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Neuroendocrine Research Laboratory, Departments of Psychiatry and Pharmacology, University of Pennsylvania, Philadelphia, Pennsylvania 19104
Address all correspondence and requests for reprints to: Eva Redei, Department of Psychiatry, Northwestern University Medical School, 303 East Chicago Avenue, 9–176, Chicago, Illinois 60611-3008. E-mail: e-redei{at}nwu.edu
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Abstract |
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and interleukin-1ß mRNA expression
increased dramatically in controls and even further in hypothyroid
animals, as measured by Northern blot analysis. In contrast,
T4-treated rats showed significant inhibition of these
inflammatory markers. Thus, the hyperthyroid state combined with
increased endogenous glucocorticoid levels is protective against
inflammatory challenges. The inverse relationship between preproTRH
expression and pituitary-adrenal function suggests the possibility of a
direct inhibitory link connecting the hypothalamic-pituitary-adrenal
and thyroid axes, and suggests alternative sites of therapeutic
intervention for rheumatoid arthritis and other inflammatory associated
disorders. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Susceptibility to RA increases if glucocorticoid levels are diminished (15), and arthritis-induced inflammation can dramatically increase after pharmacological or surgical adrenalectomy (15). Decreased endogenous glucocorticoid production also occurs in hypothyroidism (16, 17, 18). Conversely, elevated thyroid hormone levels usually increase activity of the HPA axis, resulting in elevated glucocorticoid levels (17). Thus, hypothyroidism and the subsequent lowering of glucocorticoids may result in increased inflammatory responses to SCW administration. Inversely, hyperthyroidism may be protective by increasing endogenous glucocorticoids that diminish inflammatory responses.
In the present study, we induced hypo- and hyperthyroid states by
long-term treatment with 6-propyl-thiouracil (PTU) and T4,
respectively. After establishing the appropriate steady state
condition, SCW preparation was administered, and inflammatory responses
were measured. These included messenger (RNA) mRNA levels in peritoneal
macrophages of the inflammatory cytokine interleukin (IL)-1ß and the
chemokine macrophage inflammatory protein-1 (MIP-1), which is
specifically increased in RA compared with other forms of arthritis
(19).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RNA isolation and Northern hybridization
The anterior pituitary and hypothalamus (optic chiasm to
mammillary bodies; dorsally to the anterior commisure) were rapidly
dissected on dry ice and stored at -80 C until RNA isolation.
Macrophages were harvested by injecting 5.0 ml sterile RPMI into the
peritoneal cavity, lavaging, and collecting the cells by centrifugation
(4 C, 1000 x g). Total RNA was isolated using TRIzol
reagent (Life Technologies, Gaithersburg, MD) following the
manufacturer’s protocol, with quality and quantity of RNA determined
by gel electrophoresis and spectrophotometry. Total RNA (20 µg/lane)
was electrophoresed in a formaldehyde denaturing 1.2% agarose gel,
transferred to a nylon Hybond-N+ membrane (Amersham Life Science,
Arlington Heights, IL) and UV cross-linked to the filter. Filters were
prehybridized for 4 h at 42 C with 50% formamide and 50%
Northern prehybridization solution (5 Prime
3 Prime, Boulder, CO).
Filters were hybridized for 16 h at 42 C in 50% formamide and
50% Northern Hybridization solution (5 Prime3 Prime, Boulder, CO)
with complementary DNA (cDNA) probes 32P-labeled using a
random primer labeling kit (Boehringer Mannheim, Indianapolis, IN). The
filters were washed twice for 15 min each in 2x SSC/0.1% SDS at 24 C,
twice for 30 min each in 0.1x SSC/0.1% SDS at 52 C, and exposed to
Kodak X-OMAT-5 film (Eastman Kodak, Rochester, NY). Densitometry was
carried out using an Image Analyzer, Macintosh-based BRAIN 2.1 system
(Drexel University, Philadelphia, PA) with gray scale calibration. All
comparisons were made from RNA samples hybridized on the same filter
and normalized to the housekeeping gene glyceraldehyde-3-phosphate
dehydrogenase (GAPD). Probes were stripped from filters before
rehybridization by washing in boiling water for 30 sec.
