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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: [email protected]
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.
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