Antibodies
in milk
D. B. L.
McClelland
Edinburgh
and South-East ScotlandRegional
BloodTransfusion
Service,
Royal
Infirmary,
Edinburgh
EH39HB, U.K.Introduction
Both inclinical
practice
and in the scientific literature human milkisoften treated asthough
itsonly important properties
were the volume in which it isproduced
and its nutritionalcomposition
intermsoffat,
carbohydrate
andprotein
content. Milk whichhas themisfortunetogettobabies
by
someindirect routeisfrequently
insultedby
pasteurizing
orboiling,
treatmentswhich
certainly
damage
the functionalproperties
ofmany of its constituentproteins
and cells.From an
immunologist's viewpoint,
human milk represents afascinating
blend ofbiologically
active molecules andliving
cells whichchallenge
one toexplore
theirorigin,
control andbiological importance.
It is not a new observation that milk is
important
to theimmunity
ofthe neonate. In thenineteenth century it was known thatpregnant
sheep
vaccinatedagainst
cowpox gavebirth tonewbornlambs who wereimmune and that vaccinial immunization ofmothers ledto
immunity
in their
infants,
but it was not clear how or when theimmunity
was transmitted-across theplacenta
or via themilk,
andwhether,
if it was related to antibodies in themilk,
these were absorbed into thecirculationoractedlocally
in thegut.By
the 1920's it had been shownthat,
in newborncalves,
deprivation
of colostrum was lethal.Three-quarters
of calves which weredeprived
of colostrum died ofsepticaemia
andasingle
feed of colostrumwasenough
toprotectthem. This
finding
has beenamply
confirmed for domesticanimals,
and there have beenmanystudies of human babies that show
beyond
doubt thatbreast-fed infants havealower attackrate forawidevariety
of infectiveproblems
(Chandra, 1979).
Itseemsreasonabletoconclude thatat least partof thisprotection
isa direct result of the infantreceiving
immuneprotective
factorsin milk and is notsimply
aresult of nutritional factors orthe avoidance of contaminated artificial feeds.Some of themany antimicrobial
properties
of milk andfactors in milk whicharecandidates to influence microbialgrowth
are listed in Table 1(McClelland,
McGrath &Samson,
1978).
The antibodies in milk areexciting protective
factors tostudy
becausethey
arespecific
forparticular foreign
agents and aresusceptible
tomanipulation
by
immunization,
butone should not be obsessed with antibodiestothe exclusion of other constituents. Forexample, lactoferrin,
lysozyme
andlactoperoxidase
areallpotentinhibitors ofthegrowth
ofmany bacteria andthere arenon-antibody
virus inhibitors in milk which are little studied but may bebiologically
important
(Butler, 1979).
Milk contains awiderange ofantibodiesagainst micro-organisms
andforeign proteins.
In contrast to serum, in whichIgG
predominates,
thepredominant
type ofantibody
in milk isimmunoglobulin
A(IgA).
Furthermore,
theIgA
in milk isstructurally
quite
different from that in theserum
(Tornasi
&Grey,
1972).
In serum,IgA
isstructurally
similartoIgG although
asmallproportion
ofit consists ofadimericimmunoglobulin,
i.e.2IgA
monomerscoupled
together by
apolypeptide
called the J-chain(Text-fig.
1).
MilkIgA
ispredominantly
secretory
IgA,
a dimeric molecule with two additionalpeptide
chains termed J-chain and 0022-4251/82/040537-07S02.00/0secretory
component.Secretory IgA
is found in very lowconcentrations,
ifatall,
in the serum but isamajor
constituent of milkandmostother external secretions.Table 1.
Properties
of human colostrum and milk whichmay influencemicrobial flora in the neonateandplay
arole inpreventing
infectionImmunologicalfactors
Immunoglobulins:secretoryIgA, IgM, IgG Lymphocytes, plasmacells andmacrophages
Non-immunologicalfactors
Lysozyme
Lactoferrin
Lactoperoxidase
ComplementcomponentsC3,C4,C3pro-activator pH, buffering capacity Protein concentration Salt concentration Lactose concentration Secretory IgA Secretory component J-chain
Text-fig.
