Research Communication

Macrophage Migration Inhibitory Factor is a Constitutively ExpressedCytokine in the Human Adrenal Gland

Ming Jian and C. Richard Parker Jr.

Department ofObstetrics andGynecology, TheUniversity ofAlabama atBirmingham,Birmingham,AL35233-7333,USA

Summary

Macrophage migration inhibitory factor (MIF) has been found to

be widely expressed in many cell types throughout the body and

appears to play several physiologic roles. We sought to determine the

expression and cellular distribution of MIF in human fetal (HFA)and adult (HAA) adrenals. A single band of approximately 0.8 kb

was revealed by northern blot hybridization using cDNA probes for

MIF in total RNA extracts of both HFA and HAA tissues.

Immunohistochemical analysis showed strong immunostaining forMIF in the broad fetal zone of the HFA and in both the zona

glomerulosa and zona reticularis of HAA. The cells of the zona

fasciculata of the HAA and of the neocortex of the HFA were onlyminimally, if at all, immunopositive for MIF and medullary elements

were consistently negative for MIF. These results are indicative of

local production of MIF in adrenocortical cells during intrauterine

development and also in adulthood. The role of MIF in cortical cellsof the human adrenal gland remains to be determined.

IUBMB Life, 55: 155–158, 2003

Keywords Macrophage migration inhibitory factor; adrenal cortex;immunohistochemistry; immune regulator.

INTRODUCTION

Macrophage migration inhibitory factor (MIF) originally

was discovered to be a T-lymphocyte-derived cytokine, able to

inhibit the random migration of macrophages (1). Later, MIF

was found to be a pituitary hormone, counter-regulating

glucocorticoid action in response to stress such as during

injury or infection (2 – 4). MIF also is structurally related to a

group of isomerase/tautomerase enzymes and has been found

to perform several catalytic reactions involving catecholamine

derivatives, thiols or phenylpyruvate, in vitro (5 – 7). MIF also

has been shown to overcome p53 tumor supressor activity (8).

Recently MIF has been found in many tissues (9 – 16) without

regard to their immune status. These observations suggest that

locally generated MIF could act in several different ways that

might be independently regulated from circulating MIF

secreted by the pituitary gland or T-lymphocytes.

The adrenal gland is the major source of glucocorticoids

and other steroid hormones that play various roles in

homeostasis. Stressors elicit secretory responses from both

the adrenal cortex, the site of steroid hormone synthesis, and

the adrenal medulla, a major source of circulating catechola-

mines. In view of the importance of MIF in the physiologic

responses to the stress of injury and infection, we sought to

investigate the presence of messenger RNA for MIF using

northern hybridization and MIF protein by immunohisto-

chemical methods in the human fetal adrenal (HFA) and

human adult adrenal (HAA).

MATERIALS AND METHODS

Tissues

Normal human adult adrenals (HAA) were obtained at

surgery from adults who underwent nephrectomy for renal

carcinoma and at autopsy of adults who died suddenly as the

result of accidents, etc. The donors had no signs or symptoms

of adrenal disorder. Human fetal adrenals and human fetal

kidneys were acquired at time of pathological examination of

mid trimester abortuses. These tissues were utilized for

northern blot hybridization and immunohistochemistry ana-

lyses as detailed below. The use of such tissues in these studies

was approved by the Institutional Review Board of the

University of Alabama at Birmingham.

Northern Blot Hybridization

Extraction of RNA was performed following the protocol

of Trizol reagent (Life Technologies, Grand Island, NY,

USA). Probes (25 ng, cDNA) of human MIF (9), dehydroe-

piandrosterone sulfotransferase (DST), 3 beta-hydroxysteroid

dehydrogenase (HSD) and human beta-actin were 32P-labeled

Received 30 January 2003; accepted 26 February 2003Address correspondence to C. Richard Parker, Jr, Department of

Obstetrics and Gynecology, University of Alabama at Birmingham,Birmingham, AL, 35233-7333. Tel: (205) 934-6294;Fax (205) 934-6296; E-mail: crparker@uab.edu

IUBMBLife, 55(3): 155–158, March 2003

ISSN 1521-6543 print/ISSN 1521-6551 online # 2003 IUBMB

DOI: 10.1080/1521654031000106672

to a specific activity of 108 – 109 cpm/mg. Denatured total

RNA (15 mg) was size-fractionated by electrophoresis and

blotted onto Nytran membranes. Northern blots were

hybridized with the 32P-radiolabeled cDNA probes. After

hybridization, blots were washed and then exposed to X-ray

film at 7 708C until satisfactory signals appeared. Blots were

stripped prior to reprobing.

