- Email: [email protected]
Lactoferrin: a multifunctional immunoregulatory protein?
of its deliberate policy of providing fellowship support, the Wellcome Trust was the most prominent source of personal support. Concluding remarks There has been substantial investment in immunology research in the UK in recent years, and this is reflected in outcome measures that show the UK second only to the USA in terms of output in the top immunology journals. A relatively small number of researchers were responsible for most of this output in the UK, and the impact of this productive group was high, as shown by citation analysis. The
MRC and the Wellcome Trust were the largest financial supporters of immunology research, and the MRC received most acknowledgements for intramural support, whereas the Wellcome Trust was a major source of persona1 support. As scientific research becomes progressively more expensive, there is an increasing need for funding organizations to assess the return on their investment more rigorously. This is the essence of responsible funding. However, although outcome measures and systematic analysis of trends in funding are important additions to policy debate, it must be stressed that such
techniques are best used in conjunction with more traditional modes of assessment, such as peer review. The authors dre especially grateful to Grant Lewison of PRISM for his advice on the hibliometric analyses. This work wan supported by the Wellcome
Trust.
Mairdad O’Drisd andJoe Anderson are at the Unit for Policy Research in Science and Medicine (PRISM), 210 Euston Road, Londorr, UK NW1 ZBE; Patricia Chisholm is at the Science Funding Section of the We&-ome Trust, 18.3 Euston Road, London, UK NW1 I7BE .
Lactoferrin:a multifunctional immunoregulatoryprotein? Jeremy Brock
Lactoferrin is an iron-binding protein that is closely related in structure to the plasma iron-transport protein transferrin. It is found mainly in external secretions, such as breast milk, and in the secondary granules of neutrophils. Although it has been proposed to act as an anti-infective agent, a modulator of the inflammatory response and iron absorption, and an immunoregulatory protein, there is still no consensus view on the biological role of lactoferrin. Lactoferrin structure The structure of lactoferrin has been well worked out by crystallographic studies (E.N. Baker, Massey, New Zealand) and the development of various mutants (J.W. Tweedie, Massey). It comprises a single polypeptide chain folded into two lobes. Each lobe contains a binding site for Fe3+ that is located in a deep cleft, and a site for the synergistic binding of a bicarbonate anion’. In the absence of iron, each lobe of the molecule can flex, allowing the cleft to open and shut,
Various immunoregulatory and anti-infective roles have heen proposed for lactoferrin, the ironbinding protein present in external secretions and neutrophil secondary granules. A recent meeting* updated current knowledge of the structure and function of this tinusual protein. but when iron is bound, the cleft is ‘locked’ shut (Baker). Although the overall structure of lactoferrin is very similar to that of transferrin, it is distinguished by two features that may be important functionally. First, the affinity of lactoferrin for iron is 2SO-fold greater than that of transferrin. Second, lactoferrin contains a strongly basic region close to the N-terminus, and hence this protein has a pI of approximately 9, compared with 5..5-6 for transferrin. According to Baker, this basic region is very flexible, and is probably responsible for the ability of lactoferrin to bind to a large number of acidic molecules. “The 2nd International Symposium on Lactoferrin Structure and Function was held at Honolulu, HI, USA. on 19-22 February 199.5.
Antimicrobial properties of lactoferrin One of the earliest functions ascribed to lactoferrin was the inhibition of bacterial growth. It is believed that lactoferrin helps to protect breast-fed infants against infection by iron-requiring enteric pathogens, and contributes to the antimicrobial armoury of neutrophils2. Thus, lactoferrin is often thought of as a component of innate immunity. However, it is now clear that many microorganisms can overcome the iron-withholding effect of lactoferrin, either by secreting high-affinity low-molecular-weight iron chelators (siderophores) that can compete with lactoferrin for iron, or by expressing lactoferrin receptors, which are highly species specific (A.B. Schryvers, Calgary). Recently, a second type of antimicrobial activity has been described. It is independent of iron binding and is mediated through peptides (‘lactoferricins’) that contain the basic N-terminal region of lactoferrin and are obtained by proteolytic cleavage of the protein
Prevents growth
Microorganisms Neutrophil
Fe-lactoferrin
Fe
NK-cell cytotoxicity Complement activation Gene regulation Other effects? Necrotic tissue Fig. 1. The proposed immunoregulator)l activity of lactoferrin. See text for detaris. Abbreuiatron: NK, natural killer.