Probes
Filters were hybridized with cDNA probes for preproTRH,
glucocorticoid receptor (GR), CRH, POMC, IL-1ß, macrophage
inflammatory protein-1 (MIP-1) and GAPD. The preproTRH probe was
the 1322-bp rat preproTRH cDNA insert from a plasmid kindly provided by
Dr. Stephanie Lee, Tufts University, Boston, MA (21). The GR probe was
the 2.4-kilobase insert from plasmid 6RGR containing a rat GR cDNA (22)
and kindly provided by Dr. Keith Yamamoto, University of California,
San Francisco, CA. The CRH probe was a 320-bp PstI fragment
subcloned from a rat CRH cDNA kindly provided by Dr. Robert Thompson,
University of Michigan, Ann Arbor, MI. The POMC probe was the 923-bp
insert from plasmid pMKSU16, containing a mouse POMC cDNA (23) and
kindly provided by Dr. Jim Eberwine, University of Pennsylvania. The
IL-1ß probe was a 245-bp PCR product generated with primers published
previously (24). The MIP-1 cDNA was a kind gift of Dr. Michael
Prystowsky, Albert Einstein University, New York (25). The GAPD cDNA
was a 983-bp PCR product generated using primers purchased from
CLONTECH (Palo Alto, CA).
RIAs
Trunk blood was collected into chilled polypropylene tubes
containing 0.5 ml of a 0.15 M solution of EDTA, centrifuged
at 1000 x g for 25 min at 4 C, and the plasma stored
at -80 C until assayed. All the samples were analyzed together in one
assay. ACTH was measured as described previously (26) in 25 µl
unextracted plasma with antiserum that recognizes ACTH 1–24 and ACTH
1–39 on an equimolar basis, with 125I-ACTH as the tracer
(INCSTAR, Stillwater, MN). The assay sensitivity was 0.05 pg/tube with
an intraassay coefficient of variation of 6.3%. Corticosterone (CORT)
was measured in unextracted plasma as described previously (26) using
antiserum raised against CORT-3-carboxy-methyloxime:BSA, with
125I-CORT conjugate as the tracer (ICN Biomedicals, Carson,
CA). The assay sensitivity was 0.03 ng/tube and the intraassay
coefficient of variation was 12.3%. TSH standards and specific
antiserum were obtained from the National Hormone and Pituitary Agency
(NIDDK, Baltimore, MD). Rat TSH RP-2 was used for the iodination and
standards. The assay sensitivity was 1.0 pg/tube with an intraassay
coefficient of variation of 10.5%. RIAs for T3 and
T4 were performed using ImmuChem-coated tubes purchased
from ICN Pharmaceuticals (Costa Mesa, CA), following the recommended
protocol provided with each kit. The sensitivity limits were 10 ng/tube
and 0.5 µg/tube for T3 and T4, with
intraassay coefficients of variation of 4.1% and 5.2%,
respectively.
Statistical analysis
Data were analyzed by two-way ANOVA followed by a post
hoc Newman-Keuls comparisons test. In all statistical tests,
results with P < 0.05 were considered statistically
significant. The software used was NWA Statpak (Portland, OR).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HPT axis
Treatment with PTU and T4 had highly significant
effects on plasma levels of T4
(F[2,38] = 73.3; P < 0.001)
and T3 (F[2,38] = 11.9;
P < 0.001). PTU-treated rats had decreased plasma
levels of T4 (13.4 ± 1.5 vs. 29.6 ±
2.3 ng/ml in control) and T3 (261 ± 90 vs.
595 ± 72 pg/ml in control). T4-treated rats had
elevated plasma levels of T4 (142 ± 18 vs.
29.6 ± 2.1 ng/ml in control) and T3 (711 ± 72
vs. 595 ± 72 pg/ml in control). As shown in Fig. 1A
, PTU-treated rats had plasma TSH levels that were
20-fold higher than controls (F[2,37] = 111;
P < 0.001). T4-treated rats had
significantly reduced TSH levels that were 2.5-fold below control
values (P < 0.01). PreproTRH mRNA levels in the
hypothalamus also were significantly altered by thyroid status
(F[2,38] = 6.02, P < 0.01): a
20% decrease in the T4-treated group and a 52% increase
(P < 0.01) in the PTU-treated group (Fig. 1B),
demonstrating feedback regulation of preproTRH synthesis by thyroid
hormones. No significant changes in thyroid function were observed
between saline and SCW-injected rats.
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). Although the changes in plasma ACTH levels were not
significant in saline-injected rats (Fig. 2B), they were
consistent with plasma CORT levels, as were changes in POMC mRNA
levels: T4-treated animals increased POMC expression by
50%, whereas POMC mRNA levels were less than half of controls in the
PTU rats (Fig. 3A). Thus, the hyper- and hypothyroid
states produced the expected results in HPA activity before inducing
the inflammation by SCW.
