Peripheral eosinophilia and eosinophilicgastroenteritis after pediatric livertransplantation

Liver transplantation has radically altered theclinical course of pediatric liver disease as patientand graft survivals have exceeded 80% atexperienced centers (1). This success is attribut-able to the use of potent immune modulatorsthat prevent or limit allograft rejection. Oppor-tunistic infections, dyslipidemia, and glucoseintolerance are common adverse effects of

immunosuppression (2). Recently, there havebeen reports of unusual allergic phenomenaincluding eosinophilic inflammation in the gas-trointestinal tract in pediatric liver transplantpatients (3–7). The mechanism(s) by which thelatter complications develop are unknown.One model of immune responses after trans-

plantation centers on the relative activities ofTh1/Th2 T-cell subsets (8). In this model, pro-inflammatory immune signaling is generated byTh1 lymphocytes that produce primarily interleu-kin-2 and interferon-c (8). The cells recruited by

Romero R, Abramowsky CR, Pillen T, Smallwood GA, Heffron TG.Peripheral eosinophilia and eosinophilic gastroenteritis after pediatricliver transplantation.PediatrTransplantation2003: 7: 484–488.� 2003BlackwellMunksgaard

Abstract: Reports indicate peripheral eosinophilia (PE) and gastroin-testinal eosinophilic inflammation can occur after pediatric liver trans-plantation. The incidence of these conditions, potential risk factors, andthe impact of PE and gastrointestinal eosinophilic inflammation on livertransplant outcome were determined in this pediatric liver transplantprogram. Medical records of liver transplant recipients from 1 to 97 andfrom 12 to 99 were reviewed. Fifty-seven transplants on 54 patients wereperformed during the study period. Fifty-three patients were evaluated;all had normal pre-transplantation peripheral eosinophil counts. PE of>10% developed in 28% of patients. Using this definition, all suchidentified patients had absolute eosinophil counts of >350/mm3. His-tory of immediate hypersensitivity did not differ between patients withor without eosinophilia. Gastrointestinal endoscopy and biopsy wasperformed in 23 patients with gastrointestinal complaints. Of those, sixhad eosinophilic gastroenteritis and all six had PE. Compared withpatients without eosinophilia, those with PE were younger at the timeof transplantation (p < 0.05), had more frequent rejection(p < 0.01), were more commonly managed with tacrolimus-basedimmunosuppression (p < 0.001), and experienced more frequent epi-sodes of detectable EBV viral load (p < 0.04). Patients with eosino-philic gastroenteritis were more frequently retransplanted (p < 0.006).PE associated with symptomatic eosinophilic gastroenteritis is commonafter pediatric liver transplantation. Age at transplant, frequency ofrejection episodes, tacrolimus-based immunosuppression, and EBVviral load may be associated with the development of this condition.There may be higher rates of graft loss in such patients. Whether innateimmune responsiveness or an acquired immune dysregulation accountsfor these findings merits further evaluation.

Ren� Romero1, CarlosR. Abramowsky2, Todd Pillen3,Gregory A. Smallwood4 and ThomasG. Heffron3

1Department of Pediatrics, Division of PediatricGastroenterology, Department of 2Pathology and3Department of Surgery, Division of LiverTransplantation, 4Department of Pharmacy, EmoryUniversity School of Medicine, Atlanta, GA, USA

Key words: pediatric liver transplantation –eosinophilia – eosinophilic gastroenteritis

Ren( Romero, MD, Department of Pediatrics, Divisionof Pediatric Gastroenterology, Emory UniversitySchool of Medicine, 2040 Ridgewood Dr, N.E.,Atlanta, GA 30322, USATel.: (404) 315 2832Fax: (404) 315 2631E-mail: rene.romero@choa.org

Accepted for publication 30 July 2003

Abbreviations: EBV, Epstein–Barr virus; PE, peripheraleosinophilia; PCR, polymerase chain reaction.