(M. Tomita, Zama City, Japan). This activity is also observed to a lesser extent with intact lactoferrin3, and has been shown to be mediated by binding of lactoferrins/lactoferricins to lipopolysaccharide (LPS), followed by permeabilization of the cell wall and cytoplasmic membrane (R.T. Ellison, Worcester, MA). Despite these advances in understanding the mechanisms involved in the antimicrobial activity of lactoferrin, evidence of this role in km remains scant. Therefore, it was of particular interest that S. Teraguchi (Zama City) reported that feeding lactoferricins to mice prevented translocation of coliforms from the gut to the mesenteric lymph nodes. It is not yet known if lactoferricins are liberated from lactoferrin by gastrointestinal proteolytic activity, or whether intact lactoferrin is similarly effective. Structure and function of lactoferrin receptors The nature and function of lactoferrin receptors require further clarification. A single-chain receptor of 10.5 kDa that has been previously identified on activated T cells4 appears to be present on platelets and breast carcinoma cells (G. Spik and
J. Mazurier, Lille). By contrast, the receptor present on intestinal brush border membranes comprises three 37 kDa subunits (B. Lonnerdal, Davis, CA). Another type of receptor that has not yet been fully characterized exists on hepatocytes (D.D. McAbee, Notre Dame, IN). The ability of monocytes and macrophages to bind lactoferrin has been known for many years, but the nature of the interaction involved was unclear. B.E. Britigan (Iowa City) has identified a 43 kDa protein present in the membrane that may be a lactoferrin receptor. Binding was unaffected by treating monocytes with various cytokines, including tumour necrosis factor (Y (TNF-a), interleukin la (IL-la) and IL-lB, interferon y (IFN-y) and granulocyte-macrophage colonystimulating factor (GM-CSF). This confusion over the nature of lactoferrin receptors is mirrored by a lack of understanding of their function. Only hepatocytes have been unequivocally shown to internalize lactoferrin by receptormediated endocytosiss, although Mazurier presented evidence for internalization of lactoferrin by the T-lymphoblastoid line Jurkat. There was generally little support for the
Immunology Today
4 18
idea that lactoferrin carries out an iron-transport role comparable to transferrin. Furthermore, a previous idea that lactoferrin contributes to the hypoferraemia of inflammation by short-circuiting transferrin-bound iron to macrophages now seems untenable given their slow-to-nonexistent rate of uptake of iron from lactoferrin. A problem with all studies on lactoferrin receptors is the highcapacity, low-affinity interaction of the basic N-terminal region of lactoferrin with acidic cell-surface’ molecules such as glycosaminoglycan&. This makes it difficult to establish whether bona fide receptor(s) are involved in lactoferrin-cell interactions. The cloning and sequencing of putative lactoferrin receptors is required, and their relationship, if any, to known CD antigens, as well as the metabolic consequences of interaction with lactoferrin, should be examined. Immunoregulatory functions Over the past decade, lactoferrin has been reported to affect various immunological functions, including antibody synthesis in vitro, production of IL-l, IL-2 and TNF-(Y, natural killer (NK)-cell cytotoxicity,
Vol. I6 No. V 1 VV 5
complement activation and lymphocyte proliferation (reviewed in Ref. 2). In most cases, the significance of these observations and the mechanisms involved are unknown. However, some clarification is emerging. For example, enhancement of NK-cell activity may be due to structural homology between lactoferrin and a putative NK-cell target molecule’. Furthermore, binding of LPS by lactoferrin, and a consequent reduction of LPSmediated TNF-a production, may account for the previously reported ability of lactoferrin to protect mice against experimental Eschericbia coli septicaemia*. J.H. Brock (Glasgow) reported that lactoferrin could affect T-cell proliferation by regulating iron uptake, the effect being inhibitory at low iron levels and stimulatory when excess iron was present. It was suggested that lactoferrin might protect lymphocyte function at sites of inflammation by sequestering potentially toxic iron arising from tissue destruction, and Britigan proposed that lactoferrin binding to monocytes and macrophages fulfils a similar protective function. Regulation of the lactoferrin gene Some progress has been made in understanding how expression of the lactoferrin gene is regulated. Lactoferrin can be regulated transcriptionally by oestrogen through three different regions in the promoter (C. Teng, Research Triangle Park, NC), and there may also be post-transcriptional regulation through AU-rich instability sequences in the 3’untranslated region of the mRNA (EL. Schanbacher, Wooster, OH), as occurs with some cytokines. O.M. Conneely (Houston) has used homologous recombination to produce mice heterozygous for the disrupted lactoferrin gene, and homozygotes will hopefully appear soon. This group, in collaboration with Agennix (Houston), has also developed a high-yield Aspergillus system for production of recombinant human (and eventually mouse) lactoferrin.
