis of hypothalamic CRF and pituitary POMC mRNA by in situ hybridization

In situ hybridization was performed as previously described (Young et al., 1986; Harbuz and Lightman, 1989). Briefly, the sections were warmed to room tem- perature, fixed in 4% (v/v) formaldehyde washed twice in phosphate-buffered saline and placed in 0.25% (v/v) acetic anhydride in 0.1 mol triethanolamine 1-t-0.9% (w/v) NaC1 to reduce non-specific binding and passed through 70, 80, 95 and 100% ethanol, 100% chloroform and 100% and 95% ethanol, before drying in air. The probes were synthetic 48-base oligonucleotides, com- plementary to part of the exonic mRNA sequences coding for CRF and POMC. The specificity of these probes has previously been demonstrated by the detec- tion of homogenous bands of appropriate size on Northern analysis of extracted mRNA, by use of mes- senger sense probes and by comparison with the results

of immunocytochemistry. The probes were labelled us- ing terminal deoxytransferase to add a 35S-labelled deoxyATP (1000 Ci mmol-t) tail to the 3' end of the probe. The specific activities of the probes were 9.7 x 1018 and 1.0 x 1019 dpm mol -~ for CRF and POMC, respectively. All control and experimental sections were hybridized in the same incubation reaction. The probe (in 45 /xl hybridization buffer) was applied to each section and incubated overnight at 37°C for the hy- bridization. The sections were then washed in 4 x 15 min changes of NaC1 (0.5 mol 1-1)-sodium citrate (0.015 mol 1-1) buffer (SSC; pH 7.2) at 55°C followed by 2 changes of SSC for 30 min each at room tempera- ture to remove unbound and non-specifically bound probe. The sections were rinsed in water, dried and apposed to Hyperfilm MP autoradiography film (Amersham International plc, Amersham, UK) for an appropriate exposure time. The autoradiographic im- ages were measured microdensitometrically using an image analysis system (Macintosh II computer running Image 1.22 by W. Rasband, NIH, Bethesda, MD). The optical densities were measured in dpm mg-1 by com- parison with simultaneously exposed 35S-labelled stan- dards.

The results are presented as means + SEM of the percentage change from the control which was as- signed a value of 100. Statistical analysis was per- formed by comparing control and treatment means using Dunnett's test following one-way analysis of vari- ance. A P value of < 0.05 was considered significant.

Results

Clinical course of adoptively transferred EAE The first clinical signs of EAE appeared on average

5 days after the transfer of myelin basic protein-sensi- tized T cells reaching a peak clinical score of 3 by day 7 (Fig. 1) (Leonard et al., 1990). Full recovery from disease was usually complete by day 10. Onset of

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DAYS POST TRANSFER Fig. 1. Clinical course of adoptively transferred EAE. A minimum of

eight animals were recorded at each time-point.

clinical signs was accompanied by dramatic weight loss and was scored as follows: 1, flaccid tail and weight loss; 2, hind limb hypotonia with further weight loss; 3, partial or complete hind limb paralysis. During maxi- mum clinical disease, histological examination of sec- tions of brain and spinal cord stained with haema- toxylin and eosin revealed extensive perivascular cuff- ing at the border of the white and grey matter.

Activity of the HPA axis Three days after cell transfer, prior to any clinical

signs of disease, pituitary POMC mRNA and serum corticosterone levels increased significantly in the ab- sence of any change in hypothalamic CRF mRNA

,c 2oo Z 175 ]- er-

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Fig. 2. Changes in the expression of (a) C R F m R N A in the parvocel- lular cells of the PVN and (b) P O M C m R N A in the anterior pituitary and (¢) circulating levels of corticosterone during the course of adoptivety transferred EAE. Values represent the m e a n + S E M

for n = 7 / 8 animals per group. * P < 0.05, * * P < 0.01.

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(Figs. 2 and 3). POMC mRNA and circulating corticos- terone remained elevated during the period of clinical disease. At this time of peak clinical disease and maxi- mum elevation of plasma corticosterone and POMC mRNA, hypothalamic CRF mRNA fell to levels signifi- cantly below those found prior to transfer. CRF and POMC mRNA as well as circulating corticosterone all returned to normal basal levels after recovery from the disease.

Discussion

Our results confirm previous studies which have demonstrated a close association between the develop- ment of EAE and adrenal corticosterone secretion (Leonard et al., 1990; MacPhee et al., 1989). In addi- tion we found that there was an increase in anterior pituitary POMC mRNA associated with the increase in circulating corticosterone starting well before any clini- cal signs of the disease. This clearly suggests that during the early period of lymphoproliferation and cytokine production factors are produced which result in activation of pituitary corticotrophs.

