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E L S E V I E R Transplant Immunology 15 (2005) 63 68

TransplantImmunology

w w w. e 1 sev i e r. c o m' I o ca t e/1 ri m

C4d deposition in lung allografts is associated withcirculating anti-HLA alloantibody^

D.N. Ionescu'̂ "', A.L. Gimita"^"', A. Zeevi^, R. Duquesnoy'', J. Pilewski^ B. Johnson^S. Studer'' K.R. McCurry^ S.A. YousenV^*

l. Scaife Hall. Pitlshiirgli. PA. 15213. United State's^200 Lothrop St. H'1558. Biomed Sci Twr. Pitt.sbiii-g. PA. 15213, United States"200 Lothrop St. iVI55l. Biomed Sci Twr Pimhwg, PA. 15213. United States•'300 Loihrop SI. WI552. Biomed Sci Twr. Pitishiii-g. PA. 15213. United States

"NiV 62K Monlefiort-Uiiiv Hasp. I'itlshuiyb. PA. 15213. United States"1212 Kaujmann BIdg, Piit.^hiiixh. PA. 15213. United States0. Presbyterian Unix Hasp. Piiishnrgli. PA. 15213. United States

rt^i'/, Preshyierian Uiiiv Hiisp. Pii!.\hnrgli, PA, 15213. United Siaie.s

Received 2 May 2005; accepted 9 May 2005

Abstract

The complement activation demonstrated by vasciiliir C4d deposition is used to diagnose antibody-inediiitcd rejection (AMR) in renalallogralts, but rertiains conlroversial in Iting transplantation (LTX).

Methods: C4d deposition was assessed by ininiunohistochemistry in 192 lung transplant biopsies from 32 patients. tXISA analysis wasperformed on 415 serum samples in those 32 temporally and rejection-grade matched LTX patients; 16 patients developed HLA-Ab, whilethe other 16 patients remained negative. The specificity ofC4d staining was further compared in 18 additional LTX patients wiihotit HLA-Abor acute cellular rejection (ACR), but in the presence of CMV-pneumonitis or reperfusion injury.

Results: Specific subendothelial C4d deposition was seen in 5 of 16 (31%) palients with HLA-Ab and was absent in 16 patients withoutHLA-Ab (/7<0.05). All patients with speeific C4d deposition exhibited donor-specifle HLA-Ab. There were 13 patients with bronchiolitisobliterans sytidrome in the group of 16 HLA-Ab positive patients, versus 2/16 in ELISA-negative patients (/?<0.005). One of 7 patients withCMV pneumonitis and 2 of 11 patients with reperfusion injury also showed C'4d positivity (not statistically significant).Conclusions: In this study, spceillc subendothelial C4d deposition was a marker for the involvernent of H[,A-Ab in lung allograft rejection.The patchy nature, low sensitivity, and specitlcity of C4d staining might limit clinical use in protocol biopsies. However, in patients withdecreasing pulrnonary function, refractory ACR and/or HLA-Ab, specitle C4d deposition may serve as a marker of coexistent AMR.<"• 2005 Elsevier B.V. All rights resen-'cd.

Keywords: Lung transplantation: HLA: Antibodies: Complemeni: Bronchiolitis oblilerans: Transplantation immunology

I. Introduction chrotiic rejectioti and graft loss in king transplantation(LTX) [I-3J. The "gold standard" for diagnosis of ACR is

Acttic celltjlar rejection (ACR) is considered the most perivascuiar lymphocytic infiltration, while the pathogenicimportant independent risk factor for the development of role of antibody-mediated rejection (AMR) alone or in

conjunction with cellular rejection is less well documented-—\— [4]." Tbis work was partially siinnorted bv NIH arant HL-65I8Q. . , r i • . i < • • < n^ „ , ,, T , ' , i n ^ ^ T t m - j t ^1 A^-, ̂ ^^ T,(,c, We have previously reported that the developtnent of* Corresponding autbor. Tel.: +1 412 647 6193; fax: +1 412 647 3399. i ^ i t

