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Human Lactoferrin: a Novel
Therapeutic with Broad Spectrum Potential
EUGENE
D. WEINBERG Department
of
Biology and Program in Medical Sciences, Indiana University, Bloomington,
Indiana USA
Lactoferrin (Lf), a natural defense iron-binding protein, has been
observed to possess antibacterial, antimycotic, antiviral, antineoplastic and
anti-inflammatory activities. The protein is present in exocrine secretions that
are commonly exposed to normal flora: milk, tears, nasal exudate, saliva,
bronchial mucus, gastrointestinal fluids, cervico-vaginal mucus and seminal
fluid. Additionally, Lf is a major constituent of the secondary specific
granules of circulating polymorphonuclear neutrophils (PMNs). The apoprotein is
released on degranulation of the PMNs in septic areas. A principal function of
Lf is that of scavenging 'free' iron in fluids and inflamed areas so as to
suppress free radical-mediated damage and decrease availability of the metal to
invading microbial and neoplastic cells. Mechanisms of action of Lf in addition
to iron deprivation also are described.
Administration of exogenous human or bovine Lf to hosts with various infected or inflamed sites has resulted in some prophylactic or therapeutic effects. However, an adverse response to the protein might occur if it were to stimulate antibody production or if it were to provide iron to the invading pathogen. The recombinant form of human Lf has become available. Development of the product for use in a considerable spectrum of medical conditions now can be anticipated.
The iron withholding defense system, possessed by all vertebrate species, serves to scavenge and sequester toxic quantities of the metal. Consequences of over abundant body iron include catalysis of formation of excessive hydroxyl or ferryl radicals, suppression of various leukocytic defense mechanisms, and stimulation of growth of microbial and neoplastic cell invaders (Weinberg, 1993; Kontoghiorghes and Weinberg, 1995). A paramount component of the iron withholding defense system is lactoferrin (Lf). The recombinant form of the human protein has become available; development of the product for use in a variety of medical conditions now can be anticipated.
Lactoferrin,
a 78-kDa glycoprotein, consists of a single chain of 692 amino acids folded into
two globular lobes. Each lobe is conjugated to a 3-kDa glycan chain through an
N-glycosidic linkage. The lobes each enclose a powerful iron-binding site;
active residues are two tyrosines, a histidine and an aspartate. The apo form
has an open conformation in which the iron binding site is near the protein
surface. The iron complex is a closed system in which the metal exists below the
protein surface and is inaccessible to the surrounding solution (Chug and
Raymond, 1993).
Lactoferrin
is structurally similar to transferrin (Tf) with about 44% homology. Like Tf,
lactoferrin can bind two atoms of iron. For completion of the chelate rings,
both Lf and Tf require bicarbonate ions. However, the affinity constant of
1024 for the iron complex is about 260 fold stronger than that of Tf.
Moreover, unlike Tf, lactoferrin avidly retains the metal in acidic
environments.
Iron binding in
secretions
The two transferrins, Lf and Tf, function in a complementary manner to
continuously purge body fluids of non-protein bound 'free' iron. Thus Tf is
responsible for maintaining an enviroriment void of free iron in serum, lymph
and cerebrospinal fluid. Lactoferrin is assigned to exocrine secretions that are
commonly exposed to normal flora: tears, nasal exudate, saliva, bronchial mucus,
gastrointestinal fluids, cervico-vaginal mucus and seminal
fluid.
In
addition to its iron removal function, Tf has an important second assignment
conveyance of
nutritional
amounts of the metal to and from cells throughout the body. To accomplish the
latter mission in humans, Tf normally maintains an iron saturation value of
25-35%. At values above 35%, Tf begins to lose its effectiveness as a
scavenger of hazardous iron (Kochan, 1973; Weinberg, 1974). In serious episodes
of
infection,
the iron saturation value of
Tf
can be reduced to as low as 5%. This action markedly enhances its ability to
withhold the metal from invading pathogens.
Nutritional role
(?)
In contrast to Tf, lactoferrin is not known to have a normal nutritional function (Sanchez et al., 1992). Its ability to retain the metal at mildly low pH values would prevent the protein from quickly releasing iron in acidic endosomes as occurs with Tf. In transferrin, interdomain hydrogen bonds are rapidly protonated to trigger opening of the iron cleft with prompt release of the metal (Abdallah and Chahine, 2000). In contrast, interdomain hydrogen bonds of Lf do not protonate in mild acidity and iron is retained at pH values >3.5.
