Co\v's m i l k p s o t c i n s
p i ~ l r n o n a r yh c m o s i d c r o s i s
s ; ~ ~ l i o a I l c r g o s o r t ~tests
c ~ i t (RAST)
Pccliat. Res. 11: 895-90.3 (1977)
Immunologic Studies in Cow's Milk-induced
Pulmonary Hemosiderosis
turcs found throughout this group. Upon removal of co\v's rnilk
;LIICI.
1
from tlic diet. tlie syriipt0111s i ~ i i p r o v e ~
l ~ p o 1 rei~itroductiori
Antil~odiest o cow's niilk 1)roteins (C&113) rrere studiecl I I ~ of Illilk illto tllC cliet,
byllll,tOlllS
rCtlIrIIC~,
radio:~llergosorI,enttests t o (letermine tllc quantity :lncl in~niunoiloll;lnLl cr
(()) ohscrvecl
not o n l y
lligl, inciLlcnce
glol~ulinclass oftliese antil~odicsin nine patients with precipitins chronic O r recurrelit pulmorl;lry disease. I ,;Ilso~ recurrent
~ ~
t o cow's milk. 'l'hrce of these patients hat1 puln~on:lry hen~osicle(,f ,,,ilk
tract i n ~ c c t i o n s ;Issoci;ltcd \\illl the
rosis (1'11); in o n e other patient pul111on;lry hernosiclerosis \r;ts prccil,itins. hlilk ~ , ~ ~ , ~ assOci;lted
~ ~ ~ \\.itll~ ~ i ~ i
su\pectecl, but not proven. 'l'llree patients liad n o ~ i l ~ e ~ ~ ~ o s i d c r o s i s
ancmi;l, hcl,;ltomcgaly. ;Incl splcnOfailure t o
chronic lung dise:~se and t\ro had other tliseases. A g e - n ~ a t c l ~ e t l meg;lly a n d c,lLl,;ll rclatiOns~lil, ~,et\veell[,,ilk ;Inel tilcse syml,control sul)jects Irere also studied. T h e quantity and inin~unoglo- tom,
in,plic;ltc~~,cc~,Llscimprovcmcnt of
synlptollls
bulin cI;~ssdistribution of antil~ocliest o CXlP were similar in :ill
rcnlOval
milk
fro,,,
'1ict. 1 3 ~ ) ; ~(,I.
~ (3)
patients; the cluantity of :~nti-CRII' :~ntil)odyrr.:ls signific:~ntly
\,.itll typic.ll fc;ltures (,f 1 leillcrqss y l l ~ r O m e
greater in patients than in control sul)jects (1' < 0.001). In c O n ~ ~ r r n cv;llidity
of the Hiilk prccil,itin-pulmoI1~Iry
~lcmo,i~
addition, c o n ~ p l c n i e n t - f i s i na n t i l ~ o d yt o CXlP tras not detected dcrO,is s y I 1 ~ r O I l l C;lllL1 ;llsO IIOtCel tll;lt sOlllC
tllCsC I,;ltielltS;1140
in eitlier patients' o r controls' sera.
dcvelopccl cor pulmonale zcconclary to ~ l p p c r;~ir\vayohstrucL ~ ~ ~ i p h oresponses
c~te
t o CAI]' were stutlietl in three patients tion.
with 1'11, three inclividu:~ls wit11 other ~n:~nifest:~tions
of niilk
siglli~iC~lllCe Illilk l,rCCil,itills
ill I,,l~llloll;lry
, I ()\\
hypersensitivity (positive controls), and nine neg:ltivc control hemo,iLlcrosis
;lnel rcpiratory
disorders i, cOntro,.crsi;ll,
/lndcrsubjects. Patients and positive control s111)jects responded t o son
Schloss ( 2 ) i n 923 ~ c m o n s t r a t tll;lt
e ~ m a n y cllildrcn fctl
CX11' with greater tritiated thymidine incorporation than tile
,
m i l k devclOl,ed
precil,it;lting
a n t i ~ , o i ~ i c s to Illilk I,rOtCills.
negative control sul)jccts (I' < 0.05). Ilo\r.cver, n o significant AlthOLlgllm i l k precipitins
also Ol,scrved in 5 0 5 ; (,f p;tticnts
difference in response t o CXlP rvas olbserved I)et~vcenthe 1'11 ,,.ith
~ c m O s i ~ e r o , i s rarely occurs
deficiency ), pLllmonary
patients and the positive control sulljectr.
in this group. I n adelition. ;I fciv patients with pulmorlary hemosiAntil)odies t o hunlan lung tissue Irere studied in the sera of all dcrOsis \vitllout Inilk precil,itins
a
have inlprOved \ v ~ l c l l
paticnts with PI1 and two negative control sul~jects.~\ntibotlics m i l k - f r c ctlict (,), rI.llcsc ol,,crv;ltion.;
ggc, 11,:11 ~lyl,cr,cn,itiv10 ~ I I I I ~ ~ : Ilung
II
uncl rat lung ,rere cletertecl in o n e ~):~tient
\+it11 ity to milk is import~lntin tlic p;~thogencsisof pulmonary hernoPI1.
siderosis. hut that the milk prccipitins tlicriisclves may not p l ~ ~a y
major role in this process.
Sl)eculation
TO further irlvcstigatc the possihle im~nuriologicrnccliani~riis
\Ye h:we Ijeen un:ll)le t o denlonstratc :~nyunique inin~unologie involved in the patliogencsis of milk-induced pulmonary hcmosiy.
cl;~ss.;ind sonic
mechanism associated with niilk-inducetl 1'11 in the patients clerosis. studies of the i ~ u ; ~ n t i t immunoglol~ulin
studied. l l o ~ r e v e r ,these studies d o not cornpletely rule out the 1)iologic properties o f the ;~ntihodiesto co\v's milk proteins \\ere
role of these i n ~ r ~ i u nmcchanisn~s
e
a s it is possil)lc that the undcrt;~kcnin patients tvith ( I ) milk-i~lcluecdpulmonary henloantigen o r antigens rcsponsi1)le for the relevant re:lctions niay hiderosis, ( 2 ) nonhcmosiderosis lung disc;~he,(3) milk-induccil
result fro111 partial degradation of cow's nlilk protein a n d were gastrointestin;~ldisc;~sco r cliroriic rhirlitis. and ( 4 ) iri normal
responses to co\v's milk proteins
not studied. Alternatively. increased availal~ilitgof antigens may indi\.idu;~ls.Itr 1.irr.o Iyr~~pliocyte
; ~ n d;~ritil~odics
t o Iium;~nlung tissue were ; ~ l s ostutlicd.
b e important, which nlight result fro111 exce\sive gastrointestin;ll
absorption of intact cow's milk proteins. Furthern~orc,tlie possibility exists that uniclue antigens rriay I)e present in the lungs of
kl,\'fl~RIXLS XSD klfl'fl1ODS
patients trith 1'11 lrhich cross-react either with :~ntil)odiesto
CXll' o r s e n s i t i d cells.