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) were also significantly modulated by thyroid status, but in a
manner opposite from the CORT pattern. High CORT values in the
hyperthyroid saline-injected rats were associated with lower GR and CRH
mRNA levels, whereas PTU treatment produced decreased CORT levels but
elevated CRH mRNA, indicating negative feedback regulation.
There was a significant effect of the SCW injection on POMC expression
(F[1,33] = 8.14; P < 0.01,
Fig. 3A) with a 3- to 4-fold increase in controls and hypothyroid rats,
but no increase in hyperthyroid animals (Fig. 3A). Hypothalamic CRH
mRNA levels were increased only in the control animals in response to
the inflammatory challenge (interaction
F[2,37] = 6.05, P < 0.01;
Fig. 3B).
Inflammation
Three days after injection, SCW produced slightly swollen hind
paws and ankle joints in control and PTU-treated rats. In addition, one
of eight controls and three of eight PTU-treated rats died within
48 h of SCW injection; in contrast, no T4-treated rats
died. In control rats SCW injection produced a dramatic induction of
MIP-1 mRNA (Fig. 4A). SCW-injected hypothyroid rats
showed a further increase by 30%. However, in
SCW-injected/T4-treated rats, there was a substantial
10-fold drop in MIP-1 expression compared with SCW-injected/control
rats (P < 0.01). IL-1ß mRNA levels followed a
pattern similar to MIP-1, with T4 again suppressing
the macrophage response to SCW (interaction
F[2,36] = 8.09; P < 0.01;
Fig. 4B), and PTU treatment aggravating the SCW-induced macrophage
IL-1ß inflammatory response (P < 0.01). These data
demonstrate that the hypothyroid state exacerbates, whereas the
hyperthyroid state nearly abolishes, these inflammatory responses to
SCW administration.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Intervening peptide sequences flank the five copies of TRH present within the rat preproTRH prohormone (29). Recently, our laboratory has identified that one of these peptides inhibits ACTH synthesis and secretion from anterior pituitary cells. This peptide is preproTRH 178–199 (30, 31), which has been localized to neurons within the paraventricular nucleus of the hypothalamus (32), as well as the external layer of the median eminence (33). Furthermore, this peptide has the ability to reduce stress-induced plasma ACTH and CORT levels in vivo (34). The in vitro and in vivo characteristics of this peptide suggest that it may be a physiological regulator of ACTH production: an endogenous corticotropin release inhibiting factor. The discovery of a corticotropin release inhibiting factor encoded within the same precursor as TRH establishes a potential link between the HPA and HPT axes for the first time. It has been determined that diminishing the endogenous production of glucocorticoids increases the susceptibility to SCW-induced inflammation (15). Our hypothesis was that hypothyroidism would produce the same effect. Consequently, low levels of thyroid hormones could up-regulate preproTRH synthesis with an attendant increase in the secretion of preproTRH 178–199. Conversely, increased thyroid hormones could lead to elevated levels of glucocorticoids by inhibiting preproTRH mRNA and therefore preproTRH 178–199 production. Thus, hypothyroidism may contribute to the susceptibility to RA by diminishing the activity of the HPA axis and thereby reducing glucocorticoid levels at the time of the inflammatory challenge. Manipulations of the HPT axis showed that in the hypothyroid state plasma TSH was elevated, whereas T3/T4 levels were diminished, with the reverse being found in the hyperthyroid state, as previously described (35). PreproTRH mRNA followed these tendencies suggesting that, in theory, preproTRH 178–199 could be decreased or increased appropriately to regulate ACTH. Indeed, we found an inverse relationship between preproTRH and POMC gene expression similar to a previous report (16). Despite this similarity in the POMC response, Shi et al. (16) observed a decrease in the CRH mRNA response to hypothyroidism. This difference could be due to time course (2 weeks of PTU treatment, compared with our 7-week regimen), methodology (in situ hybridization compared with Northern blots), or gender differences, because they employed young male rats. In the present study, there was a significant decrease in CORT levels in saline-injected/PTU-treated rats, similar to findings by Kamilaris et al. (17). That this HPT-derived inhibitory influence may be an important regulatory control point of POMC synthesis is suggested by the fact that T4 treatment decreased CRH but increased POMC, whereas PTU increased CRH but decreased POMC. Thus, regulation of POMC appeared to be dissociated from its primary activator CRH. Specifically, changes in hypothalamic GR and CRH mRNA levels reflected glucocorticoid negative feedback: GR and CRH expression increased when CORT was low (hypothyroid) and decreased when CORT was high (hyperthyroid). In contrast, POMC mRNA expression decreased in hypothyroid rats in the presence of low CORT and increased in hyperthyroid rats in the presence of normal to high CORT. This suggests that an additional factor other than glucocorticoid negative feedback may inhibit POMC expression.
