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PubMed1991

Association of dry eye signs and symptoms with tear lactoferrin concentration.

Diagnoses of dry eye made with the Lactoplate Immunoassay Test (which assesses tear lactoferrin concentration) were compared to diagnoses based on dry eye symptoms, tear break-up times, and rose bengal staining. For a population of 49 subjects with normal and mild to moderate dry eyes, contingency tables showed that the lactoferrin-based diagnoses were not significantly related to the diagnoses made using any of the other factors. Lactoferrin concentrations were statistically correlated with symptom scores, but there was no significant correlation between lactoferrin concentrations and either tear break-up time or rose bengal staining. These results suggest that: 1) the dry eye problems in the subject population may not have been associated with lacrimal gland dysfunction (which the Lactoplate assesses); 2) Lactoplate tests are not sensitive enough to allow accurate diagnoses for the population of mild to moderate dry eye subjects used; 3) signs and symptoms associated with dry eyes occur before lactoferrin changes are manifest so lactoferrin changes had not yet appeared in the subjects; or 4) in mild to moderate dry eye subjects, lactoferrin changes occur only in reflexive tears and not in the basal tears that were tested in this study. In summary, measurement of tear lactoferrin concentration alone has not been shown to be a sufficiently sensitive and specific test for the diagnosis of mild to moderately dry eye as defined by more common clinical techniques.

Adult↗

PubMed1989

Purification and characterization of intact lactoferrin found in the urine of human milk-fed preterm infants.

Little is known about the metabolic fate of ingested lactoferrin in human-milk-fed term or preterm infants. Enzyme-linked immunosorbent assays and sodium dodecyl sulfate/polyacrylamide gradient gel electrophoresis (SDS-PAGGE) with immunoblots have demonstrated that the urinary excretion of lactoferrin by preterm infants fed exclusively human milk exceeded that by formula-fed infants. The origin and molecular integrity of the excreted lactoferrin, however, are unclear. We have developed extraction and separation procedures involving a stationary phase of immobilized single-stranded DNA (ssDNA) that allows the efficient (greater than 80%) and rapid isolation of pure, intact lactoferrin from infants' urine. We purified lactoferrin to apparent homogeneity from infants' urine in one step, using the immobilized ssDNA with mobile phases containing up to 6 mol of urea per liter. The purified lactoferrin was evaluated by SDS-PAGGE; silver-staining revealed one protein band at 78 kDa; immunoblots confirmed the presence of intact lactoferrin. High-performance affinity chromatography with use of immobilized metal ion (Cu2+) suggested the presence of intact, iron-saturated lactoferrin. Subsequent chromatography on high-performance reversed-phase (C18) columns independently verified sample identity and purity.

Blotting, Western↗

PubMed1984

Lactoferrin stimulates the production of leucocyte migration inhibitory factor by human peripheral mononuclear leucocytes.

The effect of lactoferrin on the migration of human polymorphonuclear cells was investigated. High concentrations of lactoferrin (greater than 250 micrograms/ml) markedly inhibited the migration of granulocytes under agarose. This migration inhibition could not be neutralized by an antibody against leucocyte migration inhibitory factor (LIF), suggesting a direct effect of lactoferrin on the granulocytes. Low concentrations of lactoferrin were, however, indirectly capable of inhibiting neutrophil migration. The overnight culture of mononuclear leucocytes with low concentrations of lactoferrin (10 micrograms/ml) resulted in the release of granulocyte migration inhibiting factors in the cell free culture supernatant. Strong evidence indicating that the migration inhibiting factors were due to LIF, was obtained in experiments whereby the inhibitory activity could be completely neutralized by anti-LIF antibodies. The lactoferrin-mediated stimulation of LIF release by mononuclear leucocytes could be neutralized by an anti-lactoferrin serum, but not by an anti-albumin serum, whereas PPD-induced LIF release was not affected by either antiserum. These findings suggest that lactoferrin besides its well known anti-microbial properties, may also play a regulatory role in the migratory response of polymorphonuclear cells during inflammatory conditions.

Cell Movement↗

PubMed1982

Feedback regulation of granulopoiesis: polymerization of lactoferrin abrogates its ability to inhibit CSA production.

Neutrophil extracts were prepared from the peripheral blood of 40 normal volunteers and tested for their ability to inhibit CSA production by mononuclear leukocytes. Highly dilute neutrophil extracts inhibited CSA production/release, while extracts selectively depleted of lactoferrin by antibody affinity chromatography did not. In addition, higher concentrations of neutrophil extracts and higher doses of lactoferrin (10(-9)-10(-6) M) failed to inhibit CSA production/release. We found no evidence of CSA or CSA-enhancing factors in either our lactoferrin or our neutrophil extracts. However, using gel chromatography and rate zonal density sedimentation, we noted that lactoferrin undergoes concentration-dependent polymerization at 10(-9)-10(-10) M in tissue culture medium and that while monomeric lactoferrin effectively inhibits CSA production/release in vitro, the polymeric form does not. Thus, while we have confirmed that lactoferrin is the activity in neutrophil extracts that inhibits CSA production, we have also found that lactoferrin undergoes reversible polymerization at physiologic concentrations and that the polymerized molecule is inactive. The tendency of lactoferrin to polymerize in tissue culture medium and in vivo should be taken into account in any studies on its potential role as a physiologically relevant regulator of granulopoiesis.

Centrifugation, Density Gradient↗

PubMedDate not supplied

Neutrophil lactoferrin release during open heart surgery is unrelated to complement activation.

The relationship between neutrophil activation and complement activation during open heart surgery was evaluated by measuring plasma lactoferrin and C3a des Arg (C3a) in 30 adult patients undergoing coronary artery surgery. Measurements were made before induction of anesthesia, before cardiopulmonary bypass, at the end of bypass, and 10 min after protamine infusion. Changes in the measured parameters thus reflected activation during the three distinct phases of cardiac surgery: pre cardiopulmonary bypass, the bypass phase, and the heparin neutralization phase. A major rise in lactoferrin occurred in the pre bypass period, from 99 +/- 13 (mean +/- SEM) to 647 +/- 48 ng/ml (p < 0.001). During this time, C3a levels did not rise significantly (384 +/- 22 to 439 +/- 36 ng/ml). The bypass procedure resulted in a rise of both lactoferrin and C3a levels, with lactoferrin reaching 1,092 +/- 69 ng/ml and C3a increasing to 1,884 +/- 179 ng/ml after bypass (p < 0.001 for both). In individual patients, however, the changes in lactoferrin during bypass did not correlate with changes in C3a levels (r = -0.06). After protamine infusion, C3a levels reached 3,301 +/- 324 ng/ml, while the plasma lactoferrin levels declined to 522 +/- 57 ng/ml. Thus, during open heart surgery, neutrophil activation, as measured by plasma lactoferrin concentration, does not correlate with complement activation resulting from the bypass procedure or the protamine neutralization of heparin. The potential clinical relevance of the neutrophil granular release of lactoferrin is discussed.

Complement Activation↗

PubMed1996

Identification of lactoferrin in bovine tears.

OBJECTIVE: To determine whether bovine tear film contains the iron-binding glycoprotein, lactoferrin. ANIMALS: 40 Adult Hereford, Angus, and Simmental cattle. PROCEDURE: Protein analysis: pooled bovine tears were used for protein analysis (size exclusion high-performance liquid chromatography [HPLC] fractionation). HPLC was used for tear analysis. A diode array detector was used (215 and 280 microns) for chromatogram analysis and comparisons. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE): protein electrophoresis was performed, using 7.5% running gels with 4% stacking gels. Molecular weight of proteins in the unknown samples was determined as recommended by the manufacturer of the standards. Protein sequencing: amino acid sequencing, using automated Edman degradation of HPLC purified protein, was performed. The sequence obtained was compared with the known protein sequence of bovine lactoferrin. RESULTS: HPLC analysis of whole bovine tears resulted in a consistent chromatogram. Peak collection was performed to recover a protein from the bovine tear film with chromatogram characteristics nearly identical to purified bovine lactoferrin. Silver-stained SDS-PAGE of this peak revealed a band with molecular mass consistent with bovine lactoferrin (estimated mass of 78 kd). The first 13 amino acid residues of this protein were identical to the amino acid sequence of bovine lactoferrin. CONCLUSION: Analysis of whole bovine tears, using size exclusion HPLC, SDS-PAGE, and amino acid sequencing, provided evidence that bovine tears contain lactoferrin. CLINICAL RELEVANCE: Lactoferrin probably exerts a bacteriostatic effect in bovine tear film. Locally produced lactoferrin may bathe the ocular surface and sequester iron from potential pathogens.

Amino Acid Sequence↗

PubMedDate not supplied

Seminal plasma lactoferrin concentrations in normal and abnormal semen samples.

Although the iron-chelating protein lactoferrin is secreted by the seminal vesicles, the precise role of lactoferrin in semen is unclear. This study aimed to determine whether there is any association between seminal lactoferrin concentrations and normal and abnormal semen samples with and without leucocytospermia. Lactoferrin concentrations were measured by radial immunodiffusion of semen samples from 368 men attending a regional andrology referral center. Routine seminal analysis, including the presence of leucocytospermia, was also performed. Results showed increased seminal lactoferrin in samples showing oligospermia (13.3 mg/100 ml) and oligoasthenospermia (13.4 mg/100 ml) compared to normospermic samples (11.2 mg/100 ml). There were no significant differences in seminal lactoferrin between normospermic samples and azoospermic samples or asthenospermic samples with normal sperm density. Although there was a trend toward increased lactoferrin concentration with leucocytospermia, this was not significant. Possible causes for raised lactoferrin in association with oligospermia are discussed.

Humans↗

PubMed1997

Familial subepithelial corneal amyloidosis--a lactoferrin-related amyloidosis.

PURPOSE: To isolate the protein that collects in increased amounts beneath the corneal epithelium in familial subepithelial corneal amyloidosis (FSCA), also known as gelatinous droplike corneal dystrophy, and to identify it by N-terminal amino acid sequencing. METHODS: Peptides resulting from pepsin digestion of a unique protein isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis from frozen tissue from two corneas with FSCA were purified by high-pressure liquid chromatography followed by protein sequence analysis. The protein was identified by amino acid sequencing, Western blotting, and immunohistochemistry. RESULTS: A protein was identified in two corneas with FSCA that was not present in normal corneas or in corneas with other disorders. The amino acid sequences of two peptides derived from this protein were identical to portions of lactoferrin. The unique protein reacted with rabbit antihuman lactoferrin after Western blotting. The presence of lactoferrin in the amyloid within affected corneas was confirmed using the immunoperoxidase method on formalin-fixed, paraffin-embedded tissue sections and lactoferrin antiserum. CONCLUSIONS: Corneal tissue with FSCA contains lactoferrin, and this is the first form of amyloidosis found to be associated with this protein. Because lactoferrin is a product of lacrimal glands, the corneal lactoferrin may be derived from the tears. Because the gene for lactoferrin is on chromosome 3 (3q21-q23), this locus is a potential site for the FSCA gene.

Adolescent↗

PubMed1997

Effect of lactoferrin on proliferation and differentiation of the Jurkat human lymphoblastic T cell line.

The effect of human lactoferrin on the human lymphoblastic T cell line (Jurkat) was tested with regard to proliferation and differentiation. Lactoferrin enhanced cell proliferation in a serum-reduced (1% fetal calf serum) culture. The stimulatory effect of lactoferrin on proliferation depended on the degree of iron saturation but the amplitude of the effect was low, similar to that obtained in the presence of serum transferrin. The proliferation stimulatory effect of lactoferrin was not observed in the presence of 10% fetal calf serum (FCS) in the culture medium. These results suggest that Fe-lactoferrin can substitute for Fe-transferrin during the prolonged culture of cells in a low serum concentration. Iron-saturated lactoferrin was also shown to promote T cell differentiation. Jurkat cells, when exposed to iron-saturated lactoferrin in the presence of 10% FCS, gradually exhibited a decrease in the cell volume, cell surface density of CD71 antigen, the nuclear incorporation of [methyl-3H]thymidine, but an increase of the percentage of cell population in the G0/1 phase of the cell cycle. These modifications were accompanied by the appearance of CD4 antigen at the cell surface. Therefore, in the continuous presence of lactoferrin, proliferating cells slowly enter into quiescence state, undergoing cell differentiation.

Cell Differentiation↗

PubMed2000

The effects of local administration of lactoferrin on inflammation in murine autoimmune and infectious arthritis.

OBJECTIVE: To determine whether lactoferrin can modify articular inflammation in murine models of autoimmune and septic arthritis. METHODS: Collagen arthritis was induced in DBA/1 mice and Staphylococcus aureus septic arthritis in Swiss mice. Joints with established inflammation were injected periarticularly with 0.5 mg or 1 mg of human lactoferrin, and arthritis was monitored for 3 days. RESULTS: DBA/1 mice injected with lactoferrin showed significantly suppressed local inflammation for up to 3 days, achieving up to 71% of the effect of corticosteroid. Periarticular injection of 125I-lactoferrin confirmed that 25% of lactoferrin was retained in paws after 6 hours. Serum levels of interleukin-6, however, were not significantly reduced, suggesting a predominantly local antiinflammatory effect. Similarly, local, periarticular administration of lactoferrin into S aureus-infected Swiss mice significantly suppressed paw inflammation and did not enhance bacterial survival. CONCLUSION: Lactoferrin may have clinical utility in reducing articular inflammation, particularly in septic arthritis, in which antiinflammatory effects may be achieved without promoting bacterial survival.

Administration, Topical↗

PubMedDate not supplied

Lactoferrin and lysozyme levels in synovial fluid: differential indices of articular inflammation and degradation.

Lysozyme and lactoferrin levels were measured in 71 synovial fluids (SF) of patients with traumatic effusions, osteoarthritis, rheumatoid arthritis, pseudogout, septic arthritis, and gout, as well as in 91 synovial fluids graded according to their neutrophil count. Elevated lysozyme levels were found in all the inflammatory arthritides and also in osteoarthritis. Lactoferrin levels were not increased in osteoarthritis but displayed a close correlation to the extent of the inflammatory response as judged by SF neutrophilia. The ratio of lysozyme to lactoferrin decreased progressively with increasing SF neutrophilia. In vitro experiments showed that lactoferrin is released from neutrophils isochronously with lysozyme and beta-glucuronidase. Lactoferrin was not found in hyaline cartilage, a tissue known to contain lysozyme. These results are consistent with belief that SF lysozyme has a major derivation from both cartilage and neutrophils, and that lactoferrin arises only from neutrophils. These findings indicate that the simultaneous measurement of lysozyme and lactoferrin provides a potentially useful index of both joint inflammation and cartilage degradation.

Adult↗

PubMed1992

Lactoferrin binding sites and nuclear localization in K562(S) cells.

Lactoferrin, a single chain cationic glycoprotein, present in the secondary granules of neutrophils, acts as a negative feedback regulator of myelopoiesis. Specific receptors for lactoferrin were detected on the surface of different hematopoietic cell types. The influence of lactoferrin on cell growth in culture has been reported. Interactions of lactoferrin with DNA were also demonstrated. In the present paper we confirm the presence of lactoferrin specific binding sites on K562 cells and we estimate the number of binding sites and the dissociation constant. By Western blotting analysis performed on K562 lysates we find a band of about 120 kDa responsible for specific binding of lactoferrin. We also show that lactoferrin, after binding at the cell surface, is internalized in a temperature dependent way and is immunologically detectable as a DNA-linked protein in nuclear extracts.

Binding Sites↗

PubMed1993

Simultaneous calcium-dependent delivery of neutrophil lactoferrin and reactive oxygen metabolites to erythrocyte targets: evidence supporting granule-dependent triggering of superoxide deposition.

Optical microscopic techniques have been utilized to study the deposition of lactoferrin, a specific granule marker, and superoxide anions into target erythrocytes during antibody-dependent phagocytosis. Previous studies from this laboratory have shown that the entry of superoxide anions into erythrocytes can be sensitively monitored with Soret band transmitted light microscopy. When neutrophils were incubated with BAPTA/AM, an intracellular Ca2+ chelator, they phagocytosed IgG-opsonized sheep red blood cells (SRBC) but did not affect the microscopically detected absorption of their Soret band. When these same erythrocytes were observed after the infusion of 20 microM ionomycin, a Ca2+ ionophore, 58% of the cell-bound SRBC targets were destroyed immediately. However, neutrophils from chronic granulomatous disease (CGD) patients were unable to affect the Soret absorption of erythrocyte targets under any conditions. These results suggest that a Ca2+ signal can participate in triggering superoxide deposition in targets. Since Ca2+ signals are known to participate in the exocytic release of granules, we tested the hypothesis that specific lactoferrin-bearing granules are delivered to targets in parallel with superoxide anions. Lactoferrin delivery to phagosomes was monitored using resonance energy transfer (r.e.t.) microscopy. SRBCs were opsonized with both unconjugated and rhodamine B isothiocyanate (RBITC)-conjugated rabbit anti-SRBC IgG. After incubation with adherent neutrophils, the samples were washed, fixed with 3.7% paraformaldehyde, then labeled with fluorescein isothiocyanate (FITC)-conjugated antilactoferrin IgG. Energy transfer between FITC and RBITC was imaged microscopically and quantitated by photon counting. Significant levels of r.e.t. between antilactoferrin and anti-SRBC labels were observed after phagocytosis, but not in the absence of acceptor fluorochromes. To control for r.e.t. specificity, neutrophil membranes were labeled with FITC-conjugated, anti-HLA IgG after internalization of rhodamine B-tagged SRBCs (RSRBCs). Although r.e.t. between lactoferrin and RSRBCs labels was observed, no r.e.t. between HLA and RSRBC labels could be found. Further studies showed that treatment of neutrophils with BAPTA inhibited r.e.t. between anti-lactoferrin and RSRBCs. However, addition of ionomycin relieved this inhibition of energy transfer. These experiments show that both lactoferrin and superoxide delivery to targets are regulated in parallel by a Ca(2+)-dependent pathway. Furthermore, by combining Soret microscopy with r.e.t. microscopy, we have shown that superoxide anions and lactoferrin are delivered to the same phagosomes. We speculate that the NADPH oxidase, which produces superoxide anions, is assembled on specific granule membranes, thus accounting for their parallel Ca(2+)-dependence, activation, and delivery.

Calcium↗

PubMed1998

Induction of lactoferrin expression in murine ES cells by retinoic acid and estrogen.

Lactoferrin is an iron-binding glycoprotein present in high concentrations in milk and exocrine fluids such as bile and tears. Many functions have been attributed to lactoferrin, including antimicrobial and antiviral activities, immunomodulation, and cell growth regulation. Lactoferrin expression is controlled by different regulators, including retinoic acid and estrogen. However, the expression pattern of lactoferrin in mammalian early development has not yet been reported. Murine embryonic stem cells are pluripotent cells that can contribute to all tissues and were used for this study. We show here that while no lactoferrin protein or mRNA was detected in untreated murine embryonic stem cells, retinoic acid and estrogen can induce high levels lactoferrin expression in these cells. Expression, demonstrated by reverse transcription-polymerase chain reaction, Western blot, immunofluorescence, and ELISA assay, was dose and time dependent. Our study provides an in vitro model for examining lactoferrin expression in early development and differentiation.

Alitretinoin↗

PubMed1994

Maternal lactoferrin in the urine of preterm infants. Evidence for retention of structure and function.

Intact (i.e., 78-kDa) lactoferrin has been purified from the urine of preterm infants fed human milk. The maternal origin of this lactoferrin, and the integrity of its primary structure have been documented. Computer analyses of the circular dichroism spectra revealed a composite secondary structure for the urinary lactoferrin that was indistinguishable from that of purified human milk lactoferrin and similar to that observed in the crystal structure. Intact function was suggested by iron binding; an approximate 2:1 molar ratio of iron to lactoferrin was confirmed. Thus, maternal lactoferrin is absorbed intact by the preterm infant and appears to remain structurally and functionally intact within the circulatory system and during urinary excretion. It is possible, therefore, that maternal lactoferrin has an immunoregulatory influence in newborn infants fed human milk.

Circular Dichroism↗

PubMed1990

Stimulation of proliferation and immunoglobulin M production by lactoferrin in human-human and mouse-mouse hybridomas cultures in serum-free conditions.

The effects of growth factors, such as insulin, transferrin, lactoferrin, ethanolamine, and selenium, on proliferation and IgM production of human-human hybridomas HB4C5 cells in a serum-free enriched RDF (eRDF) medium were studied. Among them, lactoferrin markedly stimulated proliferation and IgM production of the cells. Another iron-binding protein, transferrin, stimulated proliferation of HB4C5 cells as well as lactoferrin, but its stimulatory effect on IgM production was negligible. The proliferation and IgM production of HB4C5 cells gave some detectable delays in conventional ITES-eRDF medium at low cell densities, but the delays were avoided by the addition of lactoferrin. However, eRDF medium supplemented with lactoferrin could not support proliferation and IgM production of the cells at high cell densities. For proliferation and IgM production of HB4C5 cells, eRDF medium supplemented with 25 micrograms/ml lactoferrin, 10 microM ethanolamine, 35 micrograms/ml transferrin, and 2.5 nM selenium (LETS-eRDF) gave the best result. Lactoferrin stimulated proliferation of human-human and mouse-mouse hybridomas producing IgG or IgM, but stimulation of Ig production was detected only in IgM producers. These results suggest that cell proliferation, IgM production, and IgG production of hybridomas are regulated by different mechanisms.

Animals↗

PubMed1990

Interaction of lactoferrin with Escherichia coli cells and correlation with antibacterial activity.

It has been established that the antimicrobial activity of lactoferrin towards Escherichia coli is enhanced by a direct contact between the protein and the microbial cell and that, in the case of E. coli K-12 strains, an antibacterial activity of lactoferrin unrelated to iron withdrawal is present. Evidence is now reported that lactoferrin binds to surface structures expressed in E. coli K-12 strains grown in either an "excess" or "stress" of iron. Under the experimental conditions used, lactoferrin binding both in the apo and in the iron-saturated form yields a maximum of 1.6 X 10(5) bound molecules/E. coli K-12 cell; the amount of lactoferrin bound does not depend on the expression of the iron-regulated outer membrane proteins. In contrast, lactoferrin does not bind to E. coli clinical isolates. Apo-lactoferrin (at 500 micrograms/ml in a chemically defined medium) inhibits the growth of E. coli K-12 strains but not of clinical isolates. These findings suggest that the antibacterial activity of the protein could be associated to its binding to the cell surface.

Absorption↗

PubMed1994

Effect of lactoferricin B, a pepsin-generated peptide of bovine lactoferrin, on Escherichia coli HB101 (pRI203) entry into HeLa cells.

Bovine lactoferrin and a pepsin-generated peptide of bovine lactoferrin, known as lactoferricin B, were tested for an ability to influence the cell-invasive properties of an Escherichia coli HB101 strain carrying the plasmid pRI203, which encodes the Yersinia pseudotuberculosis inv gene. At non-cytotoxic and non-bactericidal concentration (0.5 mg/ml) lactoferricin B lowered by about tenfold the cell invasion capability of E. coli HB101 (pRI203), whereas no effect was observed when bovine lactoferrin was added during the infection of HeLa cell monolayers. The step of the invasion process affected by lactoferrin B was the internalization since the adhesion of bacteria to HeLa cells was unaltered in the presence of the peptide. Latex beads coated with lactoferrin B bound to HeLa cell monolayers and induced the agglutination of bacterial cells, indicating that this highly cationic peptide interacts directly with both eukaryotic and bacterial surfaces. Moreover, we demonstrated that the anti-invasive effect induced by lactoferrin B was reversed when the medium was supplemented with Ca2+, Mg2+ and Fe2+ ions which diminished its affinity binding. Our findings suggest that lactoferrin B effectiveness towards E. coli HB101 (pRI203) invasion is correlated to its binding capability on the eukaryotic and bacterial cell surfaces.

Animals↗