P,\'III:NTS A N D C'ONTIIOL.
SUI%JIiCTS
S u III ma ry
.,
Nine cliildrcn \\.it11 one o r rnorc milk prccipitins in their scrurn
I n 1002. f4cinc.r cpt (11. (8)reported the presence o f multiple \\.ere stuclied. Three were definitely di;~gnosed;IS having milkinduced ~ > u l m o n ; ~hemosiclcrosis.
ry
In o n e atlditional child ( S C )
high titer milk precipitins in the scra o f seven infants and children with chronic cough. recurrent lung infiltrares. \vheczing. the diagnosis of niilk-induccd pulmonary hcmosidcrosis is ~11spulrnonary hemosidcrosis, pcrsihtcnt rliirlitis, g;tstroir~testir~;~Ipccted. I,ut rcrnains in clucstiorl hec;~usc of the inability to
symptorns. and a n e n ~ i a . Four of these patients dcscrihcd as tlcnionstr;tte hernosirlcrin-l;~cIcn m;icrophages in hronchinl or
~ d
pulmon;lry
having pulmonary hcmosidcrosis had Iienioptysis, iron defi- gastric \vashings. .flirec othcr children h ~ chronic
cicncy ;~rlcrni;~.
and iron-laden rnacroph;~gesdetectcil in _c;~stric disease (rionhcmosiilerosis) and t\vo hail othcr rlise;~ses (i.~.,
o r brorlchial ivashings o r at lung biopsy (0). I'recipitins t o multi- gastrointestinal dise;isc and upper rcspiri~toryinfection). Clinic;~l
plc co~lstitucntsof co\v's milk, positive intraclcrn~;~l
i~nmccli;~tc fe;iturcs and laboratory d a t ; ~relilting to this group of children arc
summarized in l';~l>lc1 . Dct;~ilctlclinic;~l i1;1[;1 on tllrec of the
hypersensitivity rc;~ctionst o v;~riousco\v's milk proteins. recurpaticnts ( L S , BW, and A E) with pulmonary hcmosidcrosis have
rent otitis media. and gro\vth retardation \vcre additional fellNOS
~
'1 0 0
STAFFORD, POLhlAII, AND BO/\T
T\vccn 2 0 and 5% norn1:tl rabbit scrunl. Reiiction mistures \vcrc
incub;~tcdat room tcmpcr:lturc for 3 hr with constant shaking.
After incubation thc supcrn;~tantfluid was removed by aspiration and the filtcr paper disc \virshcd thrcc times ivith 2 rill o f
PBS. T e n microliters of the iippropriatcly diluted r:ldioiodin;~tcd
anti-inimunoglohulin \+,as addcd t o the filtcr paper disc and the
rcaction niisturc was adjusted t o 0.2 nll with PBS containing
0 . 5 % T\vccn 3 0 and 5 8 normal rabbit serum. Reaction niisturcs were incubated at room temperature for I 8 hr wit11 shaking. after which the filter papcr discs were again \\,ashed. T h e
riidioactivity of the antigen-coated disc \vas n1c;lsurcd by counting in :) Packard autom:itic gamma spectrometer. T h e lcvcl of
nonspccific binding was determined by substituting huffcr for
serum o r scrum dilution in some reaction mixtures and processing these in an identical manner t o the esperimcntal tubes.
T h e specificity of the radioiodin;~tcd anti-immunohlobulins
was cst;iblished by binding studies using IgG-. lghl-, IgA-. IgE-,
and I3SA-coated discs. Less than 0.676 of the r:idioiodinatcd
anti-immunoglobulin preparations bound to BSA-coated discs,
\vhcrciis honiologous antihody binding ( c . g . . anti-lgG to IgGPURIFIED IhlhlUNOGLOUULlSS A N D COW'S
coated discs) ranged froni 26-53% idcpcnding upon the radioioh l l L K PROI'EISS
din;~ted antihody preparation. Hetcrologous ;intibody binding
Iluman IgG was obtaincd commercially (hliles Laboratories,
( c . g . . anti-IgG to IgM-co:itcd discs) was 6 % o r Icss.
Kankakcc, Ill.. fraction 11 powder) and further purified by
T h e total c1u;intity of antibody t o C h l P in a patient's o r conelution from D E A E (dicthyliiminoethyl)-ccllulosc columns. IgA tn)l's serum \v;is cspresscd ;is the inverse of the dilution of the
\vas prepared from human colostruni, Ighl from sera of p;iticnts serum which hound 5 0 % of the miisimuni amount of radioiodiwith \Valdenstrom's niacroglohulincn~i:~,
and IgE from serum of n ~ i t c d;1nti-im1nt111oglohuli1i
( i ~ n t i - ( F i ~ b ' )thiit
~ ) c o ~ ~ l 1txl2 b o ~ ~ n t l
an IgE myeloma paticnt ( I ' S ) . Thcsc immunoglobulins were to Chl1'-coated discs. T h e 5 0 % binding tlilutiori \\,as termed the
purified by prep;~rativcclcctrophoresis, DEAE-cellulose c o l u n ~ n B,,, of a serum. hlnsinial binding of the radioiodinated antichromatography, and gel filtration. The (Fab'), fragment of IgG immunoglobulin was determined by titration of serum from a
\\as prepared by pepsin digestion of purificd hun1;in IgG. Purity patient with I'kl that had the Iiighcst titer of precipitating antiof inimunoglobulin prepar:itions \\,as estiiblished by imniunoe- bodies to CILIP.
lcctrophorcsis and Ouchterlony double diffusion analysis.
