Selectively High Levels of Serum Interleukin 17 in a Newborn Infant With Progressive Severe Cholestasis Maho Ichikawa, Naoto Takahashi,

Yukari Yada, Yasunori Koike, Ryou Kawamata, Yumi Kono, Yoko Honma, Shigeru Yotsumoto and Mariko Y. Momoi Pediatrics 2010;126;e247; originally published online June 14, 2010; DOI: 10.1542/peds.2009-3053

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PEDIATRICS is the official journal of the American Academy of Pediatrics. A monthly publication, it has been published continuously since 1948. PEDIATRICS is owned, published, and trademarked by the American Academy of Pediatrics, 141 Northwest Point Boulevard, Elk Grove Village, Illinois, 60007. Copyright 2010 by the American Academy of Pediatrics. All rights reserved. Print ISSN: 0031-4005. Online ISSN: 1098-4275.

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CASE REPORTS

Selectively High Levels of Serum Interleukin 17 in a Newborn Infant With Progressive Severe Cholestasis
AUTHORS: Maho Ichikawa, MD, Naoto Takahashi, MD, Yukari Yada, MD, Yasunori Koike, MD, Ryou Kawamata, MD, Yumi Kono, MD, PhD, Yoko Honma, MD, Shigeru Yotsumoto, MD, PhD, and Mariko Y. Momoi, MD, PhD
Department of Pediatrics, Jichi Medical University School of Medicine, Tochigi, Japan KEY WORDS IL-17, cholestasis, immunosuppressive agent, hypercytokinemia ABBREVIATIONS ThT helper IFN-interferon ILinterleukin ASTaspartate aminotransferase ALTalanine aminotransferase Igimmunoglobulin www.pediatrics.org/cgi/doi/10.1542/peds.2009-3053 doi:10.1542/peds.2009-3053 Accepted for publication Mar 17, 2010 Address correspondence to Naoto Takahashi, MD, Department of Pediatrics, Jichi Medical University School of Medicine, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498, Japan. E-mail: naoto-t@jichi.ac.jp PEDIATRICS (ISSN Numbers: Print, 0031-4005; Online, 1098-4275). Copyright 2010 by the American Academy of Pediatrics FINANCIAL DISCLOSURE: The authors have indicated they have no nancial relationships relevant to this article to disclose.

abstract
We present here the unusual case of a male newborn infant who showed progressive severe cholestasis. The infants gestational age was 37 weeks, and his birth weight was 2134 g. His serum level of direct bilirubin gradually increased from the 6th day of life and reached 257.5 mol/L on the 22nd day of life. We could not nd any cause for his cholestasis, but his serum level of ferritin was extremely elevated at 9211.0 ng/mL. Because we felt that his clinical condition might be related to hypercytokinemia caused by an immunologic reaction, steroid pulse therapy and cyclosporine were administered. His condition improved, and his direct bilirubin and ferritin levels declined. From the investigation of his cytokine prole, we found a preferentially elevated level of serum interleukin 17 (IL-17) (96.1 pg/mL) and high level of chemokines IL-8 and macrophage inammatory protein 1. The IL-17 level gradually decreased to 7.5 pg/mL by the 124th day of life. The infant was successfully discharged from the childrens hospital but later developed epilepsy at 11 months and asthma at 1 year, 2 months of age. Although we have not yet reached a denitive diagnosis, this case may be the rst to show a relationship between cholestasis and an elevated serum IL-17 level in the neonatal period. Pediatrics 2010; 126:e247e250

PEDIATRICS Volume 126, Number 1, July 2010

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Cytokines are considered important regulators of immune function and have been associated with the development of disease. Naive CD4 T cells have been thought to differentiate into 2 main lineages: T-helper 1 (Th1) and Th2 cells.1 Th1 cells produce the signature cytokine interferon (IFN-) and interleukin 12 (IL-12) and regulate cellular immunity. Th2 cells produce IL-4, IL-5, and IL-13 and regulate antibody production. Recently, a third subset of CD4 T cells that produces IL-17, named Th17, was described.2,3 IL-17 producing T cells are responsible for autoimmune diseases in mice.24 Although the effector functions of this new helper T cell in humans are just beginning to be understood, a potential role of IL-17 in pathogenesis of several liver diseases in human adults has been reported in several articles.57 We present here the unusual case of a neonate who showed progressive severe cholestasis. The cause of his cholestasis was unknown, but his cholestasis improved with immunosuppressive treatment. His serum cytokine prole showed selectively elevated levels of IL-17 and chemokines. This case may be the rst to show a relationship between cholestasis and an elevated serum IL-17 in an infant.

