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Endocrinology Vol. 146, No. 12 5128-5134
Copyright © 2005 by The Endocrine Society

Induction of Type 3 Deiodinase Activity in Inflammatory Cells of Mice with Chronic Local Inflammation

A. Boelen, J. Kwakkel, A. Alkemade, R. Renckens, E. Kaptein, G. Kuiper, W. M. Wiersinga and T. J. Visser

Departments of Endocrinology and Metabolism (A.B., J.K., A.A, W.M.W.) and Experimental and Internal Medicine (R.R.), Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands; and Department of Internal Medicine (E.K., G.K., T.J.V.), Erasmus Medical Center, 3000 DR Rotterdam, The Netherlands

Address all correspondence and requests for reprints to: Dr. Anita Boelen, Department of Endocrinology and Metabolism, F5-171, Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. E-mail: a.boelen{at}amc.uva.nl.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
During illness, changes in thyroid hormone metabolism occur, so-called nonthyroidal illness (NTI). NTI has been characterized by a fall of serum T3 due to decreased extrathyroidal conversion of T4 into T3 by liver type 1 deiodinase (D1), without an increase in serum TSH. Type 3 deiodinase (D3) was thought not to play an important role during NTI, but recently it has been shown that D3 activity is up-regulated in liver and skeletal muscle of critically ill patients related to hypoxia. We studied D3 gene expression and activity in liver and muscle/subcutis of mice during illness, which was induced by two different stimuli: bacterial endotoxin (lipopolysaccharide) administration, resulting in an acute systemic response, and a turpentine injection in each hindlimb, resulting in a local sc abscess. Lipopolysaccharide induced a rapid decrease in liver D1 and D3 activity but not skeletal muscle of hindlimb. In contrast, local inflammation induced by turpentine did not decrease liver D1 and D3 activity but increased markedly D3 activity in the muscle/subcutis sample containing the abscess, associated with strongly increased IL-1ß and IL-6 mRNA expression. Inflammatory cells, surrounding the abscess showed D3 and T3-transporter monocarboxylate transporter-8 immunoreactivity, whereas muscle cells did not show any immunoreactivity. In conclusion, local inflammation strongly induces D3 activity in inflammatory cells, especially in invading polymorphonuclear granulocytes, suggesting enhanced local degradation of T3.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
EXTRATHYROIDAL OR PERIPHERAL thyroid hormone metabolism is predominantly mediated by deiodinases [type 1 deiodinase (D1), type 2 deiodinase (D2), and type 3 deiodinase (D3)]. D1 and D2 catalyze the conversion of T4 into T3, whereas D3 catalyzes the inactivation of T4 into rT3 and T3 into 3,3-T2. D1 is predominantly located in the liver, kidney, thyroid, and pituitary and produces most of the circulating T3 under normal conditions (1). D2 activity is found in the central nervous system, pituitary, placenta, skin, and brown adipose tissue (only in rodents). D3 is expressed in the brain, skin, placenta, pregnant uterus, and several fetal tissues and plays an important role in protecting tissues from an excess of T3. D3 expression is regulated by thyroid hormones and ERK-activated pathways. It has been shown that glucocorticoids reduce D3 activity in tadpoles and chicken embryos (2).

Whereas thyroid hormones play an important role in the regulation of D1, D2, and D3 activity under normal metabolic conditions, other regulating mechanisms might be involved in thyroid hormone metabolism during pathopysiological conditions such as starvation, severe illness, or trauma. During these conditions, a state of altered thyroid hormone metabolism occurs [nonthyroidal illness (NTI)], which is characterized by decreased serum thyroid hormones accompanied by unaltered or slightly decreased serum TSH levels. Many mechanisms at several levels have been involved in the observed alterations among which decreased hepatic D1 activity (3). D3 was not supposed to play an important role during NTI but recently Peeters et al. (4) showed that D3 activity was induced in liver and skeletal muscle of critically ill patients. This increase in D3 activity was not related to inflammation (as characterized by serum C-reactive protein levels) but associated with those disease states with poor tissue perfusion, probably resulting in cellular hypoxia (4). Furthermore, D3 activity was observed in the failing ventricle during pathological hypertrophy of the heart induced in experimental animals (5).