The in~n~unoglohulin
class t o \vhich ;I patient's tinti-ChlP heBovine y-globulin, fraction I1 ( D G G ) . \\as obtaincd from longed was determined by radio;illcrgosorl>cnt test as described 1
Sch\varz/hlann (0r:ingchurg. N . Y.). Bovine scrum albumin. ahovc and the data \verc csprcssed as the meiin percentage of
fraction V (BSA). was obtaincd from hlilcs Laboratories. Co\v's anti-immunoglobulin counts hound minus nonspecific binding.
milk protein (ChlP) prepar;itions were prcparcd from raw co\v's
Studies \\ere performed in dupliciitc. T h e pcrccntiige of antimilk by centrifugation at 17,000 rpm for I hr. T h e supernatant immunoglobulin counts rather than r a n counts \Yere used to
lipid layer was discarded and the dcfattcd Chll' \v:is stored :it
eliminiitc vari;ition due to clcc:iy of the r;iclioisotopc (I2") and to
-20" a t a concentration of 10-13 mg/ml.
permit comparisons bettveen serum samples studicd a t different
times. Statistici~l compiirisons between patient and control
group5 were c;irriccl out uhing the Stu~lcnt'sI-tcst.
been presented previously (3). Prccipitins against co\v's milk
proteins were detected by double diffusion analysis of scrum.
hfacrophagcs in smears prepared from tracheal o r gastric aspirates \\.ere stained for hemosiderin with potassiun~ferrocyanide.
lmn1unoglobulins G . A , and hl \vcre studied by ininiunoclcctrophorcsis and qu:intitnted by radial diffusion tcchniclues. IgE was
quantitated by radioimniuno:issay (13). Nasal eosinophils, hcmoglobin electrophoresis. and hcm;itocrits \Yere determined by
the routine clinical laboratory tests. Skin testing was performed
with 0.1 ml filtered unp:~stcurizcd cotv's milk containing 10.9
mg/nll protein (3).
Control subiccts consisted of a group of 30 randomly sclcctcd
age-rniitchcd children hospitalized for nonininiunolhgic, gastrointestinal, o r pulmonarv diseases. Seven adult labomtorv and
hospital personnel also served as control subjects.
Sera from patients and control subjects were stored at -20'
hcforc study. All serunl samples studied wcrc obtiiincd when
patients \\ere o n a milk-containing diet.
~
~
~~
Antiscrn to C h l P and human IgG. IgA. Ighl, IgE, and the
(Fah'), fragment of IgG were prcparcd in rabbits. These antiscra
were made specific by appropriate absorption \\.it11 human cord
scrum iind purificd imniunoglohulin prepiirations. y-Globulin
fractions of rabbit anti-Ighl, anti-IgE, and anti-ChlP were prcpared by elution from DEAE-ccllulosc. Specific anti-lgG. antiIghl, anti-lgA, and anti-(Fah'), ivere prepared by elution from
Scpharosc 4 B t o which the iippropriatc immunoglobulin h;id
been coupled by cyanogcn bromide activ;ition. A preparation of
r a d i o i o d i n a t d anti-human IgE ( N D ) \vas obtiiined from Pharmacia (I'iscata\vay. N . J.). Specificity of these antihody preparations was cstahlishcd by in~niunoelectrophorcsis:ind Ouchterlony double diffusion analysis. y-Globulin fractions and spccifically purificd antibody preparations of anti-lgG. anti-lgA. antiIghl. anti-(Fith'),. and [inti-ChlP \\ere radioiodin;itcd using the
iodine n1onochloridc method ( I ? ) . T h e y-globulin fraction o f
anti-lgE was radioiodinatcd using the chlor;iminc T method
(10).
hllCIIOCOhlPLI~hllJN1'FIXATION
ASSAY
T h e microcornplcment fixation ;issay \\.;is c;irried out in a
U-shaped well microtiter plate (Cookc Engineering). A tris(hydros)~meth)I)aminomcthanc(0.1 h1)-sodium chloride (0.14
h1)-m;igncsium sulfate ( 0 . 015 h1)-ciilcium chloride ( 0 . 01 hl)
buffer. p l l 7.4 ('l'ris-NiiCI), was user1 for dilution of all rcagents. I ' h e foIIo\ving reagents were ;iddctl to each \vcll of the
microtiter plate: cithcr 1 tlrop of appropri;itcly diluted rabl>it
anti-serum t o C h l P . patient's scruiii. o r control scrum; 1 d r o p of
Tris-NaCI buffcr; I d r o p of guinea pig serum ( c o n i p l c ~ ~ i c n' t
source) diluted 111 35; and 1 d r o p o f Chll' o r I3GG ;I conccntration of 0.25 pglml. I'hc optimal concentrations of guincii
pig serum. C h l P , and B G G were rlctcrmincd by prclirnin;~ry
cspcriments. After slow shaking a t room temperature for 5
min, the reaction misturcs \\.ere then incul>;~tedovernight at 4'.
t\fter incubation. 1 d r o p of sheep red bloocl cells that li:itl been
sensitized \vith rabbit hcn~olysin\vas adrlcd t o cach \vcll. T h e rcaction
mixtures \vcrc then incu1)atctl a t 37' for 1 hr ant1 the rcacR~\DlOi\LL.EKCiOSOIII3l~NTTEST (RAST)
tion was tcrmin;itcd hy placing the n~icrotitcrpliitcs o n ice for
C h l P was cov;~lcntly linked to cyiinogcn bromide ;~ctivated 10-15 min. The pli~tcswere then spun ;it 1.200 rpm for 10 min
filter papcr discs (\Vhatn~:in n o . 4 3 ) according to the method of and read ;iccording to the follo\ving criteria: complete hcmoCeska and Lundkvist ( 4 ) .
