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).
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)
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.
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