Immunology and Cell Biology (2003) 81, 137–143
Macrophage migration inhibitory factor exhibits a pronounced
circadian rhythm relevant to its role as a glucocorticoid
NIKOLAI PETROVSKY,1,2 LUIS SOCHA,1, 2 DIEGO SILVA,1, 2
ASHLEY B GROSSMAN,3 CHRISTINE METZ4 and RICHARD BUCALA5
1Autoimmunity Research Unit, The Canberra Hospital, 2John Curtin School of Medical Research, Australian National
University, Canberra, Australia, 3Department of Endocrinology, Saint Bartholomew’s Hospital, London, United
Kingdom, 4The North Shore-Long Island Jewish Research Institute, Manhasset, New York, 5Yale University School of
Medicine, New Haven, Connecticut, United States of America
cortisol, whereas T helper 2 cytokine responses are dominant during day-time. The pro-inflammatory cytokine,
macrophage migration inhibitory factor counter-regulates glucocorticoid-mediated immune suppression. To deter-
mine the relationship between cortisol and macrophage migration inhibitory factor, healthy volunteers had blood
drawn hourly for 24 h for measurement of plasma cortisol and basal- and stimulated-macrophage migration
inhibitory factor. Similar to cortisol, macrophage migration inhibitory factor peaked during the late morning
whereas interferon-γ, tumour necrosis factor-α, interleukin-1 and interleukin-12 demonstrated a nocturnal peak.
After oral cortisone, plasma macrophage migration inhibitory factor rose 2–4-fold, whereas the other cytokines
decreased. There was no correlation between cortisol during the insulin tolerance test and plasma macrophage
migration inhibitory factor. The late morning peak of macrophage migration inhibitory factor, by antagonizing
cortisol-mediated pro-inflammatory cytokine suppression may prolong the duration of early morning inflammation.
These observations explain the beneficial role of macrophage migration inhibitory factor neutralization in models of
In humans, maximal expression of T helper 1 cytokines coincide with the nocturnal nadir of plasma
Key words: circadian, cortisol, cytokine, diurnal, MIF, neuroendocrine, Th1, Th2.
The development and subsequent abatement of an immune
response depends upon a complex interplay of humoral and
cellular mediators. It is increasingly apparent that the immune
system exhibits extraordinary temporal compartmentalization
whereby there are marked time of day differences in the
intensity of the cellular (Th1) arm and humoral (Th2) arm of
the immune response.1 The Th1 cytokines, IFN-γ, IL-1, IL-12
and TNF-α all exhibit night-time peaks in mitogen- or
antigen-stimulated human whole blood. The IFN-γ/IL-10
ratio also exhibits circadian rhythmicity, indicating a bias
toward cell-mediated responses during the night and early
morning when the ratio is high and toward humoral immunity
during the day when the ratio is low.1 This is consistent with
the fact that delayed-type hypersensitivity responses in
humans are increased at night2 and that symptoms of chronic
inflammatory disorders including rheumatoid arthritis3 and
asthma4 are worst during the night and early morning. Circa-
dian immune rhythms are not unique to man as immune
rhythms have also been described in mice,5 rats,6 fish7 and
birds.8 Although circadian cytokine rhythms are principally
entrained by plasma cortisol, there is also influence by other
neuroendocrine factors including melatonin, growth hormone
Macrophage migration inhibitory factor (MIF) is an
important counter-regulator of glucocorticoid action that
reverses glucocorticoid-induced immunosuppression and, in
particular, glucocorticoid-induced proinflammatory cytokine
inhibition10 and is present in human serum at concentrations
ranging from 2–6 ng/mL.11,12 Although the major source of
human plasma MIF is unclear, MIF is expressed by a range of
tissues including macrophages, T, and B cells, the pituitary
gland,13–15 the liver,16 eosinophils,17 adipocytes,18 pancreatic
islet β cells,19 Leydig cells of the testes,20 ovarian follicular
cells,21 and the adrenal glands.16 Stimuli for MIF secretion
include bacterial endo- and exotoxins,13,22 TNF-α and IFN-γ,13
IL-5 and C5a17 and malarial pigment.23
Clinical features of some chronic immuno-inflammatory
disorders, for example, rheumatoid arthritis, exhibit marked
circadian fluctuation, which we postulated could be associ-
ated with circadian rhythms of pro-inflammatory cytokines.
