Lymphoid Pneumonitis in 50 Adult Patients Infected With the Human lmmunodeficiency Virus: Lymphocytic Interstitial Pneumonitis Versus Nonspecific Interstitial Pneumonitis

WILLIAM D. TRAVIS, MD, CECIL H. FOX, PHD, KENNETH 0. DEVANEY, MD, LAWRENCE M. WEISS, MD, TIMOTHY J. O’LEARY, PHD, MD, FREDERICK P. OGNIBENE, ANTHONY F. SUFFREDINI, MD, MARK J. ROSEN, MD, MICHAEL B. COHEN, MD, AND JAMES SHELHAMER, MD Lymphocytic

interstitial

stitial pneumonitis immunodeficiency of a detectable

pneumonitis

(NIP)

virus (HIV)

infection

and nonspecific

complications

infection

opportunistic

lung biopsy specimens

(LIP)

are pulmonary

from 50 adult HIV-infected

46 had NIP. The majority

four had LIF’and

inter-

plays a significant

role in the pathogenesis

of human

adult HIV-infected

patients;

a direct

that occur in the absence or neoplasm.

541.

We reviewed

(CIP;NIP),

with three showing

phase. In contrast to CIP/NIP,

four patient:5 with LIP interstitial stitium.

infiltrates

The

features of diffuse

demonstrated that extended

majority

alveolar

extensive

from

lymphocytic

into the alveolar

of the interstitial

septal inter-

lymphocytes

The etiologies

of NIP and LIP remain unknown.

opportunistic

were excluded

cial techniques,

to investigate

(EBV), or cyc-omegalovirus specimens primarily LIP

occasional

and four

NIP).

hybridization was detected of NIP, gether

in seven

similar

be caused

CMV

specimens;

to results

bp lentiviruses

Epstein-Barr

in control

nor EBV in these

RNA

we found

of HIV RNA

in the remaining were found

was found same

chain reaction specimens.

that lymphocytic in humans

centers;

HIV

(two

by in situ

specimens

EBV

in only one case These

pulmonary

and animals,

virus

in lung biopsy

of large amounts

in germinal

using the polymerase

with c.he knowledge

HIV.

By in situ hybridization,

cells expressing Neither

we sought. with spe-

could be identified

with expression

within macrophages

specimens,

methods,

whether

(CMV)

from these patients.

one LIP specimen

Since the common

by routine

either

EBV

or

CMV.

Saunders

,r. 1992 by W.B.

HUM PATHOI.

23:529-

Cornpan)

ttitirnodefic-ieti~~ s)ndtxinie (AIDS).’ Hcw~\w, sevel-al histopat hologi~ally drfined t\1)es of pne~tniottitis ttta! oc~ut~ in the absetice of a detecUtAr c~pt~ortuttistic itifec.tiott or neoplasm, including I~tttphoc~ t ic ittterstitial interslit ial lmwtnopttc.umotiitis (LIP).‘~’ nonspwitic~ ~~tieuntotiili~.” mid brotittitis (Nil’).‘.” ” dt-ug-induced c,ltiolitis oblitcwns with organizing l~tl~t~tttotli;t.“r ‘I’hrrr has heen little fi~tts ott the relatiottship twtwcwt NIP md 1,Il’ itt ttie patholci~~ literature; itt witlition. tieitlicr the etiolog tiot- the patliogeueaia of’ rhcw. ~xmditiotts i5 uttderstootl. To investigate the spec‘tt-uttt oflw~ph~~id interstitial infiltrates in adult HIV-infected pa;ients. we c.valuatecl light tiiicroscxipic findings in t~iops~~ qmitnens from 36

in both NIP

and LIP were of T-cell origin and stained for UCHL-1. infections

for

of both NIP and LIP in

our data do not demonstrate

ltrters[iti;tl lung disease is ;I III;!~OI-lifr-tl~rc~;tt~nitt~ c~oitiplic~;ttiott of’ ittfectioti with the hu11tdtt inttttunodcficirnc!~ Ctus (HIV). The vast tttajoril! t )L cpivdes of’ l~tttwtn~~nitis art due (0 01~l”)t-ttitiistic. it~fvctiorts md/ itiror Kaposi’~ ~;trconia associa(ecl ~.ith thci‘ quit-ed

damage,

the five specimens

more

role

Copyright

in contrast,

patients, of whom

(47 of 50) of specimens

from patient!5 with NIP showed mild chronic interstitial pneumonitis organizing

MD,

results, lesions

suggest

tomay

that HIV

patients with NIP and four \\ith I,IP. Opt lung biolq ~pecinietis in seven cases were studied tlsittg itntnutto1listorhetrtistt.v. in situ hybridimtion, and the polynter-ase cdiaiti reaclion (PCK) to explore the potential role 01 HIV, cytotncgalovit-us (CMV), antI Epstein-B;tt.r vim5 (ICBV) in the pathogettesis of these Iwq~hoitl inter-stitial

____

disorders.

