Archives ofDisease in Childhood 1992; 67: 657-661

REGULAR REVIEW

Biological role of lactoferrin

Lourdes Sanchez, Miguel Calvo, Jeremy H Brock

Lactoferrin is an iron-binding protein closelyrelated in structure to the serum iron transportprotein transferrin. Unlike transferrin, onlytraces are normally present in serum, and it isinstead found mainly in milk and other externalsecretions, and in the secondary granules ofneutrophils. Although lactoferrin was first iso-lated 30 years ago, its biological role remainsunclear. Some aspects of its function werediscussed about 12 years ago in this journal,'and this review will attempt to reassess thefunction of lactoferrin in the light of the largeamount of new information that has accruedsince then.Knowledge of the structure of lactoferrin has

been advanced by recent x ray crystallographicstudies, and the structure and iron bindingproperties of lactoferrin are reviewed in detailelsewhere.2 Briefly lactoferrin, like transferrin,reversibly binds two ferric ions, for whichsynergistic binding of an anion, usually bicar-bonate or carbonate, is necessary. However, itsaffinity constant for iron is 300 times greaterthan that of transferrin, and even in thepresence of a competing iron chelator such ascitrate it can retain iron down to pH 3 or lesswhile transferrin loses it at pH 5. Unliketransferrin, lactoferrin is strongly basic. Humanlactoferrin has been cloned and sequenced4 andthe recombinant protein expressed in babyhamster kidney cells.5

bearing in mind when considering its possiblebiological function.Human milk contains 3-6-12-5 ,umol/l of

iron, and of this only 60-70% is in the wheyfraction, the remainder being in the lipidfraction (11-20%) or bound to casein (2-14%). "As a consequence, milk lactoferrin is only 6-8%saturated with iron, presumably because of thedifficulty in gaining access to iron in the lipidfraction or casein micelles.

Association of lactoferrin with othermoleculesLactoferrin is very prone to binding to othermacromolecules. These include other milkproteins such as IgA, casein, secretory compo-nent, albumin, lysozyme, and f1-lactoglobulin.'2Lactoferrin also binds to DNA.'3 A report ofmultiple forms of lactoferrin in human milk,some of which exhibit ribonuclease activity,'4may arise from an interaction between lactoferrinand milk ribonuclease. Bovine milk ribonucleaseis, like lysozyme, a small basic protein (mole-cular weight 13 600),'5 and some ribonucleaseactivity in human milk was also associated witha molecule of this size. 14 So far no clearbiological role has been established for thispropensity of lactoferrin to interact with otherproteins.

Department of FoodTechnology andBiochemistry,Veterinary Faculty,University of Zaragoza,SpainLourdes SanchezMiguel CalvoUniversity Departmentof Immunology,Western Infirmary,GlasgowJeremy H BrockCorrespondence and reprintrequests to:Dr Lourdes Sanchez,Department of Immunology,Western Infirmary,Glasgow Gll 6NT.

Lactoferrin in milkHuman milk is particularly rich in lactoferrin,the concentration ranging from about 7 g/l incolostrum to about 1 g/l in mature milk, thoughit may rise again towards the end of lactation.6 7

A similar pattern is seen in the cow, butimportantly the concentration in mid-lactationmilk is very much lower, only about 0-1 g/l.8Lactoferrin concentrations were normal in irondeficient mothers,9 but lower in mothers whowere generally malnourished,'0 which suggeststhat protein energy malnutrition rather thaniron influences lactoferrin synthesis in themammary gland. Although lactoferrin is foundin the milk ofsome other species, it is completelyabsent in others, such as the rat, rabbit, anddog.6 The milk of the rat and rabbit do howevercontain significant amounts of transferrin, butthe milk of the dog contains neither protein,even though the iron content is exceptionallyhigh.6 The fact that lactoferrin is absent fromthe milk of some species is a point worth

