2004;113;e7PediatricsTai F. Fok

Cheng, Ting F. Leung, Ellis K.L. Hon, Iris H.S. Chan, Chi K. Li, Kitty S.C. Fung and Pak C. Ng, Christopher W.K. Lam, Albert M. Li, Chun K. Wong, Frankie W.T.

SyndromeInflammatory Cytokine Profile in Children With Severe Acute Respiratory

  

  http://pediatrics.aappublications.org/content/113/1/e7.full.html

located on the World Wide Web at: The online version of this article, along with updated information and services, is

 

of Pediatrics. All rights reserved. Print ISSN: 0031-4005. Online ISSN: 1098-4275.Boulevard, Elk Grove Village, Illinois, 60007. Copyright © 2004 by the American Academy published, and trademarked by the American Academy of Pediatrics, 141 Northwest Pointpublication, it has been published continuously since 1948. PEDIATRICS is owned, PEDIATRICS is the official journal of the American Academy of Pediatrics. A monthly

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Inflammatory Cytokine Profile in Children With Severe AcuteRespiratory Syndrome

Pak C. Ng, MD, FRCPCH*; Christopher W.K. Lam, PhD‡; Albert M. Li, MRCP*; Chun K. Wong, PhD‡;Frankie W.T. Cheng, MRCPCH*; Ting F. Leung, MRCP*; Ellis K.L. Hon, FAAP*; Iris H.S. Chan, PhD‡;

Chi K. Li, FRCP*; Kitty S.C. Fung, FRCPA§; and Tai F. Fok, MD, FRCPCH*

ABSTRACT. Objective. To study the inflammatorycytokine profile in children with severe acute respiratorysyndrome (SARS) and to investigate whether monoclo-nal antibody to tumor necrosis factor-� (TNF-�) could beconsidered for treatment of these patients.

Methods. Plasma inflammatory cytokine concentra-tions (interleukin [IL]-1�, IL-6, IL-8, IL-10, IL-12p70, andTNF-�) were monitored longitudinally on admission, im-mediately before corticosteroids, and 1 to 2 days and 7 to10 days after the drug treatment in a cohort of pediatricpatients (n � 8) with virologic confirmed SARS-associ-ated coronavirus infection. None of the patients requiredmechanical ventilation or intensive care treatment. Allchildren except 1 (patient 3) received corticosteroids.

Results. Plasma IL-1� levels (excluding patient 3)were substantially elevated immediately before (range:7–721 ng/L) and 7 to 10 days after (range: 7–664 ng/L)corticosteroid treatment. In contrast, the plasma concen-trations of other key proinflammatory cytokines, includ-ing IL-6 and TNF-�, were not overtly increased in any ofthe patients throughout the course of illness. In addition,plasma IL-10 concentration was significantly lower 1 to 2days and 7 to 10 days after corticosteroid treatment, com-pared with the immediate pretreatment level. Similarly,plasma IL-6 and IL-8 concentrations were significantlydecreased 7 to 10 days after the drug treatment.

Conclusions. Pediatric SARS patients have markedlyelevated circulating IL-1� levels, which suggests selec-tive activation of the caspase-1–dependent pathway.Other key proinflammatory cytokines, IL-6 and TNF-�,showed only mildly elevated levels at the initial phase ofthe illness. The current evidence does not support the useof TNF-� monoclonal antibody in this group of children.Pediatrics 2004;113:e7–e14. URL: http://www.pediatrics.org/cgi/content/full/113/1/e7; children, cytokines, SARS.

ABBREVIATIONS. SARS, severe acute respiratory syndrome;SARS-CoV, SARS-associated coronavirus; ARDS, acute respira-tory distress syndrome; TNF-�, tumor necrosis factor-�; IL, inter-leukin; RSV, respiratory syncytial virus.