MATERIALS

AND METHODS

(&es of NIP were identified fl-c,rn t\\o t AItyories of adult HIV-infected patients rnrolled in proto~x~t:; ‘kt the National Institutes of Health (NIH) desiFled I’oI- xrutlyiug HIV-assoGated pulmo~lary disease. Of‘23 iutlividuatx etlrottetl in ;I pro10~01 designed

to assess

the pulrnon~u-\

p;Lthologic

processes

HIV-infected pm-sons, I I patients (48%) wetx Cound to huvv NIP.“’ The remaining pxtirrlt~ ;iIt underwent ~ransbronchial biopsies for- evaluation of ~y~q~onlatic episodes in asyulprolnatic

(bl‘ respiraton

529

dvsfunction

or

were

az\trlptonxitic

and

were

HUMAN PATHOLOGY

Volume 23, No. 5 (May 1992)

a pl‘oIo’ol for tl‘(‘;itlllCllt studied prior IO entering of CM\‘. Thr diagnosis of’ NIP W;IS Ixwd OII thr finding of interstitial pneunionitis in transhronchial or opm lung biopsy spec-iinens in the absence of an cq~portunistic inf’ectious agent oi- neoplasm using standard iiistopathologic-. c.ytologic, and nlicrobiologic culrure and staining techniqurs. The pwscnce Iyniphoicl aggregzatm of lyniphoid interstitial inflanimation. type with or without germinal centers. alveolar nmmphages, and intrahiniinai fibrosis wis reII pneumocyrc hyperplasia, corded and gradrc~ semiquantitati~e~~ (mild, mockrate, and niarked). In ;tdciition. the presence of Kussell bociies and eosinophils was noted. Ail NIH patients who underwent transbronchial biopsy siiilliltariroiislv under-wrnr hroncho;ilveolar lavage (BAI.) with analysis of the fluid for opportunistic infectious agents.” Sincr we had oi$ one (use of1,IP at NIH, paraffin i~locks front lung biopsy specinltms ciemons~rating 1,IP in three HIVinfected patients were obtained from two other instituGons: Mount Sinai Medical Center (New York, NY, two cases) and Universitv of California (San Francisco. CA. one case). Histologic- slides f‘ronr each biopsy specinirri were reviewed Cot thr same histologic fearures as the biopsy sprcimens fr-orn NIH patients. Of rhc eight pardfi~~ blocks used fin- special studies. seven specimens were fixed in formalin; the type of’ fixativr used in one specinirn is not known.

lmmunohistochemistry Histologic sections cut Irom paraffin i~locks in eight cases (four 1.11’ and four NIP) werr applied to poly-I-lysine coated slides and stained immunoliistochenlicaiii~a~iy using an avidin-biotin-complex technique, as described previously.” Scrtions were stained with ~no~~~lo~xd antibodies LJCHI,- 1 for T lymphocyles and I.26 for B lymphocytes (DAKO, Santa Barbara. (A) in eight cases while four specimens (three NIP and one LIP) werr also stained with n~onoclonal antihodirs to I( and X inlmunoglobulin light chains (TAGO. Buriingau~r, (:A).

In Situ Hybridization In situ hybridization was perfornied on sc’vcn cases (thi-ee I.II’, four NIP) for HIV, using radiolahelcd specific rihoprobes, and for EBV (by L.M.W.) and CMV (by I..M.W.), using radiolabeled germ& DNA probes.

Human lmmunodeficiency

Virus

Tissues Mere cut from par-afin blocks at a thickness of 6 /ml. The sections were spread on an KNAase free water bath and collected on slides treated with triethoxyaminol~ropyl-silane (American HistoLabs, Gaithershurg, MD). In situ hyhridization was performed, using the detailed protocol avaiiahle from Lofstrand I,ahs I,td, Gaithershurg, MD. Probes were svnthesiLed with 1”51-laheied CTP (Amershanl Corp., Arlington heights, II.). Five sense and five antisense pmhes. in all representing 90% of the HIV genorne, were synthesized using pGem 3 subcloned restriction fragments of the clone HXB2. The sense or antisense probes were sheared according (0 the protocol and combined to yield a probe mixture. The following controls were included in each experiment: an HIV-positive control tissue confirmed hy electron microscopy ancl PCR, a negative tissue, positive cells (H9), negative cells (HLIT78). a nonsense probe control synthesized from bacteriophage DNA, and an unhyhridized emulsion control slide. Each specimen was run in duplicate with prohes in either the sense or antisense orientation. Following development, the slides were examined with incident light polarization microscopy OI- by transmitted

530

ligh1 dark field tnic rosc~op) The technique detect5 c.el1.s cxi)rmsing hetweeil 10 ant1 100 viral KNA genotlk c.opie~. Single copy proviral DNA is protmhlv no1 derecrable by tliis procedure. I:ouy open lung biopsies from HIV-inrrctecl paCents without lymphoid infihrates wvre also sllidied as conlrois.

Epstein-Barr

Virus and Cytomegalovirus

Tissue in situ Iiyl~ridization stud& bverc perfi~rnictl on Iiibtoiogic scctiona ( 111from p~u-affin-enihrtlcieci iissue according to a previously drsc-ribed riiethod. “‘.” The EBV DNA probe c.orlsisteti of the ‘Nn,r1HI-l\’ f1ragmenr of EBV DNA that htl lxxn radioiaheled with tu-“3-dCTP b\ the i-andoni hexaiiw priming technique. The (:.MV DNA p;.ot,e consisted of a ““Sradiolabeled mixturr of two c.lones of 13.3 ICI) and l(i.6 lit) insert she. Positive controls weI-e included along with cacti ruii.

