Increased interleukin-18 expression in bone marrow of a patient with systemic juvenile idiopathic arthritis and unrecognized macrophage-activation syndrome

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<ul><li><p>ARTHRITIS &amp; RHEUMATISMVol. 50, No. 6, June 2004, pp 19351938DOI 10.1002/art.20268 2004, American College of Rheumatology</p><p>Increased Interleukin-18 Expression in Bone Marrow of aPatient With Systemic Juvenile Idiopathic Arthritis and</p><p>Unrecognized Macrophage-Activation Syndrome</p><p>Nobuaki Maeno, Syuji Takei, Hiroyuki Imanaka, Kimie Yamamoto, Kazumi Kuriwaki,Yoshifumi Kawano, and Hiroshi Oda</p><p>The aberrant induction of proinflammatory cy-tokines is considered to be crucial in the pathogenesis ofsystemic juvenile idiopathic arthritis and adult-onsetStills disease. Interleukin-18 (IL-18) in particular hasbeen reported to be a candidate for the key cytokine inboth diseases; however, the origin of IL-18 is unclear. Toclarify the origin, we investigated specimens from vari-ous organs obtained during autopsy of a child withsystemic JIA and macrophage activation syndrome,using immunohistochemical staining. Our resultsshowed a high number of cells expressing IL-18 in thebone marrow but not in the other organs. This findingsuggests that bone marrow is the origin of increasedserum IL-18 and raises the possibility that other proin-flammatory cytokines are also induced by IL-18 in bonemarrow in this disease. Bone marrow may be an essen-tial organ in the pathogenesis of systemic JIA.</p><p>Systemic juvenile idiopathic arthritis (JIA), alsoknown as Stills disease, is one of the most commonsystemic and chronic inflammatory diseases in childhoodand is characterized by chronic arthritis associated witha high spiking fever, a salmon-pink evanescent rash,hepatosplenomegaly, lymphadenopathy, and serositis.The precise etiology of systemic JIA remains unclear,although the activation of macrophages is thought to be</p><p>crucial. Previous studies have demonstrated that theaberrant induction of proinflammatory cytokines such asinterleukin-6 (IL-6), IL-1, and tumor necrosis factor (TNF) may be involved in the pathogenesis of systemicJIA (1). However, it has not yet been elucidated whichmediators are the key ones, nor has the origin ofincreased cytokines in systemic JIA been determined.</p><p>Recently, we and other researchers reportedhighly elevated serum levels of IL-18 (10,000 pg/ml) inpatients with systemic JIA or adult-onset Stills disease(AOSD), which is pathogenetically identical to systemicJIA (24). IL-18 strongly stimulates T lymphocytes,natural killer (NK) cells, and macrophages to produceproinflammatory cytokines (5,6) and is thereforethought to be closely related to the pathogenesis ofsystemic JIA and AOSD. The cellular source of in-creased IL-18 synthesis in systemic JIA is unclear,although circulating or tissue monocyte/macrophagesseem the most likely candidates in AOSD (3). In thepresent study, we investigated the source of increasedIL-18 synthesis in a patient with systemic JIA.</p><p>CASE REPORT</p><p>The patient, a 20-month-old girl, was admitted tothe county hospital because of a 2-week history of highspiking fever, rash, and tender arthritis of the extremi-ties. Evaluation for bacterial and viral infections, includ-ing cytomegalovirus and Epstein-Barr virus, yieldednegative results, and a bone marrow aspirate obtained atthat time showed normal myelographic results. Thepatient was treated with intravenous antibiotics andaspirin for 1 week. However, her condition did notimprove, and she was transferred to our hospital. Uponadmission to our hospital, laboratory tests gave thefollowing results: white blood cell (WBC) count 15,500/l, hemoglobin concentration 10.0 gm/dl, platelet count</p><p>Supported in part by grant-in-aid 13670278 from the Ministryof Education, Culture, Sports, Science, and Technology of Japan.