Accordingly,
in hosts, Lf fails to provide nutritional iron. For instance, in a study in 3-10
mos infants, Lf in breast milk was found to suppress rather than enhance iron
absorption from the diet (Davidsson et al., 1994). Nevertheless, in unicellular
systems, iron saturated Lf has been observed to stimulate growth of selected
eukaryotes and prokaryotes. For example, Fe-Lf enhanced, and apo-Lf inhibited,
proliferation of human enterocyles (Caco-2 cells)(Oguchi et al., 1995).
Similarly, growth of Legionella pneumophila was stimulated
by Fe-Lf and suppressed by apo-Lf (Byrd and Horwitz, 1991). The ability of cells
of some bacterial species to bind Fe-Lf and to derive the metal from this sole
source of iron is well established (Vogel et al., 1997).
Antimicrobial
defense
Lactoferrin is a major constituent of the secondary specific granules of
circulating polymorphonuclear neutrophils (PMNs). The apoprotein is released on
degranulation of the leukocytes in septic areas. In such sites, the pH value is
lowered by catabolic acids released from metabolically active invading cells as
well as from PMNs. With its ability to chalets and retain iron at low pH values,
Lf is indeed a useful and probably an indispensable defense protein. For
disposal of iron saturated Lf, hepatocytes might serve as a major depository
(Brock et al., 1994).
Several
investigators have noted the joint presence of Lf and lysozyme (LZ) in milk
(Reiter, 1983), specific granules of PMNs (Ellison and Giehl, 1991), tears
(Leitch and Willcox 1998) and tubotympanum mucus (Lim et al., 2000). In in vitro
tests with Escherfchia
coli,
Salmonella typhimurium and Vibrio
cholerae,
each of the proteins alone was bactenostatic; together, they were bactericidal
(Ellison and Giehl, 1991). In artificial tear fluid, synergy of Lf and LZ was
observed against Staphylococcus epidermidis (Leitch and Willcox,
1998).
Some activities of Lf require prior conversion of the apoprotein to the
ferreted molecule. In these systems, the mechanism of action would most probably
be associated with the oxidant activity of the metal. Intro-macrophage killing
of Trypanosome cruzi amastigotes and Listeria monocytogenes was
enhanced by Fe-Lf (Lima and Kmerszenbaurn, 1987) as was suppression of
infra-erythrocytic growth of Plasmodium faldparum (Fritsch et al., 1987).
Human Fe-Lf arrested growth of breast carcinoma cells by inhibition of the G1 to
S transition of the cell cycle (Damiens et al., 1999). Intra-peritoneal injection of either
iron-saturated or apo-Lf suppressed growth of tumors in mice (Bezault et al.,
1994). In systems in which serum is present, apo-Lf might obtain the requisite
iron from transferrin (Fritsch et al., 1987).
Examples
of other activities of Lf that are not concerned with iron deprivation include
enhancement of (i) adherence of PMNs to endothelial cells (Oseas et al., 1981)
and (ii) functions of natural killer cells (Shau et al., 1992); Bezault et al.,
1994); Damiens et al., 1998). Lactoferrin also modulates the inflammatory
process in part by preventing endotoxin activation of macrophage cytokine
induction by binding to lipid A of lipopolysaccharide (Lee et al., 1998, Baveye
et al., 1999). Administration of Lf prior to challenge with either endotoxin or
bacterial pathogens can protect against septic shock (Lee et al., 1998; Baveye
et al., 1999).