MV feeding PSL cyclosporine

IL-17 D-bil pg/mL mol/L
100 80 60

mPSL-pulse

Ferritin
ng/mL

800

IL-17

15 000

600

ferritin
10 000

400
40 20

200

D-bil

5000

10

20

30

40

Age, day of life

FIGURE 1
The clinical course of our patient. MV indicates mechanical ventilation; feeding, enteral feeding; PSL, prednisolone; mPSL-pulse, methylprednisolone pulse therapy; D-bil, serum direct bilirubin.

CASE REPORT
A male newborn infant whose gestational age was 37 weeks and birth weight was 2134 g showed respiratory disturbance immediately after birth. He had 2 healthy siblings and no significant family history. His Apgar scores were 7 and 8 at 1 and 5 minutes, respectively. His amniotic uid volume was small and turbid. He was treated with empiric vancomycin. He also had a pneumothorax and persistent pulmonary hypertension of the newborn. His serum chemistry values on admission showed an aspartate aminotransferase (AST) level of 44 U/L and alanine
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aminotransferase (ALT) level of 10 U/L. His white blood cell count was 15 200/ L, and his platelet count was 1.89 105/L. His serum level of C-reactive protein was 0.001 mg/L and serum level of immunoglobulin M (IgM) was not increased (60 mg/L). Results of a blood culture were negative. His placenta did not show any signs of chorioamnionitis. On the 3rd day of life, his general condition gradually improved, but he experienced a generalized convulsion of unknown origin. He was transiently treated with phenobarbital, which was discontinued by the 10th day of life. He was extubated successfully on his 19th day of life. As shown in Fig 1, his serum level of direct bilirubin gradually increased from the 6th day of life and reached 2575 mol/L (151.5 mg/L) on his 22nd day of life. His serum chemistry values on the 11th day showed a total bilirubin level of 147.9 mg/L, an AST level of 14 U/L, and an ALT level of 5 U/L. On the 22nd day, he had a total bilirubin level of 228.5 mg/L, an AST level of 241 U/L, an ALT level of 80 U/L, a lactate dehydrogenase level of 529 IU/L, an alkaline phosphatase level of 592 IU/L, a -glutamyl transferase level of 88 IU/L,

and a total bile acid level of 165.5 mmol/L. Results of a blood-coagulation prole were normal, and his serum level of C-reactive protein was 0.8 mg/L. His serum amino acid prole was normal. He did not have hepatosplenomegaly. Abdominal ultrasonography showed no abnormality of either the liver or the bile ducts. Results of tests for serum IgM antibodies against cytomegalovirus, herpes simplex virus, and Epstein-Barr virus were all negative, as were the results of tests for hepatitis B virus antigens and IgG antibody against hepatitis C virus. The results of tests for antinuclear and anti-DNA antibodies were also all negative. Drug lymphocyte-stimulation tests of phenobarbital and vancomycin were negative. His stool-culture results were negative for abdominal pathogenic microorganisms. We could not nd any cause for the patients cholestasis, but his serum level of ferritin was extremely high (9211.0 ng/mL) on his 22nd day of life. Because we thought that his clinical condition might be related to hypercytokinemia caused by an immunologic reaction, we administered steroid pulse therapy (methylprednisolone 12 mg/kg per