</p><p>Nobuaki Maeno, MD, Syuji Takei, MD, Hiroyuki Imanaka,MD, Kimie Yamamoto, MD, Kazumi Kuriwaki, MD, YoshifumiKawano, MD, Hiroshi Oda, MD: Kagoshima University GraduateSchool of Medical and Dental Sciences, Kagoshima, Japan.</p><p>Address correspondence and reprint requests to NobuakiMaeno, MD, Department of Infection and Immunity, KagoshimaUniversity Graduate School of Medical and Dental Sciences, 8-35-1Sakuragaoka, Kagoshima 890-8520, Japan. E-mail: maeno@m3.kufm.kagoshima-u.ac.jp.</p><p>Submitted for publication June 4, 2003; accepted in revisedform February 4, 2004.</p><p>1935</p></li><li><p>408,000/l, serum glutamic oxaloacetic transaminase(SGOT) level 57 units/liter (normal 40), serum glu-tamic pyruvic transaminase (SGPT) level 37 units/liter(normal 35), lactate dehydrogenase (LDH) concentra-tion 995 units/liter (normal 400), erythrocyte sedimen-tation rate 108 mm/hour, and serum C-reactive proteinlevel 20.4 mg/dl (normal 0.5). Systemic JIA was diag-nosed, and the dosage of aspirin was increased to 40mg/kg/day.</p><p>Despite this week-long therapy, the patientscondition continued to deteriorate, and liver dysfunctionand pancytopenia developed. A peripheral smearshowed no abnormalities. Aspirin was then discontin-ued, and a course of parenteral steroids was instituted.However, 1 week later, her transaminase and LDHlevels had further increased (SGOT 526 units/liter,SGPT 202 units/liter, and LDH 7,891 units/liter). Inaddition, the pancytopenia and coagulation disturbancehad progressed (WBC count 2,900/l, hemoglobin con-centration 7.9 gm/dl, platelet count 38,000/l, fibrinogenlevel 109 mg/dl, and fibrinogenfibrin degradation prod-uct 27.4 g/ml [normal 1.0 g/ml]). The next day,before cyclosporin A treatment was initiated, the patientdied of a pulmonary hemorrhage. At that time, herserum levels of ferritin and proinflammatory cytokineswere extremely elevated, as follows: ferritin 12,178 ng/ml, interferon- (IFN) 4,900 pg/ml, TNF 82.3 pg/ml,IL-6 83.3 pg/ml, monocyte colony-stimulating factor627 pg/ml, and IL-18 210,288 pg/ml (IL-18 enzyme-linked immunoassay kit; MBL, Nagoya, Japan).</p><p>A postmortem examination, conducted with in-formed consent from the patients parents, revealedhemophagocytosis in the bone marrow. These findingsindicated macrophage activation syndrome (MAS), oneof the most common and severe complications of sys-temic JIA (7). The pulmonary hemorrhage observed inthis patient might be attributable to the hemorrhagicdiathesis mediated by MAS; such a finding is reportedlya characteristic of MAS (7).</p><p>In order to investigate the origin of the highlyincreased level of IL-18, we used immunohistochemicalstaining with a monoclonal antibody to IL-18 (clone25-2G; MBL) or CD68 (clone KP1; Dako, Kyoto, Japan)to examine specimens of the liver, spleen, axillary lymphnodes, lung, and bone marrow obtained from this pa-tient. Autopsy specimens from a 22-month-old boy whohad died of gastrointestinal bleeding were also studiedas a control after informed consent was obtained. Thebone marrow specimens from the patient with systemicJIA, compared with those from the control subject(Figure 1A), demonstrated prominent infiltrates of</p><p>Figure 1. Immunohistochemical staining of bone marrow with anti-CD68 monoclonal antibody or antiinterleukin-18 (antiIL-18) mono-clonal antibody. Bone marrow specimens from the control subjectwere stained with anti-CD68 (A) and antiIL-18 (B), as were thespecimens from the patient with systemic juvenile idiopathic arthritis(C and D and E and F, respectively). The specimens from thepatient were also subject to control staining by replacing the primaryantibody with an isotype-matched control reagent (mouse IgG1) (G),and by neutralizing (NT) the antiIL-18 antibody with recombinantIL-18 protein (H). Brown color indicates positivity. (Original magni-fication 100 in A, B, C, E, G, and H; 400 in D and F; 1,000 ininsets.)