However, the exact role of D3 induction during NTI is currently unknown. The aim of the present study therefore was to evaluate D3 gene expression and activity in liver and muscle of mice during illness. Two different stimuli were applied to induce NTI in mice: administration of a sublethal dose of lipopolysaccharide (LPS) and a turpentine-induced sterile abscess (6). Both stimuli result in an acute-phase response, although the time course and pattern of the acute-phase responses are different. LPS results in a systemic response, whereas turpentine injection results in a local abscess.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Female BALB/c mice (LPS) and C57BL6 (LPS and turpentine) (both Harlan Sprague Dawley, Horst, The Netherlands) were used at 6–12 wk of age. The mice were kept in 12-h light, 12-h dark cycles in a temperature-controlled room (22 C) and received food and water ad libitum. A week before the experiment, the mice were housed in groups according to the experimental set-up. Two different stimuli were used to induce nonthyroidal illness: 1) ip injection of 150 µg LPS (endotoxin; LPS, Escherichia coli 127:B8; Sigma Chemical Co., St. Louis, MO) diluted in 0.5 ml sterile 0.9% NaCl or 2) sc injection of 100 µl steam-distilled turpentine in each hindlimb.

Because of the diurnal variation of thyroid hormone-related genes, the experiments were performed using the same time schedule starting at 0900 h (t = 0). At different time points after LPS injection (t = 0, 4, 8, and 24 h) and after turpentine injection (t = 0, 8, 24, 48, and 120 h), four to five mice were anesthetized with isoflurane and killed. Blood was taken by cardiac puncture and serum was stored at –20 C until analyzed. The liver and one hindlimb were isolated. Skin and bone were removed from the hindlimb, leaving a tissue sample composed of muscle tissue and subcutis including the abscess if turpentine was administered. Both liver and muscle/subcutis/abscess were stored immediately in liquid nitrogen. The other hindlimb was isolated and fixed in 10% formaldehyde in PBS for 24 h and used for immunohistochemistry. The study was approved by the local animal welfare committee.

Serum thyroid hormones
Serum T3 and T4 were measured with in-house RIAs (7). To prevent interassay variation, all samples of one experiment were measured within the same assay.

RNA isolation and RT-PCR
mRNA was isolated from 10 mg liver and muscle/subcutis (with or without abscess) tissue of mice using the Magna Pure apparatus and the Magna Pure LC mRNA isolation kit II (tissue) (Roche Molecular Biochemicals, Mannheim, Germany) according to the manufacturer’s protocol, and cDNA synthesis was performed with the first-strand cDNA synthesis kit for RT-PCR (AMV) (Roche Molecular Biochemicals). Published primer pairs were used to amplify hypoxanthine phosphoribosyl transferase (a housekeeping gene) (8), IL-6, granulocyte macrophage-colony stimulating factor (GM-CSF), interferon-{gamma}, and IL-1ß (9). We designed primer pairs for D1 and D3 as described before (10). Real-time PCR was performed for quantitation of the above-mentioned mRNAs. cDNA standards for the different mRNAs were prepared from RNA of murine liver. For each mRNA assayed, a standard curve was generated using 10-fold serial dilutions of this target standard PCR product and the same primers used to amplify the cDNA. For each gene the standard protocol was optimized by varying MgCl2 concentrations. PCRs were set up with cDNA, MgCl2 (25 mM), SybrGreenI (Roche Molecular Biochemicals), forward and reverse primer, and H2O. The reactions were then cycled in the LightCycler (Roche Molecular Biochemicals) as described before (11). The LightCycler software generated a standard curve (measurements taken during the exponential phase of the amplification), which enabled the amount of each mRNA in each test sample to be determined. All results were corrected as to their mRNA content using hypoxanthine phosphoribosyl transferase mRNA.

