Maleic acid is a biomarker for maleylacetoacetate isomerase deficiency; implications for newborn screening of tyrosinemia type 1.

Dried blood spot succinylacetone (SA) is often used as a biomarker for newborn screening (NBS) for tyrosinemia type 1 (TT1). However, false-positive SA results are often observed. Elevated SA may also be due to maleylacetoacetate isomerase deficiency (MAAI-D), which appears to be clinically insignificant. This study investigated whether urine organic acid (uOA) and quantitative urine maleic acid (Q-uMA) analyses can distinguish between TT1 and MAAI-D. We reevaluated/measured uOA (GC-MS) and/or Q-uMA (LC-MS/MS) in available urine samples of nine referred newborns (2 TT1, 7 false-positive), eight genetically confirmed MAAI-D children, and 66 controls. Maleic acid was elevated in uOA of 5/7 false-positive newborns and in the three available samples of confirmed MAAI-D children, but not in TT1 patients. Q-uMA ranged from not detectable to 1.16 mmol/mol creatinine in controls (n = 66) and from 0.95 to 192.06 mmol/mol creatinine in false-positive newborns and MAAI-D children (n = 10). MAAI-D was genetically confirmed in 4/7 false-positive newborns, all with elevated Q-uMA, and rejected in the two newborns with normal Q-uMA. No sample was available for genetic analysis of the last false-positive infant with elevated Q-uMA. Our study shows that MAAI-D is a recognizable cause of false-positive TT1 NBS results. Elevated urine maleic acid excretion seems highly effective in discriminating MAAI-D from TT1.

Effects of Chronic Estrogen Administration in the Ventromedial Nucleus of the Hypothalamus (VMH) on Fat and Bone Metabolism in Ovariectomized Rats.

Estrogen deficiency after ovariectomy (OVX) results in increased adiposity and bone loss, which can be prevented by systemic 17-ß estradiol (E2) replacement. Studies in transgenic mice suggested that in addition to direct actions of estrogen in peripheral tissues, also estrogen signaling in the hypothalamus regulates fat distribution and bone metabolism. We hypothesized that the protective effect of systemic E2 on fat and bone metabolism in the OVX model is partly mediated through the ventromedial nucleus of the hypothalamus (VMH). To test this hypothesis, we determined the effect of systemic, central, and targeted VMH administration of E2 on fat and bone metabolism in OVX rats. Subcutaneous administration of E2 for 4 weeks decreased body weight, gonadal and perirenal fat, and bone formation rate in OVX rats. This effect was completely mimicked by intracerebroventricular injections of E2, once every 4 days for 4 weeks. Administration of E2 locally in the VMH by retromicrodialysis (3 h) acutely increased expression of the lipolytic gene hormone-sensitive lipase in gonadal and perirenal fat. Finally, chronic administration of E2 in the VMH for 8 weeks decreased perirenal fat but did not affect body weight, trabecular bone volume, or cortical thickness. In conclusion, we demonstrated that intracerebroventricular E2 replacement reduces body weight gain, ameliorates intraabdominal fat accumulation, and reduces bone formation in the OVX rats. E2 administration selectively in the VMH also reduced intraabdominal fat but did not affect bone metabolism.

Central regulation of the hypothalamo-pituitary-thyroid (HPT) axis: focus on clinical aspects.

The hypothalamus is the most prominent brain region involved in setpoint regulation of the thyroid axis. It generates the diurnal thyroid-stimulating hormone (TSH) rhythm, and it plays a central role in the adaptation of the thyroid axis to environmental factors such as caloric deprivation or infection. Many studies, including studies in human post-mortem tissue samples, have confirmed a key role for the thyrotropin-releasing hormone (TRH) neuron in the hypothalamic paraventricular nucleus (PVN) in thyroid axis regulation. In addition to their negative feedback action on TRH neurons in the hypothalamus, intrahypothalamic thyroid hormones can also modulate metabolism in adipose tissue and the liver via the autonomic nervous system. Congenital or acquired dysfunction of the hypothalamus or pituitary gland may result in central hypothyroidism (CeH). In the Netherlands, the prevalence of permanent congenital CeH as detected by neonatal screening is approximately 1 in 18000. In most neonates congenital CeH is accompanied by additional anterior pituitary hormone deficiencies, and many show clear morphological abnormalities such as a small anterior gland, a thin or absent pituitary stalk, or an ectopic posterior pituitary gland. Recently, a mutation in the immunoglobulin superfamily member 1 (IGSF1) gene was reported as a novel cause of X-linked, apparently isolated CeH occurring in neonates, children and adults. In adults, the most frequent cause of acquired CeH is a pituitary macroadenoma, usually accompanied by other pituitary hormone deficiencies. Central hyperthyroidism is a rare disorder, especially in children. In adults, it is mostly caused by a TSH-secreting pituitary adenoma.