Iysis ( n o cclls). 0; retl cell ghosts. + 1; few red cells rcm;iining,
T e n to 5 0 /*I of scruni o r herum dilution in sodium phosphate- + 2 ; tlccrc:~scd red cell pellet, + 3 ; full rcd cell pellet. + 4 . Aphuffercd saline ( P I E ) , p H 7.2, \\.as addcd to test tul>cs contain- propriate iintigcn. ;intihody, a n d complement controls were pcring ;in antigen-co:itcd filter papcr disc. T h e final v o l u n ~ e\vas formed \villi cach espcrirncnt. 'l'hc gross complcmcnt fixation
adjusted to 0 . 2 nil by the addition of PBS containing 0.576 titer of a serum was considered to he the last clilution \vliicl~
90 1
COW'S MILK-INDUCED PUL.hlONAI<Y HEhlOSlDEIlOSlS
gave a score of + 2 o r gre:ttcr. A serum's ;inticomplcrnc11t:1ry
i~ctivity\v;is clctcrnlincd by titrating tllc scruni \vith complement
but in the absence of antigen. 'l'lie difference h c t ~ v c c ~the
l gross
conlplcmcnt fixing titcr of the serum :111d its anticornplerne11t;try
activity \v;~srecortlctl as its net complement-fixing antihotly titcr.
Normals
A Pulmonar y Hemos~deros~s
x
Lung Dlseose (non-P.H.)
m Gastro~ntest~nal
and Upper
Respiratory
h4ononuclc;ir cell fractions contiiining 907; or greater Iymphocytes were isol;~tctlby Jcrl\ity gratlicnt ccntrifug;~tionusing
1:icoll-sodium mctrizo:~tc gr;~dicnts(11). The microlynlplioqtc
stin~ulationassay \v;is c;~rrictlout in microtiter I 1 culturc plates
(l,.alcon I'lastics). .l'o each \\ell of the nlicroculture pl;~tc.1 .5 x
1 0hiononucle;ir cells in 0 . I nll RI'hlI- 1610 mediunl containing
IIEI'ES Iluffcr, penicillin. streptomycin. and 20% ;~utologous
plasma \ifere addctl. CMP, I3GG. o r I3SA \\.;is ; ~ d d c dto the \vclls
at final conccntr;~tiorlsof 50. 100. 500. and 1000 pg/ml. 1-ympliocyte responses to phytohcmaggl~~tinin.
conca11av;ilirl A , :ind
pokc\vecd mitogcn were ;11s0 sti1dict1;IS ;I r1ic;isure of cell vi;ll>ility and function. Cultures containing rnitogcns \verc incuhatctl
for 3 d;~ys\vliercas tllose conti~iningantigens were incuh;itetl for
5 days at 37". Five hours Ilcforc 1i;irvcsting the cultures. 0.5 pCi
tritiated tliymidinc was atlilctl to each culturc. Culturcs \vcrc
li;~rvestcdusing the ;tutomatic microlympliocytc h;~rvcster(Otto
I liller, hladison. \Vise.). 'l'ritiatcd tliymidinc incorporation into
c;tch c ~ ~ l t was
~ ~ mcasurctl
rc
using ;I Se;irlc ;~utornaticlicluid scintillatio~isystem. All culti~reswere carrictl out in triplicate ;ind
data \\.a\ expressed as tllc m:iximum mc;m counts per mi11 in
antigen-stirnulatcil c u l t ~ ~ r cr sn i n ~ sthe mc;m counts per min in
~rnatirn~rl;~tcd
cultures. + I SI).
Lung tissue fro111tlcccascd cliiltlrcn 0 - 6 years old \vithout
pulmonary discasc \\as frozen in litluid nitrogen. cut in 1p thick
sections. ;ind mounted o n slides. Slitlcs \\.ere \v;~slicdtwice for 10
rnin at roorn tcrnpcr:lturc tvith gentle shaking with 0 . 1 1 hl N;ICI0.01 hl KI1,POl Iluffcr (NaCI h~il'fcr).pl l 7.2. and tlried. Lung
sections were ovcrl;iycrcd \vith p;iticnt's or control serum. incuIlatetl in ;I nioisturc ch;irnllcr at roonl tempcr;~turcfor 1 Ilr. and
then \vaslied t\vicc in NaCI buffer. I~luorcsceinatc~l
polyv;~lcnt
a n t i - i r n n ~ ~ i n o g l o l ~ i i(I
l i nIyland) was added to c;~clisection. The
slidcs \vcrc incub;~tedfor at least I lir as Ilcforc. \va\licd tn.ice in
NaCI I>t~ffcr.
tlricd. mounted wit11 ;I coverslip in 5 0 5 glycerin.
and stored at 4". The slidcs were read within 4S lir using a
fluorescence microscope.
QUAN7'ITt\TION OF ANI'IIIODIIJS '1'0 CO\V'S SIILK I'HOTEINS
I<I~I.ATIONSIIIPTO h l l L K I'I<I:CIPITINS
/\SD
The four paticnts \vitli pulmon;~ryhcrnosidcrosis h;td I3,,, valfrom 15-1 00 and had one to six milk prccipitins.
.fIiosc with nonhcmosidcrosis chronic lung discasc hiid I 3 , , , values from 30-04 and two to thrcc niilk prccipitin lines. 'l'llosc
with g;~atrointcstin;~l
and upper respiri~torydiscasc had 13,,, value.; of 10-1 2 ;inti one milk prccipitin. Seven patients \vho 1;icked
milk prccipiti~~s
scrvctl ;is rlcg;~tivccontrol sul>jcctb and ; i l l 11ad
I3,,, v;ilucs of less than 1 . Thcrc \vas a positive corrcl;ition ( r =
0.84. 1) < 0.01) hct\vccn the H,,, ant1 tlie rlunlhcr of milk
prccipitins (1:ig. I). i\lthough patients \\it11 p~rllnon;~ry
Ilcmosidcrosis tcnrlctl to have liiglicr I3,,, valucs anrl more milk prccipitins than the other ~xiticnts,tlicrc \\.;IS c o ~ ~ s i t I c r ; ~ovcrI;~p.
I~Ie
L I ~ ranging
S
DIi'l'lll<hllNA~I'ION 01'
IAIh1UNOC;l.OIlUI.IN CLASS 0 1 :
A S 1 IIIOIIIES
TO ('SIP
Fl'llc nine ~>:iticnts\ \ i t 1 1 B,,, valucs greater than 1 \\ere stuclietl
hy liAST to tlctcrmi~lcthe i~n~nunoglol>ulirl
cl;iss of tile :irltihotlics to CXII'. 13ccausc of a scarcity of 1,S's scrulll. a 1/40 scruln
G . hl. ant1 /\ studies
dilution \\.;I\ used for tllc immunoglol>~~lins
Dlsease
Number of M i l k Precipltlns
1:ig. 1 . Correlation o f 5 0 5 binding dilution (I3,,,) v;~lucs:ind nurnl>cr
o f milk prccipitins.