Deiodinase activities
Liver (D1 and D3) and muscle/subcutis (D3) activities were determined as described before (4). Briefly, mouse liver and hindlimb muscle/subcutis (with or without abscess) samples were homogenized on ice in 10 volumes of PED1 buffer [0.1 M phosphate and 2 mM EDTA (pH 7.2)] using a Polytron (Kinematica AG, Lucerne, Switzerland). Homogenates were snap frozen in aliquots and stored at –80 C until further analysis. Protein concentration was measured with a protein assay (Bio-Rad Laboratories, Hercules, CA) using BSA as the standard following the manufacturer’s instructions.

D1 activity was measured by duplicate incubations of homogenates (~2 µg protein) for 30 min at 37 C with 0.1 µM [3',5'-125I]rT3 (100,000 cpm) in a final volume of 0.1 ml PED10 buffer (10 mM dithiothreitol). Reactions were stopped by addition of 0.1 ml 5% (wt/vol) BSA in water on ice. The protein-bound iodothyronines were precipitated by addition of 0.5 ml ice-cold 10% (wt/vol) trichloroacetic acid in water. After centrifugation, 125I– was isolated from the supernatant by chromatography on Sephadex LH-20 minicolumns.

D3 activities were measured by duplicate incubations of homogenates (~100 µg protein) for 60 min at 37 C with 1 nM [3'-125I]T3 (200,000 cpm) in a final volume of 0.1 ml PED50 buffer. Reactions were stopped by addition of 0.1 ml ice-cold methanol. After centrifugation, 0.1 ml of the supernatant was added to 0.1 ml 0.02 M ammonium acetate (pH 4), and 0.1 ml of the mixture was applied to 4.6 x 250 mm Symmetry C18 column connected to an Alliance HPLC system (Waters, Etten-Leur, The Netherlands). The column was eluted with a linear gradient of acetonitrile (28–42% in 15 min) in 0.02 M ammonium acetate (pH 4.0) at a flow of 1.2 ml/min. The activity in the eluate was measured on-line using a Radiomatic Z-500 flow scintillation detector (Packard, Meriden, CT).

D3 and monocarboxylate transporter 8 (MCT8) staining
Fixed muscle/subcutis (including the abscess) tissue was dehydrated in increasing concentrations of ethanol followed by xylene and embedded in paraffin. Sections (6 µm) were cut and mounted on superfrost plus slides and subsequently dried for at least 2 d at 37 C. After deparaffinization in xylene and rehydration through a graded ethanol series, sections were rinsed in aqua dest and washed in Tris-buffered saline (TBS) [0.05 M Tris and 0.15 M NaCl (pH 7.6)] for 3 x 10 min at room temperature (RT). Sections for D3 staining were microwave treated in TBS for 10 min at 700 W. MCT8 staining did not require microwave treatment. After adjustment to RT the sections were incubated with polyclonal rabbit anti-D3 antiserum 676 [1:900 in supermix (0.05 M Tris, 0.15 M NaCl, 0.25% gelatin, and 0.5% Triton X-100 (pH 7.6) supplemented with 1% BSA] or polyclonal rabbit anti-MCT8 antiserum 1306 (1:500 in supermix) for 1 h at RT and overnight at 4 C.