Previous studies have demonstrated the application of SCW-induced
inflammation as a model for RA (2, 3, 14, 15), with MIP-1 shown to
be a specific marker for RA in humans (19). We found that both IL-1ß
and MIP-1 expression were increased in response to SCW
administration in control animals. Expression of IL-1ß and MIP-1
was increased in hypothyroid SCW-treated rats, whereas prolonged
T4 treatment dramatically decreased these adverse
inflammatory effects of SCW. These findings suggest T4 may
have direct antiinflammatory effects on macrophages. Hyperthyroidism
due to T4 was reported to decrease cell migration and
macrophage hydrogen peroxide production, whereas hypothyroidism
increased macrophage phagocytic activity and glucose utilization (36).
This suggests that thyroid receptors present in macrophages contribute
to this response, especially considering the profound ability of
T4 to inhibit MIP-1 and IL-1ß mRNA. In a recent
review, thyroid dysfunction, primarily hypothyroidism, was found in
female patients with RA almost three times more often than in control
women (37). Conversely, patients with subclinical hypothyroidism show
improvement of symptoms after T4 replacement (38). Similar
beneficial effects of T4 were reported in another animal
model, adjuvant-induced arthritis (39).
Neuroendocrine HPA responses to an inflammatory challenge are thought to be mediated by IL-1ß, because IL-1ß is produced in response to inflammatory agents (40, 41). Although IL-1ß is known to increase CRH secretion from the hypothalamus (42), we found that hypothalamic CRH expression in SCW-treated rats did not closely follow the IL-1ß pattern from macrophages in response to altered thyroid status, whereas the anterior pituitary POMC expression did. This suggests that IL-1ß may mediate a response to SCW by directly increasing POMC expression. Interestingly, POMC mRNA levels were high 3 days after SCW injection in control and hypothyroid rats, the same ones that showed very high levels of IL-1ß expression. In contrast, neither POMC nor the inflammatory markers changed in the hyperthyroid rats. We found no increases in plasma CORT or ACTH in response to SCW in either group. This phenomenon, together with the rather high CORT levels in saline-injected rats, suggests that an acute environmental stressor may have masked the subtle long-term effect of SCW. Because POMC mRNA levels are not expected to change rapidly, they are more accurate in reflecting HPA activity in response to the SCW challenge. Thus, the lower basal CORT levels found in controls and PTU-treated animals, allow higher POMC as well as inflammatory responses. These observations further suggest that hyperthyroid rats with their increased basal HPA activity could become refractory to an otherwise strong challenge, the inflammation. Thus, thyroid hormone-induced changes in basal HPA activity could determine susceptibility and responsiveness to inflammation.
The results of this study demonstrate that T4 affects macrophages to suppress inflammatory responses, either through a direct or indirect mechanism. Furthermore, the evidence provided here supports the existence of a direct link connecting the HPA and HPT endocrine axes, which would have great potential for determining alternative sites of therapeutic intervention for RA. Although administration of large doses of glucocorticoids are frequently the treatment of choice for reducing the inflammation associated with RA, they nonetheless have numerous side effects. Thyroid hormones may play a fundamental role in regulating the HPA axis by adjusting the levels of preproTRH and subsequently preproTRH 178–199. If this putative corticotropin release inhibiting factor proves to be an important component in the development of RA, then regulation of preproTRH may be highly relevant to the development of other autoimmune diseases that are also exacerbated by low endogenous glucocorticoid levels.
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Acknowledgments |
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Footnotes |
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Received September 9, 1996.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B in mediation of
immunosuppression by glucocorticoids. Science 270:283–290B
activity through induction of IB synthesis. Science 270:286–290: a novel chemotactic cytokine for macrophages in
rheumatoid arthritis. J Clin Invest 93:921–928
mRNA in the brain after peripheral endotoxin treatment:
comparison with interleukin-1 family and interleukin-6. Brain Res 624:291–294[CrossRef][Medline]
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