1.Simplified diagram
of the structure of theimmunoglobulin
classes. H =heavy
chain;L=
light
chain.Source of
antibody
in milk—the mucosalimmunesystemIt is
important
to know how the breast is able toproduce
anantibody
responsewhich is ofaare waysof
manipulating
thisantibody
responsetothe benefit of the infant. Todiscussthis,
it isnecessary tointroduce the concept ofa
specialized lymphoid
system which servesthe mucosal surfaces of thebody
andalsoinvolves themammarygland (Hanson
etal,
1978).
The
sub-epithelial regions
of the mucosallining
oftheintestine,
together
withother mucosal sites andglands
suchastheparotid
whichproduce
externalsecretions,
containlarge
numbers ofplasma
cells. Theseplasma
cellsproduce
IgA
in the form ofadimeric moleculecontaining
the J-chain and immunohistochemical methods show that both theplasma
cells and the surfaceepithelial
cells containlarge
amounts ofIgA.
Theepithelial
cellssynthesize
secretory component, which is alsoexpressed
on the surface ofepithelial
cells. The dimericIgA,
releasedby
theplasma cells,
binds to secretory component on the surface of theepithelial
cells and thisbinding
is followedby uptake
ofIgA
and transitthrough
theepithelial
cells to the mucosal surface.Secretory
componentiscoupled
totheIgA
moleculeduring
transit and thefinal secreted molecule is the intactsecretoryIgA.
It is also
possible
that dimericIgA
which has thiscapacity
to bind secretory componentcould be
produced
in a site suchasthegutmucosa,but instead ofbeing
secreted into the lumen could reach thedraining lymphatics,
enter the circulation and betransported
to anotherepithelial
site,
such as thebreast,
where it could bind to theepithelium
and be secreted in the same way. Thebreast,
like the intestinal mucosa, containsplasma
cells whichsynthesize
IgA,
andIgA
canalso bedetectedin theepithelial
lining
of the ducts themselves. The presenceof this distinctiveantibody-producing
system raises thequestion
of the mechanismby
whichantibody-producing
cells get into mucosal tissues and the breast and what determines thespecificity
of the antibodies whichthey
produce.
The
immunological relationships
between theantibody-producing
system
of the gut andmammary
gland
arepresented
in a verysimplified
form inText-fig.
2.Antigen
appears topenetrate
thegut
wallmainly
atspecialized
sites,
thePeyer's
patches.
These are rather likeText-fig.
2. Asimplified
scheme of the mucosal-associatedlymphoid
system.lymphocytes
are
exposed
toantigens
in the intestine and travel via thelymphocytes
and blood tosites ofsecretory
antibody production, including
the mucous membranes of thegastrointestinal
andrespiratory
tracts, and the mammaryglands.
GALT =gut-associated lymphoid
tissues(the
lymph
nodes in thegut
wall,
containing
the basicequipment
oflymph
nodes whichpermits
a flow oflymphocytes
inand out, with the architecturetoallow cellular interactionswithantigens.
The
epithelium
which overlies thePeyer's patches
is different from therestof thegutepithelium,
being
thinner and flatter and allowspenetrations
by
large
molecules and even intactmicro-organisms.
Lymphocytes
enterthePeyer's patches
from thecirculation,
encounterantigen, undergo
the cellular interactions which areneeded tobecome sensitized toproduce
antibody
tothatantigen,
and leave the
Peyer's patch
via thedraining lymphatics, travelling
through
the mesentericlymph
nodestothe thoracicduct,
and thencetothe blood. On theway ahigh
proportion
of these cells start to evolve intoIgA-producing
lymphocytes,
making
IgA
antibodiesspecifically
directedagainst
the relevantantigen.
Many
ofthesematuring,
IgA-producing
cellseventually
find theirway back to the intestinal mucosa, but
they
may also become localized in the breast and inother sites where secretory
IgA
production
occurs. TheIgA-producing
cells localized in these sitesmaturetobecometypical plasma
cells. Itis still far from certain what decides where the cell willeventually
arrive and what determines whether a cell will stay in that site and mature orproliferate.