;~ntltlicsc results \\ere not irlcludetl iri tllc stati\tical ;inalysis for
IgG. Ighl. ;~nrlIgA. For conll>arison, serum from 10 negative
co~ltrolsul>jccts\\.ere stutlietl for IgG, IgXl. ;inrl IgA a n d seruni
from four negative control sulljccts \\.ere studied for IgI:. Results
arc given as Ine;tn percentage bincling of each immunoglobulin
minus Incan nonspecific t~intling(.l'at>lc 2).
11s sho\vn in l';~l>lc2. tllc sera of;~lIpaticnts wit11 milk prccil>itins hountl more ;~nti-lgG(1) < 0 , 0 0 1 ) nnrl rnorc anti-lgi\ (1). <
0,001) tI1;111 the control sul>jects inclicatirlg the prcscncc of
lligllcr concentratio~lsof IgG and IgA anti-Chll' in the patient's
scra. 111 contrast. tlicre \vcrc no significant diffcrenccs in mean
~>crcc~it;igc
of hiritlirig of 1gh1 ;1ri~1 IgE l > e t \ v c c ~~~; ~ t i c r i ;tI sI I C ~
c.ontrol sul>jccts..l'llc diffcrcncc in Inciin percentage of bincling
I,ct\\cen the pulnion:~ry Iic~iiositlcrosispaticnts ;111tl the other
1>;1ticnts\\it11 milk ~>rccipitins\v;is not significant for immurloglol>ulins G . h l . A . ant1 li. \vlicrc;~s the tliffcrcncc in mcan
~>uwrit;igcof hirltlirig I>ct\vccn tlie pulmorlary hcnlosidcrosis
patients and control sut>jccts\\.as Iiighly significant (1) < 0.001 ) .
I3inding of labclctl anti-lg1: was tletcctctl or~lyin tllc scrulll of
one ~ ~ ~ l ~ n o Ilcmosiderosis
nary
patient. SC'.
Co~nlxirisonof percentage of hintling of tliffercnt ;inti-i~llrnunoglol>ulin reagents is very sirniliir to tllc original r;~tlioallcrgosorhcnt ~nctlioilof LC'idc. I3cnnicll. i~nrlJoll:insson (15) and is
of I3:,,, valucs for each
only scrniclunntit:~tivc. I)cterminatio~~
scruln \\it11 each raclioio~li~~:~tccl
; I ~ ~ ~ - ~ ~ I I ~ u I I ~ ~ reitgent
I ~ > I > I I I ~ I ~
llliglit give niorc ;icciIr;itc ~ ~ ~ ~ : l ~ i t i t ;of
i t i the
o r i c1;lss s~>ccificityof
tllcsc :~rltil>odics
to Ch1I'. llut this \\.;is not possible hccausc of the
for stuily.
lirnitctl amounts o f paticnta' scr;t ;ivail;~l~lc
IZAST data had indicatctl that the irnmunoglob~rinclass tlistriI?ution of ;inti-ChlP \v;is sirnil;ir in all patients. \\'e sought to
determine \vhcthcr a diffcrcncc existed in the I>iologic function
of anti-ChlP in tlie scra of patients wit11 pulrnon;~ryhernosiderosis cornp;~rctlto the anti-CX11' iintit>odics in the scr;i o f tlie
other p;~tientsstudicrl. Since tllc pulmoniiry lesions of pulmo-
003
STAFFORD. POLhlAK, AND BOAT
nary Iicmosidcrosis resemhlc Arthus reactions, the ability o f
anti-ChlP antibodies t o fix complement was studied.
T h e ability of tllc patient's scrum to fix complement upon the
l'orniation of antigen-antibody complexes with C h l P and B G G
was studicd hy the microcomplcmcnt fisl~tionassay. All paticnts
and normal (prccipitin negative) control suhjccts sho\vcd n o net
complement-fixing (CF) antibody activity titers t o C h l P o r B G G
(gross milk titer minus anti-complcnicntary titcr ( A C ) ) , \vhcrcas
the rabbit anti-CXlP scrum, used a positive corltrol, consistently showed a 4-fold o r greater difference in C F antibody titcr
with C M P o r B G G than without these antigens ( A C titcr).
Conscclucntly. n o C F antibody can be demonstrated using native
ivholc C h l P o r B G G a s the antigen. /\C titcr of greater th;ul I/
16 \\#as detected in thrcc of nine patients ivith rllilk precipitins
and in ttvo of six control subjccts.
observation is puzzling since all of the pulnionary liemosidcrosis
patients had clcv:~ted serum IgE levels ivhilc on a milk diet
except for this pi~ticnt.\Vitliin 7 months of the rcmovnl of rliilk
from their diets, the patients had norm;~lserum IgE levels (3).
T h c IgE antibody in paticnts' sera may he directed against ;In
antlgcn \vhich develops in the course of digestion of C h l P and is
not present in native C h l P preparations.
T h e ability t o fix complcnlent \v:~snot dcmonstr:~tcdin any of
the hurn;~nscra tested. T h e amount of anticornplcrncntary activity \v;~snot rcstrictccl to any specific discasc statc. Consequently,
ivc observed n o significant difference in this biologic function
bet\vccn the anti-ChlP antihodics of pulmon;try henlosidcrosis
patients and those found in the other patients with milk hypcrsensitivity disorclers without pulnlon:~ryhcmosidcrosis.
Lymphocyte stimulation responses to \vhole dckittcd ChlI'
\vcrc den1onstratc.d in all individuals studied with milk hyperscnLYhlPIIOCYTE RESPONSES TO COW'S hllLK PKOI'EINS
sitivity, i.c., pulmonitry hcmosidcrosis paticnts :und positive control suhjccts with othcr m;~nifestationsof milk hypersensitivity.