Although there was no change in hypothalamic CRF mRNA during the preclinical phase of the disease there was a paradoxical marked fall in CRF mRNA when clinical signs were maximal and plasma corticos- terone and anterior pituitary POMC mRNA were at their highest levels.

One possibility which must be considered is that the increase in anterior pituitary POMC mRNA may in- volve the CRF axis through a mechanism involving an increase in CRF precursor processing and/or release without a corresponding increase in CRF mRNA. A number of lines of evidence suggest this may not be the case. In situations such as adrenalectomy or acute stress in which an increase in CRF turnover, in CRF immunoreactivity in the median eminence, or of CRF release into the hypophysial portal blood have been reported previously, an increase in CRF mRNA has also been demonstrated (Jingami et al., 1985; Young et al., 1986; Lightman and Young, 1988; Harbuz and Lightman, 1989). Where a decrease in CRF release into the portal blood has been demonstrated for in- stance during exogenous steroid administration (Eck- land et al., 1991) we have also found a decrease in CRF mRNA (Harbuz et al., 1990). As a corollary subsequent steroid withdrawal results in a closely cor- related increase in both mRNA and peptide release (Harbuz et al., 1990; Eckland et al., 1991). Given the apparent close relationship of CRF mRNA to peptide release and turnover it would appear likely that the decrease in CRF mRNA in the present study repre- sents a genuine decrease in CRF production.

It has been reported that the Lewis rat has a defec-

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C o n t r o l D a y 7 E A E

a)

b)

Fig. 3. Autoradiograms on Hyperfilm-MP (Amersham) of representative transverse sections through (a) the anterior pituitary and (b) the hypothalamic PVN of control and day 7 EAE rats. Sections were hybridized with 35S-radiolabelled oligonucleotide probes complementary to (a)

POMC mRNA and (b) CRF mRNA. See Fig. 2 for quantification.

tive H P A axis in that this strain is unable to mount an appropriate response to acute stress. This has been localized to the hypothalamus and a defect in the synthesis and secretion of CRF. It has been inferred from these studies that the inability to regulate CRF and the subsequent lack of response of the H P A axis to acute challenge may be an important factor which influences the susceptibility of these animals to strep- tococcal cell wall (SCW)-induced arthritis (Sternberg et al., 1989). However, the responses of the H P A axis during the development of SCW-induced arthritis have not been determined. In the present study we observed a decrease in CRF m R N A at the peak of clinical symptoms of EAE. Fur thermore the return of CRF m R N A to control levels on remission of EAE suggests that the mechanism responsible for the stimulation of the H P A axis in response to acute stress (deficient in the Lewis rat) may be different to the inhibitory factor(s) acting on CRF neurons which constitute the reponse to immunological chal lenge/disease. There- fore the decrease in CRF m R N A which we observed is unlikely to be due to an inappropriate regulation of CRF in the Lewis rat. In fact, we have recently demon- strated an inhibitory mechanism regulating CRF in the

PVG rat in response to adjuvant-induced arthritis (Harbuz et al., 1992a, 1993). This strain is particularly resistant to EAE and therefore the responses to arthri- tis are not confounded by encephalomyelitis (Ben-Nun et al., 1982; Mason et al., 1990). In addition, this strain has a robust response to acute stress. Indeed it has been suggested that the robust response to stress ex- hibited by the PVG rat may be partly responsible for the resistance of these animals to EAE (Mason, 1991). The above-mentioned studies clearly show that suscep- tibility to these immune-mediated diseases is not solely dependent upon the ability of an animal to mount a response to acute stress.

The mechanism underlying the paradox of the in- crease in corticosterone and POMC m R N A associated with a decrease in CRF m R N A in EAE is unknown. We have previously reported a similar increase in cir- culating corticosterone and increased POMC m R N A in the anterior pituitary in adjuvant-induced arthritis which was associated with the appearance of inflamma- tion. At the time of onset of symptoms there was also a decrease in CRF m R N A which reached a nadir at the time of maximum severity of the disease, and corre- sponded with a marked decrease in CRF-41 peptide

131

release into the hypophysial-portal blood. We also found that there was a marked increase in AVP release into the portal circulation in these animals suggesting that in the presence of permissive levels of CRF, AVP may be an important factor in mediating the increased corticotroph activity (Harbuz et al., 1992a). These data suggest the possibility that in both EAE and adjuvant- induced arthritis activation of POMC mRNA is not mediated by CRF and, although AVP seems likely to be an important factor, other possible candidates can- not be ignored. Clearly it would be useful to obtain data on changes in parvocellular AVP mRNA in EAE; however, the presence of AVP-containing magnocellu- lar cells in the parvocellualr region of the PVN make this technically difficult.