Pediatr Transplantation 2003: 7: 484–488

Printed in UK. All rights reservedCopyright � 2003 Blackwell Munksgaard

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these signals are thought to produce cell-mediatedcytotoxic reactions such as delayed-type hyper-sensitivity and allograft rejection. Th2 lympho-cytes produce interleukin-4, interleukin-5, andother cytokines which signal the induction ofhumoral responses, eosinophilia, and presumablyare involved in the development of graft toleranceand atopic reactions (8). This controversial para-digm is likely an oversimplification as Th2 typeresponses can also participate in rejection (9, 10).Additionally, PE has been identified as an earlymarker for allograft rejection for a variety oforgans (10–12). The primary drugs utilized forimmunosupression and successful liver transplan-tation are the calcineurin inhibitors, tacrolimus,and cyclosporine. Through different molecularmechanisms, they act to inhibit the transcriptionof interleukin-2 which blocks pro-inflammatoryresponses (13, 14). However, the full consequencesof chronic inhibition of interleukin-2 transcriptionare not clearly understood.Dhawan et al., in one of the first reports of

post-liver transplant eosinophilic gastroenteritis,postulated that the treatment of acute rejectionwith bolus steroids prompted an imbalance inthe Th1/Th2 profile provoking the developmentof eosinophilic gastrointestinal inflammation (3).Similarly, Lacaille et al. suggested this mechan-ism in their report of a child who developed denovo anaphylaxis to a variety of antigens afterliver transplantation (4). However, Inui et al.failed to show in two Japanese children withsevere allergic reactions to foods, any significantdifferences in the concentration of interleukin-4or interferon-c pre- and post-liver transplanta-tion. (5). At this pediatric liver transplantcenter, we have detected a high incidence ofPE developing post-transplantation and severalchildren who have developed eosinophilic gas-troenteritis. We sought to describe potential riskfactors and mechanisms leading to the develop-ment of these conditions and whether they hadany impact on liver transplant outcome.

Methods

The medical records of all pediatric liver transplant recipi-ents at the Emory University Pediatric Liver TransplantProgram and Children’s Healthcare of Atlanta at EglestonHospital from January 1, 1997 to December 31, 1999 werereviewed. Parameters abstracted included patient demo-graphics, pre-transplant and post-transplant eosinophilcounts, personal or family histories of immediate hyper-sensitivities to drug or food allergies, prior abdominal sur-geries, immunosuppressive regimen, liver transplant typeand number, number of biopsy-proven rejection episodes,endoscopic procedures and all pathologic diagnoses. For thepurposes of this study, PE was defined by any white celldifferential count showing >10% eosinophils at any time

post-transplant up to 3 yr following transplantation.All such identified patients had eosinophil counts>350 cells/mm3. Eosinophilic gastroenteritis was defined asincreased eosinophilic inflammation of the lamina propria(>10 eosinophils per high power field) and infiltration ofsurface or mucosal gland epithelium with eosinophils. EBVviral load was monitored using a commercially availablequantitative PCR assay (EBV-PCR) (EBV Ultraquant;Specialty Labs, Santa Monica, CA, USA). For continuousdata, a two-tailed Student’s t-test was performed. Fornominal data chi-square analysis was performed exceptwhere Fisher’s exact test was appropriate. The p-values<0.05 were considered significant. Medical record reviewwas approved by the Children’s Healthcare (Atlanta atEgleston Institutional Review Board, number 00–016).

Patients

During the study period, 57 liver transplants were per-formed on 54 patients at the Emory University PediatricLiver Transplant Program. A single patient was excludedfrom this analysis, as the child died shortly after trans-plantation from idiopathic fulminant hepatitis. Theremaining 53 patients ranged from 0.5 to 18.9 yr of age, 31were females and 22 were males. The principal diagnosis in24/53 (45%) of the patients was extrahepatic biliary atresia.Other diagnoses included: autoimmune hepatitis (6), alpha-1 antitrypsin deficiency (3), tyrosinemia (3), and acute liverfailure (4). A variety of diagnoses accounted for theremaining transplanted patients, including cryptogenic cir-rhosis, neonatal hepatitis, sclerosing cholangitis, Alagille’ssyndrome, and parenteral nutrition-induced cirrhosis (13).Only five children had a history of family or personal

allergies to drug or food products. Given the numbers ofpatients with biliary atresia, 25 of 53 children had priorabdominal surgery. The types of allografts utilizeddepended on multiple variables including, but not limitedto, the condition of the child, the availability of potentialliving donors, and availability of size-matched grafts. Ofthe grafts performed, 27 were whole organ, 23 werereduced segment grafts, and six were left lateral segmentsfrom living donors. A total of four patients underwentretransplantation, two for primary non-function, and twofor chronic rejection.During the first year of the study period (January 1997 to

