(CANCER RESEARCH 47, 4184-4188, August 1, 1987)

Protective Influence of Lactoferrin on Mice Infected with the Polycythemia-inducing Strain of Friend Virus Complex1

Li Lu,2 Giao Hangoc, Allen Oliff, Li T. Chen, Rong-Nian Shen, and Hal E. Broxmeyer

Departments of Medicine (Hematology/Oncology) fL. L., G. H., H. E. B.¡,Microbiology and Immunology [H. E. B.J, Radiation Oncology [R-N. S.J, the WaltherMedical Research Institute and the Elks Cancer Center, [H. E. B.J, Indiana University School of Medicine, Indianapolis, Indiana 46223; Cancer Research, Merck,Sharp, and Dohme Research Laboratories, Westpoint, Pennsylvania 19486 ¡A.O.J; and Department of Anatomy, University of South Florida, College of Medicine,Tampa, Florida 36612 [L. T. C.J.

ABSTRACT

Purified iron-saturated human lactoferrin (LF) was assessed in vivofor effects on the survival rates of C57BL x DBA/2 f, (hereafter calledBD2F,) (Fv-2") mice and titers of spleen focus-forming viruses (SI-1-V)in BD2F, and DBA/2 (Fv-2") mice inoculated with the polycythemia-inducing strain of the Friend virus complex (FVC-P). LF prolonged thesurvival rates and decreased the titers of SFFV in mice given FVC-P.Titers of SFFV, assayed 14 days after administration of FVC-P, weremeasured by the spleen focus-forming unit assay in secondary mouserecipients. Decreases in titers of SFFV were apparent when LF was givenin vivo as a single bolus dose of 200 ¿igwithin 2 h of the Friend viruscomplex (FVC), or as a total dosage of 200 ug given on days 1, 2, 4, 7,9, and 11 after FVC-P, and to a lesser degree when LF was given as atotal dosage of 200 ¿tgon days 3, 4, 7, 9, and 11 after FVC-P. Nodecreases in titers of SFFV were detected when LF was given up to 3days before or more than 3 days after FVC-P. LF did not appear to bedirectly inactivating the viruses as it did not inactivate the SFFV or theFriend murine leukemia helper virus in vitro. The results suggest thatthe protective effect of LF in vivo is probably due to an action on cellsresponding to the FVC or to an action on cells which influence the cellsresponding to the FVC or which influence the virus. It has been shownelsewhere that LF decreases the percentage of marrow and spleen he-matopoietic progenitor cells that are in DNA synthesis in rivo and thismay be the means by which the protective effect of LF is mediated inmice given the FVC.

INTRODUCTION

Biomolecule-cell interactions are involved in the regulationof myelopoiesis and abnormalities in biomolecule-cell interactions are associated with human leukemia (1). The pathophys-iology of murine retrovirus-induced leukemias is known (2),and abnormalities in biomolecule-cell interactions, similar tothose detected in human leukemia, have been noted in miceinfected with the Abelson virus and the Friend virus complex(3-5).

LF3 is a metal-binding glycoprotein synthesized in early cells

of the neutrophilic blood series and found in the secondary(specific) granules of these cells (reviewed in Ref. l). Cells ofthe mononuclear phagocyte lineage, monocytes and macrophages, bind and have specific receptors for LF (6-10), and arole for LF in the regulation of myelopoiesis, mediated in vitrovia decreased release of growth factors from monocytes andmacrophages, has been described by several different groups of

Received 11/25/86; revised 4/8/87; accepted 5/6/87.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1Supported by Public Health Service Grants CA 36464 and CA 36740 (to H.E. B.) from the National Cancer Institute and a grant from the Little Red Door,Marion County Cancer Society, Inc. (to L. L.). G. H. was a visiting investigatorfrom the Veterinary School, Hannover, West Germany, and was supported inpart by the Deutsche Forschungsgemeinschaft during some of these studies.

2To whom requests for reprints should be addressed, at Department ofMedicine. Indiana University School of Medicine, 541 Clinical Drive. Room 379,Indianapolis. IN 46233.