Onepossibility
is that thecells whichreturn to a mucousmembrane site like thegutaredrivento matureand
proliferate
by
meeting again
theirappropriate antigen
andthereissomeevidence that this
happens.
However,
this mechanismcertainly
does notexplain completely
theability
of these cells to mature whenthey
return to a mucosal ofglandular
site.Firstly,
ithas been shown that the cells derived fromPeyer's
patch
or a mesentericlymph
node andinjected
into arecipient
animal will find their way back notonly
to the normal gut but tototally
antigen-free
fetal gut(Halstead
&Hall,
1972;
Moore &Hall,
1972). Secondly,
IgA
blast cells from thegutlymphoid
tissue will hometothe breast in the absence ofanyevidence ofantigen
inthe breast
itself,
strongly suggesting
that theremustbe otherfactors which attractthesecellstothe
glands (Lamm,
Roux,
Weisz-Carrington,
McWilliams &Phillips-Quagliata,
1977; Roux,
MeWilliams,
Phillips-Quagliata, Weisz-Carrington
&Lamm,
1977).
This behaviour oflymphoid
cellsderived from thegut-associated
lymphoid
tissues isquite
different from that of similar cells from thespleen
or aperipheral lymph
node. Whenspleen
orperipheral lymph-node
cells areinjected
into arecipient,
the great bulk of theantibody-producing
cellswillendup inthespleen
and
lymph
nodes and these cells will bepredominantly
producers
ofIgG
andIgM
antibodies andnotIgA.
The scheme in
Text-fig.
1 isover-simplified
and there isonecomplication
which should be mentioned because it is relevant to thepractical problems
ofimmunizationtoproduce
agood
antibody
response in the breast. Thegut-associated lymphoid
tissues contain acomplex
population
oflymphocytes.
These include differentpopulations
ofT-lymphocytes,
some of which arecapable
ofsuppressing
immuneresponses and someof whichhelp
thegeneration
ofantibody
production
by B-lymphocytes.
The relative activation of the suppressor andhelper
T-lymphocyte population
in thePeyer's patches depends
tosome extent onthetype
ofantigen
and the way in which it isgiven.
It isquite possible
tofeedantigen
to ananimal andproduce
apowerful suppression
of immune responses whichis,
at least in part, due totheproduction
ofsuppressor
T-lymphocytes
in thegut-associated
lymphoid
tissues(Elson,
Heck &Strober,
1979).
As an overall
concept,
it may behelpful
to thinkteleologically
of the need forpowerful
control mechanismsgoverning
theantibody
response toantigens
in the gut. Since the gut isconstantly exposed
totheantigens
oftensof millions of bacteriaand,
overtheyear,kilograms
offoreign protein,
it is obvious that without somepowerful
control mechanism the entire metabolism ofananimal could become directedtoresponding immunologically
tothisconstant massiveantigenic challenge.
The
breast,
unlike thegut, doesnotworktoits fullcapacity
allthetime,
andonly
turnsonits fullproduction
mechanismsduring
lactation. Innon-lactating
breastsecretions,
although IgA
isvolume of secretion. Incontrast, in established lactation there isaverysubstantial
output
ofIgA
intothe milk. Theremust be aneffective mechanismtoallow the breasttoincrease itsoutputofsecretory
IgA
antibody
inlactation,
eitherby
making
more antibodieswithin thebreastitselforby efficiently transporting
them from the blood into the milk(Halsey,
Johnson&Cebra,
1980).
The
non-lactating
breast in man and animals contains a small number ofIgA-containing
plasma
cells. There isa dramatic increase inlactation,
e.g.in themousethe number ofIgA
cellsincreases
by approximately
900-fold. This increase islargely
dueto anincreasedlocalization ofB-lymphocytes
derived from the gutduring
lactation. WhenB-lymphocytes
prepared
fromgut-associated lymphoid
tissue areinjected
into anon-lactating
animalthey
canbe foundinthegut mucosabutveryfewcanbe detected inthebreast.In contrast,in the
lactating
animal,
large
numbers of theinjected
gut
lymphocytes
become localized in the breast and are detected asIgA-producing plasma
cells.This effect of pregnancy can be
partly
imitatedby
treating virgin
mice with oestrogen,progesterone or
prolactin (prolactin
alonebeing
the most effectivesingle agent).