Lymphocytc responses t o tvhole d e k ~ t t c dC M P . B G G , and
H o ~ c v c r n, o diffcrencc in lynlphocytc reactivity \v:~s obscrvcd
BSA were studied by microlymphocytc stimulation assay in
bet\vccn the pulmonary liernosidcrosis patients and the control
three of the patients \vith pulnionary hcnlosidcrosis, thrcc indisuhjccts with milk hypersensitivity. B G G and BSA did not elicit
viduals (termed positive control subjects) with past clinical histosignific:lntly different lymphocyte responses in the pulmonilry
ries of hypersensitivity to milk (purpura, gastrointestinal), and
hcmosiderosis paticnts compi~redwith the negative control subnine negative control suhjccts. Results are csprcssed as the
jects.
mean rnasirnuni rcsponsc in counts per nlin minus mean backIn ;~ddition,;~ntihodiesto homologous human lung tissuc were
ground counts per min + 1 S D to ~ v h o l cC h l P , B G G , and BSA.
not demonstrated in the scrit of thrcc of the four patients \vitli
All patients with milk hypcrscnsitivity responded to C h l P with
pulmonary hcmosiderosis.
greater tritiated thyrnidinc incorporation than did the negative
T h e purpose of this sturly was to attempt to elucidate the
control subjects (1' < 0.05) (Table 3). The response of pulmoirnrnunopathologic mccharlisms involved in milk-induced pulmonary hcn~osiderosispatients does not differ significantly from the
nary hcnlosidcrosis. Four typcs of immunologic rc:~ctions arc
response of the positive control subjccts (P > 0.8). tIo\vcvcr,
thought
to be responsible for hypersensitivity diseases in the
the response of pulnlonary hemosidcrosis patient~atoCh1P docs
lungs: typc I (IgE dcpendcnt), typc I1 (cytotosic; tissue-specific
differ significantly (1' < 0.05) from that of the negative control
antilrody), typc I 1 1 (immune complex), ; ~ n dtype 1V (cell niesubjccts. N o st;ttisticnlly significant differences \\ere obscrvcd
diated-delayed hypersensitivity) ( 5 ) . Each of tlicse immune rcwith the rcsponsc against B G G and BSA.
actions induces cl~:~ractcristicpathologic changes in the lungs.
Each typc of reaction has some unique ch;~ractcristics;ho\vever,
ANTIBODIES TO IIUhIAN LUNG TlSSUE
there is also some overlap as \ifell. e.g.. typcs 11 and 111. I3y
Antibodies to human lung tissue \vcrc sought in the scra of all comparing the quantity, class and biologic function of the antihodics to C h l P and cellular immunc response to various co\v's
patients with p u l n ~ o n a r yhcmosidcrosis and in the scra of two
milk proteins in pulmonary heniosiderosis patients versus other
control subjects using an indirect in~nlunofluorcsccncctechnique. T h e serum of one patient with pulmonary hcn~osidcrosis patients, \vc ; ~ t t c ~ l l p t et do find ;I property o r m c c l i ; ~ n i s ~u in~i ( l ~ ~ c
to onc tlisc;~scstatc \vliicli \ v ; ~ snot opcrativc in t l ~ cothers.
( E n )stained pc)sitivcly \ v i t l ~ I ~ L I I ~ anil
; I ~ rat lung tissue. AntiNo immunologic mechanism studicd was found to he unicluc
bodies t o rat o r human lung tissuc were not detected in the sera
to p ; ~ t i c ~ i twith
s
niilk-induced puln~onary hcmosidcrosis. This
of the three other paticnts and the t\vo control suhjccts.
docs not complctcly rule out these mcch;~nisrns;IS being operative in this disorder since it is possible that the antigen o r
DISCUSSION
antigens rcsponsihle for the relevant reitction may result from
\Vc found no significant differences in the quantity o r immupartial degradation of co\v's milk protein and that these reactions
noglobulin class of the antihodics to C h l P in sera from paticnts could not he detected in our studies. In addition, if patients \vitli
ivith pulmonary hemosidcrosis ;IS compared to patients with
milk-induccd puln~onaryhcnlosidcrosis h ; ~ dantihodics t o ChlI'
nonhcmosidcrosis lung discasc, g:~strointcstinal o r upper respithat cross-reacted wit11 unicluc antigens present in their lungs
ratory diseases who also had milk precipitins. IgE antibodies to
(typc 11, cytotosic i~ntibodies),such itntihodies might be ahC h l P were present in only o n e of the patients studicd (SC). This
sorbed in the lung and not he dctcct:~blcin the patient's serunl.
Table 9 . hlcrrtl pcrcctlrogc of hitlt1itlg.s of i t t ~ t ~ ~ ~ r r ~ o g l oc1rr.s.s-.sl)ccijic
hrrlit~
trtlrihot1ic.s it1 cotv's t,lilk l1rotcit1.v
hlcan 7h hiniling
Group
8 (0)
IgG
18.84 2 1.81
Ighl
-. 19.50 2 0.90
3 (4)
10.00 2 1.72
18.07 t 1.01
3 (3)
18.07 t 0.12
2 (2)
.-
All paticnts wit11 milk
prccipitins
Pulmonary hemosiderosis
paticnts
Patients with nonhemosidcrosis chronic lung
disease
Patients with other iliseases uith milk prccipitins
Control subjects
' h'umhcrs in parcnthcscs
5
SI)
11I
I0 (4)
- .-- - ..--
IgA
12.32 + 3.46
Ig I!
1.28 + 2.40
+ 3.68
2.18
5
3.28
20.13 -c 0.57
13.67 2 1.08
0.50
?
0.29
10.75 + 0.45
19.70 t 0.40
13.95 2 0.15
0.45
+ 0.15
+ 1.94
17.79 + 2.29
6.37 t 1.69
0.18
+ 0.15
9.36
inilic;~tenumbcr o f iniiiviiluals used for IgE studies.
9.90
C O W ' S h1ll.K-INDU('I:I)
PUl.hlONAIIY
--
-
C;rott[>
-
~p~
-
,v
-
CXIP
.
1%Sf\
I3GG
--
-
003
lIEhlOSID~~IIOSIS
--
--
-~
7.0-15 2 2.7-1s
2 . 4 7 2 2 3,353
I .7 I 2
+
1 . 0 1 0 2 1.871
2.701
\I1 \rill1 nlilh h! pcrscll\itivity
0
';ilictit\ \$it11 [ > ~ ~ l ~ i l o ~hel;~ry
~liosiclerosi~
r o ~ i t r o l s\ v i t l ~o t l i c r t i ~ ; ~ l l i f c s -
3
3S7 2 1 8 0
3-17 2 2 2 0
3
-117
3-10
-
--
I .700
2
0 3 0 -t 0 0 5
I 3-1s
2
U n f o r t u n ; ~ t c l y . lung tissue. from o u r p a t i e n t s \v:~s n o t ; ~ v ; ~ i l ; t l > l c
Tor i r n m u n o l l u o r c \ c e n t s t u c l i c \ .