Antiserum 676 was raised against amino acids 265–278 of human D3 (12) and antiserum 1306 against amino acids 527–539 of human MCT8 (13). Specificity of antiserum 676 was recently described (14). Subsequently the slides were washed in TBS (3 x 10 min) and incubated with biotinylated goat antirabbit IgG antibody (1:400 in supermix; Vector, Burlingame, CA) for 1 h at RT. Slides were washed in TBS (3 x 10 min) and incubated with ABC-elite (avidin-biotin complex, 1:800 in supermix; Vector) for 1 h at RT. Again slides were washed in TBS (3 x 10 min) and incubated for 15 min at RT with 3,3'-diaminobenzidine in TBS containing 0.2% ammonium nickel sulfate and 0.01% H2O2. The enzyme reaction was stopped in aqua dest. Subsequently the sections were dehydrated through a graded ethanol series, cleared in xylene, and coverslipped with Entellan (Merck, Darnstadt, Germany). Adjacent sections (6 µm) were also stained with hematoxylin and eosin staining, preimmune serum, and antiserum that was preabsorbed with the specific peptide as follows: 10 µg of each synthetic peptide was dissolved in a medium containing 10% glycerol, 10% dimethylformamide, and 2.5% Nonidet P-40 (Sigma) and spotted (20 x 1 µl spots; 500 ng/µl) on 0.2% gelatin-coated nitrocellulose transfer membranes (0.1 µm; Schleicher & Schuell, Dassel, Germany) followed by overnight fixation with 4% paraformaldehyde filter paper using a press block. The membranes were rinsed in distilled water (3 x 10 min), TBS [0.05 M Tris and 0.15 M NaCl (pH 7.6); 3 x 10 min], and supermix [0.05 M Tris, 0.15 M NaCl, 0.25% gelatin, and 0.5% Triton X-100 (pH 7.6); 3 x 10 min]. The spotted homologous peptides were incubated with the first antiserum (antisera dilutions as used for immunocytochemistry) for 3–4 h at RT overnight at 4 C and again for 3–4 h at RT. This procedure was repeated one to three times until complete preabsorption was obtained, as confirmed by negative staining on the spotted nitrocellulose membrane and negative staining on muscle slides.

Statistics
Data are presented as the mean ± SEM. Differences between LPS or turpentine-treated and saline-treated mice were evaluated by ANOVA (two-way ANOVA) with two grouping factors (time and treatment). P values in the figures represent the significant effect of treatment. In case of time-related changes in the control group, times x treatment (interaction) values are also given. If the data were abnormally distributed or variances between groups were unequal, we performed a log transformation before ANOVA (15). The differences at a single time point were analyzed by the Student’s t test or the Mann Whitney U test where appropriate. Spearman’s coefficient of correlation was used for evaluation of the association between D3 activity and cytokine mRNA expression in a tissue sample of muscle and subcutis (with or without abscess). All statistical analyses were done using SPSS 11.5.1 (SPSS Inc. Chicago, IL). P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
LPS administration
LPS administration resulted in systemic illness characterized by piloerection and diarrhea within 4 h. Liver ILß mRNA expression was strongly increased (Ptreatment < 0.0001, Pinteraction < 0.0001), reaching maximal expression 4 h after LPS administration (Fig. 1Go).



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  		FIG. 1. Relative expression of IL-1ß mRNA in liver of mice after administration of LPS ({bullet}-{bullet}), compared with saline treatment ({circ}-{circ}) (left graph), and after turpentine-induced chronic inflammation ({bullet}-{bullet}), compared with saline treatment ({circ}-{circ}) (right graph). Mean values ± SEM are depicted; P value indicates treatment difference between groups by ANOVA. *, P < 0.05 and **, P < 0.01, differences between groups at a single time point. NS, Not significant.

 
Liver D1 mRNA expression (Ptreatment < 0.0001, Pinteraction < 0.0001) and activity (Ptreatment < 0.05, Pinteraction < 0.001) decreased significantly after LPS administration (Fig. 2Go). Diurnal variation was observed in the control animals, resulting in higher D1 mRNA expression 8 h after the start of the experiment. This variation was not mouse strain specific because it was also observed in C57BL6 mice under the same experimental conditions (relative D1 mRNA expression after LPS administration: t = 0, 0.41 ± 0.07; t = 4, 0.37 ± 0.09 (LPS) and 0.56 ± 0.11 (NaCl); t = 8, 0.15 ± 0.04 (LPS) and 0.59 ± 0.09 (NaCl); t = 24, 0.33 ± 0.05 (LPS) and 0.32 ± 0.09 (NaCl). Serum T3 and T4 were significantly decreased 24 h after injection, compared with t = 0 in both BALB/c and C57BL6 mice (Table 1Go). Liver D3 mRNA expression (Ptreatment < 0.0001, Pinteraction < 0.0001) and activity (Ptreatment < 0.0001, Pinteraction < 0.0001) were also significantly lower after LPS administration, compared with saline-treated animals (Fig. 3Go). D3 activity was very low in muscle/subcutis tissue of the hindlimb of both LPS-treated and control mice (Fig. 4Go).