December 1997), patients received cyclosporine and pred-nisone (n ¼ 19). Four patients in this group were eventuallyconverted from cyclosporine to tacrolimus. In the secondyear (January 1998 to November 1998), baseline immuno-suppression consisted of tacrolimus, prednisone, andmycophenolate mofetil (n ¼ 21). From November 1998,through the end of the study period, patients underwentinduction with the interleukin-2 receptor antagonist,dacluzimab, with the introduction of tacrolimus delayeduntil post-operative day 7 as recently described (15). Steroidand mycophenolate mofetil use were unaltered in this pro-tocol (n ¼ 17). Allograft rejection was determined to bepresent only when confirmed with a liver biopsy and anti-rejection therapy was initiated. Using this definition, 69episodes of acute allograft rejection occurred in 53 patients.Therapy for acute cellular rejection consisted of methyl-prednisolone boluses of 10 mg/kg which were repeated upto three consecutive days. Baseline prednisone therapy wasincreased to 0.3 mg/kg/day after documented rejection.Doses of calcineurin antagonist or mycophenolate mofetilwere adjusted as clinically indicated. During the first3 months post-transplantation monoclonal cyclosporine

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levels were maintained between 300 and 400 ng/dL. Tacr-olimus levels during the same time period were maintainedbetween 14 and 17 ng/dL. From 3 to 6 months post-trans-plant cyclosporine was maintained at 200–300 ng/dL andtacrolimus at 10–15 ng/dL. At 6–12 months after trans-plantation cyclosporine levels were maintained at200–300 ng/dL and tacrolimus at 5–10 ng/dL. After12 months post-transplantation, cyclosporine levels were100–200 ng/dL and tacrolimus maintained at approximately2–5 ng/dL. A similar approach of decreasing calcineurinlevels was utilized after documented acute rejections. A totalof six patients had steroid-resistant rejection and weretreated with monoclonal anti-CD3 antibody.Testing for EBV replication was performed when clinic-

ally indicated as a result of changes in baseline liver functiontests, or if the patient developed symptoms potentiallyattributable to systemic or localized EBV infection. A totalof 35 patients had post-transplantation EBV-PCR testing,with 20 of 35 developing detectable viral loads during thestudy period. Biopsy-proven lymphoproliferative disorderdeveloped in three patients (6%).

Results

No patients had >4% peripheral eosinophils onperipheral blood smear prior to transplantation,and all had absolute eosinophil counts of <350/mm3. PE of >10% developed in 15 of 53 patients(28%) after transplantation. All of these patientshad absolute eosinophil counts of>350/mm3. PEwas seen at a mean of 417 ± 240 days aftertransplantation. Thus, the development of PEoccurred relatively late after transplantation.During the study period, 23 patients underwentupper and/or lower gastrointestinal endoscopywith biopsies for various complaints includingvomiting, nausea, diarrhea, visible or occult bloodin the stool. Of these, six patients (26%), hadeosinophilic gastroenteritis involving the upper anlower gastrointestinal tracts. All of these patientshadPE. Significant, andoften severe and extensiveeosinophilic gastroenteritis was the single mostcommon pathologic finding in our patients under-going gastrointestinal endoscopy.Patients developing PE and unaffected trans-

plant recipients were compared, with the resultsdepicted in Table 1. Affected patients were onaverage younger than those who did not developPE (3.7 ± 5.5 yr vs. 7.3 ± 6.3 yr, p ¼ 0.05).These patients, as a group, had more rejectionepisodes, than those without PE (2.5 rejections/patient vs. 0.9 rejections/patient, p ¼ 0.01), butdid not differ in rate of steroid-resistant rejection.Differences in sex, original liver disease, priorallergic history, or type of liver graft received didnot reach statistical significance. The detection ofincreased EBV viral load was more common inthe PE group (9/15 vs. 11/38, p < 0.04), but thefrequency of lymphoproliferative disorder wasno different in the two groups.