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CASE REPORTS

dose per day for 3 days) on his 23rd day of life. Then, his direct bilirubin level decreased slightly, but the ferritin level did not decrease. On the 28th day of life, his direct bilirubin level increased again, and his ferritin level was still elevated at 11 818 ng/mL. We gave a second course of the steroid pulse therapy (methylprednisolone 30 mg/kg per dose per day for 3 days). Then, his direct bilirubin level began to decline, and his ferritin level plateaued. We presumed that the regimen might not be sufcient to reduce the serum level of direct bilirubin and ferritin to a lower level; therefore, we added cyclosporine (2.1 mg/kg per day) to the regimen during the third course of the pulse therapy (methylprednisolone 30 mg/kg per dose per day for 3 days). His ferritin and direct bilirubin levels continuously decreased once the combined therapy was introduced. His maximum serum levels of AST, ALT, alkaline phosphatase, and -glutamyl transferase were 308, 300, 1436, and 210 U/L, respectively, on his 33rd day of life, and these serum levels all decreased thereafter. His serum level of IL-17 was elevated (96.1 pg/mL) on his 22nd day of life (Table 1), and the level continuously decreased after introduction of steroid pulse therapy (Fig 1 and Table 1). His general condition improved, his body weight increased, and the cyclosporine was discontinued on his 156th day of life. The administration of prednisolone was gradually tapered, and the patient was successfully discharged from the hospital on his 167th day of life. He developed epilepsy at 11 months of age and asthma at 1 year, 2 months of age. At the time of this writing, he is 1 year, 6 months old and is still receiving low-dose prednisolone therapy. He has had mild growth retardation and mild developmental delay. Our investigation was approved by the Jichi Medical University ethics comPEDIATRICS Volume 126, Number 1, July 2010

TABLE 1 Concentrations of Serum Cytokines, Chemokines, and Growth Factors in Our Patient
Compared With Neonatal Controls
Our Patient, pg/mL DOL 22 Proinammatory cytokines TNF- IL-1 IL-6 Th1 cytokines IFN- IL-2 IL-12 Th2 cytokines IL-4 IL-5 IL-10 IL-13 Th17 cytokine IL-17 Growth factors IL-7 GM-CSF G-CSF Chemokines IL-8 MCP-1 MIP-1 0.04 0.02 4.20 0.04 8.60 16.3 0.08 0.69 10.74 1.31 96.17 0.54 18.94 20.40 78.99 228.42 443.89 DOL 29 0.04 0.66 0.04 0.66 16.70 3.13 0.01 0.41 7.04 0.29 39.61 0.21 6.92 2.03 135.72 374.00 429.30 DOL 84 0.04 1.09 2.23 5.81 0.02 1.86 0.02 0.63 1.22 0.97 11.59 0.25 0.05 7.53 24.53 233.10 235.30 Controls (N 8) (DOL 5.6 1.2), pg/mLa 6.60 5.80 2.10 1.90 4.30 3.10 5.30 5.20 0.54 1.20 2.60 4.10 0.14 0.20 2.00 0.50 1.80 2.10 1.20 1.10 7.00 7.40 0.22 0.33 17.00 13.70 11.20 7.00 20.00 10.40 487.00 221.40 185.50 73.10

DOL indicates day of life; TNF- , tumor necrosis factor ; GM-CSF, granulocyte-macrophage colony-stimulating factor; G-CSF, granulocyte-colony stimulating factor; MCP-1, monocyte chemotactic protein 1; MIP-1 , macrophage inammatory protein 1 . a The control group included 8 newborn patients who were admitted to the NICU at Jichi Medical University School of Medicine with various risks. Among them, 5 had slightly low birth weight, 2 were infants of diabetic mothers, and another had hyperbilirubinemia caused by breastfeeding. Peripheral blood was taken from patients in the control group between their 4th and 8th days of life with informed consent from their parents.5 The control data are shown as mean 1 SD.

mittee. The parents of infants included in our study as control subjects were informed of the study design, and written informed consent was obtained. We measured serum cytokine levels by using the BioPlex protein array (BioRad, Hercules, CA) and Luminex 100 (Mirai Bio, Alameda, CA) systems, as described previously.8 The BioPlex human cytokine 17-plex panel was used. The most striking nding of our patients cytokine prole was the preferentially elevated level of serum IL-17 (96.1 pg/mL) in the acute phase of his illness (Table 1). His tumor necrosis factor (TNF-), IFN-, and IL-1 levels were almost totally negative. The level of the other proinammatory cytokine, IL-6, was within normal range. The levels of the other Th1 cytokines, IL-2 and IL-12, were moderately elevated. Th2 cytokines were consistently at low lev-

els except for inhibitory cytokine IL-10. Another striking nding was the high serum levels of chemokines IL-8 and macrophage inammatory protein 1. His IL-17 level gradually declined to 11.6 pg/mL by his 84th day of life and normalized to 7.5 pg/mL on his 124th day of life (Table 1 and Fig 1).