£^»;«,7W<//('ii. yousenisaf«;iipmc.edti(S.A. Yousem). anti-HLA alloantibody (HLA-Ab) was associated with' Have equal contribution in tbe preparation of tbis manuscript. high-gradc and refractoiy ACR, lymphocytic bronchiolitis,

0966-3274/$ - see front matter C; 2005 Elsevier B.V. All riglils resen-'ed.doi:10.1016/i.lrim.2005.05.001

64 D.N. lonescii el al. ' Transplant lniinnnoit>i^v 15 (20051 63-68

and increased use of cytolytics and/or imrnunosupprcssivetherapy [5,6]. Furthermore, we and others describedsignificant associations between HLA-Ab and bronchioli-tis obliterans syndrome (BOS) [6-12]. However, astatistical association does not imply a cause-effectrelationship.

2. Objective

C4d is a tissue marker for antibody-mediated rejection inkidney allografts, but its association with liLA-Ab has notbeen docutnented in LTX [13-15]. We have previouslyfound inet eased soluble C4d in the bronchial lavage of LTXpatients with IILA-Ab [16].

In the current study, we investigate C4d deposition inlung allograft biopsies front patients with or withoutcirculating IgG HLA-Ab.

3. Materials and methods

Thirty-two patients who received lung transplantation between1999 and 2002 and had at least 2 years of follow-up were includedin the study. The Institutional Review Board of the University ofPiltsbiirgh approved the protocol and informed eonsent wasobtained trom all patients.

5.1, Deteclioii of HLA-specific antibodies

Anti-coagulated blood samples were collected betbre andafter transplantation at the time of transbronchial biopsy (TBB)procedure. A total of 415 serutn/plasma samples were tested for32 patients by enzyme-linked immunosorbent assay (ELISA)using comniereial LAT FLISA kits (One Lambda: Canoga Park.CA, USA) in aeeordanee with the manufacturer's instmctions toidentify IgG anti-class i and/or class II HLA-specifie alloanti-bodics. Briefly, the HLA-Ab were measured by a secondenzyme-linked colorimetric reaction and were replaced by 630ntii tising an ELISA reader (ELX 800, Bio-Tek). All tests wereduplicated and the positive cutotT was calculated as 20% ofaverage positive serum control and 10% of average positive IgGeontrol [5].

3.2. Traushronchiul hiop.sv procedures

Fiberoptic bronehoscopy and tluoroscopically guided TBBwere routinely pertbnned approximately 2 weeks after trans-plantation and at 3-month intervals thereafter, to detect rejection orinfection. Bronehoscopy was pertbnned whenever changes inelinicai or fijnetional parameters, such as oxyhemoglobin desatu-ralion, >10% decline in forced expiratory volume in one seeondfrom baseline or new intlltration by chest radiography occurred, aswell as 2 4 weeks af̂ er pulse methyipretlnisolone and 6 8 weeksafter anti-thymoeyte globulin. The values of FEV| were comparedto the best airflow of every patient in the tlist 100 days post-LTX.and expressed as percentage. Acute perivascuiar rejection wasgraded as previously described [4,17]. The diagnosis of BOS wasestablished according to ISIILT published eriteria and suiveillancc

spirotiietry in aeeordanee with American Thoracic Society criteria[18.19].