Immunohistochemical Analysis

Tissues were fixed in 10% buffered formalin and embedded

in paraffin. Adjacent microtome sections (5 microns) were

processed for immunohistochemical localization by the avidin-

biotin complex method (BioGenex, San Ramon, CA, USA),

using diaminobenzidine (DAB) as chromogen essentially as we

have described in the past (17, 18). A 1 : 2000 dilution of goat

anti-human MIF antibody (R&D Systems, Inc., Minneapolis,

MN, USA), a 1 : 1000 dilution of rabbit anti-human dehy-

droepiandrosterone sulfotransferase (DST) antiserum (17), or

1 : 400 of rabbit anti-human 3 beta hydroxysteroid dehydro-

genase (HSD) antiserum (18) were used. Immuno-staining of

sections from several specimens was repeated in order to

ascertain the reproducibility of results.

RESULTS AND DISCUSSION

MIF is a small protein with an increasing list of identified

functions in areas ranging from catalysis to sepsis to apoptosis

(1 – 8). It is almost ubiquitously expressed in the organs of the

human and other mammals (9 – 16), but its expression can vary

markedly among cell types within each tissue. In many cases,

MIF expression seems to be associated with differentiating and

proliferating cells (12, 16), but specific roles for MIF also have

been proposed for differentiated physiologic systems as well.

In the present study, we found a distinct single band

(0.8 kb) of MIF mRNA in HFA, HAA and fetal kidney

(Figure 1). Its size is similar to that described in the rat and

other human tissues. As we anticipated, two strong hybridiza-

tion bands of DST mRNA occurred in the HFA and less

abundant similarly sized bands of DST mRNA in the HAA.

Only the HAA was found to contain HSD mRNA. These

results support the view that MIF is expressed in many

hormone-producing tissues (2, 4, 12 – 15).

Of interest in the present study is that MIF and DST have

similar patterns of expression at mid-gestation in the HFA

(Figure 2, Panels A and B). Dehydroepiandrosterone sulfate, a

placental estrogen precursor in human pregnancy, is the major

steroid secretory product of HFA and DST is a key enzyme in

its formation (19). Cytokines such as transforming growth

factor-beta and tumor necrosis factor-alpha have inhibitory

effects on dehydroepiandrosterone sulfate production, in part

through their influence on DST expression in the HFA (19,

20). These cytokines have overlapping activities with MIF in

other systems, suggesting that MIF might be able to influence

the synthesis of dehydroepiandrosterone sulfate in a similar

way, thereby exerting effects on pregnancy and fetal develop-

ment. Such an effect would, however, not coincide with

systemic actions of MIF as a counter regulatory substance to

glucocorticoids since dehydroepiandrosterone and its major

circulating form, dehydroepiandrosterone sulfate, are believed

to enhance immune competence (21, 22).

In the HAA, MIF was noted to be present in both the zona

glomerulosa and zona reticularis, which differed from the

distribution of DST and HSD in that they are localized to only

one or the other of these cortical zones (Figure 3). Since the

zona glomerulosa is the site for production of the miner-

alocorticoid aldosterone, it has been suggested that MIF

might be able to participate in the regulation of electrolyte or

blood pressure (15). In contrast to the results from the rat (15),

in which MIF was mainly present in the zona glomerulosa but

was absent from the zona reticularis, the zona reticularis of the

human also shows prominent expression of MIF. In the adult

human, but not in the rat, the zona reticularis is the site for

synthesis and secretion of adrenal androgens such as

dehydroepiandrosterone sulfate, which serves as precursor

for the formation of active androgens and estrogens in

peripheral target tissues (19, 21, 23).

Kid

ney

HA

A

HFA

28S

18STotal RNA

hMIF

DST

HSD

Actin

Figure 1. Northern blot hybridization of MIF mRNA in

HFA, HAA and human fetal kidney. Blot was hybridized with

cDNA probes for MIF, DST, HSD and finally human beta-

actin, respectively.