DISCUSSION
Several types of congenital and acquired diseases of newborn infants can cause cholestasis. Congenital bile atresia, neonatal hepatitis, and hepatitis caused by viruses were excluded in this patient, and neonatal intrahepatic cholestasis caused by citrin deciency was excluded by the patients serum amino acid prole. Drug-induced cholestasis was excluded as well by a negative drug lymphocyte-stimulation test. Other congenital diseases such as
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Alagille syndrome were also excluded on the basis of the patients clinical presentation. Progressive familiar intrahepatic cholestasis was excluded on the basis of the patients clinical course. Occasionally, neonatologists encounter cholestasis in patients with intrauterine growth restriction or birth asphyxia, but usually the cholestasis is self-limited. In this patient, cholestasis was progressive and improved with immunosuppressive treatment. We think that this patient may have had cholestasis secondary to a dysregulation of immune response of unknown origin in the liver. Rand et al9 hypothesized that many cases of neonatal hemochromatosis are a result of maternal alloimmunity directed at the fetal liver. Maternal alloimmunity may be a possible cause of our patients disease, although the he could not be diagnosed to have neonatal hemochromatosis. His cytokine prole showed elevated levels of IL-17 and chemokines, and his REFERENCES
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serum levels of the other proinammatory cytokines were all normal. We presume that the increase in serum levels of IL-17 and chemokines was related to development of cholestasis in this patient. Ferritin is known to be an acute inammatory protein and to be induced by several cytokines. The high level of serum ferritin could be related to high levels of serum IL-17 and chemokines in this patient. Table 1 shows that chemokine levels were still increased on the 29th day of life even after improvement of the IL-17 level. It is possible that IL-17 may be more upstream to the abnormal immune reaction in this patient. To our knowledge, there has been no report that indicates IL-17 as a cause of neonatal or infantile disease. We feel that the role of IL-17 in neonatal and infantile diseases should be investigated in more detail in future studies. Acute inammation in which neutrophils are prominent is typical of IL-17 driven inammation.10 Immunity mediated by Th17 cells is particularly

important at epithelial and mucosal surfaces, as indicated by the pattern of expression of their chemokine receptors and effector cytokines.11,12 In particular, IL-17 has been linked to tissue neutrophil recruitment through the induction of IL-8.10 Pan et al13 reported that all IL-17Etransgenic mice had severe cholangiohepatitis with adenomatous hyperplasia of bile ducts. Several studies have shown a role for the liver microenvironment in the induction of Th17 in cases of liver inammatory diseases in adult humans.57 IL-17 may act on biliary epithelial cells, and neutrophils may have played a role in the inammation of the bile ducts in our patient; however, this is merely speculation and should be claried in the future.

CONCLUSION
It is important to accumulate information on neonatal patients with similar conditions in future studies to further elucidate the underlying mechanism of cholestasis in our patient.

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Selectively High Levels of Serum Interleukin 17 in a Newborn Infant With Progressive Severe Cholestasis Maho Ichikawa, Naoto Takahashi, Yukari Yada, Yasunori Koike, Ryou Kawamata, Yumi Kono, Yoko Honma, Shigeru Yotsumoto and Mariko Y. Momoi Pediatrics 2010;126;e247; originally published online June 14, 2010; DOI: 10.1542/peds.2009-3053
Updated Information & Services References including high resolution figures, can be found at: http://pediatrics.aappublications.org/content/126/1/e247.full.h tml This article cites 13 articles, 1 of which can be accessed free at: http://pediatrics.aappublications.org/content/126/1/e247.full.h tml#ref-list-1 This article has been cited by 1 HighWire-hosted articles: http://pediatrics.aappublications.org/content/126/1/e247.full.h tml#related-urls This article, along with others on similar topics, appears in the following collection(s): Allergy/Immunology http://pediatrics.aappublications.org/cgi/collection/allergy:im munology_sub Immunologic Disorders http://pediatrics.aappublications.org/cgi/collection/immunolo gic_disorders_sub Information about reproducing this article in parts (figures, tables) or in its entirety can be found online at: http://pediatrics.aappublications.org/site/misc/Permissions.xht ml Information about ordering reprints can be found online: http://pediatrics.aappublications.org/site/misc/reprints.xhtml

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PEDIATRICS is the official journal of the American Academy of Pediatrics. A monthly publication, it has been published continuously since 1948. PEDIATRICS is owned, published, and trademarked by the American Academy of Pediatrics, 141 Northwest Point Boulevard, Elk Grove Village, Illinois, 60007. Copyright 2010 by the American Academy of Pediatrics. All rights reserved. Print ISSN: 0031-4005. Online ISSN: 1098-4275.

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