Polymerase

Chain Reaction

The P(:R was performed (by T,J.O.) on \cctic,ns cut front seven formalin-fixed, l,ar;lffin-ernhetltled open lung hiolq specimens (three LIP, four NIP) to invesligatc dir presence of EBV DNA. In addition, nine autopsv Iung control specimens were tested from HIV patients where sc‘cticm~ from the paraffin block did not demonstrate lymphocytic- inter-siitial infiltrates. Our proc.etiurr for I’CR amplification from tissue srctions was nioclifed from that of Shibata et al” ‘I’ dtnd is given in detail in Wright rt al.“’ Paraffin-eIiil)edtied cells from an EBV-conraining Burkitt’s lymphoma ceil line (Raji) wvrc usrd as a positive control for EBV DNA. Negative controls for reagent COIItamination, consisting of all P(:R reagents except template DNA, were run each time a PCK assay was performeri. A segment of thr EBV IR3 region which gives r&c to a 239 base pair amplified product was selected for identification. Each case was also assayed for the single copy HER:! gene. to ensure that adequate DNA was available for an;plificatit;n. The primers for this gene give rise to a ‘L-10base pair amplified produc1.

RESULTS Clinical Features All 36 patients with NIP were lnale homosexuals with a mean age of 39.5 years (median. :17 years; range, 28-58 years). Thirty-two were synlptornatic and 14 had no pulmonary symptoms. Eight patients had HIV infection without AIDS, and 38 met criteria for AIDS.” The clinical and radiologic aspects of some of these patients have been reported previously.!‘.’ ’ Ninety-one percent of patients with NIP had no previous opportunistic pulmonary infection; the remaining patients had experienced episodes of Pneurnoc~stis rurinii pneumonia which had resolved at least 2 m&ths prior to the lung biopsy that demonstrated NIP. Extrapulmonary Kaposi’s sarcoma was present in 45% of patients. Thirteen of the 20 patients (65%) with available smoking histories were nonsmokers. Three patients with LIP were males and one was female. The mean age was 37 years (median, 47 years; range. 39-56 years). All four patients were symptomatic, two were homosexual, one was bisexual, and the fourth was an intravenous drug abuser. The two cases from Mount Sinai Medical Center are cases no. 1 and 2 from a previous report .‘, Light Microscopic

Findings

Norqtwc~fic Intrwtitiol Pneumor~itis. Fifty lung biopsy specimens (46 transbronchial and four open lung spec-

LYMPHOID PNEUMONITIS

AND HIV (Travis et al)

ittletts) UWC’ tCr\\ed I’rotn 46 NIH p:1tients with Nil’ (fi)u~ p;1lients Itad two biopsies). The tti;~jor Ix1lhologic~ I~‘.IIUII*S WC cI1tatttitatrd in Figure 1 ,Zlilcl ittterstitiA Isinl~hoid irtfiltrates were observed in 10 sptGtiietis (XIYZ.). ;iticl ttw itttiltrstes were of’ n10detXe severity in IO slk~~~ittit~tis (20%). In transbronc~hi~i~ biopsy speciitwti~. ttir I~~titphoid infiltrates were distributed ntainl!~ in ;I l~ct~il~*~c~nc~t1ic,lar;1tid peri~ascul~it- f&ion (Fig ?A). I‘ltia pal tent was also seen iii open t,iof)sy sptxitnetis; liowwer. in three of’ the four open biopsies, Ivtiiphoid iritillr~ites ~oulcl also be weit ;ilong the pletit-2 x1d intt~rlolx1l;1r fibrous septa (Fig 2K). The Iytnphoid infilttxtrs did not extend substantially into the alveolx sept:il ittrerstitiutn. NotlulatIytttphoid agpegates ~~ttd/or get‘itiiti;il cw~trrx bw-v found in I .5 speCitttetrs (30%) aiitl \G(TC~~sr~allvsitt~ated ;I~~MC’III to hrc~ttc~hiolcs (Fig 2K): I hew Irsiorts rt2ietttl~led those pre~ioualy desct-ihed 2s fi~llic~ul;ir t~t~oti~hiolitis,~~ pultiionarv I~t~iplioid hyperI)lasi;1.“,“ or hvI)erplasia of the t~rc~ri~itial-;issc,~i;ited 1vtti.:., ,, pttoid tissue.-‘ ‘Type 11 piieuitioc‘yte hyperplasia was swii in niric sI)ecititetis ( 18’:;; Fig 3). Organizing iritCrstiti;1l fibrosis, consisting of Ioosc connective tissue, \z;1s setan iii six sI~ecittirtis ( I 2’;); in three specimens this loose fibrosis w;is loc~:itecl pi-imtrily within aI\,eolar sepf;i, ;uttl in the 01 her I hree it protruder! into distal airsp;icxx .l‘hese c~)1;1tigesWC’I-etxiet- to recognize in open lung biopsic,s thati in ct.;rrtst,i-ori~hial biopsy specintens. .-III itic~rrase in alveolar ttrnctx~phages w;1s seen in 3i spc1itii~~tts (72%; Fig 3); however, they were usualI) in~o~tsl~i~uous. iZ stiiall grani1lotna consisting 01‘3 cluster 01 epitheloitl cells and several giant cells \V;IS seen in one c’;ist’. Eosittophils were focally prontinent in three spec’intetts. I’et-iodic- ;1c,id-S(.hif-positive eosinophilk hyAine glotxilrs wet7 f’ound within niacrc)ph;iges in 16 specitneiis (3t’V ); these appeared to represent ititracytophrtttic~ ac~c~itttttil;ttiotis of irtitnitt~oglo~~~i~irt or Russell tx)tiiex. In t 11rt-c spehtiietis, Itic conibiri~11iot~ of’ ;ilveoIx. st~p(A c)t.g,C1ni/ing interstitial fibrosis and type I1 pneun10cWc hyI,erplasia t~esenit~led the patter-It of ttie organizing phxta of‘difti1se Aveolw damage. (l>AI>/NIl’; Fig .+I. Howev~~r, in the remaining -I7 specitnens. the pattern w;ih pritiiaril~ that of ii miltt chronic itttcrstiti;1l pneutitc,nitis (< IP,/NlI’~. Lymphocytic