it has been shown that lactoferrin binds to specific DNA consensus sequences and can upregulate expression of reporter genes when these sequences are placed in an upstream position (P. Furmanski, New York). This implies that ex-
ogenous lactoferrin can be internalized and translocated to the nucleus. At present, the mechanism by which this might occur is unknown. Nevertheless, further support for this provocative report was provided by C. Garre (Genoa) who showed that lactoferrin could downmodulate the GM-CSF promoter in fibroblasts, particularly when they were stimulated by IL-ll3. This may be relevant to earlier reports of inhibition of granulopoiesis by lactoferrin9. Conclusions What can be concluded about the role of lactoferrin in inflammation, infection and immunity? Are its specific biochemical properties, such as its basicity and ability to bind iron, important? No clear answers have yet emerged, but the future availability of lactoferrin-knockout mice and large quantities of recombinant human lactoferrin may help to resolve these questions. Nevertheless, it is now possible to envisage a sequence of events by which lactoferrin derived from degranulating neutrophils could affect inflammatory and immunological processes (Fig. 1). Because of its ability to bind to acidic cell-surface molecules, lactoferrin could interact readily with cells such as lymphocytes and macrophages, and protect them from destructive free-radical reactions mediated by iron released following tissue damage. When lactoferrin binds iron, it becomes more resist-
ant to proteolytic degradation”‘, and iron is rendered less available to microorganisms. The more stable Fe-lactoferrin complex could then transcriptionally regulate genes that are involved in alternative defences against infection, or could mediate anti-infective roles by other mechanisms. This scheme would indeed suggest that lactoferrin is a multifunctional immunoregulatory protein. Jeremy Brock is at the Dept of Immunology, Universtty of Glasgow, Western Infirmary, Glasgow, UK G11 6NT. References 1 Anderson, B.F., Baker, H.M., Norris, G.E., Rice, D.W. and Baker, E.N. (1989) 1. Mol. Biol. 209, 711-734 2 Sanchez, L., Calve, M. and Brock, J.H. (1992) Arch. Dis. Child. 67,657-661 3 Bellamy, W., Takase, M., Yamauchi, K., Wakabayashi, H., Kawase, K. and Tomita, M. (1992) Biochim. Biophys. Acta 1121, 130-136 4 Mazurier, J., Legrand, D., Hu, W.L., Montreuil, J. and Spik, G. (1989) Eur. J. Biochem. 179,481-487 5 McAbee, D.D. and Esbensen, K. (1991) /. Biol. Chem. 266, 23624-23631 6 Mann, D.M., Romm, E. and Migliorini, M. (1994) J. Biol. Chem. 269,23661-23667 7 Hauer, J., Voetsch, W. and Anderer, EA. (1994) Immunol. Lett. 42,7-12 8 Zagulski, T., Lipinski, P., Zagulska. A.. Broniek. S. and Jariabek,‘Z. (1989) Bi /. Exp. Pathol. 70,697-704 9 Zucah, J.R., Broxmeyer, H.E., Levy, D. and Morse, C. (1989) Blood 74,1531-1536 10 Brines, R.D. and Brock, J.H. (1983)
Biochim. Biophys. Acta 759, 229-235
Regulation of other genes by lactoferrin Lactoferrin may itself act as a novel transcriptional regulator, since
Immunology
Today
4 19
Vol. 1h No. 9 199.5
MRC and the Wellcome Trust were the largest financial supporters of immunology research, and the MRC received most acknowledgements for intramural support, whereas the Wellcome Trust was a major source of persona1 support. As scientific research becomes progressively more expensive, there is an increasing need for funding organizations to assess the return on their investment more rigorously. This is the essence of responsible funding. However, although outcome measures and systematic analysis of trends in funding are important additions to policy debate, it must be stressed that such
techniques are best used in conjunction with more traditional modes of assessment, such as peer review. The authors dre especially grateful to Grant Lewison of PRISM for his advice on the hibliometric analyses. This work wan supported by the Wellcome
Trust.