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  		FIG. 2. Relative D1 mRNA expression and D1 activity in liver of mice after administration of LPS ({bullet}-{bullet}), compared with saline treatment ({circ}-{circ}) (systemic inflammation), and after turpentine-induced chronic inflammation ({bullet}-{bullet}), compared with saline treatment ({circ}-{circ}) (local inflammation). Mean values ± SEM are depicted; P values indicate treatment difference between groups by ANOVA. *, P < 0.05 and **, P < 0.01, differences between groups at a single time point. NS, Not significant.

 

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  		TABLE 1. Serum thyroid hormone levels (nanomoles per liter) at different time points (hours) after LPS or turpentine administration

 


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  		FIG. 3. Relative D3 mRNA expression and D3 activity in liver of mice after administration of LPS ({bullet}-{bullet}), compared with saline treatment ({circ}-{circ}) (systemic inflammation), and after turpentine-induced chronic inflammation ({bullet}-{bullet}), compared with saline treatment ({circ}-{circ}) (local inflammation). Mean values ± SEM are depicted; P values indicate treatment difference between groups by ANOVA. *, P < 0.05 and **, P < 0.01, differences between groups at a single time point. NS, Not significant.

 


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  		FIG. 4. D3 activity in muscle/subcutis tissue (with or without abscess) of mice after administration of LPS ({bullet}-{bullet}), compared with saline treatment ({circ}-{circ}) (systemic inflammation) (A), and after turpentine-induced chronic inflammation ({bullet}-{bullet}), compared with saline treatment ({circ}-{circ}) (local inflammation) (B). Mean values ± SEM are depicted; P values indicate treatment difference between groups by ANOVA. *, P < 0.05 and **, P < 0.01, differences between groups at a single time point. NS, Not significant.

 
Turpentine-induced sterile abscess
The sc injection with turpentine caused a sterile abscess of approximately 5 x 5 mm at the site of injection (hindlimb), resulting in serious discomfort and increasing liver IL-1ß mRNA expression, reaching statistical significance at 120 h (P < 0.05) (Fig. 1Go).

Liver D1 mRNA expression and activity did not differ after turpentine injection, compared with control mice (Fig. 2Go). Liver D3 mRNA expression (Ptreatment < 0.05, Pinteraction NS) and activity (Ptreatment < 0.01, Pinteraction < 0.05) were slightly decreased in turpentine-treated animals, compared with control mice (Fig. 3Go), whereas D3 activity in a tissue sample of skeletal muscle and subcutis including the turpentine-induced abscess increased significantly at 8, 48, and 120 h after injection (Ptreatment < 0.0001, Pinteraction < 0.0001) (Fig. 4Go) from 0.13 ± 0.04 fmol/min·mg (t = 0) to 4.13 ± 2.02 fmol/min·mg at 120 h after injection. Serum T3 and T4 were significantly decreased 8, 48, and 120 h after injection, compared with t = 0 (Table 1Go). IL-1ß and IL-6 mRNA expression were significantly increased in muscle/subcutis tissue containing an abscess, compared with control mice (IL-1ß, P < 0.001, and IL-6, P < 0.001), and expression levels increased simultaneously with D3 activity in this tissue (IL-1ß vs. D3: r = 0.40, P < 0.05, and IL-6 vs. D3: r = 0.62, P < 0.01). GM-CSF mRNA expression in muscle/subcutis tissue was also correlated with D3 activity (GM-CSF vs. D3: r = 0.49, P < 0.01; Fig. 5Go), although the turpentine-induced increase in GM-CSF mRNA expression was not significant (P = 0.078), compared with control mice. TNF{alpha} mRNA expression was not significantly increased in muscle/subcutis tissue containing the abscess and did not correlate with D3 activity (r = 0.25, NS). Muscle/subcutis interferon-{gamma} mRNA expression was not different in turpentine-treated mice, compared with control mice (data not shown).



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  		FIG. 5. Relationship between D3 activity and IL-1ß, IL-6, and GM-CSF mRNA expression in a sample of muscle tissue and subcutis of mice having a turpentine-induced abscess and control mice (n = 28). P value indicates statistical significance of Spearman’s coefficient of correlation, which was used for evaluation.