Interstitial

Percent

looir SO

60

40 20 l-7 Lymphocyllc Inflltr.

Lymphold Aggregates

Intralum.

Flbrosls

Histologic FIGURE 1. in NIP. The number of moderate,

Alveolar Macrophages

Type

II Hyperplac~la

Feature

Bar graph summarizes the histopathologic findings results are depicted as a percentage of the total biopsy specimens from patients with NIP. n , Mild: fi. 7, marked

ti~;1tisl~t~onc~hi~ilbiops) in otrl) one 5pec~irtrer1, whic.h drnionstt~;1letl extensive nlveolx septal ititiltration (Fig 7). Ilit* ;irchircctrtre of the lurtg I~;it~etich~~tn;i was always preser\x4. I.\~ttil~hoid qgregites dt~tti~;ItstI.;1tirlg get‘tiiin;il ceti(er t’orttiatioti were pt~otttint~tir it1 ;I tt-atisl~roti(.lii;tl ;itid olxw biopsy specititen i‘ix)rti 011~‘ patient (Figs fif\ ;ititl HA). These gertitinaf ~milet3 rx~tit:kinetl tiiotiottttt-feat-

wlls

t-esenihling

tttat~roph~es

(Fig

8H).

Sortie

~~ernriri~11centers showed evidence of follic~le Ivsis. L.oosc, ititt2lutttittal fit,rosis protrudittg into disc;11 airspxc3, suclt ;1s t~rori~hioles, A~eolar ducts, or Ax~~li. w;1s foiitid in three axes (Fig CC); bronchick1r in\olvcntcttt f’ornting ItGotis of l~rortchiolitis 0t)literans bxs obset-vetI in one c-ase. \‘;1rvitig degrees of ;ilveol~1r nt;icrophages bert’ Scott iti all C;~SCS(Fig 5); type II pneuttto~~~t~ h~perplasia w2s fotintl iii four spehiiens (X(1’% ). E~ck5incqh~ls were f’odIj ptxmtitient

in one

spedmen.

Kiissell

bodies

wcw

seen

in three specitiiens front two patit7itx (Fig ‘713). Viral inclusions were not seen in Ik)psy spe~~itttctts L‘t-oltt p:itietits \vith either I,Il’ or NIP. In xIdition, no I’~rcutiioc.~stis cxritiii. fungi, or xitl foist txicilli \\erc seen with spe’:i;il stains in the specintrns ot)r;1ined by lung biopsy or BAIA; neither w;is an\ trrttior swn. lmmunohistochemistry

Pneumonitis

Fivr lung biopsy specitnens (three open IrIng xtd two 1t-~tt1st,rottc,hial biopsies) wet-e reviewed f’ronn foulp;1tients with I.IP (one patient had both a tt~~1nst,ronc.hi;rl and ;~n open lung biopsy). The tn;ljor p:1thoIogic features are quantit:1ted in Fig. 5. Moderate or marked Ivtnphoicl interstitial infiltrates were observed in two specimens (40%) and three specirnens (GO%), respectivcl) (Fig GA); the distribution of the lyrnphoid infiltrates ~1s sirnilx t() N II’ with :1 l”ril,rottchiolar, perivascular. parxeptal, and IAeutA location. In contrast to NII’, suhstxttial AIveolar sept:11 infiltration wx present in all biopsy specitnens (Fig tiH); however, alveolar septal infiltration w2s sotnetintes patchy xtd did not diflusely involve the entire ‘I’tte cliagnosis of‘ L,II) was established by specirneti. 531