3The abbreviations used are: LF, lactoferrin; FVC-P, polycythemia-inducingstrain of the Friend virus complex, SFFV spleen focus-forming virus; SFFU,spleen focus-forming units; F-MuLV, Friend murine leukemia virus.

investigators (10-18). LF also acts in vivo in mice to decreaserelease of growth factors and to dampen myelopoiesis (11, 19-21). Administration of LF to mice decreases both the cyclingstatus (percentage of cells in DNA synthesis) and the absolutenumbers of granulocyte-macrophage, erythroid, and multipo-tential progenitor cells in the marrow and spleen (19-21). Sinceviral infectivity has been associated with DNA synthesis (2), weassessed the effects of LF in vivo on the course of the Friendvirus disease. LF was found to have a protective effect on thesurvival of mice infected with the FVC-P and this was associatedwith decreased endogenous levels of the spleen focus-formingvirus component of the FVC.

MATERIALS AND METHODS

Mice. C57BL/6 x DBA/2 F, (hereafter called BD2F,, Fv-2") andDBA/2 (Fv-2") female mice, 4-6 weeks old, were purchased from

Cumberland View Farms (Clinton, TN).Lactoferrin. Human milk LF, purchased from Sigma Chemical Co.

(St. Louis, MO), was purified, fully iron saturated (10), and for someof the experiments depleted of endotoxin by removing the material thatgelled in the presence of the Limulus lysate (Sigma) (10). LF wasquantitated using an ¡mmuiu»radiometrieassay for LF prior to use (10).Controls included McCoy's medium 5A (Gibco Laboratories, Grand

Island, NY) or sterile pyrogen-free saline.Friend Virus Complex. SFFV-FRE CL2/F-MuLV clone 201, a

Friend virus complex containing a replication-defective SFFV and areplicating helper F-MuLV clone 201 (5), was grown in NIH/Ola 3T3or Balb/c 3T3 mouse embryo (ihn»blastcell lines. Stocks of virus wereprepared as described elsewhere (5). The virus-containing filtered medium contained approximately 10" plaque-forming units/ml as deter

mined by the XC plaque assay for murine leukemia helper virus andabout 10s SFFU/ml. To prepare spleen extracts from erythroleukemiamice (22) a 10% w/v spleen homogenate, in cold phosphate-bufferedsaline, was centrifugea at 200 x g for 10 min and the supernatant wasfiltered by 0.45-um millipore filter. Spleen extract (0.5 ml) was usedfor the SFFU (23) and the XC plaque (24) assays. For SFFU assay,spleens from secondary mice receiving spleen extracts were fixed inBouin's solution and the yellow focal lesions on the surface of the

spleens scored using an inverted microscope (23).Electron Microscopic Study of the Splenic Tissue. The spleen slices

(1 mm thick) obtained from mice given injections of FVC-P were fixedin 5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) for 48 h. Theslices were washed in cacodylate buffer, dehydrated through gradedctHanoi, and embedded in Epon. Thin tissue sections were stained withuranyl acetate and lead citrate and studied under the electron microscope (Philips 301).

Statistical Analysis. Significant differences between samples weredetermined with the use of the Student's t test. A comparison of survival

curves (2-wcek intervals) was undertaken using the procedure outlinedby Colton (25) with P values given for a 1-tailed test.

RESULTS

Influence of LF on the Survival Rates of BD2F| Mice GivenInjections of FVC-P. Fully iron-saturated purified LF was assessed in 3 separate experiments for its effect on the survival

4184

Research. on February 25, 2020. © 1987 American Association for Cancercancerres.aacrjournals.org Downloaded from

PROTECTIVE EFFECT OF LACTOFERRIN ON FVC-P-INFECTED MICE

rate of BD2F, mice (Fv-2sr) each given a lethal dosage of FVC-

P. In the first experiment, 16 mice were given injections i.p. ofFVC-P. Eight of these mice received LF diluted in McCoy's

medium and administered i.p. for a total dosage of 200 Mgovera period of 12 days in equal concentrations of LF given on day0, 4 h after the FVC-P, and then on days 2, 4, 7, 9, and 11.Control mice were given equal volumes of McCoy's medium