,?..?. Imnntnoliistochetnistiy for C4d

TBB available in the 32 patients described above were seleetcdfrom the paraftln archives of the University of Pittsburgh MedicalCenter {A'=192 TBB and approximately 900 biopsy fragments).The first biopsy following HLA-Ab detection ("sentinel biopsy")was assessed in 16 cases and compared to 16 temporally andrejection grade matched biopsies from the HLISA-negative group.Ilcmatoxylin and eosin sections were analyzed for the presence ofACR, which was graded as minimal (Al). mild (A2). moderate(A3), and severe (A4), as previously described [4,17]. Moderateand severe grades are referred to as high-grade acute rejection(HACR) in this paper. C4d immunohistochemical stain wasperfonned in fonnalin fixed, paraffin-embedded tissue sections.C4d deposition was assessed initially on the first TBB followingHLA-Ab detection (^ = 32. 16 in HLA-Ab positive and 16 inELIS.^-negative patients, matched for rejection grade and post-transplant period) and was further assessed within the HLA-Abpositive group in all available TBB {N= 160, total A'= 192). Four-micron-thick sections were deparaffinized and rehydrated. Antigenretrieval was performed in a microwave, where the slides werepreheated in citrate buffer (10 tnmol/L. pH 6.0) for 12 min.followed by cooling at room temperature for 20 min and washingin PBS three times. The slides were quenehed with H^O^ for 5 minto block the endogenous peroxidase, and then washed with TBST(50 mmol/L, pH 7.6). Non-specific background staining wasblocked by incubating the tissue at room temperature with 10%Goat Serum in Protein blue bloek for 10 min, followed by a PBSwash, with Avidin Bloek for 15 min followed by three PBS washesand with Biotin Block for 15 min. I he slides were then incubatedat 4 °C overnight with 1:30 dilution of C4d primary rabbit anti-human antibody (C4d. American Products Company, Windham,NH. ()04-BI-RC"4d) followed by three washes in TBS'l" bath.Secondary antibody (biotinylated goat anti-iabbit IgG, 1:200) wasapplied tbr 30 min at room temperature, followed by three washesin TBST. Horseradish peroxidase-avidin (1:1000) was applied andthe slides were ineubatcd for 30 min at room tetnperature in ahumidity chamber, followed by three washes with TBST AEC wasapplied for 10-15 min, followed by washing with distilled water.The slides were then eounterstained with dilute aqueous hematox-ylin. dehydrated with graded alcohols, cleared in xylene. and coverslipped.

C4d endothelial deposition was assessed independently by twopathologists (SAY and DNI) with an inter-observer variability ofless than 5%. Immunoreactivity was defined as linear, continuoussubendothelial deposition identified in capillaries (Fig. I A),arterioles (Fig. IB), and venules. Some C4d staining was seen inthe alveolar walls and stronger and more ditTiise staining in thebronchial and non-subendothelial blood vessels walls (Fig. IC).This staining pattern corresponding to the distribution of the elasticlibers in the vessels, airways, and intcrstitium was considered non-specific and therefore interpreted as negative. Positive controlsconsisted of myocardial allograft biopsies and the negative controlswere adequate.

To assess the specitlcity o'i the staining, C4d deposition wasalso studied in LTX recipients with diagnosis of CMV pneumonitis{N = l) or diffuse alveolar datnage (A'=ll) and without ACR.CMV-pneumonitis was defined by the presence of viral inclusions

lone.\cu I't al. • Transplanl 15 (2(105) rt.V 65

F'ii;. I. (A. \i) C4d cndolhclial posilivity defined ;is linear, conlimioiissubcndoihclial dcposiiion was identified in both capillaries (A) andarterioles (B). (C) More diffuse C4d staining involving the elastic layerof the arteriolcs was considered non-spceific and therefore interpreted asnegative.

idenlillett on H&E and/or on special stains tor CMV accompaniedby at! inflammatory reaction. These patictits {N= 18) were ELISA-negative over the study period (173 post-tratisplant scmni samplesnegative for HLA-Ab).

3.4. Skili.slical analysis

Contingency tables and tbe chi-2 test (/7-vakics expressed astwo-tailed Kishet's Exact Test and/or Yates con^eetion for smallnumbers, with a significance level of 0.05 - STATISTICAsoftware from StatS()li"r") were used [20].

4. Results

4.1. Antibody deleclion

Afler the analysis of 415 semm samples for the presence ofHLA-Ab in 32 lung allograft recipients. 16 palients exhibitedmultiple positive results, while 16 patients remained KLISA-negative. In 65% of cases, tbe first detection of HLA-Ab was in thefirst 6 months after transplantation, while one third appeared

between 6 montbs and 2 years post-LTX. The donor specificity ofIILA-Ab was proved in 8 otit of 16 patients (50%).