;\lthough both B cells and ‘I‘ WIIS w~7.c‘ itletttifietl in the I~ntpltoid infilttxtes, ;1I,retlontittatlt,~, of7‘ (Il(:HI.I positive) cells WIS f’ound in ;A I’otrt~ l.IP attd Iour NII’ (xscs (Fig 9). A polyc~lotlal I~~1ttt3~ttof st;iining w;1s ()I)ser-ved ilt plania cells xtd Russell bodies in fi)ur t)ioI>sv specimcxs (tltree NIP, one 1,IP) ttt;1t wt’rc’ staitted f’or K 01’ X ititmunoglot~itli~i light ch;iiti4. In Situ Hybridization H~rrrcc~fmrn/rrrodf~frrif~,~c~ I’im.5. 011~’ 01‘ three open lung biopsy specitiiens of IJI’ cc~ntaitied Ixge .nnounts of’ HI\’ Rh’A. The HIV signal \%‘:Isconc.entt-atcbd in the ~erriiiri;il centers and most 0I‘ the cells \vithin the gertttin;1l centers expressed HIV RNA (Fig 10). Outside the qxtitin~il centers, occasic~rial positive trll~ c~ould be de1ec.ted ;1ntong Iyntphoid cells. Morpt~oIc~gic~;1lly, AI of’ t he

HUMAN

PATHOLOGY

Volume 23, No. 5 (May 1992)

FIGURE 2. (Top) Transbronchiol biopsy from a patient with NIP demonstrates a mild perivascular lymphoid infiltrate. (Hematoxylin-eosin stain; magnification >‘lOO.) (Bottom) Open lung biopsy from a patient with NIP demonstrates moderate interstitial lymphoid infiltrates distributed primarily along lymphatic routes around bronchioles (arrowhead), vessels, interlobular septa (double arrows), and the pleura (single arrow). There is minimal alveolar septal infitration. (Hematoxylin-eosin stain: magnification v 42.)

Polymerase

cells expressing HIV were mononucle;u cells resembling either macrophages or lymphocytes. No evidence of HIV-infected epithelium was found in any of the specimens. The remaining two LIP and four NIP specimens fniled to show a consistent distribution of HWexpressing cells, and populations of infected cells were fewel than one/cmL) of section. The strongly HIV-positive case differed histologically from the remaining specimens that did not demonstrate prominent germinal center formation. No HIV was detected in any of the four control open lung biopsy specimens from HIV-infected patients that did not demonstrate lymphoid infiltrates. l$steiwBurr Virus curl CytomegcllovillLs. No reactivity for either EBV or CMV was found in these specimens.

Chain Reaction

Epstein-Barr virus DNA was detected in one of seven (three LIP. four NIP) open lung biopsy specimens using PCR; this specimen was from a patient with NIP and very mild interstitial lymphoid infiltrates. It was also identified in one of nine autopsy lung specimens from HIV patients where sections from the paraffin block tested did not demonstrate lymphocytic interstitial infiltrates.

DISCUSSION Lymphocytic interstitial pneumonitis and CIP/NIP appear to represent a spectrum of lymphoid interstitial

532

LYMPHOID PNEUMONITIS

AND HIV (Travis et al)

FIGURE 4. This transbronchial biopsy from a patient with NIP shows features of the organizing phase of diffuse alveolar damage with organizing airspace fibrosis, interstitial chronic inflammation, and large. reactive, proliferating type II pneumocytes (Hematoxylin-eosin stain; magnification ‘* 192.)

FIGURE 3. rhls open lung biopsy from a patient with NIP demonstrates a mild lymphocytic infiltrate around a subpleural lymphatic In addition, the surrounding alveolar parenchyma demonstrates a mild increase in alveolar macrophages and hyperplasla of type II pneumocytes. (Hematoxylin-eosin stain: magnification 188.)

IeGons in ;~tlult H I\:-inf’ec (~(1 patients. in ;ttlult HIVilllrc-ted p;itiriits 1,lP is rare,“-’ but NIP is relativel!, ~ommon.‘~~’ ’ 111contrast. in HIV-infec?ed children LIP is ~oninio~i 2nd NIP has not been \vell characterizcd.“~‘.““~“’ Compared with NIP, patients with I,Il’ xc more liket!, to have siqiifkant respiratory symptoms, radio~rapliic p~ilnionary infiltrates, pleural kffusions. , and a subset of PA:n~ct gener;ilir.etl lympl~adenopathy tic,nts ~nay haves ;i S,jiiq-en’+like syndrome (Table 1). interstitial pneumonitis” flie term “nonspe~ifk WIS proposed fi)r episodes of respiratory dysfunction in HIV-inferted patients in the AsenCe of that OC.CUI’ 311 opport~~nistic infec,tion or neoplasm detec.tahle I,> lung l>iOp!j\ 2nd BAI,.” Several studies had previously suggested the cxistenc.e of 2 similar form of pneumonitis in HIV-iliftxtcd adult patients.“.‘” Nonspecific interstiti;ll pneurnonitis was found in up to 33% of Ixttients with respiratory symptoms evaluated at the N IH. ‘.!‘L’’ That NIP w;ls found in 48% of‘asymptomatic H IV-infec.tcd ;xIult individmtls in :i subsequent NIH an proto supports the ccmcept that NIP represents e;lrl\ pulmonary manifestation of‘ HIV infection.‘” Since NIP WAS not ori,$nally defined ;IS 2~pathologk entity, but rather as ;I clinicopathologi~ cxmdition characterized primarily by the absence of an infectious pneumonia, tile pathologic findings in biopsy specimens from these patients can be somewhat heteroge533