Biological functions of lactoferrinBACTERIOSTATIC ACTIVITYAlmost all bacteria require iron for growth, andbecause of their iron sequestering properties,the iron free (apo) forms of lactoferrin andtransferrin are able to impede iron utilisation bybacteria. A large number of studies, reviewedelsewhere,'6 have demonstrated a bacteriostaticeffect and in some cases a bactericidal effect oflactoferrin in vitro on a wide range of micro-organisms, including Gram positive and Gramnegative bacteria, aerobes, anaerobes, andyeasts. However, mechanisms other than simpleiron withholding may be involved in the anti-bacterial action of lactoferrin, such as blockadeof microbial carbohydrate metabolism'" ordestabilisation of the bacterial cell wall, perhapsthrough binding of calcium and magnesium.'8Lactoferrin may synergise with other anti-bacterial proteins such as lysozyme, which isalso present in milk. In this case even the ironsaturated form is active, and inhibition resultsfrom agglutination by lactoferrin of bacteria

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whose cell wall has been modified by lysozyme. 19Antibodies can also enhance the bacteriostaticaction of lactoferrin,20 probably by blockingproduction or uptake of microbial siderophores.

Lactoferrin may also possess antiviral activity,as it could protect mice against polycythaemiadue to Friend virus.2' This appears to be anindirect effect, perhaps via a reduction in targetcell proliferation, and is probably related to theproposed inhibitory role of lactoferrin inmyelopoiesis (see below).

In vivo, lactoferrin in milk might exercise itsinhibitory effect on microbial growth in themammary gland, in the intestine of the newborn,or both. Its role as a defence against infection inthe human mammary gland appears not to havebeen investigated, but in cattle concentrationsof lactoferrin increase during intramammaryinfection,22 suggesting a possible role as amammary non-specific defence mechanism.Conditions in the bovine mammary gland im-mediately before parturition and during involu-tion favour antimicrobial activity of lactoferrinas concentrations of bicarbonate are higher andthose of citrate lower than in milk.22 23 Thuslactoferrin might perform a role in preventinginfection of the mammary gland, particularly atparturition and involution.

It has frequently been suggested that theantimicrobial activity of lactoferrin plays a partin the selection of the intestinal flora of thenewborn and preventing colonisation by entero-pathogenic organisms. The conditions in theintestine of the newborn may be more favourablefor lactoferrin than those in the lactating mam-mary gland, as citrate is rapidly absorbed andintestinal fluid has a high concentration ofbicarbonate, although this function might beadversely affected by the proteolytic enzymespresent in the intestine, as discussed below.Nevertheless, despite the wealth of in vitrodata, attempts to establish an in vivo anti-microbial role for lactoferrin have generallyyielded disappointing results. The much quotedexperimental study of Bullen et al,20 whichsuggested a protective effect of lactoferrin innewborn guinea pigs infected with Escherichiacoli is open to other interpretations, as discussedpreviously in this journal.' However, additionof chicken ovotransferrin to cows' milk was ableto reduce mortality and bacterial counts inyoung guinea pigs infected with enteropatho-genic E coli.24 Two more recent clinical studieshave analysed the faecal flora of infants fedbovine lactoferrin (in one case in conjunctionwith bovine IgG) and failed to find any signifi-cant difference between infants fed lactoferrin-supplemented formula and controls fed un-supplemented formula.25 26 However, differ-ences between bovine and human lactoferrins(such as resistance to proteolysis, discussedbelow) may mean that bovine lactoferrin is notan appropriate substitute for the human protein,and recombinant DNA technology now offersthe possibility of obtaining sufficient humanlactoferrin for clinical trials.Thus although it is now well established that

breast feeding offers protection to the newborninfant against gastrointestinal infection, the partplayed by lactoferrin must still be considered

unproved until the wealth of in vitro datashowing antimicrobial activity is supported byconvincing in vivo data, particularly fromclinical trials. The possibility that lactoferrinmight exert a systemic protective effect alsodeserves further investigation, as studies inmice have shown that lactoferrin has a protectiveeffect in experimental E coli septicaemia that isindependent of its iron content.27