Fasting-induced changes in hepatic thyroid hormone metabolism in male rats are independent of autonomic nervous input to the liver.

During fasting, profound changes in the regulation of the hypothalamus-pituitary-thyroid axis occur in order to save energy and limit catabolism. In this setting, serum T3 and T4 are decreased without an appropriate TSH and TRH response reflecting central down-regulation of the hypothalamus-pituitary-thyroid axis. Hepatic thyroid hormone (TH) metabolism is also affected by fasting, because type 3 deiodinase (D3) is increased, which is mediated by serum leptin concentrations. A recent study showed that fasting-induced changes in liver TH sulfotransferases (Sults) and uridine 5'-diphospho-glucuronosyltransferase (Ugts) depend on a functional melanocortin system in the hypothalamus. However, the pathways connecting the hypothalamus and the liver that induce these changes are currently unknown. In the present study, we investigated in rats whether the fasting-induced changes in hepatic TH metabolism are regulated by the autonomic nervous system. We selectively cut either the sympathetic or the parasympathetic input to the liver. Serum and liver TH concentrations, deiodinase expression, and activity and Sult and Ugt expression were measured in rats that had been fasted for 36 hours or were fed ad libitum. Fasting decreased serum T3 and T4 concentrations, whereas intrahepatic TH concentrations remained unchanged. D3 expression and activity increased, as was the expression of constitutive androstane receptor, Sult1b1, and Ugt1a1, whereas liver D1 was unaffected. Neither sympathetic nor parasympathetic denervation affected the fasting-induced alterations. We conclude that fasting-induced changes in liver TH metabolism are not regulated via the hepatic autonomic input in a major way and more likely reflect a direct effect of humoral factors on the hepatocyte.

A novel role for the thyroid hormone-activating enzyme type 2 deiodinase in the inflammatory response of macrophages.

Deiodinase type 2 (D2) is a thyroid hormone-activating enzyme converting the prohormone T4 into the active hormone T3. In the present study, we show for the first time that D2 is up-regulated in the mouse liver during acute and chronic inflammation, in close correlation with the proinflammatory cytokine IL-1ß and independently of serum T3. Inflammation-induced D2 expression was confirmed in macrophages, in conjunction with selective thyroid hormone transporter (monocarboxylate transporter 10) and thyroid hormone receptor (TR)α1 stimulation, and was absent in hepatocytes. Moreover, D2 knockdown in macrophages resulted in a clear attenuation of the lipopolysaccharide (LPS)-induced IL-1ß and GM-CSF expression, in addition to aberrant phagocytosis. Locally produced T3, acting via the TRα, may be instrumental in this novel inflammatory response, because LPS-treated TRα(0/0) mice showed a markedly decreased LPS-induced GM-CSF mRNA expression. We now propose that hepatic D2 favors the innate immune response by specifically regulating cellular thyroid hormone levels in macrophages.

NFκB signaling is essential for the lipopolysaccharide-induced increase of type 2 deiodinase in tanycytes.

The enzyme type 2 deiodinase (D2) is a major determinant of T3 production in the central nervous system. It is highly expressed in tanycytes, a specialized cell type lining the wall of the third ventricle. During acute inflammation, the expression of D2 in tanycytes is up-regulated by a mechanism that is poorly understood at present, but we hypothesized that cJun N-terminal kinase 1 (JNK1) and v-rel avian reticuloendotheliosis viral oncogene homolog A (RelA) (the 65 kD subunit of NFκB) inflammatory signal transduction pathways are involved. In a mouse model for acute inflammation, we studied the effects of lipopolysaccharide (LPS) on mRNA expression of D2, JNK1, and RelA in the periventricular area (PE) and the arcuate nucleus-median eminence of the hypothalamus. We next investigated LPS-induced D2 expression in primary tanycyte cell cultures. In the PE, the expression of D2 was increased by LPS. In the arcuate nucleus, but not in the PE, we found increased RelA mRNA expression. Likewise, LPS increased D2 and RelA mRNA expression in primary tanycyte cell cultures, whereas JNK1 mRNA expression did not change. Phosphorylation of RelA and JNK1 was increased in tanycyte cell cultures 15-60 minutes after LPS stimulation, confirming activation of these pathways. Finally, inhibition of RelA with the chemical inhibitors sulfasalazine and 4-Methyl-N¹-(3-phenylpropyl)benzene-1,2-diamine (JSH-23) in tanycyte cell cultures prevented the LPS-induced D2 increase. We conclude that NFκB signaling is essential for the up-regulation of D2 in tanycytes during inflammation.