neous. In our study, the majority of lung hiops): spec.imens f’rom patients iuith NIP showed a mild to moderate OIJI!~ been well chat-ac.leriz&l in ad11lt HIV:infected patienta. M’hether NIP. as desc~rihed in atl~~l~s, correspontts to the PI .H 1qx)rted in c~hildreii is not clear. The cxistencx~ 0f’NI I’ in children Ius been briefly alluded to in sevrra1 I>;llients,‘.~“,“” hut p;ithologic dctails were riot prcnidtvl 10 de~eriiiine whether these cases resembled adult N It’. The diffrrential diagnosis of NIP and LIP in lung I,iops\, specin1eus frcm HIV-iufectecl patitmts includes sever& entities. Marked Ivtuphocyric inrcmtiti;ll infil1rates ill+ be associated &th infectious pneunioni~rs, and 1nay c,\~rJ3tladow the t5peciallv I’ 01v/a/2 pneunionia, organisnis in biopsy sl~ecinie11s.‘~““~“” l,~mphoprolifel-ati\c disorders and nialignant Iyniptiornas may also present in the IJrngs of HIV-infected patients; however, rhe lytiiphoitl c.ells in such cases will generally denionstrate atypical cytologic~ features that are no1 prt-sent in either CIP/NIP 01‘ LIP.‘~‘“’ With iiiany ilew experinieut~il drugs being wed in the treatment of Ii IV irifec%on or

HUMAN PATHOLOGY

Volume 23, No. 5 (May 1992)

FIGURE 8. (Left) Lymphoid aggregate with architecture resembling a germinal center. (Hematoxylin-eosin stain; magnification X192.) (Right) Within this germinal center prominent mononuclear cells resembling macrophages are present. (Hematoxylin-eosin stain; magnification x940.)

its complications, the possibility of a drug-induced interstitial pneumonitis should be kept in mind when an opportunistic infection cannot be found in biopsy specimens demonstrating an unexplained lymphocytic interstitial infiltrate. Diphenylhydantoin, which can be associated with IJP,‘“’ has been reported as a cause of infiltrates in an HIV-infected patient.” Finally, NIP may be underdiagnosed due to dismissal of subtle lymphocytic interstitial infiltrates or lymphoid follicles as nondiagnostic findings in lung biopsy specimens from HIVinfected patients. However, we are convinced that these findings represent genuine pathologic abnormalities that may cause symptomatic episodes of pneumonitis in HIVinfected patients. Persuasive support for the existence of CIP/NIP can be found in an interesting series of reports characterizing the Iymphocytic alveolitis (LA) detected by BAL in adult HIV-infected patients.x~‘i7-“” Although LA can be found in 78% of patients with lung infections or tumors, it is also present in up to 69% of patients without lung infections or tumors and in up to 59% of patients from the latter group who are asymptomatic and without abnormality on chest x-ray.x.“x This latter observation appears analogous to NIP, which can be found in both symptomatic and asymptomatic HIV-infected adult patients. It is not surprising, that the percentage of comparable groups of patients found to have LA is higher than that of those found to have NIP since BAL samples greater numbers of alveoli than transbronchial biopsy 536

specimens. Furthermore, the pathologic. findings describedin fouropen lungbiopsyspecimensol>tainedf~-onI patients with LA appear to resemble those seen in our cases of NIP.X The lymphocytes of LA are primarily CD8,CD44-positive (+) c‘ytotoxic ‘I‘ lymphocytes ((:TI.)““; these lymphocytes can recognize and kill HIVinfected alveolar macrophages in vitro.‘“’ This led to speculation that interactions between HIV-specific (:TI, and infected macrophages may play a major role in the pathogenesis of LA.‘“’ Further studies of the clearance of !“‘“‘Tc-diethylene triamine pentaacetate (DPTA) demonstratecl increased epithelial permeability in patients with LA, indicating that pulmonary epithelial cell injury may occur.“7 A high positive correlation was found hetween the value of epithelial permeability and the number of CD8+,CD44+ lymphocytes, and DPTA clearance was increased only in patients whose alveolar lymphocytes displayed a cytotoxic activity on alveolar rnacrophages.“7 It is interesting that the characteristics of BAI, lymphocytes of LA (CD8+,CD44+) are similar whether an associated infection is present or not.‘.“’ These studies provide support for the concept that lymphoid lung disease in HIV-infected patients results in altered trafficking of lymphocytes and recruitment of primarily CD8+ cells to the interstitium. Our histopathologic observation of epithelial abnormalities in biopsy specimens from patients with CIP/ NIP provides morphologic evidence to support the data of Meignan et al, analyzing DPTA clearance in patients

LYMPHOID PNEUMONITIS

AND HIV (Travis et al)