Given the role of MIF as a glucocorticoid counter-regulator,
we sought to determine the circadian relationship between
plasma MIF and cortisol in normal human subjects. We
Correspondence: Associate Professor Nikolai Petrovsky, Head,
Autoimmunity Research Unit, The Canberra Hospital, Yamba Drive,
Woden, ACT, Australia 2606. Email: firstname.lastname@example.org
Received 5 June 2002; accepted 22 November 2002.
N Petrovsky et al.
performed insulin tolerance tests (ITT), and administered
normal human subjects an oral dose of cortisone acetate to
determine whether there was a causal relationship between
cortisol and the level of plasma MIF.
Materials and Methods
After institution ethics committee approval and informed consent,
five healthy young adult male and female subjects had blood (10 mL)
taken each hour for 24 h for measurement of tetanus- or LPS-
stimulated whole blood cytokine production and for measurement of
plasma MIF, cortisol and melatonin. The subjects ranged in age from
20 to 45 years. During the study period the subjects maintained their
normal sleep/wake cycle and activity patterns. Blood samples were
taken hourly via an indwelling venous cannula inserted in a forearm
cubital fossa vein. On separate occasions, subjects were each given an
oral dose of cortisone acetate 25 mg at 21.00. Immediately after
venesection, blood was centrifuged and the plasma aspirated, ali-
quoted and frozen at –20°C until assayed for cytokines, MIF, cortisol
Pituitary function tests
For the insulin tolerance test (ITT), healthy subjects (n = 5) had an in-
dwelling forearm cannula inserted at 08.30; the subject remained
supine for the remainder of the study. At 09.00 (0 min), 0.15 U/kg
insulin (Actrapid, NovoNordisk, Denmark) was given as an intrave-
nous bolus. Blood was taken for measurement of plasma cortisol and
MIF at –30, 0, 30, 45, 60, 90 and 120 min.
Whole blood assay
The whole blood assay was performed as previously described (30).
Briefly, heparinized venous blood was aliquoted in quadruplicate at
280 µL/well into 96-well tissue culture plates (Falcon, Becton Dick-
inson, San Diego, USA) containing tetanus toxoid (10 Lyon floccu-
lating units/mL) or LPS (1 µg/mL) in 20 µL of human tonicity (HT)-
RPMI-1640 medium. The plates were incubated at 37°C in 5% CO2
atmosphere for 48 h and the plasma supernatants were then removed
and stored at –20°C until assayed for cytokines.
Plasma MIF levels were measured blindly with a routine MIF-
specific sandwich ELISA using recombinant human MIF as stand-
ard.11,12 Briefly, 96-well plates (Immulon II, Dynex Technologies,
Chantilly, France) were coated with anti-MIF monoclonal antibody
(R & D Systems, Minneapolis, MN, USA) 2.5 µg/mL in PBS 100 µL/
well overnight at 4°C. The plates were then washed and blocked with
Superblock (Pierce Chemical Co., Rockford, IL, USA) containing 2%
goat serum. After an additional wash in Tris-buffered saline contain-
ing 0.05% Tween-20, the samples were plated in duplicate and
incubated overnight at 4°C. After washing, plates were incubated
with rabbit anti-MIF polyclonal antibody (1 : 250) (Picower Institute,
New York, NY, USA), rewashed and incubated with alkaline phos-
phatase conjugated goat antirabbit IgG (Chemicon; 1 : 4000). The
concentration of MIF was visualized using p-nitrophenyl phosphate
(pNPP/ethanolamine) (Pierce Chemical Co.) as chromogen substrate.