Lactoferrin
has been reported to inhibit replication of such viruses as cytomegalo, hanta,
hepatitis C, herpes simplex human immunodeficiency and poliomyelitis apparently
by interfering with attachment of infectious particles to host cell receptors
(Hanmsen et al., 1995; Marchetti et al., 1998; Tanaka et al., 1999; Voriand,
1999; Murphy et al., 2000). Inasmuch as metal binding causes a conformational
change in Lf, the antiviral effect might be expected to vary with the percentage
of iron saturation. In replication of human herpes virus in green monkey kidney
cells, the ID(50) of bovine Lf saturated 10% with iron was 0.36然;
with 90% iron, 0.15然 (Manchetti et al., 1998). Iron, alone, had no antiviral
effect
An
antimicrobial function that does not involve iron trapping has been suggested
for specific fragments of Lf. Pepsin digestion of human or bovine apo-Lf yields
basic peptide sequences, distinct from the iron binding regions, that apparently
after cytoplasmic membrane permeability of bacteria, fungi and protozoa. The
peptides, termed lactoferricins, range in length from 10-47 amino acid residues
(Voriand, 1999). Release of the peptides in vivo might occur upon exposure of Lf
to gastric pepsin or to pepsin-like proteases in neutrophilic
phagolysosomes.
In
in vitro tests, synthetic peptides corresponding to the first eleven residues of
the N terminus of
hLf
have been observed to have strong bactericidal (Nibbering et al., 2001) and
furgicidal (Lupetti et al., 2000; Ueta et al., 2001) activity. However, the size
and tertiary structures of the synthetic peptides may differ considerably from
those of natural peptides derived tam hLf (Nibbering et at., 2001). Possibly,
potent antimicrobial peptides are formed from hLf at specific sites of invasion.
Thus, in chemotherapy, hLf might be more effective than synthetic peptides
(Nibbering et al., 2001). In a study in mice cited below, peroral hLf was more
active than a synthetic peptide in reducing the level of urinary tract
infections (Haversen et al., 2000).
Examples of the concentrations of apo-Lf in fluids of healthy and
infected humans are contained in Table 1. The large amount of Lf in human milk
suppresses growth of such iron-dependent bacteria in the infant intestine as
Bacteroides, Clostridium, Escherichia, Salmonella and Staphylococcus
(Weinberg, 2001). Accordingly, the gut of the breast-fed infant, in the absence
of supplemental iron, develops a predominantly natural flora of non-pathogenic
Lactobacillus
and
Bifidobacterium. The former totally abstains from use of iron; its
enzymes utilize manganese and cobalt in place of iron (Weinberg, 1997). Growth
of Lactobacillus
results
in a gut pH value of 5, whereas the gut pH of formula-fed infants is 5.9-8.2.
Although Bifidobacterium
requires
iron, it has developed a unique ferrous iron acquisition system that can
function at pH 5 and which, to a considerable extent, is resistant to iron
withholding by apo-Lf. The fungistatic action of human milk has been shown to
depend solely on its content of apo-Lf; the action is abolished by addition of
iron (Andersson et al., 2000).
The
large concentration of apo-Lf in tears, together with LZ, efficiently protects
ocular tissues from most bacterial pathogens. These two natural (non-immune)
proteins permit much lessen reliance on secretory antibody (IgA) for
antibacterial defense. Accordingly, possible scanning of delicate ocular tissues
due to antigen-antibody reactions is minimized.
Note
also in Table 1 the remarkable increase in concentration of Lf in serum during
severe bacterial infection. The protein is derived from degranulating
neutrophils. One million neutrophils have been estimated to contain 3痢 Lf
(Bennett et al., 1973) Inherited inability to produce specific granules and
neutrophlic Lf is associated with recurrent infection and, in untreated persons,
death (Breton-Gorius et al., 1983).
Moreover, appearance of Lf in increasing amount in serum is a very early
indication of an inflammatory reaction to invasion or trauma. For example,
intravenous injection of E. colt in piglets caused a rise in serum Lf from 0.01
然 at zero time to 0.1 然 at 1 h and 0.2 然 at 2 h (Gutteberg et al., 1988).
Similarly, in the initial phase of infection with Neisseria
meningitides,
serum
Lf in humans increased at a rate of 0.15 然/h (Gutteberg et al.,
1984).