The severe acute respiratory syndrome (SARS)is a newly discovered infectious disease causedby a novel coronavirus.1,2 Hong Kong is one of

the most severely affected cities.3,4 The outbreaks ofSARS at the Prince of Wales Hospital and in adensely populated housing estate, Amoy Gardens,have affected 1755 local residents and claimed 299lives (as of July 16, 2003). Similar to the H5N1 “avianflu” influenza infection, patients with SARS-associ-ated coronavirus (SARS-CoV) infection develop pri-mary viral pneumonia and lymphopenia, and severecases may involve acute respiratory distress syn-drome (ARDS).5,6 The H5N1 influenza virus hasbeen shown to be a potent inducer of proinflamma-tory cytokines.6,7 In particular, there is substantialupregulation in tumor necrosis factor-� (TNF-�) pro-duction.7 Whether SARS-CoV may induce a similarinflammatory cytokine pattern and contribute to theunusual severity of human disease is not known.Recently, it was proposed that cytolysis may be as-sociated with viral amplification in the early stage ofSARS, which is then followed by an adaptive immu-nologic response with clearance of virus and severetissue inflammation.8 The use of immunomodulatingdrugs, such as corticosteroids or monoclonal anti-body to TNF-�, may be useful in suppressing thehost immune response. Thus, knowing the cytokinepattern could assist the clinicians in understandingthe disease mechanism and in designing a treatmentstrategy most effective for the management of thiscondition. We measured prospectively a panel ofproinflammatory and anti-inflammatory cytokines,using the flow cytometric technique during the acutephase of illness, to investigate whether monoclonalantibody to TNF-� is indicated for treatment ofSARS. This report describes the inflammatory cyto-kine profile in a cohort of children with SARS.

METHODS

PatientsDuring the SARS outbreak between March 13 and May 17,

2003, 8 children with virologic confirmed SARS-CoV infectionwere admitted to the pediatric unit of the Prince of Wales Hospi-tal. Their clinical characteristics are summarized in Table 1.

Drug RegimenAll febrile children with suspected SARS were initially given a

broad-spectrum antibiotic, cefotaxime, and an antimicrobial,erythromycin or clarithromycin, for treatment of atypical pneu-monia. Oral ribavirin (40–60 mg/kg/d) was also started when thechild had a definitive contact history of SARS or had high fever

From the Departments of *Paediatrics, ‡Chemical Pathology, and §Micro-biology, Prince of Wales Hospital, Chinese University of Hong Kong, Sha-tin, New Territories, Hong Kong.Received for publication Jul 31, 2003; accepted Oct 1, 2003.Reprint requests to (P.C.N.) Department of Paediatrics, Level 6, ClinicalSciences Bldg, Prince of Wales Hospital, Shatin, New Territories, HongKong. E-mail: pakcheungng@cuhk.edu.hkPEDIATRICS (ISSN 0031 4005). Copyright © 2004 by the American Acad-emy of Pediatrics.

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(�38.5°C) and abnormal radiographic changes suggestive ofpneumonia on admission. When the patient remained febrile 48 to72 hours after commencement of antibiotics and antiviral treat-ment, oral prednisolone (0.5–1 mg/kg/d) was added. Intravenousribavirin (20 mg/kg/d) and pulse methylprednisolone (10 mg/kg/d, for 2–3 days) were reserved for those who had persistentfever, continuing clinical deterioration, or progressive consolida-tion on chest radiographs or computed tomography scan of thethorax. Thereafter, a dose-tapering course of oral prednisolonewas prescribed in the following 2 weeks. This pediatric treatmentregimen was extrapolated from the adult experience,3,8 and thedose and timing of commencement of these medications wereconstantly under review during the outbreak.

Laboratory InvestigationsIn view of the difficulty in diagnosing SARS at the initial stage

of clinical presentation, serial blood tests for monitoring lympho-cyte counts, biochemical enzymes, and inflammatory cytokineswere performed in all children with probable SARS.9 Blood sam-ples for cytokine measurement were obtained 1) on admission, 2)immediately (ie, within 24 hours) before commencement of corti-costeroids, 3) 1 to 2 days after the drug treatment, and 4) 7 to 10days after the drug treatment. All specimens were collected dur-ing the routine blood sampling procedure to minimize the distur-bance to the patients. On each occasion, 0.6 mL of venous bloodwas collected in a chilled container. The blood samples wereimmediately immersed in ice and transported to the laboratory forprocessing.