</p><p>1936 MAENO ET AL</p></li><li><p>macrophages stained with CD68, and some macro-phages showed hemophagocytosis (Figures 1C and D).Furthermore, it is noteworthy that strong IL-18 stainingwas observed in these bone marrow cells (Figures 1Eand F). In contrast, few cells expressing IL-18 wereobserved in the bone marrow from a control subject(Figure 1B). The infiltration of CD68-positive cells inthe patients liver was not so significant and was local-ized around the portal tract, while CD68-positiveKupffer cells were widely seen in sinusoids in the controlsubject (Figures 2A and C). IL-18 was expressed in theliver cells of neither the patient with systemic JIA northe control subject (Figures 2B and D). In addition, nocells with significant IL-18 staining were observed in thespleen, lung, or lymph nodes of the patient with systemicJIA (data not shown).</p><p>DISCUSSION</p><p>Our results suggest that highly elevated serumIL-18 levels originate in bone marrow, raising the pos-sibility that bone marrow contains abundant proinflam-matory cytokines induced by IL-18 in this disease. This</p><p>hypothesis may be supported by reports that severalkinds of viruses, such as varicella virus, measles virus, orparvovirus B19, can induce bone marrow suppressionthat may lead to transient clinical remission in childrenwith intractable systemic JIA (8,9). Furthermore,Schnedl et al reported that diffuse bone marrow edemawas characteristically detected by magnetic resonanceimaging and biopsies in patients with AOSD, and thatthis might be the result of increased permeability of thecapillary endothelium in the bone marrow mediated byinflammatory cytokines (10). In addition, quantitativeand qualitative abnormalities in bone marrow cells weredemonstrated in patients with JIA, and these changeswere considered to be consequences of the inflamma-tory milieu, including cytokines (11). Likewise, the eti-ologic association between bone marrow cells and rheu-matoid arthritis (RA) in adults has been discussed.Remarkable elevations of IL-6, IL-8, and TNF in bonemarrow were reported in patients with RA (12,13).Another study demonstrated that nurse-like cells fromthe bone marrow of patients with severe RA haveunique characteristics and may play an important role inthe pathogenesis of RA (14). The immunologic role ofbone marrow cells in the pathogenesis of systemic JIAneeds to be elucidated by further studies.</p><p>In this study, the significant expression of IL-18was observed only in bone marrow and not in otherorgans. We at first supposed that the liver, especially itsKupffer cells, might be the source of the high elevationof IL-18, because Kupffer cells have been shown toproduce IL-18 in a mouse model of endotoxin shock (5)and in children with biliary atresia (15). In our patient,however, immunohistochemical staining revealed fewinfiltrated Kupffer cells and no expression of IL-18 inthe liver tissue. Therefore, the origin of IL-18 mightdiffer among diseases. Accordingly, the expression ofother cytokines, including IL-6, TNF, and IFN, inliver and bone marrow should be also studied. Wecannot exclude the possibility that our findings are adistinctive feature of MAS. However, considering thathighly elevated serum IL-18 levels were detected inalmost all patients with systemic JIA/AOSD regardlessof the presence of MAS (24), our results may becommonly observed in patients with systemic JIA orAOSD.</p><p>It was recently reported that depressed NK cellfunctions might account for inadequate control of T celland macrophage activation in patients with systemic JIAcomplicated by MAS (16), because NK cells and theperforin-based systems are normally involved in thedown-regulation of cellular immune responses (17).