Table
1: Examples of concentrations of apo-Lf in human body fluids
(然)
Underlying
Colostrum 100
normal
Sanchez et al., 1992
Milk
20
normal
Hamosh, 1998
40
normal
Fond et al., 1977
60
normal
Zavaleta et al., 1995
Tears
25
normal
Hunt et al., 1996
Seminal
fluid 1.4
normal
Buckett et al., 1997
Vaginal
fluid
2.0
just after menses
Cohen et al., 1987
0.1 just
prior to menses
ibid
<0.25
oral contraceptive users
ibid
Saliva
0.11
normal adults Tenuvuo et
al., 1986
0.05
normal children
Smith et al., 1981
0.25
children: cystic fibrosis
ibid
Amniotic
fluid 0.02
non-infected
Pacora et al., 2000
0.0
infected
ibid
Cerebrospinal 0.00
normal children
Maffei et. al., 1999
Fluid
0.01
Children: aseptic meningitis
ibid
0.13
Children: bacterial meningitis ibid
Synovial
0.014
non-inflammatory
Bennett et al., 1973
fluid
0.338
inflammatory arthritides ibid
Serum
0.005
normal
Kelver et. al., 1996
2.5
acute sepsis
Vorland, 1999
The considerable spectrum of activities and locations of Lf suggest that
the protein might be developed for a variety of prophylactic and therapeutic
applications. A number of pilot studies are available that provide information
on this possibility. For instance, intraperitoneal (i.p.) injection of rhLf in
mice followed 10 h later by i.p. inoculation of E. coli decreased
mortality from 43% to 0% (Wand et al., 1995). Administration of topical 1%
bovine Lf prior to inoculation of herpes simplex type 1 on mouse cornea
suppressed but did not eradicate the infection (Fujihara and Hyashi,
1995).
In Table 1, it may be seen that inflamed body joints might be another
appropriate site for testing exogenous Lf. Examination of synovial fluid from 25
humans with inflammatory synovltis showed that 30% of the specimens contained
'free' iron. In these samples, concentrations of Lf were significantly lower
than in those with no 'free' iron (Guillen et al., 1998). Addition of exogenous
human apo-Lf to the samples consistently reduced the amount of 'free' iron. In a
subsequent study, collagen arthritis was induced in DBA/1 mice and S. aureus
septic arthritis was established in Swiss mice (Guillen et al., 2000). In each
set, peri-articuiar injection of human Lt significantly reduced
inflammation.
The
systemic mechanism of action of orally administered Lf is not well understood.
The extent to which available iron in the intestine might modulate the amount of
Lf digested and/or absorbed is unclear.
In 24 mice inoculated with E. coli by bladder installation and fed
0.5 mg hLf, serum samples at 24 h contained 0.02-1.1 nM Lf in eleven animals,
none in thirteen (Haversen et al., 2000). Urine specimens, 2 h after feeding Lf,
contained 0.5-1.0 nM; 5 h after feeding, 0.2-0.4 nM. In this investigation,
bovine Lf and synthetic peptide sequence 16-40 also were
tested.
perorally. The hLf-treated group showed the strongest reduction in numbers of
kidney and bladder bacteria.
Prefeeding,
on intravenous injection, of human and bovine Lf reduced kidney infections in
mice inoculated with S. aureus by 40-60% and lowered viable counts 5-12
fold (Bhimani et al., 1999). In this study, apo- and holo-Lf were found to be
equipotent, but hydrolyzed Lf was inactive. Daily feeding of bovine Lf to guinea
pigs infected with dermatophytes failed to prevent onset of symptoms during the
early phase of infection but facilitated clinical improvement of skin lesions
after the peak of symptoms had occurred (Wakabayashi at al.,
2000).
Development
of various tumors in the colon, esophagus and lungs of rats exposed to chemical
carcinogens was partially suppressed by feeding bovine Lf (Ushida et al., 1999).
Orally administered bovine Lf inhibited angiogenesis in adult rats (Norrby et
al., 2001). Prefeeding
bovine
Lf to germ free piglets provided significant protection against lethal shock
induced by intravenously administered endotoxin (16.7% vs 73.7%
mortality-, p <0.001)(Lee et al., 1998).
Human patients with
hepatitis C were fed bovine Lf for
eight weeks (Tanaka et al., 1999). Three of four patients with low
pretreatment levels of viremia
experienced a decrease in serum values of HCV-RNA and alanine transaminase.
However, no significant changes occurred in seven patients who had high
pretreatment levels of viremia.