156 JIAN AND PARKER

There also is an anatomic basis for possible paracrine

actions of MIF, as has been postulated for other cytokines

(24) in the human adrenal. Since blood flow in the adrenal

proceeds in a centripedal fashion, the thin-walled sinusoids in

HAA direct flow from zona glomerulosa into the zona

fasciculata, as well as from the zona reticularis into the

medulla. Therefore, MIF secreted by the cells of the zonae

glomerulosa and reticularis could exert influence in the zona

fasciculata and medulla, respectively. MIF is a glucocorticoid-

induced regulator (2), which also has been speculated to have

an important role in catecholamine metabolism by virtue of its

enzymatic activity (5 – 7). Interestingly, glucocorticoids and

catecholamines share some common modulators and re-

sponses (25, 26). Thus, there may be a basis for MIF to

regulate the systemic effects of both glucocorticoids and

catecholamines in stress circumstances and perhaps also to

locally modulate their production. Indeed, the apparent

pleiotropy of MIF raises many possibilities for functions in

HAA and HFA and roles in response to stress and disease

states as well as embryonic development and reproduction.

ACKNOWLEDGEMENTS

We thank CathyMercer for assistance with tissue processing

and W. E. Grizzle and O. Faye-Petersen for providing

pathological specimens for our studies. We also thank Graeme

Wistowfor theMIFprobe.Apreliminaryreportof thisworkwas

presented at the 82nd annual meeting of the Endocrine Society.

These studies were supported by Grant N00014-96-1-0255.

REFERENCES1. George, M., and Vaughan, J. H. (1962) In vitro cell migration as a

model for delayed hypersensitivity. Proc. Soc. Exp. Biol. Med. 111,

514 – 521.

2. Bucala, R. (1996) MIF rediscovered: cytokine, pituitary hormone, and

glucocorticoid-induced regulator of the immune response. FASEB

Journal 10, 1607 – 1613.

3. Calandra, T., Bernhagen, J., Metz, C. N., Spiegel, L. A., Bacher, M.,

Donnelly, T., Cerami, A., and Bucala, R. (1995) MIF as a

glucocorticoid-induced modulator of cytokine production. Nature

377, 68 – 71.

4. Waeber, G., Calandra, T., Bonny, C., and Bucala, R. (1999) A role for

the endocrine and pro-inflammatory mediator MIF in the control of

insulin secretion during stress. Diab/Metab. Res. Rev. 15, 47 – 54.

5. Rosengren, E., Bucala, R., Aman, P., Jacobsson, L., Odh, G., Metz,

C. N., and Rorsman, H. (1996) The immunoregulatory mediator

macrophage migration inhibitory factor (MIF) catalyzes a tautomer-

ization reaction. Mol. Med. 2, 143 – 149.

Figure 2. Immunohistochemical analysis of MIF and DST in

a HFA and kidney at 18 weeks gestation. Panels A and B show

the immunolocalization of DST- and MIF-positive cells,

respectively, indicated by brown staining; Panel C, negative

control section. Original magnification: 6 20.

Figure 3. Immunolocalization of MIF-, DST- and HSD-

positive cells on sequential paraffin sections of normal adrenal

(20-year-old female). Positive reactions are indicated by brown

staining. Panels A, B, and C are high power (6 100) views of

immunostaining for MIF, DST, and HSD, respectively. ZG-

zona glomerulosa; ZF-zona fasciculata; ZR-zona reticularis.

157MIF LOCALIZATION IN THE HUMAN ADRENAL GLAND

6. Matsunaga, J., Sinha, D., Pannell, L., Santis, C., Solano, F., Wistow,

G. J., and Wearing, V. J. (1999) Enzyme activity of macrophage

migration inhibitory factor toward oxidized catecholamines. J. Biol.

Chem. 274, 3268 – 3271.

7. Esumi, N., Budarf, M., Ciccarelli, L., Sellinger, B., Kozak, C. A., and

Wistow, G. (1998) Conserved gene structure and genomic linkage for

D-dopachrome tautomerase (DDT) and MIF. Mam. Genome 9, 753 –

757.

8. Hudson, J. D., Shoaibi, M. A., Maestro, R., Carnero, A., Hannon, G.

J., and Beach, D. H. (1999) A proinflammatory cytokine inhibits p53

tumor suppressor activity. J. Exp. Med. 190, 1375 – 1382.

9. Paralkar, V., and Wistow, G. (1994) Cloning the human gene for

macrophage migration inhibitory factor (MIF). Genomics 19, 48 – 51.

10. Magi, B., Bini, L., Liberatori, S., Marzocchi, B., Raggiaschi, R.,

Arcuri, F., Tripodi, S. A., Cintorino, M., Tosi, P., and Pallini, V.