The decrease in CRF mRNA in response to chronic immunological stress is surprising. Acute stress results in an increase in CRF mRNA within 4 h (Lightman and Young, 1988; Harbuz and Lightman, 1989). In response to repeated stress (daily injections of hyper- tonic saline for 12 days), we have also demonstrated the maintenance of increased levels of CRF mRNA (Lightman and Young, 1989). However, it must be borne in mind that the effects of chronic stress on the activation of the HPA axis differs in many respects from the effects of acute stress and this may, in part, explain our findings. Recent evidence has implicated a major role for AVP in repeated stress paradigms, for example De Goeij and co-workers (1991) have shown that repeated stress selectively increases vasopressin, rather than CRF, stores in the median eminence. Simi- larly, the importance of vasopressin has recently been demonstrated in response to sustained HPA activation. Following repeated stress exogenous CRF was unable to stimulate pituitary-adrenal activity while exogenous AVP was still active (Scaccianoce et al., 1991). Further- more, chronic stress in the immune-mediated model of adjuvant-induced arthritis resulted in increased portal vasopressin levels (Harbuz et al., 1992a). The decrease in CRF mRNA in the PVN is probably not simply due to increased corticosteroid feedback from the raised circulating corticosterone concentrations since follow- ing removal of endogenous corticosterone by adrenal- ectomy, CRF mRNA levels in arthritic animals are still suppressed below those seen in control non-arthritic adrenalectomized animals (Harbuz et al., 1993).

At a central level there is good evidence that cate- cholaminergic pathways can influence hypothalamic-re- leasing factors. Lesions of ascending catecholamine pathways to the PVN result in decreased portal CRF-41 release (Guillaume et al., 1987; Eckland et al., 1988), but do not affect vasopressin release (Eckland et al., 1988). However, stimulation of ACTH release by the al-agonist methoxamine can be reduced by the vaso- pressin antagonist dPTyrMeAVP but not by an equipo- tent dose of the CRF-41 antagonist a-helical CRF-41

(AI-Damluji et al., 1990) suggesting that vasopressin is more important than CRF-41 in mediating the effects of a l adrenoceptors. Stress itself has been shown to release noradrenaline within the hypothalamus (Yokoo et al., 1990), and in EAE itself there is evidence for a strong inverse relationship between corticosterone and hypothalamic noradrenaline content in the stressful clinical stages of disease (Leonard et al., 1991).

The interactions of the interleukins with the HPA axis are currently the subject of much research activity but the findings to date remain somewhat controver- sial. It is however generally agreed that interleukin (IL)-l/3 is able to stimulate the HPA axis through a CRF mRNA mediated mechanism at the hypothalamic level (Suda et al., 1990; Harbuz et al., 1992b). The action of IL-la on the HPA axis is less well established but its importance to the pathogenesis of EAE has been recently reported. EAE was found to be exacer- bated by IL-la injection, whereas soluble IL-1 recep- tor (which binds both IL-la and IL-1/3) delayed the onset, reduced the severity, and shortened the duration of the disease (Jacobs et al., 1991). We have recently shown that IL-2, a product of activated T lymphocytes, increases POMC mRNA at the pituitary level without evoking any change in CRF mRNA (Harbuz et al., 1992c). It is possible that the preclinical rise in POMC mRNA and plasma corticosterone in EAE could be effected through such a mechanism without affecting CRF. At this stage, however, the precise role of the cytokines must remain speculative although the effect of local cytokines on all these changes deserves further detailed investigation.

In conclusion, we have shown that in EAE there are short-term changes in pituitary POMC mRNA as well as corticosterone which can be detected before the development of clinical disease. At the time of peak clinical symptoms when plasma corticosterone and POMC mRNA are maximal, hypothalamic CRF mRNA is suppressed. The factor(s) inhibiting CRF mRNA are unknown and are currently under active investigation.

Acknowledgements

This study was supported by the Multiple Sclerosis Society of Great Britain and Northern Ireland. M.S.H. is supported by the Arthritis and Rheumatism Council.

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