The detection limit of plasma MIF using this ELISA is 1–2 ng/mL
with a detection limit for this study of 1.9 ng/mL.
Interferon-γ (sensitivity 0.1 U/mL) and IL-6 (sensitivity 2 pg/mL)
were measured by ELISA from CSL Australia Ltd and Biosource
International, respectively. The IL-10 ELISA (sensitivity 10 pg/mL)
used rat monoclonal antibodies (mAb) 9D7 and 12G8-NIP (Phar-
Mingen, San Diego, USA) for cytokine capture and detection,
respectively. The TNF-α ELISA (sensitivity 12.5 pg/mL) used mouse
mAb pair, 18631D and biotinylated 18642D (PharMingen) for
cytokine capture and detection, respectively. For both the IL-10 and
TNF-α ELISA capture mAb was coated overnight at 4°C onto Nunc
Maxisorb plates at a concentration of 5 µg/mL. The plates then were
washed and blocked with 10% BSA for 1 h at room temperature
(RT). Samples (50 µL) were added to wells and incubated overnight
at 4°C followed by washing and incubation with a secondary detec-
tion mAb at 1 µg/mL for 1 h at RT. This was followed by incubation
with 100 µL streptavidin-peroxidase (1 : 500) for 1 h. Colour was
developed with 100 µL tetramethyl benzidine (TMB) peroxidase
substrate for 30 min followed by addition of 1 M H2SO4.
Plasma cortisol was measured with the Orion Diagnostica cortisol
Plasma melatonin was measured using a direct radioimmunoassay
(RIA) based on tritium-labelled melatonin (Amersham International
Laboratories, Amersham, UK) and antiserum 8483 from J. Arendt,
Stockgrand, UK.24 The tracer concentration was 24 pmol/L and
antiserum dilution 1 : 45 000 in the reaction mixture with solid-phase
second antibody separation and direct liquid scintillation counting of
the bound fraction in the assay tube. The assay detection limit in
plasma was 17 pmol/L (3.9 pg/mL).
For group analysis, each cytokine measurement was expressed as the
percentage of the 24 h individual mean. Cosinor analysis was per-
formed with Chronolab, a software package for chronobiologic time
series analysis, kindly provided by L. Fernida, Bioengineering and
Chronobiology Laboratories, ETSI Telecommunie, University of
Vigo, Spain. Differences in cytokine production pre- and post-
cortisol were compared using the Student’s t-test.
Circadian variation in plasma MIF
Data were standardized by expressing each individual’s
results as a percentage of their 24 h mean. Plasma MIF
exhibited significant circadian variation by Cosinor analaysis
(P < 0.01) in each subject. Macrophage migration inhibitory
factor was very different in phase to the stimulated rhythms of
the other pro-inflammatory cytokines, namely IFN-γ, IL-1,
IL-12, and TNF-α, which all peaked during the late evening
and early morning at a time when plasma MIF was near its
nadir (Fig. 1). When group data were analysed, mean MIF
production peaked at 08.00 and reached a nadir at 03.00
(Table 1). In individual subjects, the time of peak MIF
production ranged from 06.00 to 09.00 and the time of the
nadir from 00.00 to 03.00. Peak MIF levels were significantly
(up to 4-fold) higher than nadir levels with a range from
approximately 2–8 ng/mL. The mean MIF peak to nadir
interval for the group was 18 h.
Circadian MIF rhythm
Relationship between plasma MIF and cortisol
There was a close temporal relationship between the rhythms
of MIF and plasma cortisol (Fig. 2). The MIF peak coincided
with or followed the plasma cortisol peak. A phase delay may
be anticipated between the rise in plasma cortisol and any
effect on the production or secretion of MIF. To see whether
there was a phase shift between the rhythms of cortisol and
MIF, the phases between the two cycles were progressively
adjusted to see whether a positive phase shift would maxi-
mize the correlation coefficients between the two cycles
(Fig. 3). This showed that there was a maximum positive
correlation between plasma MIF and cortisol levels (r = 0.53,
P < 0.001), when MIF levels were phase advanced by
between two and three hours, consistent with a lag between
changes in plasma cortisol and the effect on MIF.