, \ l t c r n a t i v c l y . t l i c ; ~ v a i l : ~ l l i l i t of
y a n t i g e n m i g l i t pl;~ya c r u c i a l
role in d c t c r m i n i r l g \ v l i c t h c r o r n o t p u l m o n a r y I l c r n o s i d c r o s i \ \\.ill
d c v c l o p . I t i s p o s \ i l > l c tIi;lt c s c c z s i v c a l > s o r l > t i o n o f c o \ v ' s n i i l l i
p r o t e i n f r o m t h e g ; t \ t r o i n t c \ t i l i a I t r a c t may occur in ~ x t t i c n t s\\ itli
I l e i n c r ' s a y n c l r o r n c ~ > r o v i i l i l l gt h e n c c e s s i t r y a n t i g e n f o r t h c \ c
I c.t'1011s t o o c c i ~ r .
i r i i r i i u n o l o g i c r c-;
1\11 o f t h e s e p o \ \ i l > i l i t i c \ r c c l i r i r c i n \ . c a t i g ; l t i o n s i n c e t h e i r n m u ~ l o p ; t t I i o g c c i sof ~ i i i l l i - i n c l u c c d pulmonary h c n ~ o s i i l c r o s i si s s t i l l
urlkno\vn.
1. A111ni;lnn. I\. J . . ;lnJ 1lr111g.I<.: Sclccti\c Igr\ deficiency: I'rc\cnt.~tiot~of 3 0
c ; ~ \ c \;tnJ ;I rc\ic\v of the Iltcr;~t~lrc.
hlcd~cinc.SO: 223 ( 1971).
2 , t111dcr\on, ,\, I..,. and Scl~lo\\.0. %I.: Allergy to co\\'\ 111iIk in I I I ~ ; I I I ~\ \\ i t t i
nutrition;~ltfi\ordcr\: ,1 prsllrnin.iry report. Anicr. J . I)!\. Child.. 26: 45 I
(1023).
3. Ho;~t.T. I:.. c.1 111.: Il)pcrre:~ctivity t o CON'\ rliilh ill young chllclrcn uitli
pt~In~ciri:~ry
I i c ~ ~ i ~ i ~ ~ :~~lncdcc)r
r o ~1>ulnic11i;1lc
i\
\cco11<1;1ryt o r i : ~ \ o ~ ~ I ~ ; i r y n g c ; ~ l
o l ~ \ t r u c t i ~ i J.
n . Pccli;it.. 87: 23 ( 1 0 7 5 ) .
4 . C'c\h;~,h l . . ;inJ I.unclh\i\t, LJ.: h nc\\ ;ind \ ~ n i p l cr;~dioimmuno;~\\.~y
n1cthcid
for the dctcrrnin;~t~oll
111 IgII. I l i ~ ~ ~ ~ u n o c l ~0:
c m1021
~ ~ t (1'172).
q,
of ;~llcrgicr c ; ~ c t i o ~ i \
5 . C.oon~h\. I<. I<. ti.. ;IIIJ GIII, 1'. ( i . 11.: C'l;~\\ific;~tio~)
rc\pon\~ldcfor cllnlc;~lh)pcr\cn\it~vity ; ~ n dd ~ r c a \ c 111:
. C'linic;~lr\\pcctr of
Immunology. p. 575 (131;1ck\\cIlS c i c n t ~ f ~I'uhlic:~tion\.
c
O\ford. 1'16X).
0 . Ilcincr. I). C'.: Pulmi1n;lry hc~iio\iclcro\i\. In: I<. L . Kcndlg. Jr.: I'ulrnon:iry
C'ONCLUSION
l)i\orclcr\ of the IIc\nir;itorv 'l'r;~ct in ('l~ilclrcn. Vol. I.. an. 345 ( \ V . 13.
S;~undcr\C'o.. I'h~I.~cIclphi;~.
1072).
A n t i l > o c l i c s t o C'hlI' wcrc stucliccl b y R / \ S P T t o c l c t c r m i n c t l l c
7 , I l c l n c r , I ) , (.,: I ~ , l l l l l , l n ~ l q I)i,,,rder,, \,,,I,
p , 37h ( \ V , 13, S;lunJcr\.
clu;lritity ;lnel i m m u n o f l o l ) ~ t l i nc l a s s o f t h c s c ; ~ n t i h o c l i c si n s c r ; ~o f
~ ' ~ I ~ : I ~ ~ ~1072).
~ ~ ~ ~ I I ; I .
X . llciner. I). C'.. Se;ir. J . W . . ;Inel Kniker, W . T.: hlultiplc prccip~Iln\t o co\\'\
cllrollic
( 1 ) f o u r P ; l t i e n t s \\.ith milk-irlduccd 1'1 1, ( 2 ) tllrcc
milk in chronic r c \ p i r ; ~ t o ndi\c;~\c.Arncr. J. Ili\. Child.. 103: 4 0 (1062).
l u r l g clisc;lxc \ v i t I i o u t I'll. ;Inel ( 3 ) t \ \ o \\it11 o t h e r c l i \ o r d c r s tvitli
I l , , l l a n d . N.
Ilnllg.
I),l\i,. N , '.,. ;II1ll
(., ,),: SIglIIIIe~lnee
,,,
riiilli p r c c i p i t i n s ;IS \vcll ;ts in t h e sera o f ( 4 ) 1 0 a g e - m a t c l i c d
precipitilting c ~ n t ~ l i o d ~t oc \mill. protein\ in the \crum of inf;~nt\; ~ n dchilr;lndomlv s c l c c t e d c o n t r o l z u h i c c t s . .l'he c ~ u ; l r i t i t v ; ~ n dimmunodrcn. J . I'cdi:~~..61: 1 x 1 1 1 o h 2 ) .
I". I I u n t ~ \V.