Optimal management of Graves orbitopathy: a multidisciplinary approach.

Graves' thyroid disease is a relatively common disorder in endocrinology and general internal medicine practice. Graves' hyperthyroidism is mediated by circulating stimulating autoantibodies. Up to 60% of patients with Graves' hyperthyroidism develop some form of Graves' orbitopathy. Immune reactivity to the thyroid-stimulating hormone receptor is also thought to play a role in the immunopathogenesis of Graves' orbitopathy. Graves' orbitopathy is characterised by a wide open eye appearance, caused by upper eyelid retraction and soft-tissue swelling that causes exophthalmus. Symptoms include photophobia, sandy feeling in the eye, painful eye movements and diplopia. Visual acuity may be reduced. In some cases emergency treatment is necessary to prevent irreversible vision loss. Smoking should be stopped. Mild to moderate Graves' orbitopathy may be an indication for corticosteroid treatment or radiotherapy. Once inflammatory signs and symptoms have waned, surgery can be performed to correct residual diplopia, exophthalmus or lid retraction. The presence of Graves' orbitopathy has consequences for the management of Graves' hyperthyroidism. Adequately controlled Graves' thyroid dysfunction is likely to improve Graves' orbitopathy, while radioactive iodine treatment can worsen the condition. Due to the wide variety in clinical presentation and the possible interference between treatment of thyroid disease and eye disease, the management of more complicated patients with Graves' orbitopathy can best be performed in combined thyroid-eye clinics, in which the patient is seen simultaneously by the ophthalmologist and the endocrinologist.

Acute inflammation increases pituitary and hypothalamic glycoprotein hormone subunit B5 mRNA expression in association with decreased thyrotrophin receptor mRNA expression in mice.

The biological function of thyrostimulin, consisting of the GPA2 and GPB5 subunit, is currently poorly understood. The recent observation that pro-inflammatory cytokines up-regulate the transcription of GPB5 in vitro suggested a role for thyrostimulin in the nonthyroidal illness syndrome, a state of altered thyroid hormone metabolism occurring during illness. In the present study, we used GPB5 knockout (GPB5(-/-) ) and wild-type (WT) mice to evaluate the role of GPB5 in the pituitary and hypothalamus during acute inflammation induced by lipopolysaccharide (LPS, bacterial endotoxin) administration. We evaluated serum thyroid hormones and mRNA expression of genes involved in thyroid hormone metabolism in the pituitary and in two hypothalamic regions; the periventricular region (PE) and the arcuate nucleus/median eminence region. As expected, LPS administration increased deiodinase type 2 mRNA in the PE, at the same time as decreasing pituitary thyrotrophin (TSH)ß mRNA and serum thyroxine and triiodothyronine both in GPB5(-/-) and WT mice. GPB5 mRNA, but not GPA2 mRNA, markedly increased after LPS in the pituitary (200-fold) and hypothalamus of WT mice. In addition, we found large (>50%) suppression of TSH receptor (TSHR) mRNA in the pituitary and hypothalamus of WT mice but not in GPB5(-/-) mice. In conclusion, our results demonstrate in vivo regulation of central GPB5 transcription during acute illness. The observed differences between GPB5(-/-) and WT mice point to a distinct role for GPB5 in pituitary and hypothalamic TSHR suppression during acute illness.

Skeletal muscle deiodinase type 2 regulation during illness in mice.