4.2. Acitlc and chronic ft'jeclion in patients with or withoutHI A-Ah

No differences were seen between the bisfological features ofACR in FLISA-positivc versus ELISA-negative patients (Table I).

Although the two groups exhibited the similar prevalence ofHACR. 13/16 patients (82%) ultimately developed chronicrejection in Ihe ELlSA-posilive group, cotiipared to only 2/16(13%) in the ELISA-negative (/;-^0.005. Table 2).

4.3. C4d deposition was associated with cniti-HLA alUntnlihodifs

C4d positivity was defined as linear, continuous subendothelialdeposition in capillaries (Fig. lA) and aiierioles (Kig. IB). In ihepositive biopsies, the C4d positivity was patehy. involving onlysome of the lissue fraginents from a TBB. and only some bloodvessels within a biopsy fragtiient. In some eases, a non-specificC4d staining was seen in the intcrstitium: in addition, a stronger,more dilTuse staining was also seen in the elastica of the bronchialwail, as well as in the elastic layer of the arteries, arterioles, andveniiles (Fig. IC). This pattern was considered as non-specific C4ddeposition (negative). Overall, a non-specific C4d slaining waseneotintered in 41% of TBB.

The prevalence of C4d deposition was significantly higher inpatients with HLA-Ab (5/16, 31.25%) as compared to post-transplant period and ACR-grade matched patients without IIL.A-Ab (0/16, /?<0.()5). All C4d positive eases were encountered inpatients with documented donor-speeific HLA-Ab. A summary ofHLA-Ab and C4d positivity and their association with differentgrades of ACR ean be seen in Tables 2 and 3.

The Jh'iitiency of C4d deposition was 4.7% (9/192 routine andindication biopsies). When perfonncd in biopsies showingACR>A2 from HLA-Ab positive patients, the C4d stainingfreqtiency was 12.9% (S/62 biopsies). Furthermore, in thesubgroup of HACR biopsies (grade>A3, ;V = 28 indicationbiopsies), 6 were C4d positive (frequency=2l.5%), while inbiopsies showing HACR from HLA-Ab positive patients, thesensitivity was the highest (6/18 = 33%).

Table 1Histologieal featiire.s of transbrondiial allograli biopsies in patients with orwithout anti-HLA alloantihody

lli;;tological featiire.s

Large airwayinflaniination

Small airwayinflammation

HndolheliitisPlasma cellsNeutrophilsEosinophilsPerivascular infiltrateFibrinoici necrosis/

va.sculitis

IfLA-Ab" (,V-16)''

15 (93.7%)

14 (S7.5%)

'i (56.2%)10(62.5%)11 (6S.7%)13 (81.2%)4 (25%)0

No HLA-Ab'(.V= 16)"

16 (IOO"nl

16(100%)

12(75%)15 (93.7%)12(75%)11 (68.7%)3(18.7%)0

" HLA-Ab = ELISA-detccted anti-HLA alloaiitibody.^ The two groups of patients were matched for ihe pre\alenee of aciile

and high-grade actite eellular rejection.

66 D.i\. toncscii i'l al. / Transplanl lininiinoliitiv 15 12005) 63

Table 2

Distribution of acute cellular rejection, chronic lung altograft dysfiinclion.circtiiating IILA-Ab. and C4d deposition in individual palients

Paticni' number C4d HLA-Ab" AC'R'' grade

234567891011121314151617181920212223

242526272829303132

NoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoPositi\eNoPositiveNoNoNoNoNoNoPositivePositiveNoPositiveNoNo

NoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoYesYesYesYesYesYesYesYesYesYesYesYesYesYesYesYes

A3A3A3A3A2A3A3A3A3A3A2A3A3A3A3A3A3A2A2A4A3A3A3A3A3A3A3A4A3A3A3A3

NoNoNoNoNoYesNoNoYesNoNoNoNoNoNoNoYesYesYe.sYesYesYesNoYesYesNoYesYesYesGrafi lossYesYes

•' Palients with and without anii-HLA alioaiilibody were matched for theprevalence of acute and high-i;rade acute cellular rejeclion.