 
Immunohistochemistry of the inflamed hindlimb showed an abscess above the skeletal muscle as indicated by hematoxylin and eosin staining (Fig. 6AGo). The inflammatory cells surrounding the abscess showed D3 immunoreactivity (Fig. 6BGo), whereas no staining was present in myocytes. Incubation of slices with D3 preimmune serum and antiserum that was preabsorbed with the specific peptide did not result in any staining (Fig. 6Go, D and E). The T3 transporter MCT8 was also present in the cells surrounding the abscess as indicated by a positive MCT8 staining (Fig. 6CGo). Incubation of slices with MCT8 preimmune serum and antiserum that was preabsorbed with the specific peptide also did not result in any staining (Fig. 6Go, F and G). A detailed picture of the abscess showed that part of the D3-positive cells are polymorphonuclear granulocytes (Fig. 7Go).



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  		FIG. 6. Hindlimb section showing a turpentine-induced abscess in the subcutis. A, Hematoxylin and eosin staining. Note necrosis in the middle of the abscess, surrounded by a large number of granulocytes, lymphocytes, and macrophages. B, D3 immunocytochemistry (ab 676). C, MCT8 immunohistochemistry (ab1306). D, Staining with preabsorbed D3 anti-serum. E, D3 preimmune staining. F, Staining with preabsorbed MCT8 anti-serum. G, MCT8 preimmune staining. Bar, 500 µm.

 


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  		FIG. 7. Inflammatory cells that were stained with anti-D3 antibody as indicated by dark-colored cytoplasm. These inflammatory cells are part of the turpentine-induced abscess in the subcutis. Polymorphonuclear granulocytes, indicated by an arrow, could be observed.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Illness induces an acute-phase response, which is a major mechanism of the body to restore homeostasis and is heavily regulated by inflammatory cytokines (16). NTI might be viewed as an adaptive mechanism to protect the organism against excessive catabolism during illness. Involvement of cytokines in its pathogenesis classifies NTI as part of the acute-phase response (6). Proinflammatory cytokines decrease hepatic D1 gene expression and activity (17, 18), which contributes to a major extent to decreased serum T3. However, it has been shown recently that induction of hepatic D3, possible by cellular hypoxia, might also be an important contributor to the development of NTI (4). The aim of the present study therefore was to evaluate the role of D3 in liver and muscle of mice in two defined models of an acute-phase response, which differ in time course and cytokine pattern. Turpentine-induced sterile abscess was used because this experimental model corresponds closely to local tissue injury and inflammation and results in serial activation of specific inflammatory cytokines (19), whereas LPS administration induces very rapidly several inflammatory mediators from a variety of cells, resulting in a potent systemic response (20). Both stimuli result in decreased serum T3 and T4 levels (6, 10). Because thyroid hormone metabolism has a circadian rhythm that results in significantly higher liver D1 and thyroid hormone receptor-ß1 mRNA expression at the end of the day than in the morning, each time point has his own control group if necessary (21, 22).

Systemic illness, as induced by LPS, did not result in increased D3 mRNA expression and activity in the liver, an important organ involved in the acute-phase response. Turpentine injection, however, resulted in a strong increase in D3 activity at the inflammation site (subcutis above the skeletal muscle of the hindlimb) and also in markedly increased expression of IL-1ß, IL-6, and GM-CSF mRNA in this tissue. Immunohistochemistry showed D3 and MCT8 [a very active and specific thyroid hormone transporter (13)] staining of cells (predominantly granulocytes, lymphocytes, and macrophages) surrounding the turpentine-induced abscess, whereas muscle cells did not stain. A detailed picture of the abscess showed that part of the cells that stain positive for D3 are polymorphonuclear leukocytes (granulocytes). D3 activity therefore might be induced in granulocytes but also in lymphocytes and macrophages by cytokines or growth factors.