Plasma was separated by centrifugation (1900 � g for 5 min-utes) at 4°C and stored in 200-�L aliquots at �80°C until analysis.A panel of proinflammatory and anti-inflammatory cytokines—including interleukin (IL)-1�, IL-6, IL-8, IL-10, IL-12p70, and TNF-�—were simultaneously quantified by the Human InflammatoryCytokine Cytometric Bead Array Kit (BD Pharmingen, San Diego,CA) using flow cytometry (FACSCalibur system, Becton Dickin-son Corp, San Jose, CA). The assay sensitivities for IL-1�, IL-6,IL-8, IL-10, IL-12p70, and TNF-� were 7.2, 2.5, 3.6, 3.3, 1.9, and 3.7ng/L, respectively. In cytometric bead array, 6 bead populationswith distinct fluorescence intensities were coated with specificantibodies for capturing different cytokines in plasma. The cyto-kine-captured beads were then mixed with phycoerythrin-conju-gated detection antibodies to form sandwich complexes. Afterincubation, washing, and acquisition of sample data, the resultwas generated in a graphic format using the BD cytometric beadarray analysis software. The coefficients of variation for all cyto-kine assays were �10%.

In addition, microbiologic investigations were performed todetect common bacterial and viral pathogens associated with com-munity-acquired pneumonia. Throat swab, sputum samples, andblood samples were taken for routine bacterial cultures. Throatswab in infants and throat gargle samples from older childrenwere also obtained for 1) antigen detection of influenza A and B;respiratory syncytial virus (RSV); adenovirus; and parainfluenza1, 2, and 3, using the commercial immunofluorescence assay kits;2) virus isolation using different culture cell lines to recover com-mon respiratory viruses and SARS-CoV; and 3) reverse-transcrip-tase–polymerase chain reaction for detecting influenza A and B,RSV, enteroviruses, and SARS-CoV.10 Tracheal aspirate samplewas not available for virologic or cytokine analysis, as none of thepatients required intubation or mechanical ventilation. Pairedacute and convalescent serum samples were tested for Chlamydiapneumoniae, C psittaci, Mycoplasma pneumoniae, and SARS-CoV an-tibodies.11 It was estimated that reverse-transcriptase–polymerasechain reaction for all types of clinical specimens identified approx-imately 62% of SARS patients, whereas paired viral titers forSARS-CoV is considered to be the gold standard test for diagnos-ing SARS. More than 90% of patients demonstrate a significantincrease in titer when the convalescent sample is taken 4 weeksafter the onset of illness.11

Statistical AnalysisWilcoxon signed rank test was used to assess the difference in

plasma cytokine concentrations immediately before and 1 to 2days or 7 to 10 days after commencement of corticosteroids. Allstatistical tests were performed by SPSS for Windows (Release 10;SPSS Inc, Chicago, IL). The level of significance was set at 5% in allcomparisons.

Ethical ApprovalThe study was approved by the research ethics committee of

the Chinese University of Hong Kong. Informed consent wasobtained from the parents or guardians for all patients.

RESULTS

The clinical features; laboratory, radiology, andvirology findings; and treatment outcomes are sum-marized in Table 1. All patients had significant in-crease in their convalescent viral titers specific forSARS-CoV. All except 1 child (patient 3) receivedoral prednisolone. In most cases (except patients 2and 6), subsidence of fever and improvement in chestradiographic appearances occurred within 48 hoursof corticosteroid treatment (Table 1). Patient 6 alsoreceived intravenous ribavirin because of persistenthigh fever and severe pneumonia. Patients 2 and 6required pulse methylprednisolone for treatment ofprogressive lung condition, and both patients re-sponded with substantial clearance of chest radio-graph 72 hours after treatment. None of the childrenrequired mechanical ventilation, and all patients sur-vived.