C4d posilive = eontinuous. linear, subendolhelial t'4d deposilion.'• HLA-Ab = ELISA-detected anii-HLA alloantibody.'' ACR = acute cellular rejection: AO-A4 = ISHLT grade of aeule cellular

rejeelion.= bronchioli(is oblitcrans syndrome.

Temporal relationship (Table 4): in 4/5 (80%) patictits, thespecific C4d deposition was idcntitled in the time-period whencirculating HLA-Ab were present, while in one patient (#28), theC4d preceded the identification of HLA-Ab. In three patients (#18,

Table 3Relationship beiwecn specilk C4d deposition and ihc grade of biopsy-proven rejection in patients with anii-HLA alloantibody

Rejection grade"

AOAlA2A3A4C

No. of TBB' '

7242292265

C4d+"

I01421

Table 4Temporal concordance between deteetion of anti-HLA alloantibody .xspeeitlc C4d deposition

Paticninumber

Period of HLA-Ab" detection (POD*") C4d positive'-' (POD)

27IK203028

0 to 52212 to 70129 10 188370 to 459385 to 513

10 and 293! and 13217843210. 20. 31

•' A0-A4^ISHLr grade of acute cellular rejection, C = obliterativebronchiolitis.

'' TBB = trans-bronchial biopsy.'^ C4d+ ^continuous, linear, subendothclial C4d deposition.

•' HLA-Ab = ELISA-detected anti-HLA alloantibody.^ POD = post-operative day.

C4d positive ̂ continuous, linear, subendothclial C4d deposition.

27. 28), the specific C4d staining was observed in the first 6months post-LTX. while two patients (#20, 30) exhibited C4ddeposition after 6 months and in the second post-operative year,respectively. All patients with C4d and HLA-Ab developed BOSand/or graft loss.

Subendothelial C4d deposition was also detected in one out of 7TBB frotn 7 patients with CMV-pneumonitis. in the absence ofACR and HLA-Ab, and in two out of 11 TBB from 11 patientswith diffuse alveolar damage due to harvest/reperfttsion injury.

5. Discussion

In this study, we describe the specific C4d stainingpattem in lung allografts. the sensitivity atid specillcity ofC4d itntnutiohistochemieal stain in parafftn-etnbeddedtissue, and its association with circulating HLA-Ab.

The specific C4d deposition in kidney allografts isconsidered as continuous, linear subendothelial staining ofperitubular capillaries and is incorporated into the BantTclassification [15,21]. Sitnilarly. in lung allografts, weconsidered speeifie the continuous, linear, subendothelialC4d staining. As in kidneys, the C4d deposition in our casesinvolved the basement rnembranc of blood vessels (arteries.aiterioles. capillaries, and venules) and was very foeallydistributed, affecting only a few capillaries and/or arterioles.

Different frequencies of positive C4d staining arereported in the literature when considering protocol versusindication biopsies. In combined (protoeol and indieation)cardiac biopsies. Chantranuwat et al. reported a prevalenceof 11% C4d positive staining (35 out of 315 cardiac protoeolbiopsies), similarly with data published by Behr et al. (14 of155 biopsies) [22,23]. In both studies, specific C4ddeposition was associated with impaired cardiac functionand/or grafl loss. They also found diffuse, non-specific C4dstaining in a significant proportion of eases, without anyclinical significance. Similar to our observations, the C4dpositivity in all protocol renal biopsies (A'^551) of aEuropean collaborative study was low (4.4% eompared to4.7% in our study) [24]. In contrast, the frequency ofspecific C4d deposition in indication biopsies (A'^337) wassignificantly inereased (20.7%) [24]. We have also deteetedincreased frequency of C4d positivity in LTX patients with