A few in vitro studies describe the stimulating effects of epidermal and fibroblast growth factors [epithelial growth factor and fibroblast growth factor (FGF)] on D3 mRNA expression and activity in primary cultures of various rat cells (such as brown preadipocytes and astrocytes) (23, 24). It has also been described that hepatic and cutaneous hemangioma (a vascular tumor consisting of myofibroblasts and endothelial cells) often express high levels of D3 activity, resulting in (consumptive) hypothyroidism. The proliferative phase of a hemangioma is characterized by increased expression of angiogenic growth factors such as basic FGF, which might in turn stimulate endothelial cells to produce substantial amounts of D3 (25, 26). These growth factors induce via their cellular receptors several intracellular downstream signaling cascades. Pallud et al. (24) found that the D3-inducing effect of basic FGF was at least partly mediated by activation of the Raf/MAPK kinase (MEK)/ERK signaling cascade. The MEK/ERK cascade is activated by many stimuli, i.e. growth factors (27), IL-1ß (28), and LPS (29), and phosphorylates and activates transcription factors like nuclear factor-{kappa}B and activator protein-1 (29). The MEK/ERK cascade is one of the three MAPK cascades, which is a major signaling system shared by various cell types. Activation of distinct MAPK subtypes is dependent on cell type and applied stimuli. It has been shown in human neutrophils that GM-CSF activates the MEK/ERK cascade strongly, whereas IL-1ß and TNF{alpha} activates this cascade weakly (30). It is also known that IL-1ß stimulation of HepG2 cells (a human hepatoma cell line) results in a mild induction of the of MEK/ERK cascade (31). Differences in cell types involved (granulocytes, lymphocytes, and liver cells) and tissue-specific factors thus might account for the difference in D3 activity between LPS-induced systemic inflammation and turpentine-induced local inflammation.

Our results show that a turpentine-induced abscess, one of the stimuli to induce NTI in mice as characterized by a marked decrease of serum T3 and T4, results in an induction of D3 activity in inflammatory cells that migrate to the site of inflammation. Part of the D3 protein is expressed by granulocytes and MCT8, a novel thyroid hormone transporter, is also present at the site of inflammation. It is, however, unlikely that the induced D3 activity was responsible for the observed decrease in serum T3 and T4 levels. Although the abscess shows an intense D3 staining and activity, the relatively small size of the lesions makes it unlikely that the increase in D3 activity was fully responsible for the decrease of serum thyroid hormones. Furthermore, the time course of the D3 activity increase does not correlate with the decrease in serum thyroid hormone concentrations. The observed decrease in serum thyroid hormone levels after turpentine injection might be due to diminished food intake during the first days of illness resulting in, for example, decreased T4 secretion by the thyroid (32, 33). A recent finding has been the recognition of constitutive androstane receptor-mediated induction of enzymes other than deiodinases involved in the clearance of thyroid hormone during fasting and illness (34). However, the increased D3 expression may contribute to the decrease in systemic T3 levels during severe illness as well as have an important effect locally on inflammatory cells as the resultant decrease in T3 levels may favor proliferation of these cells. Thus, the finding that D3 is significantly increased in an abscess containing inflammatory cells is very relevant in understanding NTI more fully.


   Acknowledgments
 
We thank J. Daalhuisen (Department of Experimental Internal Medicine, Academic Medical Center, Amsterdam) for expert biotechnical assistance; Dr. O. Bakker (Department of Endocrinology and Metabolism, Academic Medical Center) for expert immunohistochemical assistance; and the staff of the Laboratory of Endocrinology and Metabolism (Academic Medical Center) for measuring thyroid hormones. Dr. M. Tanck (Department of Clinical Epidemiology, Academic Medical Center) is thanked for his advice on the statistical analyses.


   Footnotes
 
First Published Online September 8, 2005

Abbreviations: D1, Type 1 deiodinase; D2, type 2 deiodinase; D3, type 3 deiodinase; FGF, fibroblast growth factor; GM-CSF, granulocyte macrophage-colony stimulating factor; LPS, lipopolysaccharide; MCT8, monocarboxylate transporter 8; MEK, MAPK kinase; NTI, nonthyroidal illness; RT, room temperature; TBS, Tris-buffered saline.

Received May 19, 2005.

Accepted for publication August 29, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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