ROLE OF LACTOFERRIN IN INFLAMMATION ANDTHE IMMUNE SYSTEMA number of studies suggest that lactoferrinmay mediate some of the effects of inflammationand have a role in regulating various componentsof the immune system. It was proposed someyears ago that lactoferrin released by degranu-lating neutrophils mediated the hyposideraemiaof inflammation by removing iron from plasmatransferrin and short circuiting it to macrophagesof the reticuloendothelial system, where it wasincorporated into ferritin.28 However, ironuptake by macrophages from lactoferrin is atbest extremely slow,29 and there is no recyclingof the protein as occurs with transferrin, aslactoferrin that has bound to monocytes cannotsubsequently rebind to these cells.30 In addition,the rate of exchange of iron between transferrinand lactoferrin at physiological pH is likely tobe extremely slow, and finally neutrophil-derivedlactoferrin differs from milk lactoferrin, whichwas used in most studies, in lacking theterminal fucose residues in its glycan chains thatare required for binding to macrophages.3' Ithas also been found in mice that interleukin- 1induces hypoferraemia even in the presence ofneutropenia,32 suggesting that lactoferrin isunimportant. Thus it now seems unlikely thatlactoferrin plays a significant part in the hypo-ferraemia of inflammation.

Lactoferrin might also contribute to thebactericidal activity of neutrophils by twoopposing mechanisms. In the apo form it mayperform an iron withholding function andprevent growth of phagocytosed bacteria.33 Onthe other hand iron-lactoferrin may provide ironthat can catalyse the production of free radicalswhich lead to microbial killing within thephagolysosome of phagocytic cells.34 The abilityof lactoferrin to promote formation of freeradicals is probably confined to acidic conditionssuch as those within the phagolysosome, as atphysiological pH it is more likely that lactoferrininhibits radical production by scavengingcatalytic 'free' iron.35 A role for lactoferrin inthe antimicrobial activity of neutrophils issupported by the finding that patients whoseneutrophils lack specific granules suffer fromrecurrent infections.36

Lactoferrin may also act as an inhibitorof secretion of granulocyte-monocyte-colonystimulating factor (GM-CSF).37 This complexarea has been intensively investigated and isreviewed fully elsewhere.38 In summary, lacto-ferrin appears to act by decreasing synthesis ofinterleukin-l which is necessary for GM-CSFproduction. Iron-lactoferrin was more effectivethan apo lactoferrin but it is not known what ifany part is played by iron. Lactoferrin isolated

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from neutrophils lacking receptors for the Fcregion of immunoglobulin or from neutrophilsof leukaemia patients was inactive, though thereason for this is unclear as possible differencesbetween the active and inactive forms of lacto-ferrin were not investigated.The role of lactoferrin as an inhibitor of

myelopoiesis is somewhat controversial, how-ever, as some reports have failed to find anyinhibitory effect.39 40 Some aspects of theoriginal studies such as the extremely lowconcentrations oflactoferrin required for activity(10-'7 M) and a discrepancy between theisoelectric point of the active component (6-5)37and purified lactoferrin (8 7)41 require explan-ation. Further work is needed, particularly witha view to establishing how lactoferrin itselfinitiates these events at the molecular level,before its role in myelopoiesis can be regardedas fully established.

Lactoferrin has been reported to affect variousaspects of the immune system, such as inhibitingin vitro antibody synthesis,42 regulating mono-cyte/macrophage cytotoxic activity,43 44 andaffecting lymphocyte proliferation.458 In mostof these studies the mechanism by whichlactoferrin carries out these functions was notestablished. The effect on lymphocyte prolifer-ation is unclear, as some authors have reportedenhancement45 and others inhibition.46 47 Pos-sibly this may depend upon culture conditions,as recent work has suggested that apolactoferrincan overcome the inhibitory effect of 'free' ironon lymphocyte proliferation, but itself inhibitsproliferation when iron is bound to transferrin.48Activation of T lymphocytes induces thepresence of lactoferrin-binding molecules in thecell membrane,45 49 and binding to B cells hasalso been reported.50 However, further work isrequired before an immunoregulatory role forlactoferrin can be clearly established.