5A i.p. at the same times. All 8 mice given FVC-P and thencontrol medium were dead by 12 weeks after FVC-P inoculation, but 4 of 8 mice receiving FVC-P and then LF were stillalive 52 weeks after FVC-P inoculation, when the experimentwas terminated (survival curve not shown). The differencesbetween the 2 groups were statistically significant (z value of2.62, P< 0.01). In the second experiment (Fig. \A) the protocolwas similar except that the total final dosage of 200 ^g LF wasgiven over the time period of days 1,2,4, 7, 9, and 11. Micegiven inoculations of FVC-P and control medium all were deadwithin 32 weeks of the FVC-P injection; but in the group givenLF, 8 of 15 mice were still alive after 52 weeks, when theexperiment was terminated. The 2 survival curves were significantly different (z value of 2.85, P < 0.01). In the third experiment (Fig. IB) the protocol was similar to the second experiment except that the FVC-P, LF, and control medium wereadministered i.v. rather than i.p. All 20 mice receiving the FVC-P and control medium were dead by 11 weeks after FVC-Pinoculation, but 6 of 20 mice receiving FVC-P and LF werealive 30 weeks after the FVC-P injection when the experimentwas stopped. The two survival curves were statistically different(z value of 2.64, P < 0.01).

Effect of LF on SFFV in BD21 , Mice Given Injections ofFVC-P. Virus particles were detected in the spleens of micegiven FVC and then control medium (Figs. 2 and 3). In orderto better understand the effects of LF on prolongation of the

O—-O FV (Day 0} •LF (200*ig total, days 0-14)

FV (Day 0) * Control Medium

Weeks after Injection with Friend Virus Complex (FVC-P) i.p. with and without

Lactoferrin (LF) A

o—o FV (Day 0] «LF (200(ig total, days 0-14)

FV (Day 0) »Control Medium

6. ^— "20

Weeks after Injection with Friend Virus Complex (FVC-P) i.V. with and without

Lactoferrin (LF) B

Fig. 1. Comparison of the survival curves of HI)2I , mice given injections i.p.(A) or i.v. (B) with the FVC-P and then treated with either control (McCoy's)medium or a total dosage of 200 tig fully iron-saturated purified human LF givenon days 0, 2, 4, 7, 9, and 11. When LF was given on day 0, it was 4 h after theadministration of the FVC-P. Fractions at the end of the survival curves, numberof mice surviving at the end of the experiment over the number of mice in thatgroup.

survival of mice given the FVC-P, LF was assessed in vivo forits effects of SFFV. This was determined by the numbers ofSFFU in secondary BD2F, mice given injections i.v. of extractsof spleens from primary mice first given FVC-P and then giveneither control medium or LF. Mice were given injections ofFVC-P i.v. and then of LF or control medium i.v. as describedin the legend to Table 1. LF decreased significantly the numbersof SFFU when given in a total dosage of 200 ng over days 0-11, 1-11, or 3-11 (Table IA) to the primary mice. DecreasedSFFU in the spleens of the secondary mice correlated well withthe decreased weight of the spleens of these secondary mice(Table \B). After these experiments were performed, it wasfound that both the control McCoy's medium and the LF,which had been diluted in the McCoy's medium, contained

approximately 0.1 to 1.0 ¿igendotoxin per ml, as determinedby the Limulus lysate test. The iron-saturated purified humanLF was depleted of endotoxin (<0.1 ng per injection), dilutedin sterile pyrogen-free saline, and assessed for its effects onspleen focus-forming viruses in mice 15 days after they weregiven FVC-P in comparison to mice given FVC-P and thensterile pyrogen-free saline. Endotoxin-depleted LF had a sup-pressive effect on numbers of SFFU only when administeredfor a total dosage of 200 ^g from days 1 to 11 (days 1, 2, 4, 7,9, and 11) or days 3 to 11 (days 3, 4, 7, 9, and 11) after FVC-P. Starting the administration of LF on day 1 after FVC-P wasmore effective by 1 log unit of virus titer than starting theinjections on day 3 after FVC-P. There were no differences innumbers of SFFU in comparison to mice treated with controlmedium when LF was given on days 7 to 11 after FVC-P, onday 11 after FVC-P, or on days 14 to 3, days 7 to 3 or day 3prior to FVC-P inoculation. When endotoxin-depleted LF wasgiven as a single bolus injection of 200 iig/mouse within 2 hafter inoculation of the FVC-P, LF decreased the numbers ofSFFU (Table 2).

Influence of LF on SFFV in DBA/2 Mice Given Injections ofFVC-P. After the above results were obtained, several groupsof BD2F, mice were found to be ultrasensitive to the FVC-Pand all of these mice died within 1-2 weeks after injection ofFVC-P in comparison to the 11-32 weeks noted in Fig. 1, Aand A, for the survival rate of mice given FVC-P. The differential sensitivity of these BD2Fi mice could not be attributedto the virus preparation since the same frozen stock of viruswas used at the same dilution. Administration of a single bolusdosage of 200 n% LF given within 2 h after the FVC-P or asmultiple injections starting on day 0 had no effect on thesurvival of these latter batches of BD2F, mice that were dyingwithin 2 weeks after FVC-P injection. At this time, we switchedto using DBA/2 mice which are homozygous at the Fv-2 locus(Fv-2ss) for sensitivity to the FVC. Studies with these mice

(Table 3) were similar to our initial studies with the BD2F,mice. In 2 separate experiments giving FVC-P-inoculatedDBA/2 mice a total of 100 MgLF 6 h prior to FVC-P and ondays 2, 4, 6, 9, and 11 after FVC-P and in 4 other separateexperiments giving a bolus dosage of 100 ng LF 2 h prior toFVC-P, endotoxin-depleted LF significantly reduced the numbers of SFFU in these mice.

Effect of LF in Vitro on SFFV and on the F-MuLV. In orderto evaluate whether LF could directly inactivate SFFV or F-MuLV, NIH 3T3 cells producing SFFV and F-MuLV wereincubated for 18 h with McCoy's medium, sterile pyrogen-freesaline, up to 10 ¿tgLF/ml diluted in McCoy's medium, or upto 10 Mgendotoxin-depleted LF/ml. The virus-containing conditioned medium was assessed undiluted and at dilutions of10"' to 10~4 for numbers of SFFU (3 mice/point for each of 2

4185

Research. on February 25, 2020. © 1987 American Association for Cancercancerres.aacrjournals.org Downloaded from

PROTECTIVE EFFECT OF LACTOFERRIN ON FVC-P-INFECTED MICE

Fig. 2. Friend virus particles (arrow) adjacent to a leukemia cell in the spleen of a KD2I ,mouse given injections i.v. 15 days earlier withthe FVC-P and control medium. A, x 14,000,B, x 70,000.

Fig. 3. Budding of a virus particle (arrow)at the surface of a leukemia cell undergoingmitosis in the spleen of a HI>-I , mouse giveninjections i.v. 15 days earlier with the FVC-Pand control medium. A, x 14,000, B, x 70,000.

B

experiments) and undiluted for numbers of plaque-formingunits using the XC plaque assay (2 experiments). LF had noinfluence on numbers of SFFV. Control numbers of SFFU wererespectively too numerous to count, 5±2, 0, and 0 for 10°to10 ' dilutions of virus-containing conditioned medium. The

carry-over effect of LF, which was 1 /¿g/mouse,had no effectin vivo on numbers of SFFV in mice infected with the FVC-P.LF also had no effect on numbers of plaque-forming units/mlwhich were in the range of IO5 for all treatments.

DISCUSSION

The protective effect of LF on survival of mice given injections of FVC-P was closely associated with decreased levels ofSFFV. These results are consistent with morphological studieson the spleens of mice receiving FVC-P (26). Leukemia cells

were found in the red, but not in the white, pulp of the spleen.The white pulp was greatly enlarged and LF decreased greatlythe numbers of leukemia cells in the spleens of these mice.