. hl.. ; I I I ~C i r c c n u o ~ ~I:.~ lC. . : N;lturc. 104: 405 (1002).
g l o l > u l i n cl;15s cli5tril>ution of antilloclies t o C h l P \\.ere similar ill
I..: % l i c r o c o ~ ~ ~ p l e r nt~\;ition.
cnt
In: I). h1. \Vcir: ll;~ndllool.of Expcri~ o l l l ~ l l e l lof
t~y
tile I t . l.c\inc,
l , ~ l t i e t l t s , ; + ~ l l l t i ~ , ~ ) l ~ i e s ch11,
mcnt.ll I r n ~ ~ ~ u n < i l p.
< ~ 707
g ) . (13l;1ch\rcll Sclcnt~licI'uhlic:~tion\. O\ford.
IcG and IcA c l ; l \ \ c s . The c l u a n t i t y of anti-CPvll' ; ~ r i t i h o c l y was
1. 0. ..6 7 ).
zignific;lrltly g r c ; I t c r in ~ x t t i e n t stli;ln in c o ~ l t r o ls u l l j c c t s ( I ' <
1 2 . % I ~ L I ~ I ; II\.I I s.:
~ , h l ; ~ t ~ i ~ ~ ~ ;lBrotcin
~ I i ; i t i hlct;1I>oli\11i,I: 331 ( 1 0 0 4 ) .
13. I'oln1:ir. 5 . 11.. \V:lld~l~:lnn.
7'. 11.. ;lnd Terry. W . I).: ,\ comp;~ri\onof thrcc
( ) . ( ) ( ) I ) . In a d c l i t i c ) l l . C[: ;lnti[>oJy t o C h l l ' \,,;I.; l l o t clctectctl in
r ; ~ c l l o ~ n i l n u n o ; ~tcchniclucr
\~~iy
for the nic;i\urcmcnr 111 w r u m I ~ l lJ.. Irnn~ue i t h e r p a t i e n t s o r c o n t r o l s ' ser;i.
not.. 110: 1253 (1'173).
111 1.itr.o l y m p h o c y t e r c s p o r i w s t o ChlI'. I3GG. a n c l I 3 S A t v c r c
.I.hOr,l,y, I!,, and Hr;lt;llie.
A r;lpid methc,d
prcp;lr;lti,ln of
s t u d i c c l in ( I ) t h r c c p a t i c r l t s \\.it11 I ' l l , ( 2 )t h r e e i r i c l i v i i l u a l s n i t h
I.~ r n. p l ~.r i c ~\u\pcn\i(~n\.
tc
In: 1'. I. l'crc\:~ki: Ili\tcicomp:~tihility Tc\tinp.
1970, p. h55-hi() ( h l u n k ~ g ; ~ ; ~Copcnh;~gcn.
rJ.
1070).
o t h e r m ; ~ n i f c s t a t i o n s of m i l k h y p e r s e n s i t i v i t y ( p o s i t i v e c o r l t r o l
15. \Vlde. I... Hcnnlch. 11.. and Joh;~n\\on.S. <i. 0.:Di;~gno\i\of;lllergy hy iinrpl
z,l,jects
) t l l l e l l e g l t v e e , l l t r , sLl,,,ec l t e l l t s
1,irn) tc\l for ;~llcrgcna n t i l ~ i d i c \ .L;~ncct,ii: 1105 ( l ' l h 7 ) .
l w s i t i v c c o n t r o l s u l > j e c t \ r c s p o n c l c c l t o ChlI' \\.it11 g r c ; l t c r t r i t i a t e d
I,,. hl,,,, ,,( ,I,,,study ,;,, d,,lle rclr,,,pcct,vely, c ~ l n \ c l l , l c n t l yinfl,rnlcd
,
t h y n l i c i i n c i n c o r p o r i l t i o n tll;ln d i l l t h e n e g a t i v e c o n t r o l z u l > j e c t s
\\;I\ ncic ncce\\:~ry.Vcrh;~linformed con\cnt \\;I\ o l > t : ~ ~ nfor
e d the rcrn;~i~iclcr
of the \tudy.
(I' < 0.05). I l o \ v c v c r . n o s i g n i l ' i c ; ~ n tc l i f l ' c r c n c e in respon\c t o
17. 11. A . St:~lforJi\ recipient of :I Northern ()hi<>I.ung r\\\oci;~tion Student
chip
ot)\erved I ) ~ ~ tllc\ \1'11~ l,;lticllts
~ ~
tllc
itive
l I c \ c ; ~ r c ll:clIo\\~liip.
~
c o n t r o l s ( I ' > 0 . S ) . N o s t a t i s t i c ; l l l s i g n i f i c i ~ n td i f f e r e n c e s i v c r e
18, .This rc\e;lrch \\a, \upported 1,). gr;lnt, fro,,,
C'y,tie Flhro\i\ I:ound;ltil,n
o h s c r v c c i wit11 t l l c r c s l > o r l s c s ; r ~ a i n s tUGG and I 3 S A h c t \ \ c c n all
(Clc\cl;~ndC h : ~ p t c r )and the h';~tion:~l
In\titutc\ of Ilc;~lth( I l l . 13885 : ~ n d
grolrps s t i ~ d i c i l .
111. ooooo).
10. IIcquc\t\ for reprint\ s110uId lie :idJrc\\cd to: S. 11. I'oIn~:ir, l ' l i . l ~ . ,h l . l l . ,
si t u d i e~d ill ~
~
~
~ t o nornlal
i
~ ~llm
, ~ ll ul~nl g tissuc
~
~
Di\i\ion of P c d ~ ; ~ t r iIrnmu~~ology.
c
1l;linllow Ii;lliic\ and Cli~ldrcnllo\pit;~l.
scr;i of four p ; l t i c r l t s \\.it11 I'H a n d t \ v o n c g ; l t i v c c o n t r o l s u l > j c c t s .
2101 Adelhcrt IIJ.. Clc\cl;ind. Ohhi 44106 ( U S / \ ) .
i \ n t i l ~ o c l i e s t o I i u m a n ;1nc1 r a t lung \ y e r e t l c t c c t c d in one p ; ~ t i c n t 20. Ilccci\cd for puhlic:~tionJ U I ~25. 1076.
21. Accepted for pul~lic;ition l:clvu;~ry 9 . 1077
\vitll 1'1 1.
,,,
.',.
'
,
,(,,
Copyrig111 $3 1977 I r ~ t c r n : ~ t ~ o nI'eJi;itr~c
;~l
IIcsc;ircIi I;tiund.~ti<rn.Inc.