Mairdad O’Drisd andJoe Anderson are at the Unit for Policy Research in Science and Medicine (PRISM), 210 Euston Road, Londorr, UK NW1 ZBE; Patricia Chisholm is at the Science Funding Section of the We&-ome Trust, 18.3 Euston Road, London, UK NW1 I7BE .
Lactoferrin:a multifunctional immunoregulatoryprotein? Jeremy Brock
Lactoferrin is an iron-binding protein that is closely related in structure to the plasma iron-transport protein transferrin. It is found mainly in external secretions, such as breast milk, and in the secondary granules of neutrophils. Although it has been proposed to act as an anti-infective agent, a modulator of the inflammatory response and iron absorption, and an immunoregulatory protein, there is still no consensus view on the biological role of lactoferrin. Lactoferrin structure The structure of lactoferrin has been well worked out by crystallographic studies (E.N. Baker, Massey, New Zealand) and the development of various mutants (J.W. Tweedie, Massey). It comprises a single polypeptide chain folded into two lobes. Each lobe contains a binding site for Fe3+ that is located in a deep cleft, and a site for the synergistic binding of a bicarbonate anion’. In the absence of iron, each lobe of the molecule can flex, allowing the cleft to open and shut,
Various immunoregulatory and anti-infective roles have heen proposed for lactoferrin, the ironbinding protein present in external secretions and neutrophil secondary granules. A recent meeting* updated current knowledge of the structure and function of this tinusual protein. but when iron is bound, the cleft is ‘locked’ shut (Baker). Although the overall structure of lactoferrin is very similar to that of transferrin, it is distinguished by two features that may be important functionally. First, the affinity of lactoferrin for iron is 2SO-fold greater than that of transferrin. Second, lactoferrin contains a strongly basic region close to the N-terminus, and hence this protein has a pI of approximately 9, compared with 5..5-6 for transferrin. According to Baker, this basic region is very flexible, and is probably responsible for the ability of lactoferrin to bind to a large number of acidic molecules. “The 2nd International Symposium on Lactoferrin Structure and Function was held at Honolulu, HI, USA. on 19-22 February 199.5.
Antimicrobial properties of lactoferrin One of the earliest functions ascribed to lactoferrin was the inhibition of bacterial growth. It is believed that lactoferrin helps to protect breast-fed infants against infection by iron-requiring enteric pathogens, and contributes to the antimicrobial armoury of neutrophils2. Thus, lactoferrin is often thought of as a component of innate immunity. However, it is now clear that many microorganisms can overcome the iron-withholding effect of lactoferrin, either by secreting high-affinity low-molecular-weight iron chelators (siderophores) that can compete with lactoferrin for iron, or by expressing lactoferrin receptors, which are highly species specific (A.B. Schryvers, Calgary). Recently, a second type of antimicrobial activity has been described. It is independent of iron binding and is mediated through peptides (‘lactoferricins’) that contain the basic N-terminal region of lactoferrin and are obtained by proteolytic cleavage of the protein
Prevents growth
Microorganisms Neutrophil
Fe-lactoferrin
Fe
NK-cell cytotoxicity Complement activation Gene regulation Other effects? Necrotic tissue Fig. 1. The proposed immunoregulator)l activity of lactoferrin. See text for detaris. Abbreuiatron: NK, natural killer.