LF did not inactivate SFFV or F-MuLV directly, as determined by the lack of effect of LF in vitro on these viruses. Thissuggests that the effects of LF in vivo were on the cells that canbe infected by the viruses, and/or on other cells which theninfluence the viruses themselves, or on the cells that can beinfected by the viruses. Since LF decreases the percentage ofmarrow and spleen hematopoietic progenitors in DNA synthesis (19-21) and virus infectivity is associated with the DNA-

synthesis phase of the cell cycle (2), the protective effects of LFprobably relate to its capacity to decrease the cycling status ofthe hematopoietic progenitor cells in vivo. It has been suggestedthat the action of LF in vivo on hematopoietic progenitor cellsis mediated by decreased release of growth factors, since LF

4186

Research. on February 25, 2020. © 1987 American Association for Cancercancerres.aacrjournals.org Downloaded from

PROTECTIVE EFFECT OF LACTOFERRIN ON FVC-P-INFECTED MICE

Table 1 EffectofLF on spleenfocusforming virusesin primary BD2Ftmicegiven injectionsof the FVC-P

BD2F, micewere given injections i.v.of the FVC-P and then givenMcCoy'smedium i.v. on days 0, 2, 4, 7, 9, and 11 or a total final dosage of 200 ng fullyiron-saturated purified human LF i.v. on days 0, 2, 4, 7, 9, and II, days 1, 2, 4,7, 9, and 11,or days 3, 4, 7, 9, and 11. When LF was givenon day 0, it was 4 hafter the administration of FVC-P. Mice were sacrificed 14 days after receivingthe FVC-P and spleen extracts were given to secondaryBD2F| mice in order toevaluatethe numbersof SFFU and the spleenweight.Resultsare the averagesof4 primary mice per group. The spleen extracts from each primary mouse wereassessedin 3 secondarymice.

SFFU per dilution of spleenextractDilution

ofextractA)

SFFU10°IO'1io-2io-3io-4IO"'io-4B)

Weight (g)10°io-1io-2io-3io-io-5io-"FVC-P

+control

mediumTNTC°TNTCTNTCTNTCTNTC15±702.7

±0.12.1±0.21.7±0.10.8±0.10.2

±0.020.1±0.010.1

±0.01FVC-P

+LF(days0-

11)TNTCTNTC6

±2»0*0*0*01.8

±0.1*0.7±0.1*0.1±0.01*0.1

±0.01*FVC-P

+ LF(days1-11)TNTCTNTC7

±3*0*0»0*02.3

±0.1*1.5±0.2*0.1±0.01*0.1

±0.01*FVC-P

+ LF(days3-11)TNTCTNTC24

±4*0*0»0*02.3

±0.1*1.2±0.1*0.2

±0.01*0.1±0.01*

•TNTC, numbersof SFFU were too numerous to count.*Significantlydifferent (/' at least < 0.001) from mice givencontrol medium

when usingsame dilution of spleenextract.

Table 2 Influenceof a singleinjectionof endotoxin-depletedLF givenon day 0on spleenfocusforming virusesinprimary BD2F,micegiven injections

of the FVC-PBD2F, mice were given injections i.v. of the FVC-P and then given one

injection i.v. of either sterile pyrogen-freesaline or 200 *igendotoxin-depletedfully iron-saturated human LF within 2 h of the FVC-P. Six primary mice ineach group were sacrificed15 days later and spleen extracts from each primarymouse were givenby injection i.v. into 2 secondary HI)2F, recipients for measurement of numbersof SFFU.

Dilution ofextract10°io-1

io-2io-3io-1SFFUFVC-P

+salineTNTC*

TNTCTNTC35

±44±1FVC-P

+LFTNTC32

±7*6 ±2»

0.3 ±0.2*0*

' TNTC, SFFU are too numerous to count.*Significantlydifferent (/' at least < 0.001) at that specificdilution of spleen

extract from SFFU of micegivensalinecontrol.

decreases the release in vitro of granulocyte-macrophage colony-stimulating factor ( 12) and of a monokine (13-15), recentlyidentified as interleukin-1 (27-29), which then trigger othercells such as fibroblasts, endothelial cells, and T-lymphocytesto release factors that stimulate colony formation in vitro bygranulocyte-macrophage progenitor cell and in the presence oferythropoietin stimulates colony formation by multipotentialprogenitor cell and enhances colony formation by erythroidprogenitor cell. Mice treated with LF have lower levels of serumcolony-stimulating factors than do control mice, and organsremoved from mice pretreated with LF release fewer colony-stimulating factors in vivo than do organs removed from controlmice (11). The inability of LF to protect mice from the FVC-P, when LF is given to mice up to 3 days prior to inoculationwith the FVC-P, reflects the reversible action of LF in vivo.The effects of LF on cycling status of hematopoietic progenitorsrequires concentrations of LF greater than 10 ¿¿g/mouse,whichis gone by 48 h after LF administration (19,20). The decreasinginability of LF to protect mice from the FVC-P, when LF is

Table 3 Effectof endotoxin-depletedLF on spleenfocus-formingvirusesinprimary DBA/2 mice given injectionsof the FVC-P

Two different protocols were used. In Part A, DBA/2 mice were given injectionsi.v.witheither sterilepyrogen-freesalineor a finaltotal dosageofendotoxin-depletedLF givenon day 0, 6 h prior to i.v. injectionof FVC-P and then also ondays 2, 4, 6, 9, and 11. Mice were sacrificed 15 days after receivingthe FVC-P.The numbersof SFFU shown are the averagesof data from spleen extracts of 8primarymicein both control and LF groups(in a total of 4 separateexperiments),each assessed in 2 secondary DBA/2 mice. In Part B, DBA/2 mice were giveninjections i.v. of either sterile pyrogen-freesaline or 200 *tgendotoxin-depletedLF 2 h prior to injection i.v. of the FVC-P. Mice were sacrificed 15 days afterreceivingthe FVC-P. The SFFU shown are the averages of data from spleenextractsof 6 primary micein both control and LF groups (in a total of 2 separateexperiments),each assessedin 2 secondaryDBA/2 mice.

Dilution olextractPart

Aio-1

io-2io-'

io-4PartBio-1

io-2io-3io-4SFFUFVC-P

+salineTNTC"(JV=6)*

TNTC (N = 6)35 ±6 (N = 5) TNTC (N = 1)5 ±2 (N =6)TNTC

(N = 8)TNTC (N = 6) 28 ±2 (N = 2)33 ±9 (N = 8)3±1 (N=8)FVC-P

+LF32

±10(A'=5)TNTC(Ar =^±9(^=6)2 ±2 (N = 6)

0(^ =6)54±6(JV=6)TNTC(;V

=13 + 5(^=8)2±1(/V=8)

0(JV=8)•1)2)*TNTC, SFFU are too numerous to count.*Numbers in parentheses,numberof primary mice used for each point.

given to mice 3 or more days after the inoculation of FVC-P,probably relates to the infectivity already beginning and theinsensitivity of the target cells, monocytes, and macrophagesfrom FVC-injected mice, to the actions of LF in vitro with timeafter FVC-P infection (5).

Another possibility for the protective effects of LF in micegiven injections of the FVC-P that cannot yet be ruled out isthat LF is causing the release in vivo of factors which thendirectly inactivate the FVC-P or one of its component virusesor that it is priming certain cells to attack tumor cells. It isclear from studies in vitro that 1 molecule can have more than1 action, including suppressing or enhancing the release ofseveral biomoiecules from cells (1), and LF has numerousactivities in vitro (10) including the enhancement of monocytenatural killer activity (30), enhancement of hydroxyl radicalproduction (31), and modulation of activation of the complement system (32).

ACKNOWLEDGMENTS

We wish to thank Dr. Douglas Williams and David Bicknell fordepletion of endotoxin from the samples of lactoferrin, Dr. LeonMcKenzie for statistical evaluation of the mouse survival curves, andShirley Duke and Stephanie Moore for typing the manuscript.