(1998) Charge heterogeneity of macrophage migration inhibitory

factor (MIF) in human liver and breast tissue. Electrophoresis 19,

2010 – 2013.

11. Arcuri, F., del Vecchio, M. T., de Santi, M. M., Lalinga, A. V., Pallini,

V., Bini, L., Bartolommei, S., Parigi, S., and Cintorino, M. (1999)

Macrophage migration inhibitory factor in the human prostate:

identification and immunocytochemical localization. Prostate 39,

159 – 165.

12. Arcuri, F., Cintorino, M., Vatti, R., Carducci, A., Liberatori, S., and

Paulesu, L. (1999) Expression of macrophage migration inhibitory

factor transcript and protein by first-trimester human trophoblasts.

Biol. Reprod. 60, 1299 – 1303.

13. Meinhardt, A., Bacher, M., Wennemuth, G., Eickhoff, R., and

Hedger, M. (2000) Macrophage migration inhibitory factor (MIF) as

a paracrine mediator in the interaction of testicular somatic cells.

Andrologia 32, 46 – 48.

14. Wada, S., Fujimoto, S., Mizue, Y., and Nishihira, J. (1997)

Macrophage migration inhibitory factor in the human ovary: Presence

in the follicular fluids and production by granulosa cells. Biochem Mol

Biol Int. 41, 805 – 814.

15. Bacher, M., Meinhardt, A., Lan, H. Y., Mu, W., Metz, C. N.,

Chesney, J. A., Calandra, T., Gemsa, D., Donnelly, T., Atkins, R. C.,

and Bucala, R. (1997) Migration inhibitory factor expression in

experimentally induced endotoxemia. Am. J. Pathol. 150, 235 – 246.

16. Kobayashi, S., Satomura, K., Levsky, J. M., Sreenath, T., Wistow, G.

J., Semba, I., Shum, L., Slavkin, H. C., and Kulkarni, A. B. (1999)

Expression pattern of macrophage migration inhibitory factor during

embryogenesis. Mech. Dev. 84, 153 – 156.

17. Parker, C. R. Jr, Falany, C. N., Stockard, C. R., Stankovic, A. K.,

and Grizzle, W. E. (1994) Immunohistochemical localization of

dehydroepiandrosterone sulfotransferase in human fetal tissues. J.

Clin. Endo. Metab. 78, 234 – 236.

18. Parker, C. R. Jr, Faye-Petersen, O., Stankovic, A. K., Mason, J. I.,

and Grizzle, W. E. (1995) Immunohistochemical evaluation of the

cellular localization ontogeny of 3b-hydroxysteroid dehydrogenase/

delta 5-4 isomerase in the human fetal adrenal gland. Endocr. Res. 21,

69 – 80.

19. Parker, C. R. Jr (1999) Dehydroepiandrosterone and dehydroepian-

drosterone sulfate production in the human adrenal during develop-

ment and aging. Steroids 64, 640 – 647.

20. Parker, C. R. Jr, Stankovic, A. K., Faye-Petersen, O., Falany, C. N.,

Li, H., and Jian, M. (1998) Effects of ACTH and cytokines on

dehydroepiandrosterone sulfotransferase messenger RNA in human

adrenal cells. Endocr. Res. 24, 669 – 673.

21. Baulieu, E. E. (1996) Dehydroepiandrosterone (DHEA): A fountain

of youth?. J. Clin. Endo. Metab. 81, 3147 – 3151.

22. Araneo, B., and Daynes, R. (1995) Dehydroepiandrosterone functions

as more than an antiglucocorticoid in preserving immunocompetence

after thermal injury. Endocrinology 136, 393 – 401.

23. Longcope, C. (1995) The metabolism of dehydroepiandrosterone

sulfate. Sem. Repro. Endocrinol. 13, 270 – 274.

24. Ehrhart-Bornstein, M., Hinson, J. P., Bornstein, S. R., Scherbaum,

W. A., and Vinson, G. P. (1998) Intraadrenal interactions in the

regulation of adrenocortical steroidogenesis. Endo. Rev. 19, 101 – 143.

25. Koob, G. F. (1999) Corticotropin-releasing factor, norepinephrine,

and stress. Biol. Psych. 46, 1167 – 1180.

26. Meduri, G. U. (1999) New rationale for glucocorticoid treatment in

septic shock. J. Chemotherapy 11, 541 – 550.

158 JIAN AND PARKER