We have previously shown that skeletal muscle deiodinase type 2 (D2) mRNA (listed as Dio2 in MGI Database) is upregulated in an animal model of acute illness. However, human studies on the expression of muscle D2 during illness report conflicting data. Therefore, we evaluated the expression of skeletal muscle D2 and D2-regulating factors in two mouse models of illness that differ in timing and severity of illness: 1) turpentine-induced inflammation, and 2) Streptococcus pneumoniae infection. During turpentine-induced inflammation, D2 mRNA and activity increased compared to pair-fed controls, most prominently at day 1 and 2, whereas after S. pneumoniae infection D2 mRNA decreased. We evaluated the association of D2 expression with serum thyroid hormones, (de-)ubiquitinating enzymes ubiquitin-specific peptidase 33 and WD repeat and SOCS box-containing 1 (Wsb1), cytokine expression and activation of inflammatory pathways and cAMP pathway. During chronic inflammation the increased muscle D2 expression is associated with the activation of the cAMP pathway. The normalization of D2 5 days after turpentine injection coincides with increased Wsb1 and tumor necrosis factor alpha expression. Muscle interleukin-1beta (Il1b) expression correlated with decreased D2 mRNA expression after S. pneumoniae infection. In conclusion, muscle D2 expression is differentially regulated during illness, probably related to differences in the inflammatory response and type of pathology. D2 mRNA and activity increases in skeletal muscle during the acute phase of chronic inflammation compared to pair-fed controls probably due to activation of the cAMP pathway. In contrast, muscle D2 mRNA decreases 48 h after a severe bacterial infection, which is associated with local Il1b mRNA expression and might also be due to diminished food-intake.

Iron deficiency among children of asylum seekers in the Netherlands.

OBJECTIVES: To investigate, in asylum seekers' children in the Netherlands, biochemical iron status and the prevalence of iron deficiency (ID) and anemia in relation to age, region of origin, length of stay in the Netherlands, body mass index (BMI), and dietary iron intake. PATIENTS AND METHODS: Hemoglobin (Hb) and plasma ferritin concentrations were determined in 122 asylum seekers' children (median age, 7.1 years; range, 2-12 y). ID was defined by plasma ferritin levels <15 microg/L. Anemia was defined by Hb levels <6.8 mmol/L (11 g/dL) for children <6 years of age and Hb levels <7.1 mmol/L (11.5 g/dL) for children between 6 and 12 years of age. Nutritional status of the children was assessed by BMI and dietary intake of iron was estimated by 24-hour recall. RESULTS: Twenty percent of the children had compromised iron status (16% with ID, 4% with ID anemia [IDA]). Another 6% of the children had anemia caused by thalassemia. ID was significantly more prevalent in children <6 years of age and in children of African origin. The iron status was not significantly correlated with the length of stay in the Netherlands (r = 0.6; P = 0.48). Higher BMI z scores were positively correlated with iron status. Adequate or marginal dietary iron intake was not significantly related to the presence of ID (r = 0.02; P = 0.9) or anemia (IDA and thalassemia; r = 0.15; P = 0.9). CONCLUSIONS: Iron deficiency is highly prevalent among the children of asylum seekers in the Netherlands. Our data indicate that systematic biochemical screening for ID is warranted in asylum seekers' children.

High incidence of hypermethioninaemia in a single neonatal intensive care unit detected by a newly introduced neonatal screening programme.

From 1 January 2007 an expanded neonatal screening programme was initiated in the Netherlands, including homocystinuria with methionine as the primary marker. During the first 2 months hypermethioninaemia was detected in 14 newborns who, after proper evaluation, were demonstrated not to have classical homocystinuria. Remarkably, all these children were admitted to a single neonatal intensive care unit (Academic Medical Center, Amsterdam (AMC-NICU)). We evaluated the possible causes for this finding. The cohort of newborns with hypermethioninaemia (group 1) was compared with the cohort of newborns with normal screening results admitted to the AMC-NICU in the same time period (group 2). In addition, parenteral nutrition protocols from all NICUs in the Netherlands were compared. Mean birth weight and gestational age were significantly lower in group 1 than in group 2. All patients in group 1 received parenteral feeding (TPN) at the time of screening and received a higher mean amino acid intake per kilogram body weight than patients receiving TPN in group 2. Also, the AMC-NICU uses a different amino acid mixture for TPN than the other Dutch NICUs, containing more than twice the amount of methionine per gram of amino acids compared with other mixtures. The high incidence of hypermethioninaemia in the AMC-NICU is explained by a combination of low birth weight, low gestational age, and high protein intake supplied by a specific parenteral amino acid mixture containing large amounts of methionine. To prevent hypermethioninaemia, the use of high-methionine containing solutions for TPN should be reconsidered.