The primary immunosuppresion regimen util-ized was highly statistically related to the devel-opment of PE in our population. Patients withPE were much more likely to have receivedtacrolimus as primary immunosuppression(11/15 vs. 10/38, p < 0.001). The use of cyclo-sporine was not statistically associated with thedevelopment of PE. Although tacrolimus wasstill utilized after induction with dacluzimab in13 patients, PE developed in only one suchpatient. Although the rate of retransplantation inthose with and without PE was not statisticallydifferent, the sub-population of patients with PEthat developed eosinophilic gastrointestinalinflammation was indeed retransplanted at ahigher frequency (3/6 vs. 1/38, p < 0.006).

Discussion

Reports of eosinophilic inflammation in thegastrointestinal tract and allergic phenomenaafter liver transplantation have increased inrecent years (3–7). In these studies, potential riskfactors for the development of these conditionsare not identified. Dwahan et al. in their reportof two patients with eosinophilic gastroenteritis,postulated that therapy for rejection with pulsedsteroids could potentially cause a shift to a Th2response, promoting tissue eosinophilia (3).Spanish researchers have recently linked thedevelopment of severe food allergy in five chil-dren after liver transplant to prednisone weaningand tacrolimus use (16). Recent reports fromcenters in the US have also been published (6).However, it is difficult to draw firm conclusionsas control populations were not identified and

Table 1. Clinical factors assessed relative to development of peripheraleosinophilia after pediatric liver transplantation

Characteristic

<10%Eosinophils(n ¼ 38)

>10%Eosinophils(n ¼ 15) p-value

Age at transplant 7.3 € 6.3 3.7 € 5.5 <0.05Rejections per patient 0.9 2.47 <0.01Steroid resistant rejection 4 2 nsMale 16 6 nsFemale 22 9 nsBiliary atresia 15 9 nsReduced segment 14 10 nsPrior allergies 4 1 nsDetectable EBV viral load 11 9 <0.04Lymphoproliferative disorder 2 1 nsCyclosporine (n ¼ 19) 16 3 nsTacrolimus (n ¼ 21) 10 11 <0.001Dacluzimab (n ¼ 13) 12 1 nsRetransplantation (n ¼ 4) 1 3 nsRetransplantation in those witheosinophilic gastroenteritis

3/6 <0.006

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evaluated for comparison. Whether eosinophilicinflammation of the gastrointestinal tract andpotentially associated allergic phenomena areclinically important entities in this settingdepends upon an assessment of the prevalenceof the conditions and their relationship to clinicaloutcomes after transplantation.Our study indicates that significant PE is a

relatively common occurrence after liver trans-plant (28% of our patient population) andusually occurs late in the transplant course. Thishigh prevalence is notable as a relatively high cutoff point was chosen: >10% eosinophils on theperipheral smear, a level that consistently correlatedwith an absolute eosinophil count of >350/mm3.No other reports of pediatric liver transplantrecipients have indicated the prevalence of PE,however, a very similar frequency of PE andeosinophilic gastroenteritis has been reported inabstracts among heart transplant recipients at theUniversity of Pittsburgh (17, 18).Within our patient population, young trans-

plant recipients appeared to develop PE morefrequently. Very few of our patients had personalor family histories of allergic problems identifiedprior to transplant and there was no difference infrequencies of these histories among the PE andunaffected groups. In our study, the presence ofatopic disorders such as eczema and asthma werenot ascertained. The retrospective nature of thestudy did not accurately reflect these histories. Itwould therefore appear that at least a familialpredisposition for immediate hypersensitivity isnot a prerequisite for the development of PE aftertransplant. Whether this would be true regardingfamily or patient histories of eczema or asthma isuncertain. Pre- and post-transplantation cytokineprofiles or IgE levels were not obtained as part ofthis study, therefore one cannot completelyexclude the possibility of an atopic predispositionin those developing PE. Although not systemat-ically analyzed in all patients due to the retro-spective nature of this report, three of the sixpatients with eosinophilic gastrointestinal inflam-mation had elevated IgE levels and positiveradioallergosorbent testing to specific allergen(milk). The results do confirm however, that PE isan acquired condition as it was not present priorto transplantation in any patient.If the condition is common and acquired, what