infected patients with IA.” There is little l~uhtisl1ed inforniation regarding the ii1iiiiunophenot~~iii~ of the lymphoc! tes in LIP associated \vith HIV infection; howe\er, in one pediatric case a inixture of 13 cells and ‘I‘ cells was fi)~md.“~ In another pediatric case the infiltrate consisted of l”‘etlo1ninantl~ T lymphoc~\~~ea of the CDH subset,” .md data in several adult cases (two of which are also inctudect in the current study) have shown ;I predominance 0fT cells Ivith either iii&Xl\ (:1X c,ells or a mixture of (;D8 aiict CD4 cells.’ Since the common opportunistic. inf’tctions werca excluded by routine methods, we sought IO investigatr whether HIV. EBV. or CMV could be identified in lung biopsy specimens from these patienls. 13~in situ Iiybridization, \ve found large quantities of‘ HIV primarily in macrophages present within germinal centers, which were prominent in one wedge hiops\. specimen from ;I patient with IAlP. Interestingty, only a sma]t airioiirit of HIV was detected in the remaining open lung hiopsl specimens analyzed in which lymphoid aggregates and germinal centers were inconspicuoiis or ahsenr. It is possible that in these cases the amount of HIV present ma; be undcI-ectiniated due to sampling l~robleiiis aid timing of the biopsy relative 10 the stage of‘ infection. our finding of suh&ntial amounts of HIV iii lung gci-minat centers can be comparetl with the pre\,iour demonstraCon ofHI1’ in lymph node germinal (enters from patients with the per&tent lymphadenopal lip, syndromt~ (PM) using electron mici-oscol~w,~“~‘!’ iriiii~uiioliistochemistr\ to the HI\: : formed. M” Th~ls, in contrast to cr)ther viruses that directly infect pulmonary epithelial ~~clls, such as (:ML’ and Herpes simplex, HIV appears to primaril\ infecl tymphoreticular cells of the lung:. and aiiy epithelial changes seen in NIP or LIP are probably the result of indirrct effects of the immunologic response to HIV infection in the lung. C:sing several difierent merhods. HIV has been recovered from BAI, specimens from a substantial number of HIV-infected patients with a varietv 01’ puln~o~larc

FIGURE 9. lrnmunohistochemistry for UCHL-1 on this lung biopsy from a patient with LIP demonstrates positive staining of the vast majority of the lymphocytes in this lymphoid aggregate situated adjacent to a bronchiole and blood vessel. (UCHL-1; magnificatiorl 8235.)

lvitli 1,A.“’ \2’e l’orintt evidence of mild epitlieliat injuq in the majority (X0%) of biopsy specimens showillg‘I,IP and, less cc~mmonl~. in specimens from pat&its with (:II’/NIP (1 ~li%~,.These changes were easier to recognize in open lung biopsies than in ti-anshroii~hial biops) specimens. Two main signs ofepitlieliat cell injury were observed: type I I prieuniocyte hyperplasia ant1 intraluminal fibrosis. Intraluniinal or airspace fibrosis has previously been shown I o be associated with destrucGon of resp&tc~ry epithelium and subepitlielial basement membranes with subsequent proliferation of connective tissue into the lunnen of distal airspaces.” It is difficult 11)be certain whether the alveolar macrophages observed in our biopsb speciniens are a significant component of Nil’ and/or I,Il’, since alveolar macrophage acc~iii~uIation is nonspecific and may occur in association with a wide variety of causes. However, alveolar macrophages are a rich source’ of cytokines, which may play a role in the pathogenesis of interstitial pulmonary disorders.” The results of immunohistochemistry in our cases demonstrate that the lymphocytes in both NIP and I,IP in adult HIi’-infected patients are predominantly T cells. This observation in biopsy specimens of NIP is consistent with findings in patients with LA who have a predominatIce of c:ytotoxic. T lymphocytes in BAI. speciIJlenS,H.:~!~..IO. i:s.41 It also correlates with the results of (;uillon et al, who found predominantly (X)8+ tymphoc‘ytes in optan lung biopsy specimens from four HIV537

HUMAN PATHOLOGY

Volume 23, No. 5 (May 1992)

FIGURE 10. Dark field microscopy demonstrates numerous silver grains that indicate positive in situ hybridization for HIV-RNA in the center of this lymphoid aggregate. (In situ hybridization for HIV-RNA, dark field microscopy; magnification X250.)

complications of’ AIDS, including I,II’.~“~5’ Human iminunodeficiency virus has also hceii clenionstratecl in a lung biopsy specinwn of another HIV patient with I,Il”H as well as in a lung biopsy specimen from an aclult HIVinfected patient with ;I f’~~~l-i~~ii-~~sso(.iateclLIP-like process where no germinal center forniation was found.“” These data, in conjunction with our own, suggest that

538

the lvmphoicl proliferation in both NIP and LIP in adult HIV-infected patients is related to pulmonary infection with HIV. We cannot exclude the possibility that other infecrious agents, such as chlaniyclia. or other viral agents or cofactors that we have not investigated may be important in the pathogenesis of these forms of lyrnphoid l~rieuinonitis.!‘~~!

LYMPHOID PNEUMONITIS

AND HIV (Travis et al)

that

chlam)dia, other viruses, or tliftrerlt inlectious could also play a role. Our conclusion is supported bv the recovery of HIV from the lungs of Ijatients with LIP in previous studies,“~‘” m addition to evidence that lymphoid pneumonitis can be associated with pulmonary inl’ectioll by other lentiviruscs closely related to H I~‘.“““‘i