Interleukin-1beta modulates endogenous thyroid hormone receptor alpha gene transcription in liver cells.

One of the main characteristics of nonthyroidal illness (NTI) is a decrease in serum triiodothyronine, partly caused by a decrease in liver deiodinase type 1 (D1) mRNA and activity. Proinflammatory cytokines have been associated with NTI in view of their capability to decrease D1 and thyroid hormone receptor (TR)beta1 mRNA expression in hepatoma cells. Proinflammatory cytokine induction leads to activation of the inflammatory pathways nuclear factor (NF)kappaB and activator protein (AP)-1. The proinflammatory cytokine interleukin (IL)-1beta decreases thyroid hormone receptor (TR)beta1 mRNA in an NFkappaB-dependent way. The aim of this study was to unravel the effects of IL-1beta on endogenous TRalpha gene expression in an animal model and in a liver cell line. The TRalpha gene product is alternatively spliced in TRalpha1 and TRalpha2, TRalpha2 is capable of inhibiting TRalpha1-induced gene transcription. We showed that both TRalpha1 and TRalpha2 mRNA decreased not only after lipopolysaccharide administration in liver of mice, but also after IL-1beta stimulation of hepatoma cells (HepG2). Using the NFkappaB inhibitor sulfasalazine and the AP-1 inhibitor SP600125, it became clear that the IL-1beta-induced decrease in TRalpha mRNA expression in HepG2 cells can only be abolished by simultaneous inhibition of NFkappaB and AP-1. The IL-1beta-induced TRalpha1 and TRalpha2 mRNA decrease in HepG2 cells is the result of decreased TRalpha gene promoter activity, as evident from actinomycin D experiments. Cycloheximide experiments showed that the decreased promoter activity is independent of de novo protein synthesis and therefore most likely due to posttranslational modifications such as phosphorylation or subcellular relocalization.

Differential involvement of nuclear factor-kappaB and activator protein-1 pathways in the interleukin-1beta-mediated decrease of deiodinase type 1 and thyroid hormone receptor beta1 mRNA.

One of the hallmarks of the sick euthyroid syndrome or non-thyroidal illness is a decrease of serum triiodothyronine, caused mainly by a decrease in liver deiodinase type 1 (D1) mRNA and activity. Proinflammatory cytokines like interleukin (IL)-1beta are likely involved in this disease, but are also known to inhibit thyroid hormone receptor (TR)-beta1 gene expression, which is of interest as the D1 promoter contains TREs. The aim of the present study was to evaluate whether the IL-1beta-induced decrease of D1 and TRbeta1 mRNA is mediated by the same cytokine signalling pathways in a human hepatoma cell line (HepG2). We observed a downregulation of both D1 and TRbeta1 mRNA after 4 h of incubating the cells with IL-1beta. Sulfasalazine was used to inhibit the nuclear factor-kappaB (NFkappaB) pathway and SP600125, a chemical inhibitor of the c-Jun N-terminal kinase, was used as an inhibitor of the activator protein-1 (AP-1) pathway. AP-1 inhibition did not affect the decrease of D1 and TRbeta1 mRNA, but the TRbeta1 mRNA decrease was completely abolished after inhibiting NFkappaB, while D1 mRNA was unaffected. Only simultaneous inhibition of both the NFkappaB and AP-1 pathways abolished the D1 mRNA decrease. We concluded that IL-1beta stimulation of HepG2 cells results in a marked decrease of D1 and TRbeta1 mRNA. The decrease of TRbeta1 mRNA is exclusively mediated by the NFkappaB pathway, while the decrease of D1 mRNA requires inhibition of both the AP-1 and the NFkappaB pathways.

Induction of type 3 deiodinase activity in inflammatory cells of mice with chronic local inflammation.

During illness, changes in thyroid hormone metabolism occur, so-called nonthyroidal illness (NTI). NTI has been characterized by a fall of serum T(3) due to decreased extrathyroidal conversion of T(4) into T(3) 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-1beta and IL-6 mRNA expression. Inflammatory cells, surrounding the abscess showed D3 and T(3)-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 T(3).