Exogenous
Lf might be useful as an adjunct in antimicrobial therapy. In in vitro tests,
effective concentrations of diverse antifungal drugs were lowered against
Pneumocystis cannii (Cirioni et al., 2000) and Candida sp. (Kuiper
et at., 1997; Wakabeyashi et al., 1998) by combination with either bovine on
human Lf. Similarly, bacteriostatic and bactericidal concentrations of rffampin
and doxycycline were lowered against Pseudomonas aeruginosa and
Burkholderia cepacia by combination with
rhLf (Alkewash et al., 1999). Of
course, only pathogen: unable to employ Lf as an iron carrier could be safely
attacked in this manner. To date, fungi are not known to extract iron from
transferrins such as Lf. However, Trichomonas protozoa (Weinberg, 1999) and
Helicobacter pylori bacteria (Dhaenens et al., 1997) as well as
various members of the bacterial family, Neisseriaceae (Vogel et al., 1997) can
acquire iron from human Lf.
Production of human recombinant Lf has been reported in a variety of
organisms. These include baby-hamster kidney cells (Stowell et al., 1991),
Aspergillus nidulans
and
A. awamori (Wand et al., 1992, 1995), Saccharomyces cerevisiae (Liang and
Richardson, 1993), transgenic dairy animals (Krimpenfort, 1993) and transgenic
potatoes and tomatoes (Arakawa et al., 1999). In the A. awamori
fermentation, quantities in excess of 25 然 have been obtained. The protein
molecules are glycosylated and have excellent metal binding and antibacterial
activity.
A major advantage of human Lf over other chelating drugs is that it is a
natural product of humans and thus should be biocompatible. However, an
important potential hazard of therapeutic use of hLf in human patients is
possible induction of an antibody response. Antibodies to endogenous Lf have
been detected in patients with such autoimmune diseases as systemic lupus
erythematosis, rheumatoid arthritis and primary scerosing cholangitis (Skogh and
Peen, 1993; Alfetra et al., 1996). In some patients with rheumatoid arthritis,
antibodies to Lf are present in synovial fluid as well (Guillen et al., 1998),
Unfortunately, anti-Lf IgG can cause lactoferrin bound iron to become reactive
in the bleomycin assay (Guillen at al., 1998).
A
second possible hazard of exogenous human Lf is stimulation of growth of
specific pathogens. As mentioned above, Trichomonas vaginatis obtains
iron from human Fe-Lf. The protozoan grows mainly in the Lf-rich environment of
human vaginal mucus. Disease symptoms begin or exacerbate during menses at which
time the vaginal concentrations of Lf and iron are notably grater than at
midcycle. In the male urethra, the illness is self-limited or
asymptomatic;
seminal
fluid contains Lf but is vary low in iron, as is urine. Fortunately, T.
vaginalis cannot obtain iron from Tf and thus fails to cause systemic
infections in either women or men (Weinberg, 1999 Were human Lf to be used in
therapy of vaginal yeast infections, the patients would first need to be
carefully evaluated for freedom from trichomoniasis.
Another
human pathogen that can specifically derive iron from human Lf is
Helicobacter
pylori.
This
bacterium is the major etiologic agent of chronic gastritis and is a component
of the eptiology of gastric ulcers and carcinomas. Cells of this pathogen form a
70 kDa hLf-binding protein. H.
pylori
also can obtain iron from heme but mot from human Tf nor from bovine or equine
Lf or Tf (Dhaenens et al., 1997). Thus bovine Lf can suppress H.
pylori
infection in mice (Dial et al., 1998; Wada et al., 1999).
The
singular location of H.
pylori
in human
gastric epithelium apparently is a consequence of the availability of human Lf
and iron in gastric juice. In a set d 30 H. pylori positive and 14 negative
patients with chronic gastritis (Nakao et al., 1997), the average level of
endogenous Lf in the former was 4.25 fold greater than in the latter
(p<0.0007).
In in vitro susceptibility tests of H.
pylori
to
apo-rhLf, 5 of 13 strains required 10 然 for inhibition, 3 required 20 然, and 5
needed >40 然 (Miehlke et al., 1996). No strains were sensitive to low
concentrations of the protein. Thus human apo-Lf might not be a successful
therapeutic agent for H.
pylori
and, indeed, could intensify the infection. In a recent study, six adult humans
with H.
pylori
infection
were fed 1.25 g and six were fed 5 g of rhLf over a 24 h period. The amount of
endogenous Lf had not been ascertained. Not surprisingly, none of the 12
subjects cleared the infection. Fortunately, no adverse effects were observed
(Opekun et al., 1999).