ROLE OF LACTOFERRIN IN THE ABSORPTION OFIRONThe greater bioavailability of iron and higherconcentration of lactoferrin in human milkcompared with cows' milk suggests that lacto-ferrin might promote iron absorption in breastfed infants. In a previous review in this journalit was suggested that lactoferrin actually had anopposite effect, and served as an additionalcontrol of iron absorption during the neonatalperiod.' More recent clinical trials have failed todemonstrate any improvement in iron absorp-tion in infants fed formula milk supplementedwith (bovine) lactoferrin.5' 52 A study in ratsdid show that feeding lactoferrin improved ironstatus53 but the rat is a poor model for suchstudies as the milk of this species does notcontain lactoferrin. Studies in piglets,54 wean-ling mice,55 or infant rhesus monkeys56 showedthat the bioavailability of iron bound to lacto-ferrin was no better than that of inorganic iron.

If lactoferrin were to have a role in ironabsorption one might expect a receptor to existin mucosal cells. Lactoferrin-binding proteinshave been identified in the microvillus mem-branes of rabbit,57 mouse,58 and rhesus monkeyenterocytes.59 The molecular weights and bind-

ing characteristics of these proteins differbetween species. However, there is so far noevidence that these membrane proteins actuallymediate uptake or transport of lactoferrin-boundiron in mucosal cells. Given that there are alsostudies suggesting that lactoferrin may inhibitiron absorption, reviewed previously,' ourearlier conclusion that lactoferrin controlsrather than promotes iron absorption in theneonate still seems to be valid.

PROTEOLYTIC DEGRADATION OF LACTOFERRINAND SURVIVAL IN THE GASTROINTESTINAL TRACTA role for lactoferrin in iron absorption orestablishment of the microbial flora of the gutpresupposes a certain resistance to the conditionswithin the gastrointestinal tract. In vitro studieshave found that apolactoferrin is more sensitiveto the action of trypsin than iron-lactoferrin,although human apolactoferrin was less sensi-tive than bovine.60 Lactoferrin is also degradedby gastric fluid from premature babies, thoughunlike transferrin or casein more degradationoccurred at pH 3-2 than at pH 18.6' Howeverthe gastric pH will be raised by the bufferingcapacity of milk proteins, and the intestinalflora and the velocity of transit also affectdigestion of lactoferrin.9 Studies of lactoferrinisolated from the faeces of newborn infants fedcows' milk supplemented with human lacto-ferrin or bovine lactoferrin showed that iron-lactoferrins survived better than the apo formsand, surprisingly, bovine lactoferrin was moreresistant than human.62 Partly digested proteinsmaintained their capacity to bind iron afterexcretion, and fragments of human lactoferrinobtained by acid hydrolysis were still able tobind to the putative rhesus monkey mucosalreceptor, albeit with lower affinity than intactlactoferrin.63

Intact lactoferrin and fragments have beendetected in the urine of premature infantsreceiving human milk,' arising apparentlyfrom lactoferrin absorbed across the gut. '3 Thissuggests that lactoferrin is partially degraded inthe gut and that significant absorption of thisprotein occurs in the premature infant. How-ever, some fragments may be present beforeingestion, as in contrast to a previous report65we have recently found lactoferrin fragments inhuman and bovine milk.' It seems less likelythat lactoferrin absorption occurs in terminfants, as no difference in plasma lactoferrinconcentrations was found between breast andbottle fed infants.67

ACTIVITY OF LACTOFERRIN AS A GROWTH FACTORRecent studies have suggested that lactoferrinmay promote cell growth. As well as possiblypromoting lymphocyte proliferation (see above),human lactoferrin also stimulated growth ofvarious cell lines.29 68 It also enhanced prolifer-ation of rat mucosal crypt cells,69 which sug-gests that it might play a part in maturation ofthe intestine in the newborn. The possibilitythat lactoferrin acts as a growth factor issupported by the presence ofhigh concentrationsof lactoferrin in some tumours,70 although the

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reasons for these high concentrations areunclear. In some cases growth promotion re-quired iron68 whereas in others iron wasunimportant.29 69 The mechanism by whichlactoferrin is able to stimulate cell growth istherefore unclear, but seems unlikely to involveiron transport in an analogous manner totransferrin, as lactoferrin is not internalised andlittle or no iron is taken up from lactoferrin.29 Itmight involve activation of membrane oxido-reductase activity.7'