Simultaneous changes in central and peripheral components of the hypothalamus-pituitary-thyroid axis in lipopolysaccharide-induced acute illness in mice.

During illness, major changes in thyroid hormone metabolism and regulation occur; these are collectively known as non-thyroidal illness and are characterized by decreased serum triiodothyronine (T(3)) and thyroxine (T(4)) without an increase in serum TSH. Whether alterations in the central part of the hypothalamus-pituitary-thyroid (HPT) axis precede changes in peripheral thyroid hormone metabolism instead of vice versa, or occur simultaneously, is presently unknown. We therefore studied the time-course of changes in thyroid hormone metabolism in the HPT axis of mice during acute illness induced by bacterial endotoxin (lipopolysaccharide; LPS).LPS rapidly induced interleukin-1beta mRNA expression in the hypothalamus, pituitary, thyroid and liver. This was followed by almost simultaneous changes in the pituitary (decreased expression of thyroid receptor (TR)-beta2, TSHbeta and 5'-deiodinase (D1) mRNAs), the thyroid (decreased TSH receptor mRNA) and the liver (decreased TRbeta1 and D1 mRNA). In the hypothalamus, type 2 deiodinase mRNA expression was strongly increased whereas preproTRH mRNA expression did not change after LPS. Serum T(3) and T(4) fell only after 24 h. Our results suggested almost simultaneous involvement of the whole HPT axis in the downregulation of thyroid hormone metabolism during acute illness.

Immunoneutralization of interleukin-1, tumor necrosis factor, interleukin-6 or interferon does not prevent the LPS-induced sick euthyroid syndrome in mice.

The sick euthyroid syndrome is a state of altered thyroid hormone metabolism which occurs during illness. The pathogenesis is incompletely understood but recent studies indicate a role of cytokines. It is unknown if cytokines released during illness are directly responsible for the changes in thyroid hormone metabolism. Therefore we studied if previous immunoneutralization of cytokines can prevent endotoxin (lipopolysaccharide LPS), induced sick euthyroid syndrome. LPS administration resulted in systemic illness, an increase in serum tumor necrosis factor (TNF alpha) and interleukin (IL)-6 and a decrease in serum triiodothyronine (T3) and thyroxine (T4). Immunoneutralization of the effects of cytokines was accomplished by administration of monoclonal antibodies against mouse IL-1 type-1 receptor (IL-1R), TNF alpha, IL-6 or interferon (IFN gamma) prior to LPS. The LPS-induced release of cytokines was affected by previous immunoneutralization as compared with control experiments with normal immunoglobulin (IgG): anti-IL-1R did not affect serum TNF alpha but decreased serum IL-6, anti-TNF alpha decreased serum TNF alpha but not IL-6, anti-IL-6 did not affect serum TNF alpha but hugely increased IL-6 and anti-IFN gamma decreased both serum TNF alpha and IL-6. Specific immunoneutralization of IL-1, TNF alpha or IFN gamma did not prevent the LPS-induced decrease in serum T3, T4 and liver 5'-deiodinase mRNA. However, immunoneutralization of IL-6, although not preventing the fall in serum T3 and T4, did mitigate the LPS-induced decrease in liver 5'-deiodinase mRNA. In view of possible non-specific effects of the huge dose of immunoglobulins (1 mg), used only in the immunoneutralization of IL-6, we repeated the experiment with F(ab')2 fragments of anti-IL-6 antibodies. Compared with F(ab')2 fragments of control IgG, anti-IL-6 F(ab')2 did not affect the LPS-induced rise in serum TNF alpha or the decrease in serum T3 and T4 and liver 5'-deiodinase mRNA. Serum IL-6 levels induced by LPS were, however, cleared more rapidly from the circulation when anti-IL-6 F(ab')2 fragments rather than intact anti-IL-6 were administered. In conclusion, immunoneutralization of IL-1, TNF alpha or IFN gamma did not prevent the LPS-induced sick euthyroid syndrome in mice; immunoneutralization of IL-6, however, transiently inhibits the LPS-induced decrease of liver 5'-deiodinase mRNA.

Induced illness in interleukin-6 (IL-6) knock-out mice: a causal role of IL-6 in the development of the low 3,5,3'-triiodothyronine syndrome.