</p><p>Figure 2. Immunohistochemical staining of liver specimens with anti-CD68 monoclonal antibody or antiinterleukin-18 (antiIL-18) mono-clonal antibody. Liver specimens from the control subject were stainedwith anti-CD68 (A) and antiIL-18 (B), as were those from the patientwith systemic juvenile idiopathic arthritis (C and D). (Original magni-fication 100.)</p><p>ELEVATED IL-18 IN A PATIENT WITH SYSTEMIC JIA 1937</p></li><li><p>Considering that NK cells are strongly stimulated byIL-18, the relationship between the highly elevatedIL-18 level and the NK cell dysfunction in patients withsystemic JIA is interesting and should be investigated. IfNK cells produce no negative feedback despite highlyelevated IL-18 levels, the sustained macrophage activa-tion will result in further production of high levels ofproinflammatory cytokines. In addition, autologousstem cell transplantation for severe systemic JIA wasrecently shown to restore the reduced perforin expres-sion in CD8 T cells and NK cells (18) and to induce asignificant and drug-free remission of the disease(19,20). Bone marrow ablation and subsequent reconsti-tution may remove the activated cells and then allowregrowth of the normal cell population. Therefore,strategies that target activated bone marrow cells, in-cluding antiIL-18 therapy, may lead to a more satisfac-tory treatment for systemic JIA.</p><p>In conclusion, we demonstrated that bone mar-row was the probable source of highly elevated IL-18levels in a patient with systemic JIA. Further investiga-tions are needed to determine the role of bone marrowIL-18 in the pathogenesis of systemic JIA or AOSD.</p><p>REFERENCES</p><p>1. De Benedetti F, Martini A. Is systemic juvenile rheumatoidarthritis an interleukin 6 mediated disease? J Rheumatol 1998;25:2037.</p><p>2. Maeno N, Takei S, Nomura Y, Imanaka H, Hokonohara M,Miyata K. Highly elevated serum levels of interleukin-18 insystemic juvenile idiopathic arthritis but not in other juvenileidiopathic arthritis subtypes or in Kawasaki disease: comment onthe article by Kawashima et al [letter]. Arthritis Rheum 2002;46:253941.</p><p>3. Kawashima M, Yamamura M, Taniai M, Yamauchi H, TanimotoT, Kurimoto M, et al. Levels of interleukin-18 and its bindinginhibitors in the blood circulation of patients with adult-onsetStills disease. Arthritis Rheum 2001;44:55060.</p><p>4. Kawaguchi Y, Terajima H, Harigai M, Hara M, Kamatani N.Interleukin-18 as a novel diagnostic marker and indicator ofdisease severity in adult-onset Stills disease. Arthritis Rheum2001;44:17167.</p><p>5. Okamura H, Tsutsi H, Komatsu T, Yutsudo M, Hakura A,</p><p>Tanimoto T, et al. Cloning of a new cytokine that induces IFN-production by T cells. Nature 1995;378:8891.</p><p>6. McInnes IB, Gracie JA, Liew FY. Interleukin-18: a novel cytokinein inflammatory rheumatic disease. Arthritis Rheum 2001;44:14813.</p><p>7. Stephan JL, Zeller J, Hubert P, Herbelin C, Dayer JM, Prieur AM.Macrophage activation syndrome and rheumatic disease in child-hood: a report of four new cases. Clin Exp Rheumatol 1993;11:4516.</p><p>8. Bateman HE, Kirou KA, Paget SA, Crow MK, Yee AM. Remis-sion of juvenile rheumatoid arthritis after infection with parvovirusB19. J Rheumatol 1999;26:24824.</p><p>9. Saulsbury FT. Remission of juvenile rheumatoid arthritis withvaricella infection. J Rheumatol 1999;26:16068.</p><p>10. Schnedl WJ, Lipp RW, Trinker M, Ranner G, Schreiber F, KrejsGJ. Bone scintigraphy and magnetic resonance imaging in adult-onset Stills disease. Scand J Rheumatol 1999;28:2579.</p><p>11. Yetgin S, Ozen S, Saatci U, Bakkaloglu A, Besbas N, Kirel B.Myelodysplastic features in juvenile rheumatoid arthritis. Am JHematol 1997;54:1669.</p><p>12. Tanabe M, Ochi T, Tomita T, Suzuki R, Sakata T, Shimaoka Y, etal. Remarkable elevation of interleukin 6 and interleukin 8 levelsin the bone marrow serum of patients with rheumatoid arthritis.J Rheumatol 1994;21:8305.</p><p>13. Jongen-Lavrencic M, Peete...</p></li></ul>