is the driving factor in its occurrence? Biopsyproven rejection episodes occurred in all patientsdeveloping PE and occurred more frequentlythan in those not developing PE. In fact, PEnever occurred in the absence of a precedingrejection episode. The relationship of rejectionwith PE and eosinophilic gastrointestinal inflam-

mation is further strengthened when the fre-quency of retransplantation is examined. Threeof the six patients with carefully defined eosino-philic gastrointestinal involvement were retrans-planted compared with one of 38 in theunaffected group (p < 0.006). The importantrole of rejection in setting the stage for thedevelopment of PE and gastrointestinal inflam-mation is further suggested in our data. Rejectionrates decreased after the introduction of daclu-zimab induction (15). Although it did not reachstatistical significance, there was a reduced rateof PE and no documented gastrointestinal eosi-nophilic inflammation in the dacluzimab-treatedpatient population. This did coincide with lowermean tacrolimus levels at day 28 post-transplan-tation in the dacluzimab-treated patients asrecently reported by our group. Over the longterm, dacluzimab-treated patients had slightlylower tacrolimus levels (15).Although the presence of prior rejection is

apparently required to produce PE, it is quitepossible that it is the treatment of rejection incertain patients that leads to PE and subse-quently to gastrointestinal eosinophilic irritation.As noted by Dwahan et al., the acute manage-ment of rejection is frequently treated with bolussteroid therapy. The patients described in theirseries did not have increases in baseline steroidtherapy after the boluses and they postulatedthat a relative Th1/Th2 imbalance caused bybolus steroid therapy was driving the develop-ment of eosinophilic inflammation (3). Ourpatients� rejection episodes were treated withbolus steroid and increases in baseline prednisonetherapy and still PE developed in this setting.Further, patients who did not develop PE andhad rejection were treated in a similar manner.It is therefore unlikely that bolus steroid therapyafter rejection is alone responsible for the devel-opment of PE after transplant.There was a strong association of baseline

tacrolimus immunosuppression and the develop-ment of PE and eosinophilic gastroenteritis.Asante-Korang et al. demonstrated a similarprevalence of PE on tacrolimus therapy in hearttransplant recipients, and Sutton et al. documen-ted eosinophilic gastroenteritis in these patients(17, 18). The reason for the association betweentacrolimus, PE, and eosinophilic gastroenteritis isnot clear. Tacrolimus increases gastrointestinalpermeability, possibly causing greater dietaryantigenic exposure and thus may be a predispo-sing factor for the development of an atopic state(19). Compared with cyclosporine, tacrolimus isa more potent inhibitor of interleukin-2, andtherefore of Th1 cellular responses (13, 14). It is

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possible that rejection occurring in the face ofbaseline tacrolimus therapy when treated withsteroid bolus therapy augments Th-2 responsesand eosinophilia. The apparent decrease in PEafter induction therapy with dacluzimab in ourpatients may be attributable to lower tacrolimuslevels in dacluzimab-treated patients (18).The role of EBV viral load in the development

of PE is not known. Younger age at the time oftransplantation may be an adequate explanationfor an association between viral load and PE.Our study indicates that PE and eosinophilic

gastrointestinal inflammation are common afterliver transplantation in children. Case reportsfrom across the world suggest that this is not apeculiar problem to our center. It is very likelythat these conditions are under-recognizedbecause peripheral white blood cell differentialcounts may not be routinely monitored, especi-ally later in the post-transplant course. Theprevalence of PE and its association with gastro-intestinal inflammation suggests that monitoringwhite blood cell differentials could be important.Furthermore, the management of eosinophilicgastrointestinal inflammation can be challenging.Our data suggests multiple, potentially interre-lated factors participate in producing these con-ditions. Younger patients with reactive immunesystems are at risk. The precise role of baselineimmunosuppression, therapy of rejection andviral infection are still to be determined. Thisstudy offers a starting point for various hypo-theses that can be tested both in the laboratoryand clinic that may offer insight into eosinophilicgastrointestinal inflammatory states in both thetransplant and non-transplant setting.

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