Combined treatmentwith the three hormones leadstoalarge
increaseinthe number ofIgA plasma
cellsin the mammaryglands
in intactanimals,
and also causes localization of transferred gutlymphocytes
in theglands.
In contrast, treatment with testosterone stops the normalaccumulation of
IgA
cells in thelactating
gland
(Weisz-Carrington,
Roux, McWilliams,
Phillips-Quagliata
&Lamm,
1978).
Thesignal by
whichreproductive
hormones cause thesechanges
inthelymphoid
cellmigration
tothe breast isnot known.Presumably
thetargetof the hormones is theepithelial
cells. Whether theseareinducedtoproduce
aremotely
acting
factor,
or whethersomelocalchange
occursin the breasttopromotelocalization of thelymphocytes
is notknown.Normal human milk contains secretory
IgA
antibodiesagainst
a wide range ofmicro-organisms,
e.g.bacteria,
bacterial toxins andviruses,
aswell asantibodiesagainst dietary
proteins
such as bovine albumin. Thetype
ofantibody
in the milkreflects,
at least to someextent, the
immunological experience
of the mother. Forexample,
the milk secretions ofEuropean
and Pakistani mothers have asimilar level ofantibody against
commonsero-types
of Escherichiacoli,
but the Pakistani mothers have muchhigher
levels of antibodies to certainenteropathogenic
strains ofE.coli and Cholera vibrios and Cholera toxin(Carlsson
etal, 1976;
Holmgren,
Hanson, Carlsson,
Lindblad &Rahimtoola,
1976).
These differencespresumably
reflect the different microbial exposureof the two groups. In individual
patients,
rather than inpopulation
studies,
theeffect ofgutinfectionorcolonizationwith the bacteriacanalso be shown.For
example,
Salmonella infection in a pregnant mother leads to theproduction
ofspecific
antibody
in the milk when lactationbegins (Allardyce
etal,
1974).
Similarly, experimental
colonization ofa mother's intestine with
non-pathogenic
E.coli,
in the lasttrimester,
leads toproduction
ofspecific
IgA
antibodiestothatorganism
inthemilk(Goldblum
etal,
1975).
Immunizationto
produce specific
antibodies in milkLooking
at these observations and theunderlying physiological
mechanisms,
it is hard toresistthe conclusion thatnaturehas
equipped
thelactating
mother with asystem
forproducing
in her milkspecific
IgA
antibody
directedagainst micro-organisms
which she encounters in her environment and which arethereforelikely
to pose a threattothe infant. It would therefore belogical
tolook forwaysofimmunizing
motherstoimprove
thequality
andquantity
ofprotective
antibody
in themilk,
atleast inenvironments whereinfectionisamajor
hazardtoyounginfants.However,
ifthisapproach
is to be apractical
possibility,
it is necessary to find a convenient method of immunization whichdoes notrequire
the mother toundergo
intestinal colonization with themicro-organisms,
or actualinfection;
preferably
this would involve the use of conventionalvaccines.In animal
studies,
it is sometimespossible
to boostspecific
secretoryIgA
antibody
production
inthemilkby
conventionalparenteral
immunization. Thisapproach
has beenusedtoincrease levels of milk
antibody
to Cholera in Pakistani women.Lactating
women were immunizedparenterally
with killedCholera vaccine. After 14days,
therewas amarkedincrease in milkantibody
content, all of which wasIgA,
accompanied by
a strong serumantibody
response. Incontrast,
antibody production
of Swedishmothers couldnotbe boostedinthisway,suggesting
that therewas aneed for'priming'
of theimmunesystemby
anearlierexposuretotheorganism (Hanson
etal,
1979).
However,
there are somecomplications
in thisapproach
to immunization of thebreast,
because of thepowerful
control mechanismswhichgovernthe mucosal immunesystem and this isemphasized by
someunexpected
andcautionary
results ofanattempt
toimmunize motherstoproduce polio
antibodies inthe milk. Mothers who havepreviously
received conventionalpolio
immunization were
given
either intramuscular vaccine or oral live vaccine. The mothers who received intramuscular vaccineproduced
an increase in milkantibody
topolio.