D.N. lonescu el al. I Tninsplanl Immiinotogv 15 (2005) 63 - 6H 67

indieation biopsies—HACR (21.5%). Furthermore, tbehigbest ineidence of C4d positivity (33%) was observed inLTX patients wbo had both indication biopsies (HACR) andcirculating, donor-specitlc HLA-Ab. Several investigatorshave also repoiied increased frequency ot" specific C4d stai-ning in itidication kidney and cardiac biopsies [15,22-25].Feucht et al. found positive C4d staining in about 30% ofrenal indication (rejection) biopsies and distinguishedrejections with a humoral component from "pure" cell-mediated rejections [15]. In two large cohort studies(Manniyycdi ct al. and Nickeleit et al.). a frequency o'i30% C4d positive staining in kidney recipients withindication biopsies (ACR) was reported [26.27]. Theinvestigators bave also found a strong association betweenthe presence of HLA-Ab and positive C4d staining. Inaddition, circulating HLA-Ab was associated with positiveC4d staining in tbe multicentric European renal transplantstudy, as well as in ours [24]. Consistent witb our data, in allprevious three renal studies, a significantly higher rate ofgraft failure was encountered in tbe group with ACR andC4d deposition, versus ACR withotit C4d deposition[24.26,27].

In LTX, Magro et al. reported frequent findings ofhumoral immunity against endothelial-based alloantigens[28]. These were characterized by a necrotizing pauci-intlammatory capillary injury syndrome and, clinically, bydeereased pulmonary function [29]. However, the authorsdid not find an association between HLA-Ab and AMR[28]. The association between C4d deposition in chondro-cytes, basement membrane zone of the bronehial epithelium,bronchial wall microvasculature, and BOS, as described byMagro et al., was not observed in our cases [30]. Althoughall 5 patients with positive C4d deposition ultimatelydeveloped BOS/graft loss, at tbe time of BOS, they showedonly non-specific bronchial C4d staining, related to elasticfiber binding.

hiteipretation of C4d immunohistoehemical stain wascomplicated by non-speeifie staining of elastic tissue in thewall of vessels, alveolar septa, and submueosa of airways.The low sensitivity of C4d detecfion in lung tissue couldpartially be explained by the technieally challengingimmunohistocbemical stain on paraffin-embedded tissuewhen compared to immunotluoresenee staining perfonnedon frozen tissue in other solid allograft biopsies. C4ddeposition was also encountered in a limited number ofpatients without ACR. but with barvest/reperfusion injury orCMV pneumonitis. These results indicate that non-specificcomplement activation may occur under different conditions.Therefore, the specificity for this staining as a clinical test,especially wben a concomitant disease other than rejection ispresent in the lung allograft should be considered. Tbespecificity of C4d deposition in heart allografts wasaddressed by Baldwin et al., who described a non-specificC4d deposition early post-transplantation, in associationwitb ischemic injury or following administration of poly-clonal anti-lympbocytic antibody [31-33]. A multivariate

analysis involving C4d deposition, HLA and non HLA-Ab,barvest/reperfusion injury, or CMV-pneumonitis requiresstudies on larger cohorts. The number of cases (/V = 36) couldbe considered a statistieal limitation of our investigation.However, the number of samples analyzed in this cohort,including 415 serum samples for detection of HLA-Ab andappro.ximately 900 biopsy fragments with C4d stainingrepresented a eomprchensive immunopathological analysis.

Although about 30% of renal indieation biopsies arepositive for C4d, all renal allografts biopsies are regularlystained, since capillary C4d can appear and disappearanytime post transplantation, persisting for approximately3 weeks in tissues [15]. In our study. 80% of specific C4dstaining was detected at the same time with circulating,donor-specific HLA-Ab. We have previously reported theassociation of donor-specific HLA-Ab with increased levelsof soluble C4d in temporally matched bronehial lavage fluidof LTX patients [16].

Specific subendothelial C4cl deposition demonstratesthat the classic complement activation is involved in thepathogenesis of AMR of LTX patients with circulatingHLA-Ab. The patcby nature, low frequency, and specific-ity of C4d staining are limitations lor routine clinical usein lung protocol biopsies. However, in patients withdecreasing pulmonary function, HLA-Ab and/or HACR,C~4d deposition might ser\e as a marker of in tissueantibody-mediated lesion and makes the diagnosis ofcoexistent AMR highly-probable.

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