Four children (patients 4, 5, 6, and 8) did not haveblood taken for cytokines on admission. The longi-tudinal profile of the inflammatory cytokines areillustrated in Fig 1. Plasma IL-1� concentrations (ex-cluding patient 3) were substantially elevated bothimmediately before (range: 7–721 ng/L) and 7 to 10days after corticosteroid treatment (range: 7–664ng/L; Fig 1A). In contrast, the plasma levels of otherkey inflammatory cytokines, including IL-6 (range:2.5–99 ng/L) and TNF-� (range: 0–18 ng/L), werenot overtly increased in any patient throughout thecourse of illness (Fig 1B–F).

The cytokine results before and after corticosteroidtreatment are summarized in Table 2. Plasma IL-10concentrations were significantly lower 1 to 2 daysand 7 to 10 days after corticosteroid treatment, com-pared with the immediate pretreatment level (P �.028, for both time points). Similarly, both plasmaIL-6 and IL-8 concentrations were significantly de-creased 7 to 10 days after the drug treatment (P �.018 and P � .043, respectively). The overall trend ofthese cytokines suggests elevated levels at the initialphase of the disease (ie, the first 2 time points), whichwas then followed by a decline in plasma concentra-tion with time (Fig 1B–D , Table 2). The reductions inplasma cytokine levels 7 to 10 days after corticoste-roid therapy paralleled improvements in the clinicalconditions. Plasma TNF-� concentrations, however,were not significantly different before and after cor-ticosteroid treatment (P � .08, for both time points).

Patient 6 was the sickest child in our cohort. Hiscytokine profile indicated that he had the highestproinflammatory cytokine levels—IL-1� (721 ng/L),IL-6 (88 ng/L), IL-8 (26.7 ng/L), and TNF-� (18 ng/L)—immediately before corticosteroid treatment.The decline in plasma cytokine concentrations wasalso much slower compared with other patients (Fig1).

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Fig 1. A, Change in plasma IL-1� concentrations in 4 different time points after the onset of illness—on admission, immediately beforecorticosteroid treatment, and 1 to 2 days and 7 to 10 days after treatment—in 8 children with mild and moderate SARS. All patients (exceptpatient 3) received corticosteroid treatment. B, Change in plasma IL-6 concentrations in 4 different time points after the onset ofillness—on admission, immediately before corticosteroid treatment, and 1 to 2 days and 7 to 10 days after treatment—in 8 children withmild and moderate SARS. All patients (except patient 3) received corticosteroid treatment.

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Fig 1. C, Change in plasma IL-8 concentrations in 4 different time points after the onset of illness—on admission, immediately beforecorticosteroid treatment, and 1 to 2 days and 7 to 10 days after treatment—in 8 children with mild and moderate SARS. All patients (exceptpatient 3) received corticosteroid treatment. D, Change in plasma IL-10 concentrations in 4 different time points after the onset ofillness—on admission, immediately before corticosteroid treatment, and 1 to 2 days and 7 to 10 days after treatment—in 8 children withmild and moderate SARS. All patients (except patient 3) received corticosteroid treatment.

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Fig 1. E, Change in plasma IL-12p70 concentrations in 4 different time points after the onset of illness—on admission, immediately beforecorticosteroid treatment, and 1 to 2 days and 7 to 10 days after treatment—in 8 children with mild and moderate SARS. All patients (exceptpatient 3) received corticosteroid treatment. F, Change in plasma TNF-� concentrations in 4 different time points after the onset ofillness—on admission, immediately before corticosteroid treatment, and 1 to 2 days and 7 to 10 days after treatment—in 8 children withmild and moderate SARS. All patients (except patient 3) received corticosteroid treatment.