Conclusion: the biological function oflactoferrin-a unifying hypothesisOver the past 10 years there has been consider-able research on the function of lactoferrin,much of it in areas unrelated to the traditionalfields of iron absorption and antimicrobialactivity. Despite this, no clear biological role forlactoferrin has been established. Its anti-microbial activity, so well documented in vitro,still awaits convincing in vivo evidence. Newstudies of iron absorption have generally failedto show that iron in lactoferrin has a higherbioavailability than iron salts, and there is stillno direct evidence that lactoferrin actuallymediates uptake across the mucosa. Althoughlactoferrin-binding proteins have been identifiedin mucosal membranes, these could just aseasily serve to regulate iron transport as enhanceit. A role in hypoferraemia now seems unlikely,and effects on myelopoiesis and the immunesystem are either controversial or essentiallyphenomenological, with little information onhow lactoferrin actually mediates these effects.Although lactoferrin receptors or bindingproteins have been reported from a wide varietyof cells and tissues the molecular structure andbinding characteristics of these putative recep-tors varies considerably, and in many casesbinding is relatively non-specific. It may well bethat the various apparently unrelated effects oflactoferrin on the immune system arise from itsability to bind to macrophages and lymphocytes.This could alter the surface charge of the cellmembrane, with consequent effects on thevarious cell-cell interactions that have a crucialrole in many immunological mechanisms.We believe that current evidence suggests

that lactoferrin, despite its structural similarityto transferrin, does not act as an iron transportprotein. Instead, we propose that its role is thatofa specialised iron-scavenging protein, designedto act particularly under conditions wheretransferrin would be less effective at bindingiron due to reduced pH, such as exist in thegastrointestinal tract or inflammatory lesions.By binding iron under these conditions it wouldrender harmless 'free' iron that might otherwisecause free radical-mediated damage to sensitivetissues, reduce absorption of unwanted iron inthe immediate postnatal period, and decrease itsavailability to micro-organisms. The ability oflactoferrin to bind to a wide variety of cells andtissues, perhaps without the need for expressionof a truly specific receptor, would enhance itsability to prevent free radical-mediated damageor microbial invasion. It would also help toensure its eventual removal and catabolism via

the reticuloendothelial system or by biliaryexcretion. Further studies, particularly of lacto-ferrin-cell interactions, may help to establishthe validity of this proposal.

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3 Anderson BF, Baker HM, Norris GE, Rice DW, Baker EN.Structure of human lactoferrin: crystallographic structureanalysis and refinement at 2-8A resolution. J Mol Biol1989;209:71 1-34.

4 Powell MJ, Ogden JE. Nucleotide sequence of humanlactoferrin cDNA. Nucleic Acids Res 1990;18:4013.

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6 Masson PL, Heremans JF. Lactoferrin in milk from differentspecies. Comp Biochem Physiol 1971;39B: 119-29.

7 Hennart PF, Brasseur DJ, Delogne-Desnoeck JB, DramaixMM, Robyn CE. Lysozyme, lactoferrin, and secretoryimmunoglobulin A content in breast milk: influence ofduration of lactation, nutrition status, prolactin status, andparity of mother. Am J Clin Nutr 1991;53:32-9.

8 Sanchez L, Aranda P, Perez MD, Calvo M. Concentration oflactoferrin and transferrin throughout lactation in cow'scolostrum and milk. Biol Chem Hoppe Seyler 1988;369:1005-8.

9 Prentice A, McCarthy A, Stirling DM, Vasquez-VelasquezL, Ceesay SM. Breast milk IgA and lactoferrin survival inthe gastrointestinal tract-a study in ruralGambian children.Acta Paediatr Scand 1989;78:505-12.

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13 Hutchens TW, Henry JF, Yip T-T, et al. Origin of intactlactoferrin and its DNA-binding fragments found in theurine of human milk-fed premature infants. Evaluation bystable isotopic enrichment. Pediatr Res 1991;29:243-50.

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24 Antonini E, Orsi N, Valenti P. Effetto delle transferrine sullapatogenicita delle enterobacteriaceae. Giormale di MalattieInfettive e Parassitarie (Milano) 1977;29:481-9.

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