Effect of low dose glucocorticoids on plasma MIF
Glucocorticoids induce macrophage secretion of MIF, in-
vitro. It is possible therefore that the circadian rhythm of
plasma MIF is directly entrained by plasma cortisol. Oral
cortisone acetate, 25 mg at 21.00, resulted in a rise in plasma
MIF although with a lag of 1–2 h, consistent with a causal
relationship (Fig. 4). This was in sharp contrast to the effects
of cortisone on the other pro-inflammatory cytokines, IFN-γ,
IL-12 and TNF-α, which were all markedly suppressed
Effect of high dose glucocorticoids on plasma MIF
Supraphysiological levels of glucocorticoids (dexamethasone
administered intravenously at 1 mg/h over four hours) resulted
in greater than 70% suppression of plasma MIF levels with
this suppression persisting at 24 h (Fig. 6). This is similar to
the bimodal effects of glucocorticoids on Th2 cytokines
where medium doses enhance whilst high doses suppress,
stimulated whole blood proinflammatory cytokine production
(IFN-γ [?], IL-12 [?], and TNF-α [✧]) and plasma macrophage
migration inhibitory factor (MIF, [
cytokines all peak during the late evening and early morning at a
time when plasma MIF and cortisol (?) are near their nadirs. To
allow comparison, data for each cytokine is presented as a per-
centage of the 24 h mean.
Relationship between the circadian rhythms of LPS-
]). The proinflammatory
(MIF) levels (mean and standard deviation) for a group of five healthy
subjects under normal conditions (second 2 columns) or with
administration of cortisone acetate 25 mg orally at 21.00 (last 2
Circadian plasma macrophage migration inhibitory factor
TimeBasal With cortisone at 21.00
MIF (ng/mL) MIF (ng/mL) Std. Dev. Std. Dev.
inhibitory factor (MIF) and its relationship to plasma cortisol for a
representative male subject. ?, MIF (ng/mL); ?, Cortisol (nM).
Circadian rhythm of plasma macrophage migration
N Petrovsky et al.
Relationship between plasma melatonin and MIF
The rhythm in plasma melatonin is in reverse phase to that of
cortisol; melatonin peaked at approximately 04.00 and was
low or unmeasurable during day-time, whereas cortisol
peaked during late morning around 09.00 and reached a nadir
at 04.00. The nadir in plasma MIF coincided temporally with
the peak in plasma melatonin (data not shown).
Origins of plasma MIF
To determine whether activated macrophage secretion con-
tributed to circulating MIF levels, the circadian levels of MIF
in LPS-stimulated whole blood in healthy subjects was
compared to the level of MIF in plasma. Incubation of whole
blood with LPS (1 µg/mL) for 24–48 h did not increase
plasma MIF levels (data not shown), suggesting that the
major source of plasma MIF was not peripheral blood
macrophages or T cells. This is in contrast to the other
proinflammatory cytokines such as IFN-γ, IL-1 and TNF-α,
whose secretion is markedly enhanced by incubation of whole
blood with LPS.1
Response of MIF to insulin-induced hypoglycaemia
To assess the contribution of the pituitary gland to overall
plasma MIF levels we performed ITT on healthy subjects
(n = 5). As expected, in response to insulin-induced hypo-
glycaemia, there was a significant rise in plasma cortisol and
growth hormone. This was not associated, however, with any
consistent change in plasma MIF (Fig. 7).
phage migration inhibitory factor (MIF) in a hypophysectomized
male subject on cortisone replacement. The correlation between
plasma MIF and cortisol was maximal when MIF levels were
phase advanced by between two and three hours relative to
plasma cortisol, consistent with a short delay between changes in
plasma cortisol and its effect on plasma MIF.