However,
mothers immunized with oralpolio
vaccineconsistently
showedastriking
andprolonged
fall inthemilk
antibody
content(Hanson
etal,
1979),
presumably
because the live vaccinein thegut had activated aninhibitory
control mechanism. It seems that any effortstoproduce protective
antibody
in milk willrequire
carefulstudy
of thetypeofantigen
and theway it is administeredbeforeone canbe confident of
stimulating production
oftherequired
milkantibody.
Importance
of antibodiesinhumanmilkDespite
the attractiveness of theconcept
that human milk antibodiesareimportant
tothe humaninfant,
onemustask whatevidence supportsit.Secretory
IgA
from milk has unusualproperties
which,
teleologically,
seem welladapted
to make itan effectiveprotectorof thegut(Tornasi
&Grey, 1972).
Theseproperties
can be demonstrated in in-vitro systems and to some extent in animal models. There isnodoubt thatin manyexperiments
with non-humananimals,
colostrum and milk antibodiescaneffectively
protectinfantsagainst challenge
with virulentorganisms
such asgastroenteritis
viruses and bacteria which invade themucosa oractby
producing
entertoxins.In
humans, however,
the evidence for the clinicalefficacy
of milkantibodies,
although
verysuggestive,
is still not clear-cut. If breast-fed and bottle-fed infants in many differentenvironments are
compared
there are fewer infective andallergic problems
in the breast-fed infants. Forexample,
Chandra(1979) compared
breast- and bottle-fed infants inCanada,
attempting
to control for as many other factors aspossible,
and showedstrikingly
fewer infectionproblems
inthe breast-fedgroup.Thefindings
weresimilarinIndian infants.However,
these studies showonly
that breast milkisadvantageous;
they
donot provethat the benefits are due to theantibody
contentofthe milk. Milk contains a host of anti-infectiveagents and to assess the clinical
importance
of milkantibody,
its effect must be isolated fromthat ofall the other factors.
Ideally,
astudy
toestablish theimportance
of milkantibody
wouldcompareinfectionin the infants oftwo groupsof
breast-feeding
mothers who differonly
inthat one group has andonegroup doesnothave antibodiestotheoffending
organism.
Anintriguing
alternative
study
wouldbetomakeuseof the fact thatapproximately
0-2% ofthepopulation
areIgA-deficient.
Mothers withIgA
deficiency
lactatenormally
andproduce
milkwhich containsnoIgA
but has normalamounts of all the other anti-infective factors.Acomparison
oftheratesof infection in their infants withanappropriate
control group,could be aneffectivewayofisolating
thebiological
effects of milkantibody,
but becausethey
arerather difficult tofind this groupof mothers has received little attention.However,
in-vitro studies of the anti-bacterialproperties
ofIgA-deficient
breastmilk showthat it isveryeffectiveatinhibiting
thegrowth
ofmicro-organisms
and the addition ofspecific
secretoryIgA antibody
from normal breastmilk does not seemtoincrease the anti-bacterial effect of
IgA-deficient
breast milk(Samson,
Mirtle &McClelland,
1979).
In summary, the
lactating
human breast is alarge
scaleproducer
ofa form ofantibody
which isparticularly adapted
toprotecting
mucosal surfaces. The breast ispart ofageneralized
mucosal-associatedlymphoid
system whichpermits
immunization of the breastagainst
organisms experienced by
the motheras a result of colonization of the intestine and other sites. This secretoryantibody-producing
system
in the breast is conservedthoughout
a very widerange of
species
and therefore appears to be ofevolutionary importance.
Thepossibility
now exists formanipulating
theproduction
of antibodies in breast milkby
immunization.Many
other constituents of milk are alsoimportant
inprotecting
the neonateagainst
infection. Thesefactors,
likeantibodies,
maybedamaged by processing
milk: if milkmustbe fedtoinfantsby
an indirect route, it should be treated in ways which preserve thesefragile
butimportant
components(Björksten
etal,
1980).
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