Neitlit7 (:&IV nor EBV was found b). in hitii hybridization in seven specimens; in these same specimens EBV could not he detected using PCR except in one c;IW of NIP. The positive finding of EBV by PCR in the latter (‘ase was not felt to be significant since a siniilat result was fi)untl in a group of control specimens, and the nmllbe-1. of EBV-infected B lymphocytes is known IO be frequrntly increased in HIV-infected patients.“” In contrast to o;lr cases of LIP in adults, several studies in children havr suggested that EBV plays a role in the pat hogenesia of LIP in pediatric HIV-infected pa(ients,l,~‘.‘” Ii:!.The absence of EBV in lung biopsy specimens 1.ron.l adult HIV-infected patients with LIP was c.onfirmed in allother study by in situ h\‘bridization.“~ It is not certain whether the presence c’,f EB\’ in the lungs of pediatric \‘ersus adult patients with I.IP is related to the apparently greater severity of Iymphoid inliltration obser\4 in cases of LIP in children compared with adults. Epstein-Barr virus has been reported to be associated with a fatal IvmI>hoproliferative disorder in an adult HIV-infected patient who had widespread organ infiltration, including pulmonary involvelnent.“” Thus, P:BV c’an play a role in lymphoproliferativc disorders that ma\’ aflect the lmlg in both adult and pediatric HI\‘-inf&tcd patients;“5.“” however, it does not appear to be important in adult NIP or LIP. Evidence in animals arid humans that lymphocytic pneumcmitlls mav be induced by viruses similar- to HIV sul)ports the concept that NIP and LIP are induced b) HIV in adults. Patients with human T-cell lymphotropic virus type l-associated tropical spastic paraparesis have bechn showr~ to have a BAL CDXS lymphocytosis similar to that seen in HIV-infected patients.“” In addition. pulmonar\ lesions similar to NIP and LIP occur in sheep infected with thy ovine lentivirus (OvLV)““~““; these lymphoc.ytcs also are primarily of T-cytotoxic/suppressol lyr1lphocytcs.““This observation is significant since HIV has been placrtl within the lentiviruses on the basis of viral genome sequence homology, viral morpholog, and viral replicarive mechanisms.‘” The severity of the pulmonary lymphoid infiltrate seems to vary depending on the strain of 0vI.i’; lambs infected with lytic strains develop a LIP-like picture with frequent extrapulmonar) disease, while lambs infected with nonlytic strains of WI,\ develop mild lymphoid pulmonary infiltrates that bear a closer resemblance to our cases of NIP.“” It is also intriguing that transgenic mice infected with HIV proviral DNA developed mild lymphocytic interstitial infiltrates similar to those seen in NIP, although it is difficult to compare the disease observed in these mice these data are with NIP in hunlans.‘i7 Taken together, consistent ,rvith the concept that pulmonary Iymphoid interstitial infiltrates similar to those seen in NIP and LIP can be produced by HIV and several closely related lwtiviruse? in humans anti animals. In su~mmmy, (:IP/NIP and LIP appear to represent a spectrunl of lymphoid pneumonitis in adult HIV-infected patients. with CIP/NIP representing an early manifestation of pulmonary HIV infection. Our data suggest that HIV is either the primary causative agent or plays an important role in the pathogenesis of both NIP antl I ,I 1’: however. we cannot exclude the possibility

agents

Wh) pulmonary 1ymphoid inliItrates in some HIVinfected patients manifest as NIP rather- than LIP OI vice versa is not known. In hght of the observation that (>vLV isolates induce strain-depender~t patterns of pulmonary Iymphoid infiltrates, “” it mav be that differing strains of HIC’ determine whether- a patient develops NIP or I,IP or a different type of‘ pulmonary c,omplication. The idea that the occurrence of‘ 1,IP in HIVinfected patients rriay be related to different strains of the virus has been su’ggested previouslv due to the rare occurrence of LIP among white homosexual American males and its striking frequency in Haitialls.“‘.“” Other possibilities include potential different-es in host susceptibility or cofactors that could afttic t the type of pulmonary manifestation that occurs in ;I gi\,en HIV-infected patient. Host diffewnces codcl he due to dissimilar mechanisms of lvmphocvte recruitment to the lung, which might involvt lytrlpt;c,~\itt,-entloth~.lial interactions, sue h as lymphocyte lxtm’ing receptors and vascular addressins, as well as lvmphwyte (.llemc)attraCtant factors. such as interle&n-1, itltcrlcukin-2, and Iymphocyte chemoattractant factor.“” These represent potential areas for future research to fiirthCr investigate the pathogenesis of pulmonarv lvmphoid pneumonitis in HIV-infected patients.

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HUMAN PATHOLOGY

Volume 23, No. 5 (May 1992)

IO. Ognibene Fl’, h,lasur H. Rogers P. ct itI: konbp~( ihc intet.stitial pneutnonitis without evidence of f’nrumocysti.t ccrrrnii in asymptomatic patirnts infected with hutll;m illltrlUtlc,tl;fic.irll(.). virus (HIV). Ann Intel~n Med 109:874-x79. I!)XX I I. Simmons ]‘I’, Suftrrdini AF, 1.xk I3:. ct al: Nonspecific interstitial pnrumon~tis in patients with AIDS: Rxliologic fe;tturrs. ;?JR I -1026.G26X. 1087 I?. Hostrttlrr (1. .4mundson I). O‘(2mtror S: l’hrtt!toitl hypcrsensitivity with pulmonxy invc)Ivement in a hemophiliac patient with hut~l~m ilnmutlodrfic-irrlc c virus inf’ection. Drug Intell (:lin Phar-m ?I : X75-X76. 10x7 I: