• View in gallery
    Figure 1—

    Median ± median deviation for the 3H-thymidine incorporation index in PBMCs incubated with lactoferrin (200 ng/mL [black bar]), LPS (1 μg/mL [cross-hatched bar]), or lactoferrin (200 ng/mL) followed 1 hour later by LPS (1 μg/mL [diagonal-striped bar]). The PBMCs were obtained from 8 calves. The 3H-thymidine incorporation index was determined by subtracting the CPM for the control cells from the CPM for the treated cells and was standardized on the basis of the CPM for the control cells. *Value differs significantly (P < 0.05) from values for other treatments.

  • View in gallery
    Figure 2—

    Mean ± SD fold increase in gene expression for COX-2 (A) and MMP-9 (B) in PBMCs incubated with no treatment (control group [gray bar]), lactoferrin (200 ng/mL [black bar]), LPS (1 μg/mL [cross-hatched bar]), or lactoferrin (200 ng/mL) followed 1 hour later by LPS (1 μg/mL [diagonal-striped bar]). The PBMCs were obtained from 5 and 4 calves for panels A and B, respectively. Results represent the fold increase in gene expression and were standardized on the basis of gene expression for a housekeeping gene (GAPDH). *,†Value differs significantly (*P = 0.006; †P = 0.005), compared with the value for PBMCs treated with LPS alone.

  • View in gallery
    Figure 3—

    Median ± median deviation PGE2 production by PBMCs incubated with lactoferrin (200 ng/mL [black bar]), LPS (1 μg/mL [cross-hatched bar]), or lactoferrin (200 ng/mL) followed 1 hour later by LPS (1 μg/mL [diagonal-striped bar]). The PBMCs were obtained from 6 calves. Results are reported as the fold increase for the PGE2 stimulation index. Data were standardized on the basis of values for PGE2 production in control cells by use of the following equation: PGE2 production = (PGE2 for treated PBMCs – PGE2 for control cells)/PGE2 for control cells. *Value differs significantly (P < 0.05), compared with the value for PBMCs treated with LPS alone.

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In vitro effects of lactoferrin on lipopolysaccharide-induced proliferation, gene expression, and prostanoid production by bovine peripheral blood mononuclear cells

Maisie E. DawesDepartment of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri, Columbia, MO 65211.

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Jeff W. TylerDepartment of Veterinary Medicine and Surgery, College of Veterinary Medicine, University of Missouri, Columbia, MO 65211.

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Antoinette E. MarshDepartment of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri, Columbia, MO 65211.

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Robert L. LarsonDepartment of Veterinary Extension, College of Veterinary Medicine, University of Missouri, Columbia, MO 65211.

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Barry J. SteevensDepartment of Animal Sciences, College of Agriculture, Food and Natural Resources, University of Missouri, Columbia, MO 65211.

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Jeffrey LakritzDepartment of Veterinary Medicine and Surgery, College of Veterinary Medicine, University of Missouri, Columbia, MO 65211.

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Abstract

Objective—To evaluate the effect of lactoferrin on lipopolysaccharide (LPS)-induced proliferation of bovine peripheral blood mononuclear cells (PBMCs), gene expression of inflammatory mediators, and production of prostanoids in vitro.

Sample Population—PBMCs isolated from 15 Holstein bull calves.

Procedures—Mixed populations of PBMCs were isolated by differential centrifugation. Proliferation assays were conducted in 96-well plates designed to allow addition of lactoferrin (200 ng/mL) with and without LPS (1 μg/mL) in a checkerboard design. Incorporation of 3H-thymidine was used to determine proliferation of PBMCs. Prostaglandin E2 production was determined in culture-conditioned medium by use of enzyme immunoassay. Effects of lactoferrin on LPS-induced gene expression of cyclooxygenase (COX)-2 and matrix metalloproteinase (MMP)-9 were monitored by use of PCR assays.

Results—Lactoferrin supplementation significantly reduced LPS-induced incorporation of 3H-thymidine and production of prostaglandin E2 by PBMCs. Lactoferrin reduced LPS-induced expression of COX-2 and MMP-9 mRNA.

Conclusions and Clinical Relevance—Lactoferrin reduced LPS-induced cellular proliferation, inflammatory mediator gene expression, and prostaglandin E2 production by bovine PBMCs in vitro. These effects may be beneficial in reducing the impact of endotoxemia in neonates.

Abstract

Objective—To evaluate the effect of lactoferrin on lipopolysaccharide (LPS)-induced proliferation of bovine peripheral blood mononuclear cells (PBMCs), gene expression of inflammatory mediators, and production of prostanoids in vitro.

Sample Population—PBMCs isolated from 15 Holstein bull calves.

Procedures—Mixed populations of PBMCs were isolated by differential centrifugation. Proliferation assays were conducted in 96-well plates designed to allow addition of lactoferrin (200 ng/mL) with and without LPS (1 μg/mL) in a checkerboard design. Incorporation of 3H-thymidine was used to determine proliferation of PBMCs. Prostaglandin E2 production was determined in culture-conditioned medium by use of enzyme immunoassay. Effects of lactoferrin on LPS-induced gene expression of cyclooxygenase (COX)-2 and matrix metalloproteinase (MMP)-9 were monitored by use of PCR assays.

Results—Lactoferrin supplementation significantly reduced LPS-induced incorporation of 3H-thymidine and production of prostaglandin E2 by PBMCs. Lactoferrin reduced LPS-induced expression of COX-2 and MMP-9 mRNA.

Conclusions and Clinical Relevance—Lactoferrin reduced LPS-induced cellular proliferation, inflammatory mediator gene expression, and prostaglandin E2 production by bovine PBMCs in vitro. These effects may be beneficial in reducing the impact of endotoxemia in neonates.

Gram-negative bacterial infections are common in cattle. These infections are associated with the release of a wide spectrum of proinflammatory mediators, including cytokines and prostanoids, which are manifested clinically as endotoxemia.1–3

Lactoferrin is an 80-kd non–heme-associated ironbinding glycoprotein of the serum transferrin gene family. Lactoferrin is capable of maintaining environments unfavorable for bacterial growth through its ability to bind and sequester iron.4 Lactoferrin also has high affinity for lipid A, which ultimately limits interaction of bacterial cell wall components with host pattern-recognition receptors.5–8 Lactoferrin is a key component of glandular epithelial secretions, including those of the mammary glands, reproductive tract, salivary glands, and lacrimal glands. Lactoferrin is also a component of neutrophil granules and provides a source of preformed lactoferrin.5–9 In mastitis in cattle, mammary gland concentrations of lactoferrin are increased as a result of increased production by mammary gland epithelia and recruited neutrophils.5–9 In contrast, increases in serum concentrations of lactoferrin have been linked to neutrophilia.9

The purpose of the study reported here was to determine whether in vitro treatment with lactoferrin would inhibit LPS-induced cellular proliferation, gene expression, and prostanoid production in bovine PBMCs. We hypothesized that lactoferrin would inhibit LPS-induced cellular proliferation of PBMCs and alter the host inflammatory responses commonly detected for gram-negative bacterial infections. In our in vitro experiments, we targeted mechanisms that included binding of LPS and PBMC production of proinflammatory mediators. Our objective was to provide the basis for further studies aimed at establishing lactoferrin as a therapeutic alternative for attenuating LPS-induced systemic disease in cattle. Natural products, such as lactoferrin, that modulate neonatal bovine immune responses to LPS may be beneficial for use in controlling inflammation.

Materials and Methods

Sample population—Blood samples collected from 15 Holstein bull calves were used in the study. Birth of each of these calves was observed, and each calf was provided colostrum by use of an orogastric feeder shortly after birth. Calves were raised in the Food Animal Clinic of the College of Veterinary Medicine at the University of Missouri. Calves were housed separately and fed 2 L of a commercial milk replacer twice daily until they were 8 weeks old. Calves were allowed ad libitum access to choice alfalfa hay, a commercial calf starter ration, and water. Blood samples were collected from calves for use in lymphocyte proliferation analysis (calves 1 to 8; 1 to 4 months of age), PCR assay of COX-2 expression (calves 9 to 12 and 15; 4 to 8 months of age), PCR assay of MMP-9 expression (calves 9 to 12; 4 to 8 months of age), and measurement of PGE2 concentrations (calves 9 to 14; 4 to 11 months of age). The experiments were evaluated and approved by the University of Missouri Institutional Animal Care and Use Committee.

Isolation of PBMCs and initial culture—Blood samples (50 mL) were collected by jugular venipuncture into tubes containing acid citrate dextrose (acid citrate dextrose-to-blood ratio, 1:9). Blood was centrifuged at 1,000 × g for 15 minutes at 23°C. After centrifugation, PBMCs were isolated by initial harvest of buffy coats.10 Buffy coats were suspended in HBSSa and layered over an equal volume of nonionic, tri-iodated, water-soluble, low-density gradient mediumb (specific gravity, 1.083). Samples were centrifuged at 342 × g for 30 minutes at 23°C, and cells suspended between the HBSS and nonionic, tri-iodated, water-soluble, lowdensity gradient medium were aspirated with a pipette. Following harvest, cells were washed twice, resuspended in HBSS, and placed on ice. Viability of PBMCs was determined by exclusion of 0.1% trypan blue dye.11 Differential cytologic examination was performed by counting 200 cells prepared by use of a cytocentrifuge systemc and stained with Wright stain.

PBMC treatment—Mixed populations of PBMCs (4 × 106 cells) isolated from each of the calves were placed into 12-well culture plates in 2 mL of RPMI 1640d supplemented with 10% heat-inactivated FBS,e 0.1mM nonessential amino acids,f 2mM L-glutamine,g 55μM 2-mercaptoethanol,h and penicillin-streptomycini (50 U/mL and 50 μg/mL, respectively). After isolation, cells were cultured for 24 hours at 37°C in an atmosphere of 5% carbon dioxide. After culture for 24 hours, medium in wells was removed and replaced with 1 mL of fresh RPMI 1640 with FBS and supplements. Nonadherent cells in the removed media were pelleted by centrifugation (117 × g for 5 minutes at 23°C), and the pellet was resuspended in 1 mL of RPMI 1640 with FBS and supplements. Viability of nonadherent cells was evaluated by trypan blue dye exclusion, and the remaining suspension was added to the appropriate wells (final volume, 2 mL). Viability of adherent cells was assessed subjectively by visual examination of these cells in the tissue culture plates.

Cell treatments included a control treatment (no treatment), lactoferrinj (200 ng/mL), LPS stimulation (Escherichia coli strain O55:B5k; 1 μg/mL), and treatment with lactoferrin (200 ng/mL) followed by LPS stimulation (1 μg/mL). For the latter, lactoferrin was added to wells 1 hour before LPS was added; lactoferrin remained in the wells for the duration of the experiment.

Incorporation of 3H-thymidine by proliferating PBMCs—The PBMCs from calves 1 to 8 were used for proliferation studies, as described12 but with modifications. After addition of each treatment, cells remained in culture for 48 hours, after which 3H-thymidinel (1 μCi/well) was added to each well. Twenty-four hours later, contents of each well were thoroughly mixed by pipetting, and 100 μL of medium and cells were transferred to 96-well tissue culture plates (in triplicate). Cells were harvested onto filter matsm by use of an automated cell harvester.n Wells were not scraped prior to transfer of suspended cells to ensure evaluation of nonadherent cellular proliferation. Radioactivity retained in the filter mat, expressed as mean CPM, was determined by use of a workstation.o Incorporation of 3H-thymidine by PBMCs (in CPM) was converted to the incorporation index by standardizing each treatment to results of control cell incubations conducted concurrently by use of the following equation:

article image
where CPM for treated PBMCs is 3H-thymidine incorporation for each cell treatment (ie, control treatment, lactoferrin, LPS stimulation, and lactoferrin followed by LPS stimulation), and CPM for control cells is 3H-thymidine incorporation into PBMCs incubated in tissue culture medium alone. The proliferation index was used as a means to standardize fold increases in proliferation among calves.

Gene expression—The PBMCs from calves 9 to 12 and 15 were used to evaluate COX-2 gene expression, whereas PBMCs isolated from calves 9 to 12 were used to evaluate MMP-9 gene expression. Cells were cultured by use of the same conditions as described for the proliferation experiments, except that isolated PBMCs were cultured for 24 hours in medium after addition of treatments.13,14 Twenty-four hours after addition of treatments (control treatment, lactoferrin, LPS stimulation, and lactoferrin followed by LPS stimulation), culture medium was aspirated and centrifuged (117 × g) to enable collection of nonadherent cells; supernatant was aspirated to leave only the pellets of the nonadherent cells. Adherent cells were immediately lysed with a monophasic solution of phenol and guanidine isothiocyanatep and incubated for 5 minutes on ice. The solution for the lysed adherent cells was aspirated and added to each tube containing the nonadherent cell pellet. The resulting total RNA preparation was purified by use of phenol-chloroform extraction and ethanol precipitation, as described elsewhere.13,15 Concentration and purity of RNA was determined by use of a spectrophotometer, and 2 μg of RNA was used to generate cDNA in reverse transcriptase reactions.13,14 Expression of COX-2, MMP-9, and GAPDH was determined by use of PCR assays, as described elsewhere.13,14 Briefly, cDNA was prepared by addition of 0.5 μg of oligo-(dT)12–18, PCR buffer (20mM Tris HCl [pH, 8.4] and 50mM KCl), 2.5mM MgCl2, 10mM dithiothreitol, 0.2mM of each dNTP, and 50 units of MuLV–RT (final volume, 50 μL). A negative control sample was assayed simultaneously by substituting diethylpyrocarbonate-treated water for the MuLV–RT to control for genomic DNA. Cycling conditions for the cDNA synthesis were 2 minutes at 42°C, 50 minutes at 42°C, 15 minutes at 70°C, and 5 minutes at 5°C.13 Cycling conditions for PCR assays were as described elsewhere,13,14 with minor modifications (Appendix). The PCR assays for each calf and each gene were conducted simultaneously. The PCR amplification consisted of 5 μL of reverse transcription template or water, 1.5mM MgCl2, 1 × PCR buffer, 0.2mM of each dNTP, 10μM of each primer, and 1.5 units of Taq polymerase (final volume, 100 μL), with the aforementioned thermocycler conditions.13,14 Products of PCR amplification (COX-2, 449 bp; MMP-9, 658 bp; and GAPDH, 468 bp) were analyzed on ethidium bromide–stained 2% agarose gels. Scanned gels were analyzed by inverting each gel image such that product bands appeared black on a white background. Stored images were analyzed by use of an image-analysis program.q Gels were evaluated such that the gene product of each inflammatory mediator and corresponding GAPDH were scanned concurrently. Band density was determined as peak area for each gene and standardized on the basis of the peak area for GAPDH. The ratios of COX-2 to GAPDH or MMP-9 to GAPDH were adjusted by dividing the GAPDH-standardized gene expression for COX-2 and MMP-9 to provide fold increases (or fold decreases) in gene expression by use of the following equation13:

article image

Production of PGE2—For samples obtained from calves 9 to 14, supernatants of cell cultures of PBMC incubations used for gene expression experiments were evaluated for PGE2 production by use of a competitive enzymeimmunoassay.13,r The assay was based on the addition of sample or standards containing PGE2, which compete with PGE2-acetylcholinesterase conjugate (tracer) for binding to a monoclonal anti-PGE2 antibody. Plates coated with goat polyclonal anti-mouse antibody were used, and capture monoclonal antibody bound to PGE2 or PGE2-acetylcholinesterase conjugate were added to each well. Absorbance of each well was proportional to the concentration of bound PGE2-acetylcholinesterase conjugate, which was inversely proportional to the PGE2 concentration in the sample or standard.r

Each sample was diluted 1:100 with assay sample buffer, and aliquots (50 μL) were analyzed in triplicate in accordance with the manufacturer's recommendations. An 8-point standard curve was established with known concentrations of PGE2 by serial dilution of a stock solution (10 ng/mL; provided with the kit); serial dilutions were achieved by use of assay sample buffer. To each well, 50 μL of PGE2-acetylcholinesterase conjugate and 50 μL of monoclonal anti-PGE2 were added. Values for standards and samples were determined simultaneously in an identical manner. After addition of culture supernatants to wells, plates were covered with plastic film and incubated for 18 hours at 4°C in the dark. Wells were emptied and rinsed 5 times with wash buffer, after which binding of PGE2-acetylcholinesterase conjugate was determined by use of 200 μL of an acetylcholinesterase substrate (5,5′-dithio-bis-[2-nitrobenzoic acid]).

Blank wells (empty wells) and wells for total activity (5 μL of PGE2-acetylcholinesterase conjugate), NSB (50 μL of PGE2-acetylcholinesterase conjugate), and maximal binding (ie, B0; 50 μL of PGE2-acetylcholinesterase conjugate and monoclonal anti-PGE2 antibody) were included for calculation of PGE2 concentrations. Values for B0 were corrected by determining the mean absorbance values for duplicate wells and subtracting the mean of the NSB wells. For each standard concentration and each sample, the ratio of the percentage of PGE2 bound versus the Bo value was determined by subtracting absorbance for NSB from the absorbance for the standard or sample and dividing the difference by the corrected value for B0 (ie, B0 – NSB). These ratios were then multiplied by 100 to determine the percentage of bound PGE2. The data were entered into a spreadsheet and subjected to 4-point logistic regression analysis in accordance with the manufacturer's recommendations. Data were standardized on the basis of values for PGE2 production in control cells to account for animal-to-animal and day-to-day variation by use of the following equation: PGE2 production = (PGE2 for treated PBMCs – PGE2 for control cells)/PGE2 for control cells. The stimulation indices were logarithmically transformed for data analysis.

Statistical analysis—Normality of proliferation data was evaluated by use of the Kolmogorov-Smirnov test, and equal variance was evaluated by use of the Levene median test. Proliferation indices were logarithmically transformed and compared by use of a Kruskal-Wallis 1-way ANOVA on ranks. Differences between treatments were considered significant at values of P < 0.05. The LPS-induced gene expression and effects of lactoferrin on basal and LPS-induced gene expression were adjusted on the basis of results for GAPDH. The ratio of control or treated cellular gene expression was logarithmically transformed and compared by use of a 1-way ANOVA, with post hoc testing with the Holm-Sidak test. Concentrations of PGE2 were compared by use of a 1-way ANOVA.

Results

Cell culture, cell viability, and differential cell counts—Mean ± SD cell viability after isolation was approximately 89.4 ± 7.3% (range, 74% to 98%). Cellular differential counts for lymphocytes and monocytes revealed approximately 85 ± 9% (range, 70% to 97%) and 15 ± 9% (range, 3.5% to 22%), respectively. Cellular viability of nonadherent cells after 24 hours of culture was 94.4 ± 4% (range, 89% to 100%).

Effect of lactoferrin on 3H-thymidine incorporation—Stimulation indices (standardized on the basis of results for control cells analyzed simultaneously) revealed that LPS induced bovine PBMCs to incorporate 3H-thymidine, compared with results for lactoferrin-treated cells (Figure 1). In PBMCs treated with lactoferrin followed by LPS, significantly lower amounts of 3H-thymidine were incorporated, compared with results for LPS-treated cells. The proliferation index for lactoferrin-treated PBMCs did not differ significantly from that for the control wells.

Figure 1—
Figure 1—

Median ± median deviation for the 3H-thymidine incorporation index in PBMCs incubated with lactoferrin (200 ng/mL [black bar]), LPS (1 μg/mL [cross-hatched bar]), or lactoferrin (200 ng/mL) followed 1 hour later by LPS (1 μg/mL [diagonal-striped bar]). The PBMCs were obtained from 8 calves. The 3H-thymidine incorporation index was determined by subtracting the CPM for the control cells from the CPM for the treated cells and was standardized on the basis of the CPM for the control cells. *Value differs significantly (P < 0.05) from values for other treatments.

Citation: American Journal of Veterinary Research 69, 9; 10.2460/ajvr.69.9.1164

Effect of lactoferrin on LPS-induced gene expression—Resting PBMCs cultured in vitro revealed variations in COX-2 gene expression, which was markedly less than that for LPS-treated PBMCs (Figure 2). Cells treated with lactoferrin did not differ significantly from control PBMCs with regard to COX-2 gene expression. Stimulation of PBMCs by LPS was associated with significant increases in COX-2 gene expression, compared with gene expression for control cells (mean ± SD increase of 5 ± 3-fold; P = 0.006) and lactoferrin-treated PBMCs (mean increase of 4.5 ± 1.8-fold; P = 0.006). Treatment with LPS induced a significant (P = 0.005) increase of 9 ± 6-fold in COX-2 gene expression, compared with gene expression in PBMCs treated with lactoferrin followed by LPS.

Figure 2—
Figure 2—

Mean ± SD fold increase in gene expression for COX-2 (A) and MMP-9 (B) in PBMCs incubated with no treatment (control group [gray bar]), lactoferrin (200 ng/mL [black bar]), LPS (1 μg/mL [cross-hatched bar]), or lactoferrin (200 ng/mL) followed 1 hour later by LPS (1 μg/mL [diagonal-striped bar]). The PBMCs were obtained from 5 and 4 calves for panels A and B, respectively. Results represent the fold increase in gene expression and were standardized on the basis of gene expression for a housekeeping gene (GAPDH). *,†Value differs significantly (*P = 0.006; †P = 0.005), compared with the value for PBMCs treated with LPS alone.

Citation: American Journal of Veterinary Research 69, 9; 10.2460/ajvr.69.9.1164

Furthermore, LPS treatment resulted in a significant increase (> 2-fold) in MMP-9 gene expression, compared with gene expression for control cells (Figure 2). The MMP-9 gene expression for PBMCs treated with lactoferrin followed by LPS was significantly (P = 0.005) reduced, compared with gene expression of PBMCs treated with LPS alone. Peripheral blood mononuclear cells treated with nothing (control) and lactoferrin each expressed significantly (P = 0.006) less MMP-9 gene than that expressed by cells treated with LPS. The MMP-9 gene expression for PBMCs treated with lactoferrin followed by LPS did not differ significantly from gene expression for PBMCs incubated with lactoferrin alone or from gene expression for control cells.

Effect of lactoferrin on production of PGE2—Concentrations of PGE2 produced by control cells ranged from 13 to 49 ng/mL. Addition of lactoferrin to PBMCs did not significantly alter basal PGE2 production. The LPS-treated PBMCs produced significantly more PGE2 than did control cells and lactoferrin-treated PBMCs (Figure 3). Treatment with lactoferrin followed by LPS stimulation significantly attenuated production of PGE2, compared with production by PBMCs treated with LPS alone.

Figure 3—
Figure 3—

Median ± median deviation PGE2 production by PBMCs incubated with lactoferrin (200 ng/mL [black bar]), LPS (1 μg/mL [cross-hatched bar]), or lactoferrin (200 ng/mL) followed 1 hour later by LPS (1 μg/mL [diagonal-striped bar]). The PBMCs were obtained from 6 calves. Results are reported as the fold increase for the PGE2 stimulation index. Data were standardized on the basis of values for PGE2 production in control cells by use of the following equation: PGE2 production = (PGE2 for treated PBMCs – PGE2 for control cells)/PGE2 for control cells. *Value differs significantly (P < 0.05), compared with the value for PBMCs treated with LPS alone.

Citation: American Journal of Veterinary Research 69, 9; 10.2460/ajvr.69.9.1164

Discussion

Gram-negative bacterial infections are common in livestock and are often severe in neonates. The processes leading to morbidity result from exaggerated responses of the host innate immune system to gramnegative bacteria or their cellular products.16 Lipopolysaccharide is a component of the outer membrane of gram-negative bacterial cell walls, is mitogenic, and has profound effects on the humoral and cellular immune systems.17 During endotoxemia, systemic generation of proinflammatory molecules subsequent to activation of mononuclear cells and the culmination of a myriad of intracellular processes, including gene and protein expression, protein secretion, and activation of other cellular processes, lead to septic shock.18–20 Septic shock has been associated with cell-generated cytokines, chemokines, and eicosanoid mediators.18,19 These soluble molecules also play roles in cellular proliferation.21

The objectives of the study reported here were to determine the ability of lactoferrin to alter LPS-induced 3 H-thymidine incorporation by PBMCs as a measure of cell proliferation. We also evaluated whether lactoferrin would modify LPS-induced production of proinflammatory cytokines and lipid mediators in vitro. The experimental design was based on the cellular effects of bacterial LPS in the presence of lactoferrin. Several factors were assumed during the performance of these experiments. First, 3H-thymidine incorporation into PBMCs subsequent to LPS stimulation requires antigen-presenting cells (monocytes) and lymphocytes. Incorporation of 3H-thymidine was used as an end point indicative of DNA replication. Second, PBMC proliferation requires iron provided through interactions with transferrin in vitro. Third, interactions of LPS with the acute-phase reactant, LPS-binding protein, enhance LPS-induced activation of monocytes, which are the most efficient activator of differentiated effector lymphocytes and one of the major antigen-presenting cells of hosts.17,18 Fetal bovine serum is a natural source of both transferrin and LPS-binding protein.22,23 The in vitro assay included all factors required for cellular proliferation to determine whether lactoferrin modulates LPS-induced proliferation of bovine PBMCs.22,23 Fourth, the mixed population of PBMCs was appropriate for evaluation of 3H-thymidine incorporation of nonadherent cells, which are primarily lymphocytes. Gene expression and prostanoid evaluations represented contributions of all PBMCs in each sample. We assumed that lymphocytes would proliferate, and this proliferation was the result of gene expression and prostanoid production of all PBMCs.

Results of our PBMC proliferation experiments differed from those of other investigations in terms of maximal proliferation indices after addition of LPS.17 We attributed these differences to several factors. Maximal LPS stimulation is believed to require serum LPS-binding protein. Because our study used heat-inactivated FBS, reduction of the serum activity of LPS-binding protein could be expected.23 To improve the interaction of LPS with cellular receptors, we dissolved LPS in FBS to solubilize aggregate LPS prior to incubation with cells, as described elsewhere.24 Furthermore, the concentration of LPS (1 μg/mL) used in our incubations was relatively high, which should have improved exposure of cells to this stimulus. Despite our efforts to limit the impact of heat-treated FBS, these manipulations may have resulted in lower 3H-thymidine incorporation indices. However, the general response of LPS-treated PBMCs was to induce 3H-thymidine incorporation, gene expression, and prostanoid production. The general response to treatment of PBMCs with lactoferrin followed by LPS stimulation was to significantly reduce LPS-induced effects.

In our study, lactoferrin (200 ng/mL) significantly reduced 3H-thymidine incorporation induced by LPS. The reduction in LPS-induced 3H-thymidine incorporation associated with lactoferrin treatment of PBMCs in vitro suggested that lactoferrin may be capable of inhibiting LPS-stimulated proliferation of PBMCs in vivo. We attributed this to the reported binding affinity of lactoferrin for LPS, which prevents interaction between LPS and cells bearing appropriate receptors.25 These results are strengthened by results of another study26 in which supplemental lactoferrin provided immediately after birth was absorbed systemically. Increases in serum concentrations of lactoferrin may be useful in reducing the systemic effects of LPS on neonates.26

The assay was not able to discriminate between the effects of lactoferrin on LPS binding and lactoferrin-mediated iron sequestration on reduced cellular incorporation of 3H-thymidine. To evaluate whether lactoferrin sequestration of iron in the media results in reduced 3H-thymidine incorporation, it would have been necessary to quantitate the amount of iron and transferrin in the media and determine the saturation of transferrin and lactoferrin prior to addition of LPS.27,28 Furthermore, it would have required us to conduct experiments to evaluate unscheduled DNA synthesis in the absence of cell division. This was beyond the scope of the study reported here.

The ability of lactoferrin to interfere with LPS-induced 3H-thymidine incorporation by PBMCs was also associated with diminished expression of COX-2, which is the inducible form of COX responsible for PG production. These results were supported by the reduction in the LPS-induced production of PGE2 by lactoferrin-treated PBMCs. The LPS-induced expression of COX-2 and production of PGE2 by bovine alveolar macrophages in vitro have been reported.13 Induction of COXs is associated with availability of substrates resulting from activation of membrane phospholipases.13 Activation of phospholipase is greatly enhanced by bacterial LPS.13,29,30 Results of our in vitro study support the possibility that lactoferrin interferes with LPS-induced production of prostanoids through a mechanism involving the expression of an LPS-inducible gene (ie, COX-2). In other studies, investigators have reported that prostanoids play a role in physiologic and morphologic changes associated with gram-negative bacterial infections.31,32 Inhibition of LPS binding may reduce the systemic effects of endotoxin on a host, including release of arachidonic acid from membranes and production of bioactive prostanoids. Studies evaluating the amount (if any) of inducible COX protein would provide information regarding this mechanism.

In another study14 conducted by our laboratory group, we determined that the expression of the MMP-9 gene and MMP-9 protein by bovine alveolar macrophages in vitro is induced by exposure to bacterial LPS. The production and release of this protease in cattle have been associated with pneumonia caused by gramnegative bacteria.33 In the study reported here, we determined that treatment of PBMCs with lactoferrin followed by LPS stimulation inhibited LPS-induced MMP-9 gene expression. Unfortunately, we could not easily study the protein expression of MMP-9 by PBMCs for reasons related to cellular growth requirements. The experimental design hinged on a source of LPS-binding protein, a natural constituent of FBS. Serum concentrations of MMP-9 are usually low but can be measured zymographically and immunologically. However, we were not convinced that detection of differences in MMP-9 protein produced by PBMCs, compared with MMP-9 protein contained in FBS, would have sufficient sensitivity to warrant further evaluation of this protein.

In another study21 conducted to evaluate the effects of human lactoferrin on lymphocyte proliferation, investigators reported that lactoferrin mitigates the proliferative effects of high concentrations of iron, which suggests that lactoferrin has a protective effect mediated through binding of iron. Because we were more interested in determining the ability of lactoferrin to modulate the effect of LPS, similar investigations were beyond the scope of the study reported here. The role of lactoferrin in mitigating LPS-induced cellular production of cytokine has been evaluated,21 and analysis of results indicates that lactoferrin has the ability to prevent LPS interaction with cells.

Data for the study reported here supported a role of lactoferrin in antagonism of LPS-induced 3H-thymidine incorporation, gene expression, and mediator production in PBMCs. We presumed this to be via the ability of lactoferrin to bind LPS. However, other mechanisms in which expression of inflammatory mediators may be inhibited should not be disregarded without further investigation.34 Lactoferrin inhibition of LPS-induced cellular effects is not fully understood; however, binding and sequestration of lipid A may be an important and natural way to reduce the systemic effects of endotoxin. Lactoferrin inhibition of bacterial growth by sequestration of iron may also limit the effects of sepsis on neonates.35 Analysis of our results suggests that lactoferrin modulates LPS-induced cellular effects and corroborates results of other studies4,34,35 in which investigators indicated that lactoferrin has multiple functions in vitro. Results of the study reported here provide in vitro data that suggest lactoferrin may be effective in limiting some aspects of gram-negative bacterial inflammation in cattle.

ABBREVIATIONS

COX

Cyclooxygenase

CPM

Counts per minute

dNTP

Deoxynucleoside triphosphate

FBS

Fetal bovine serum

GAPDH

Glyceraldehyde-3-phosphate dehydrogenase

HBSS

Hanks' balanced salt solution

LPS

Lipopolysaccharide

MMP

Matrix metalloproteinase

MULV–RT

Muloney murine leukemia virus–reverse transcriptase

NSB

Nonspecific binding

PBMC

Peripheral blood mononuclear cell

PG

Prostaglandin

a.

Hanks' balanced salt solution, Invitrogen Corp, Carlsbad, Calif.

b.

Accu-Paque lymphocytes, mammalian & fish, cell separation media, Accurate Chemical & Scientific Corp, Westbury, NY.

c.

Cytospin, Shandon Thermo-Fisher Scientific, Waltham, Mass.

d.

RPMI-1640, GIBCO Invitrogen cell culture media, Invitrogen Corp, Carlsbad, Calif.

e.

Fetal bovine serum, certified, heat-inactivated (US), Invitrogen Corp, Carlsbad, Calif.

f.

MEM non-essential amino acids solution, 10mM (100×) liquid, Invitrogen Corp, Carlsbad, Calif.

g.

L-glutamine, 200mM (100×) liquid, Invitrogen Corp, Carlsbad, Calif.

h.

2-mercaptoethanol (1,000×) liquid, 55μM in D-PBS, Invitrogen Corp, Carlsbad, Calif.

i.

Penicillin-streptomycin, liquid containing 10,000 units of penicillin and 10,000 μg of streptomycin, Invitrogen Corp, Carlsbad, Calif.

j.

Colostral lactoferrin, affinity purified, New Zealand Milk Products North America Inc, Santa Rosa, Calif.

k.

Lipopolysaccharide, Escherichia coli O55:B5, TCA purification, Sigma-Aldrich Co, St Louis, Mo.

l.

3H-Thymidine, Sigma Chemical Co, St Louis, Mo.

m.

TOMTEC, Harvester 96 accessories, TOMTEC Inc, Camden, NH.

n.

TOMTEC, Harvester 96, TOMTEC Inc, Camden, NH.

o.

Wallac Microbeta, GMI Inc, Ramsey, Minn.

p.

TRI-zol reagent, Invitrogen Corp, Carlsbad, Calif.

q.

Scion Image for Windows (release Beta 3b), Scion Corp, Frederick, Md.

r.

Prostaglandin E2 EIA kit—monoclonal, Cayman Chemical, Ann Arbor, Mich.

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Appendix

Appendix

Primers and cycling conditions used in PCR assays to detect gene expression for COX-2, MMP-9, and the housekeeping gene GADPH in RNA isolated from PBMCs obtained from 4 calves.

GeneGenBank accession No.Product length (bp)Primer sequenceCycling conditions
COX-2AF004944449Sense: ACC AAC TGG GAC GAC ATG GAG; Antisense: TCT TTG ACT GTG GGA GGA TAC A1 cycle at 94°C for 10 minutes; 35 cycles at 94°C for 1 minute, 60°C for 1 minute, and 72°C for 2 minutes; and 1 cycle at 72°C for 10 minutes
GADPHU85042468Sense: GAT GCT GGT GCT GAG TAT GTA GTG; Antisense: ATC CAC AAC AGA CAC GTT GGG AG1 cycle at94°C for 10 minutes; 32 cycles at94°C for 1 minute, 54°C for 1 minute, and 72°C for 2 minutes; and 1 cycle at72°Cfor 10 minutes
MMP-9X78324658Sense: CGA CGA TGA AGA GTT GTG GT; Antisense: GTA CAT GGG GTA CAT GAG CG1 cycle at 94°C for 10 minutes; 30 cycles at 94°C for 45 seconds, 55°C for 30 seconds, and 72°C for 90 seconds; and 1 cycle at 72°C for 7 minutes

Contributor Notes

Dr. Dawes' present address is College of Veterinary Medicine, Western University of Health Sciences, Pomona, CA 91766.

Dr. Marsh's present address is Department of Veterinary Preventive Medicine, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210-4007.

Dr. Larson's present address is Department of Clinical Sciences, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506.

Dr. Lakritz's present address is Department of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210-4007.

Supported by grants from the University of Missouri Department of Veterinary Medicine and Surgery Committee on Research, University of Missouri College of Veterinary Medicine Committee on Research, and USDA National Research Initiative Competitive Grants Program (agreement No. 2001–35204–10799).

Address correspondence to Dr. Lakritz.