Phase relationship between plasma cortisol and macro-
plasma macrophage migration inhibitory factor (MIF) for a repre-
sentative 18-year-old male subject (n = 5). ?, MIF (ng/mL); ?,
Effect of oral cortisone acetate, 25 mg at 21.00, on
21.00, on plasma macrophage migration inhibitory factor (MIF)
and LPS-stimulated whole blood cytokine production. Interferon-γ,
TNF-α, IL-6 and IL-10 production is shown immediately before
and one hour postcortisone whereas plasma MIF is shown imme-
diately before and three hours postcortisone when the MIF
response was greatest. Data shown for one representative 18-year-
old male subject (mean ± SD of duplicate samples). ?, Pre-
cortisone; ?, Post-cortisone.
Comparison of effects of oral cortisone, 25 mg at
tory factor (MIF) (mean ± SD of duplicate samples) in response to
dexamethasone infusion (1 mg/ h) over four hours for a represent-
ative 42-year-old male subject.
Suppression of plasma macrophage migration inhibi-
Circadian MIF rhythm
Effect of hypophysectomy on plasma MIF
To further explore the contribution of the pituitary to plasma
MIF, we investigated the circadian profiles of MIF and
cortisol in a hypophysectomized patient who was on cortisol
replacement therapy (25 mg at 08.00 and 12.5 mg at 18.00)
(Fig. 8). Following his morning dose of cortisone there was
prolonged, at times complete suppression of antigen-
stimulated IFN-γ, TNF-α and IL-1 production. Plasma MIF
levels showed a maximum positive correlation with plasma
cortisol with a two hour phase difference between the two
cycles (r = 0.34, P < 0.05).
Relationship between plasma MIF and white cell count
The circadian rhythm in plasma MIF could reflect underlying
changes in the number of circulating MIF-producing lym-
phocytes. Although the peripheral blood white cell count
(WBC) and lymphocyte count demonstrated significant circa-
dian rhythmicity, both peaking at approximately 23.00, these
were negatively, rather than positively correlated with plasma
MIF (data not shown).
Plasma MIF exhibits a significant circadian rhythm in normal
human subjects, with a peak around 08.00 at a similar time to
the peak of plasma cortisol. Plasma MIF levels rose several
hours after oral cortisone ingestion. This finding is consistent
with data obtained in rats where plasma MIF levels rose after
administration of corticosterone.25 Macrophage migration
inhibitory factor is therefore unusual in being a pro-
inflammatory mediator whose secretion is increased rather
than decreased by physiological levels of glucocorticoids.
While studies of rodent or human monocytes/macrophages
in vitro have established a prominent MIF release response
to low-dose glucocorticoid stimulation, this effect is lost at
high-dose glucocorticoid levels (> 107 M), which act to
inhibit MIF expression.25 This would explain our observations
that low dose cortisone acetate stimulated, whereas high-dose
dexamethasone inhibited plasma MIF levels. Macrophage
migration inhibitory factor release into plasma therefore
appears to demonstrate a bimodal sensitivity to glucocorti-
coids depending whether they are at physiological or pharma-
The major tissue source of circulating plasma MIF is not
currently known. Pre-formed MIF accounts for ∼0.05% of
total protein in the anterior pituitary gland.15 In keeping with
the colocalization of MIF with adrenocorticotrophic hormone
(ACTH), stimulation of cultured pituitary cells with cortico-
trophin releasing hormone (CRH), the major stimulus for
ACTH release, results in the dose-dependent release of MIF.26
The secretion of MIF was found to occur at lower CRH
concentrations than those required to induce ACTH secretion.