Interleukin-6 (IL-6) administration to human subjects or experimental animals induces changes in thyroid hormone metabolism resembling those in the sick euthyroid syndrome. Furthermore, the decrease in serum T3 during illness is significantly related to serum IL-6 concentrations. These findings suggest, but do not prove, a causal role for IL-6 in the development of the low T3 syndrome. The aim of the present study was to evaluate the role of IL-6 in the development of the sick euthyroid syndrome in IL-6 knock-out (IL-6(-/-)) mice compared to that in wild-type mice (IL-6(+/+)). Illness was induced in IL-6(-/-) and IL-6(+/+) mice by 1) administration of bacterial endotoxin (LPS), 2) infection with Listeria monocytogenes, and 3) turpentine injection in both hind limbs. Food intake was kept similar in both groups in all three experiments. Serial measurements were made of serum IL-6, tumor necrosis factor-alpha, T3, T4, corticosterone, and liver 5'-deiodinase (5'-DI) messenger RNA (mRNA) for 24 h (LPS), 96 h (L. monocytogenes), and 48 h (turpentine). Serum IL-6 increased in response to all stimuli in IL-6(+/+) mice, but not in IL-6(-/-) mice. Serum tumor necrosis factor-alpha was induced by LPS in both groups to a similar extent, but did not rise after L. monocytogenes or turpentine administration. Serum T3 and T4 decreased after all three stimuli. The decrease in serum T4 in IL-6(-/-) was similar to that in IL-6(+/+) mice. The decrease in serum T3, however, was smaller in the IL-6(-/-) mice than in the IL-6(+/+) mice; T3 levels were 1.56 +/- 0.29 and 0.99 +/- 0.15 nmol/liter, respectively, 24 h after LPS treatment (P < 0.01), 2.39 +/- 0.17 and 1.75 +/- 0.24 nmol/liter 96 h after L. monocytogenes treatment (P < 0.01), and 1.46 +/- 0.18 and 1.10 +/- 0.25 nmol/liter 48 h after turpentine treatment (P < 0.05). The smaller fall in serum T3 in IL-6(-/-) mice could not be attributed to differences in the severity of the induced illness, food intake, or corticosterone response, which were all similar in IL-6(-/-) mice and IL-6(+/+) mice. Liver 5'-deiodinase mRNA decreased after all three stimuli; the decrease after LPS and L. monocytogenes infection was smaller in the IL-6(-/-) mice, but the difference in IL-6(+/+) mice just failed to reached statistical significance. Liver 5'-deiodinase activity after turpentine injection administration decreased in the wild-type animals by 36%, but did not change in the knock-out mice. In conclusion, acutely induced illness generates the low T3 syndrome, which is less marked in IL-6 knock-out mice than in wild-type mice. The data suggest a causal role of IL-6 in the development of the low T3 syndrome.

Selective macrophage depletion in the liver does not prevent the development of the sick euthyroid syndrome in the mouse.

A decreased serum triiodothyronine (T3) level is one of the main characteristics of the sick euthyroid syndrome, caused mainly by a decreased 5'-deiodination of thyroxine (T4) in the liver. Cytokines have been implicated in the pathogenesis of the changes in thyroid hormone metabolism during illness. We therefore investigated the role of cytokines produced by the liver macrophages (Kupffer cells) in the development of the sick euthyroid syndrome, which was induced in mice by a single injection of bacterial endotoxin (lipopolysaccharide) or by 24-h starvation. Experiments were carried out with or without previous selective depletion of liver macrophages by intravenous administration of liposome-encapsulated dichloromethylene diphosphonate. Relative to saline-injected pair-fed controls, the administration of lipopolysaccharide caused a decrease of serum T3 and T4 and liver 5'-deiodinase mRNA. Selective depletion of liver macrophages, did not affect these changes. Starvation for 24h decreased serum T3 and T4, associated with a slight decrease of liver 5'-deiodinase mRNA. There were no differences between macrophage-depleted and non-depleted animals in this respect. In summary, selective depletion of liver macrophages did not affect the decrease in serum T3, T4 or liver 5'-deiodinase mRNA induced by lipopolysaccharide or 24-h starvation in mice. We conclude that cytokines produced by Kupffer cells are not involved in the pathogenesis of the sick euthyroid syndrome in this experimental model.

The role of cytokines in the lipopolysaccharide-induced sick euthyroid syndrome in mice.