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DISCUSSIONThis study was designed to investigate prospec-

tively the longitudinal cytokine profile of childrenwith SARS. Similar to H5N1 “avian flu” pneumonia,SARS-CoV–induced atypical pneumonia is also asso-ciated with pulmonary epithelial cell proliferation,infiltration of alveolar macrophages, early hyalinemembrane formation, hemophagocytosis, and otherhistopathologic features suggestive of ARDS.6,12 Thepresence of hemophagocytosis supports the notionthat cytokine dysregulation may be, at least partly,responsible for an exaggerated and persistent inflam-matory response causing diffuse alveolar damageand alveolar fibroproliferation.6,12,13 Marked in-crease in TNF-� and other proinflammatory cyto-kines, including IL-6 and interferon-�, have beendocumented in human influenza A H5N1 infec-tion.6,7 To date, the cytokine pattern in children withSARS has not been reported. Our results revealed apredominant upregulation of IL-1� but not TNF-� orIL-6 after SARS-CoV infection in children. In exper-imental respiratory infection of pig lungs, Van Reethet al14 showed that IL-1 but not TNF-� or interfer-on-� was increased after porcine reproductive-respi-ratory syndrome virus infection, whereas all 3 proin-flammatory cytokines were upregulated in responseto swine influenza virus. Similarly, IL-1� but notTNF-� was secreted from macrophages infected witha neurotropic strain of coronavirus in irradiatedmice.15 A unique feature of IL-1� production is thatthis pyrogenic cytokine is synthesized as an inactiveprecursor (pro-IL-1�), and virus-induced activationof caspase-1 (IL-1�–converting enzyme) is requiredfor converting this precursor protein into its biolog-ically active form (IL-1�).16 Pirhonen et al16 foundthat influenza A and Sendai virus could infect hu-man macrophages, and induced the activation ofcaspase-1–dependent pathway resulting in enhance-ment of IL-1� production. Hence, one plausible ex-planation of our observation is that there has been aselective activation of the caspase-1–dependentpathway in SARS-CoV infection. Nonetheless, theexact mechanism that leads to SARS-CoV–inducedimmunomodulation remains to be elucidated. Ourresults also suggest that other key circulating proin-flammatory and anti-inflammatory cytokines—TNF-�, IL-6, and IL-10—on admission and immedi-ately before corticosteroid treatment were notovertly elevated as observed in H5N1 infection.Monoclonal antibody to TNF, therefore, is unlikely tobe useful in this group of children, and the proin-

flammatory and antiviral action of normal TNF-�response may even be adversely attenuated by thistreatment. However, we must point out that the cir-culating TNF-� levels may not necessarily reflect thelevels produced locally in the lungs, although a sub-stantial increase in regional TNF-� level is likely tooverspill into the circulation. Hence, more researchinto pulmonary cytokine production and in severecases is required for clinicians to understand betterthe immunologic aspect of this new disease.

More important, previous studies on cytokinesand ARDS suggested that nonsurvivors who re-ceived intensive care treatment had 1) significantlyhigher circulating TNF-�, IL-6, and IL-8 levels onadmission; 2) persistent elevation of proinflamma-tory cytokine levels over time; and that 3) such levelsremained elevated despite corticosteroid treatment,compared with survivors.13,17 Again, our findingsindicate a decreasing trend in most key inflamma-tory cytokines with time and a significant decrease inplasma concentrations of IL-6, IL-8, and IL-10 after 7to 10 days of corticosteroid treatment (Fig 1). Thereduction in plasma cytokine levels correspondedclosely with improvement in the clinical conditionand resolution of inflammatory shadows on chestradiographs. It has been suggested that effective con-tainment of host inflammatory response, as a resultof either spontaneous improvement or suppressionby anti-inflammatory drugs such as corticosteroids,was associated with a reduction in circulating in-flammatory cytokine levels. The reduction in cyto-kine levels also correlated with improvement in gen-eral clinical condition, pulmonary function, andsurvival.13,17 In view of rapid deterioration in lungfunction in many adult patients during the acutephase of SARS-CoV infection,3,8 it would be deemedunethical to withhold corticosteroid treatment in anypatients who had persistent fever or progressive ra-diographic changes at the onset of the outbreak.8Hence, all except 1 patient in this cohort were treatedwith corticosteroids. Consequently, we are unable toconclude with confidence that the reduction in cir-culating cytokine levels was associated with cortico-steroid usage or just the natural recovery process ofillness independent of any drug treatment. The ab-sence of overtly elevated plasma IL-6 and TNF-�levels, together with a progressive reduction in con-centrations of other cytokines, IL-8 and IL-10, didpredict a favorable outcome for our patients. Suchfindings concur with our previous clinical observa-tion that children tend to have milder disease and a