Anterior pituitary cells also secrete MIF when stimulated with
LPS.15 However, although the pituitary gland contains signif-
icant quantities of MIF stored in secretory granules,26 we
observed no increase in plasma MIF in response to pituitary
activation during the ITT. This is supported by other recent
findings that also suggest the pituitary is not the major source
of circulating MIF.27 It was noticeable that plasma MIF levels
did not rise in parallel with plasma cortisol levels during the
ITT. This could be explained by the fact that plasma cortisol
levels were already high at the time of performing the ITT
and may have been sufficient to maximally stimulate MIF
production. Alternatively, because of the phase delay between
rises in cortisol and induction of MIF, the ITT may not have
been of sufficient duration to see a subsequent rise in plasma
MIF. The lack of a rise of MIF in response to pituitary
stimulation is consistent with the finding that a hypophysect-
omized patient had normal circulating levels of MIF despite
the absence of a functioning pituitary gland and demonstrated
a circadian rhythm of MIF that correlated with time of
administration of exogenous cortisone acetate. Instead, it
seems likely that pituitary MIF serves a local (autocrine)
rather than systemic role, perhaps by regulating pituitary axis
function.27,28 Other potential sources of circulating MIF
include macrophages, T-lymphocytes, endothelial cells,
Figure 7 Cortisol, growth hormone and macrophage migration
inhibitory factor (MIF) responses to insulin-induced hypo-
glycaemia (ITT) in healthy subjects (n = 5).
inhibitory factor (MIF) in a previously hypophysectomized male
subject receiving cortisone acetate replacement therapy (25 mg at
08.00 and 12.5 mg at 18.00). , Plasma MIF (ng/mL); ?, Plasma
Circadian variation of plasma macrophage migration
N Petrovsky et al.
eosinophils, hepatocytes, adipocytes, pancreatic islet β cells,
the Leydig cells of the testes and ovarian follicular cells.13–21
Although, LPS-simulates MIF release by macrophages in
vitro, LPS-stimulation had no effect on plasma MIF levels
in whole blood. This suggests that activated macrophages are
not likely to be the major source of circulating MIF.
What is the significance of the circadian rhythm in plasma
MIF? Given the role of MIF as a counter-regulator of the
immunosuppressive effects of cortisol, the early morning rise
in plasma MIF will tend to offset the immunosuppressive
effects of the early morning increase in plasma cortisol. The
major function of the morning rise in cortisol is to increase
sodium and water retention, increase blood pressure and
prepare for the transition from an inactive supine to active
vertical posture. There is a need to separate these effects of
cortisol from its effects on immune function. We would
propose therefore that the circadian rhythm in plasma MIF
acts to offset the extent of the cortisol-mediated circadian
variation of immune function.
Neutralizing antibodies to MIF (anti-MIF) have been
shown to inhibit delayed-type hypersensitivity (DTH) in
mice and to inhibit the generation of antigen-specific T- and
B-cell responses, in vivo.14,29 Circulating TNF-α levels were
reduced by 69% in mice protected from lethal Escherichia
coli peritonitis12 by anti-MIF. At least part of the pro-
inflammatory effects of MIF may be explained by its ability
to induce release of the pro-inflammatory cytokine TNF-α by
macrophages.13 In fact, MIF and TNF-α appear to form a
positive feedback loop, as TNF-α is itself able to induce MIF
secretion via a tyrosine-kinase dependent pathway. Similarly,
the pro-inflammatory cytokine IFN-γ stimulates MIF secre-
tion.13 Consistent with a pro-inflammatory role, MIF expres-
sion is increased in sepsis,12 renal allograft rejection,30 acute
uveitis,31 atopic dermatitis,32 adjuvant arthritis33 and human
rheumatoid arthritis.11 Anti-MIF treatment leads to profound,
dose-dependent inhibition of adjuvant arthritis in the rat
model.33 In an inflammatory setting the morning peak of MIF,
by antagonizing cortisol-mediated pro-inflammatory cytokine
suppression, may act to prolong the duration of early morning
inflammation. This may help explain the beneficial role of
MIF neutralization in arthritis models, given that one of the
major features of inflammatory arthritis is a disease flare in
the early morning that coincides temporally with the morning
rise in plasma MIF.
DS is a recipient of a scholarship from The Canberra Hospital
Salaried Specialists Private Practice Fund. Supported by NIH
grant 1RO1-AR049610-01 (RB).
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