(M. Tomita, Zama City, Japan). This activity is also observed to a lesser extent with intact lactoferrin3, and has been shown to be mediated by binding of lactoferrins/lactoferricins to lipopolysaccharide (LPS), followed by permeabilization of the cell wall and cytoplasmic membrane (R.T. Ellison, Worcester, MA). Despite these advances in understanding the mechanisms involved in the antimicrobial activity of lactoferrin, evidence of this role in km remains scant. Therefore, it was of particular interest that S. Teraguchi (Zama City) reported that feeding lactoferricins to mice prevented translocation of coliforms from the gut to the mesenteric lymph nodes. It is not yet known if lactoferricins are liberated from lactoferrin by gastrointestinal proteolytic activity, or whether intact lactoferrin is similarly effective. Structure and function of lactoferrin receptors The nature and function of lactoferrin receptors require further clarification. A single-chain receptor of 10.5 kDa that has been previously identified on activated T cells4 appears to be present on platelets and breast carcinoma cells (G. Spik and
J. Mazurier, Lille). By contrast, the receptor present on intestinal brush border membranes comprises three 37 kDa subunits (B. Lonnerdal, Davis, CA). Another type of receptor that has not yet been fully characterized exists on hepatocytes (D.D. McAbee, Notre Dame, IN). The ability of monocytes and macrophages to bind lactoferrin has been known for many years, but the nature of the interaction involved was unclear. B.E. Britigan (Iowa City) has identified a 43 kDa protein present in the membrane that may be a lactoferrin receptor. Binding was unaffected by treating monocytes with various cytokines, including tumour necrosis factor (Y (TNF-a), interleukin la (IL-la) and IL-lB, interferon y (IFN-y) and granulocyte-macrophage colonystimulating factor (GM-CSF). This confusion over the nature of lactoferrin receptors is mirrored by a lack of understanding of their function. Only hepatocytes have been unequivocally shown to internalize lactoferrin by receptormediated endocytosiss, although Mazurier presented evidence for internalization of lactoferrin by the T-lymphoblastoid line Jurkat. There was generally little support for the
Immunology Today
4 18
idea that lactoferrin carries out an iron-transport role comparable to transferrin. Furthermore, a previous idea that lactoferrin contributes to the hypoferraemia of inflammation by short-circuiting transferrin-bound iron to macrophages now seems untenable given their slow-to-nonexistent rate of uptake of iron from lactoferrin. A problem with all studies on lactoferrin receptors is the highcapacity, low-affinity interaction of the basic N-terminal region of lactoferrin with acidic cell-surface’ molecules such as glycosaminoglycan&. This makes it difficult to establish whether bona fide receptor(s) are involved in lactoferrin-cell interactions. The cloning and sequencing of putative lactoferrin receptors is required, and their relationship, if any, to known CD antigens, as well as the metabolic consequences of interaction with lactoferrin, should be examined. Immunoregulatory functions Over the past decade, lactoferrin has been reported to affect various immunological functions, including antibody synthesis in vitro, production of IL-l, IL-2 and TNF-(Y, natural killer (NK)-cell cytotoxicity,
Vol. I6 No. V 1 VV 5
complement activation and lymphocyte proliferation (reviewed in Ref. 2). In most cases, the significance of these observations and the mechanisms involved are unknown. However, some clarification is emerging. For example, enhancement of NK-cell activity may be due to structural homology between lactoferrin and a putative NK-cell target molecule’. Furthermore, binding of LPS by lactoferrin, and a consequent reduction of LPSmediated TNF-a production, may account for the previously reported ability of lactoferrin to protect mice against experimental Eschericbia coli septicaemia*. J.H. Brock (Glasgow) reported that lactoferrin could affect T-cell proliferation by regulating iron uptake, the effect being inhibitory at low iron levels and stimulatory when excess iron was present. It was suggested that lactoferrin might protect lymphocyte function at sites of inflammation by sequestering potentially toxic iron arising from tissue destruction, and Britigan proposed that lactoferrin binding to monocytes and macrophages fulfils a similar protective function. Regulation of the lactoferrin gene Some progress has been made in understanding how expression of the lactoferrin gene is regulated. Lactoferrin can be regulated transcriptionally by oestrogen through three different regions in the promoter (C. Teng, Research Triangle Park, NC), and there may also be post-transcriptional regulation through AU-rich instability sequences in the 3’untranslated region of the mRNA (EL. Schanbacher, Wooster, OH), as occurs with some cytokines. O.M. Conneely (Houston) has used homologous recombination to produce mice heterozygous for the disrupted lactoferrin gene, and homozygotes will hopefully appear soon. This group, in collaboration with Agennix (Houston), has also developed a high-yield Aspergillus system for production of recombinant human (and eventually mouse) lactoferrin.