TABLE 2. Changes in Cytokine Profile of Children With SARS (Excluding Patient 3) Before and After Corticosteroid Treatment

Plasma CytokineConcentrations

(ng/L)

Immediately BeforeCorticosteroid Treatment

(n � 7)

1–2 Days AfterCorticosteroid Treatment

(n � 7)

7–10 Days AfterCorticosteroid Treatment

(n � 7)

NormalReferenceRange19–23

IL-1� 127 (105–400) 130 (58–390) 100 (39–433) �1.5IL-6 29 (24–56) 20 (3–74) 3 (3–27)* �4.3IL-8 6.4 (3.6–24.4) 7.5 (3.6–26.7) 3.6 (3.3–11.8)* �30.4IL-10 9.2 (6.9–15.9) 5.3 (4.9–6.5)* 3.3 (2.0–3.3)* �2.0IL-12p70 12.1 (6.2–125) 13.0 (5.5–121) 10.5 (5.9–126) �9.0TNF-� 3.7 (3.7–7.2) 3.7 (2.1–5.8) 3.7 (2.4–7.4) �3.5

Results are median (interquartile range).* P � .05

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less aggressive clinical course than adult SARS pa-tients.4 The cytokine results, therefore, cast doubt onthe liberal use of corticosteroids in pediatric SARSpatients, as the host immunologic response did notseem to be as severe as initially anticipated. How-ever, it is important to note that our patients hadrelatively mild symptoms, and none required me-chanical ventilation or intensive care. Thus, the re-sults may not be generalizable to cases with severedisease. Nonetheless, it is reassuring that there wasno fatality among the pediatric patients in HongKong. It is interesting that corticosteroids were notused in Canada, and all children recovered withsupportive treatment.19 Corticosteroids should prob-ably be reserved for patients with severe disease, inparticular, those who require oxygen supplementa-tion and mechanical ventilatory support.

CONCLUSIONSPediatric SARS patients had markedly elevated

plasma IL-1� levels, which suggested selectivecaspase-1–dependent pathway activation in infectedmacrophages. In contrast to H5N1 influenza infec-tion,6,7 other key proinflammatory cytokines, includ-ing IL-6 and TNF-�, were only mildly elevated at theinitial phase of the illness. Most of these cytokinelevels fell with time and coincided with improve-ment in clinical conditions and radiographic appear-ances. Thus, the current evidence does not supportthe use of monoclonal antibody to TNF-� for treat-ment of children with SARS. A randomized, con-trolled study would be useful to determine the effec-tiveness and adverse effects of corticosteroids inpediatric patients with severe SARS-CoV infection.

ACKNOWLEDGMENTWe are grateful to Kelly Cheng of Greater China Technology

Group Ltd for the donation of a multifluorescence flow cytometerfor SARS research.

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2004;113;e7PediatricsTai F. Fok

Cheng, Ting F. Leung, Ellis K.L. Hon, Iris H.S. Chan, Chi K. Li, Kitty S.C. Fung and Pak C. Ng, Christopher W.K. Lam, Albert M. Li, Chun K. Wong, Frankie W.T.

SyndromeInflammatory Cytokine Profile in Children With Severe Acute Respiratory

  

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