In
contrast to H.
pylori,
Helrcobacter
felis
does appear to be susceptible to the antibacterial action of human Lf. In mice
infected with 3x109 viable cells of the latter bacterium, rhLf
partially reversed both the infection-induced gastritis and the infection rate
(Dial et al., 2000).
In
this system, the efficacy of Lf was comparable to that of amoxacillin as well as
to the combination of metronidazole, tetracycline and bismuth
subsalicylate.
As rhLf increasingly becomes available, it may be appropriate for best
results to substitute this product for bovine Lf in studies in humans. However,
in some systems, bovine Lf has been observed to be more effective than human Lt.
For instance, the ability of Prevotella nigrescens, a bacterium
associated with dental infections, to adhere to am enamel component,
hydroxapatke, was suppressed to a greater extent by bovine than by human Lf
(Yasuyuki et al., 2001). In a study of the cytopathic sited of human
immunodeficiency virus-1 on MT4 cells, the IC(50) of bovine Lf was 0.5 然; of
human Lf, 1.0 然 (Harmsen et al., 1995).
Furthermore,
as with any iron chelator, it will be essential to recognize and monitor the
iron background of the system under investigation. Effective doses of Lt would
be expected to vary with the level of iron available to the protein. The degree
to which, if any, iron might be required by Lf for the specific activity also
should be determined. With the exception of the investigations on synovial fluid
(Guillen et al., 1998), the pilot studies cited above failed to ascertain tissue
or fluid values for endogenous Lf and iron.
In
systems in which Lf were to be employed as an adjunct to, or a replacement for,
antibacterial drugs, consideration should be given to pairing it with lysozyme.
However, disease states that probably would not be helped by lysozyme include
fungal and viral infections, cancers, and sterile inflamed
areas.
Ten years ago, Sanchez et al. (1992) proposed that the 'biologic role of
Lf is that of a specialized iron-scavenging protein, designed to act
particularly under conditions where Tf would be less effective at binding iron
due to reduced pH, such as exist in the gastrointestinal tract or inflammatory
lesions. By binding iron under these conditions it would render harmless 'free'
iron that might otherwise cause free radical-mediated damage to sensitive
tissues, reduce absorption of iron in the immediate post-natal period, and
decrease its availability to microorganisms'. During the past decade, successful
research applications of administration of exogenous Lf have tended to validate
this proposal. In the coming decade, some of these applications might well be
developed into prophylactic or therapeutic products.
Lactoferrin, a 78 kDa iron binding protein, provides antioxidant and
antimicrobial activity in secretions of lacrimal and mammary glands and of
respiratory, gastrointestinal, and genital tracts. It is released also from
neutrophils at sites of infection and can scavenge non-protein bound iron in
areas that have lowered as well as neutral pH values. Recombinant human Lf is
becoming available for evaluation for possible prophylactic or therapeutic use
in a wide variety of human medical conditions. As a human natural product, it
should be efficiently metabolized with no side effects. Precaution is needed,
however, to avoid antigenic sensitization as well as introduction of the protein
to tissues that may be infected with specific protozoa or bacteria that utilize
Lf in their acquisition of host iron.
This
review is dedicated to Dr. Bruno Reiter in recognition of his pioneering
research on lactoferrin.
4. Andersson, Y., Lindquist, S., Lagerquist, C., Hernell, O. (2000) Lactoferrin is responsible for the fungistatic action of human milk Early Hum'. Dev. 59:95105.
16. Cohen, M.S., Britigan, B.E., French, M., Bean, K (1987) Preliminary observations on lactoferrin secretion in human vaginal mucus. Am. J. Obstet. Gynecol. 157:1122-1125.
17. Damiens, E., Mazurier, J., Yazidi, I.E., Masson, M., Duthille, I., Spik, G., Biolly-Marer, Y. (1998) Effects of human lactoferrin on NK cell cytotoxicity against haemochromatosis and epithelial tumor cells. Biochim. Biophys. Ado 1402:277-287.