it has been shown that lactoferrin binds to specific DNA consensus sequences and can upregulate expression of reporter genes when these sequences are placed in an upstream position (P. Furmanski, New York). This implies that ex-
ogenous lactoferrin can be internalized and translocated to the nucleus. At present, the mechanism by which this might occur is unknown. Nevertheless, further support for this provocative report was provided by C. Garre (Genoa) who showed that lactoferrin could downmodulate the GM-CSF promoter in fibroblasts, particularly when they were stimulated by IL-ll3. This may be relevant to earlier reports of inhibition of granulopoiesis by lactoferrin9. Conclusions What can be concluded about the role of lactoferrin in inflammation, infection and immunity? Are its specific biochemical properties, such as its basicity and ability to bind iron, important? No clear answers have yet emerged, but the future availability of lactoferrin-knockout mice and large quantities of recombinant human lactoferrin may help to resolve these questions. Nevertheless, it is now possible to envisage a sequence of events by which lactoferrin derived from degranulating neutrophils could affect inflammatory and immunological processes (Fig. 1). Because of its ability to bind to acidic cell-surface molecules, lactoferrin could interact readily with cells such as lymphocytes and macrophages, and protect them from destructive free-radical reactions mediated by iron released following tissue damage. When lactoferrin binds iron, it becomes more resist-
ant to proteolytic degradation”‘, and iron is rendered less available to microorganisms. The more stable Fe-lactoferrin complex could then transcriptionally regulate genes that are involved in alternative defences against infection, or could mediate anti-infective roles by other mechanisms. This scheme would indeed suggest that lactoferrin is a multifunctional immunoregulatory protein. Jeremy Brock is at the Dept of Immunology, Universtty of Glasgow, Western Infirmary, Glasgow, UK G11 6NT. References 1 Anderson, B.F., Baker, H.M., Norris, G.E., Rice, D.W. and Baker, E.N. (1989) 1. Mol. Biol. 209, 711-734 2 Sanchez, L., Calve, M. and Brock, J.H. (1992) Arch. Dis. Child. 67,657-661 3 Bellamy, W., Takase, M., Yamauchi, K., Wakabayashi, H., Kawase, K. and Tomita, M. (1992) Biochim. Biophys. Acta 1121, 130-136 4 Mazurier, J., Legrand, D., Hu, W.L., Montreuil, J. and Spik, G. (1989) Eur. J. Biochem. 179,481-487 5 McAbee, D.D. and Esbensen, K. (1991) /. Biol. Chem. 266, 23624-23631 6 Mann, D.M., Romm, E. and Migliorini, M. (1994) J. Biol. Chem. 269,23661-23667 7 Hauer, J., Voetsch, W. and Anderer, EA. (1994) Immunol. Lett. 42,7-12 8 Zagulski, T., Lipinski, P., Zagulska. A.. Broniek. S. and Jariabek,‘Z. (1989) Bi /. Exp. Pathol. 70,697-704 9 Zucah, J.R., Broxmeyer, H.E., Levy, D. and Morse, C. (1989) Blood 74,1531-1536 10 Brines, R.D. and Brock, J.H. (1983)
Biochim. Biophys. Acta 759, 229-235
Regulation of other genes by lactoferrin Lactoferrin may itself act as a novel transcriptional regulator, since
Immunology
Today
4 19
Vol. 1h No. 9 199.5