To evaluate the role of cytokines in the sick euthyroid syndrome, we tried to establish an animal model of non-thyroidal illness in mice by the administration of a sub-lethal dose of bacterial endotoxin (lipopolysaccharide; LPS) which induces a variety of cytokines, including tumour necrosis factor (TNF alpha), interleukin-1 (IL-1 alpha), interleukin-6 (IL-6) and interferon-gamma (IFN gamma). When compared with pair-fed controls, a single dose of LPS resulted in (a) systemic illness, (b) induction of TNF alpha and IL-6 and (c) a decrease of liver 5'-deiodinase mRNA from 4 h onwards followed by a decrease of serum tri-iodothyronine (T3) and thyroxine (T4) at 8 h and of serum free T3 (fT3) and free T4 (fT4) at 24 h; serum TSH remained unchanged. We then studied whether a single dose or a combination of IL-1 alpha, TNF alpha, IL-6 or IFN gamma could induce the sick euthyroid syndrome in mice, again using pair-fed controls. None of the cytokines except IL-1 alpha caused systemic illness, and IL-1 alpha was the only cytokine that decreased liver 5'-deiodinase mRNA transiently. IL-1 alpha, TNF alpha or IL-6 did not decrease serum T3, T4 and TSH, but administration of IFN gamma decreased serum T4, T3 and fT3 in a dose-dependent manner without changes in serum TSH. Administration of all four cytokines together had no synergistic effects; observed changes were of a smaller magnitude than after LPS. The following conclusions were reached.(ABSTRACT TRUNCATED AT 250 WORDS)

Soluble cytokine receptors and the low 3,5,3'-triiodothyronine syndrome in patients with nonthyroidal disease.

Cytokines have been implicated in the pathogenesis of the low T3 syndrome during illness. This is supported by our recent observation of a strong negative relationship between serum T3 and serum interleukin-6 (IL-6) in nonthyroidal illness (NTI). In the last few years, soluble cytokine receptors and cytokine receptor antagonists have been discovered in human serum. These proteins have the potential to further regulate cytokine activity. Therefore, we now studied the association between serum T3 and serum levels of soluble tumor necrosis factor-alpha (sTNF alpha R p55 and sTNF alpha R p75), soluble interleukin-2 receptor (sIL-2R), and the interleukin-1 receptor antagonist (IL-1RA) in 100 consecutive hospital admissions with a wide variety of nonthyroidal diseases. Patients were divided into group A (T3, > or = 1.30 nmol/L; T4, > or = 75 nmol/L; n = 41), group B (T3, < 1.30 nmol/L; T4, > or = 75 nmol/L; n = 46), and group C (T3, < 1.30 nmol/L; T4, < 75 nmol/L; n = 13). Serum sTNF alpha R p55, sTNF alpha R p75, sIL-2R, and IL-1RA were lower in group A than in groups B and C [median values; sTNF alpha R p55, 1.25, 2.25, and 3.55 ng/mL (P < 0.001); sTNF alpha R p75, 2.02, 4.56, and 7.00 ng/mL (P < 0.001); sIL-2R, 184, 259, and 272 U/mL (P = 0.0004), respectively]. Serum IL-1RA levels were not different in the three groups (median values, 122, 193, and 258 pg/mL, respectively). Taking all patients together, a significant negative relation was found among serum T3 and sTNF alpha p55 (r = -0.59; P < 0.0001), sTNF alpha R p75 (r = -0.55; P < 0.0001), sIL-2R (r = -0.54; P < 0.0001), IL-1RA (r = -0.38; P = 0.001), and IL-6 (r = -0.56; P < 0.0001). A remarkable high correlation (r = -0.70; P < 0.0001) was found between serum T3 and a newly designed total score based on the summation of serum levels of IL-6 and the four soluble cytokine receptor proteins. IL-6 and the four cytokine receptor proteins were all significantly related to each other. Stepwise multiple regression indicated IL-6 and sTNF alpha R p75 as independent determinants of T3 [serum T3 = 2.09-0.32ln (sTNF alpha R p75) -0.15ln (IL-6); r = 0.70]. The variability in serum T3 was accounted for 35% by changes in ln (sTNF alpha R p75) and 14% by changes in ln (IL-6).(ABSTRACT TRUNCATED AT 400 WORDS)