REFERENCES

1. Broxmeyer,H. E. Biomolecule-cell'interactionsin the regulation of myelo-poiesis.Int. J. Cell Cloning,4: 378-405, 1986.

2. Schiff,R. D., and Oliff,A.The pathophysiologyof murineretrovinis-inducedleukemias.CRC Crit. Rev. Oncol. Hematol., 5: 257-323, 1986.

3. Broxmeyer,H. E., Ralph, R. Gilbertson, S., and Margolis, V. B. Inductionof leukemia-associatedinhibitory activity and bone marrow granulocyte-macrophageprogenitor cell alterations during infectionwith Abelsonvirus.Cancer Res., 40: 3928-3933, 1980.

4. Marceletti,J., and Furmanski, P. A murine model for the study of leukemiaassociatedinhibitoryactivity.Blood,56: 134-137, 1980.

5. Lu, L., Broxmeyer,H. E., Moore, M. A. S., Sheridan, A. P., and Gentile, P.Abnormalitiesin myelopoieticregulatoryinteractionswith acidicisoferritinsand lactoferrin in mice infectedwith Friend viruscomplex:associationwithaltered expressionof la antigenson effectorand respondingcells. Blood,65:91-99, 1985.

6. Van Snick, J. I... and Masson, P. L. The binding of lactoferrin to mouseperitonealcells.J. Exp. Med.. 144: 1568-1580, 1976.

7. Bennett,R. M., and Davis,J. Lactoferrinbindingto human peripheralblood4187

Research. on February 25, 2020. © 1987 American Association for Cancercancerres.aacrjournals.org Downloaded from

PROTECTIVE EFFECT OF LACTOFERRIN ON FVC-P-INFECTED MICE

cells: an interaction with a B-enriched population of adherent mononuclearcells. J. Immunol., 127: 1211-1215, 1981.

8. Steinman, G., Broxmeyer, H. E., DeHarve.n, E., and Moore, M. A. S.Immunoelectron microscopic tracing of lactoferrin, a regulator of myelo-poiesis, into a subpopulation of human peripheral blood monocytes. Br. J.Haematol., 50: 75-84, 1982.

9. Bergens, H. S., Hansen, N. E., Karle, H., and Kristensen, L. O. Receptorbinding of lactoferrin by human monocytes. Br. J. Haematol., 54: 383-391,1983.

10. Broxmeyer, H. E., Bicknell, D. C, Gillis, S., Harris, E. L., Pelus, L. M., andSledge, G. W., Jr. Lactoferrin: affinity purification from human milk poly-morphonuclear neutrophils using monoclonal antibody (II2C) to humanlai Kid-rein, development of an immunoradiometric assay using II2C, andmyelopoietic regulation and receptor-binding characteristics. Blood Cells,//: 429-446, 1986.

11. Broxmeyer, H. E., Smithynmn. A., Eger, R. R., Meyers, P. A., and deSousa,M. Identification of lactoferrin as the granulocyte-derived inhibitor of colonystimulating activity (CSA)-production. J. Exp. Med., 48: 1052-1067, 1978.

12. Broxmeyer, H. E., and Platzer, E. Lactoferrin acts on I-A and I-E/C antigen*subpopulations of mouse peritoneal macrophages in the absence of T-lym-phocytes and other cell types to inhibit production of granulocyte-macro-phage colony stimulating factors in vitro. J. Immunol., 133: 306-314, 1984.

13. Bagby, G. C., Riga, V. D., Bennett, R. M., Vandenbark, A. A., and Garewal,H. S. Interaction of lactoferrin, monocytes and T-lymphocyte subsets in theregulation of steady state granulopoiesis in vitro. J. Clin. Invest., 68: 56-63,1981.

14. Bagby, G. C., McCall, E., and Layman, D. L. Regulation of colony stimulating activity production. Interactions of fibroblasts, mononuclear phagocytesand lactoferrin. J. Clin. Invest. 71: 340-344, 1983.

15. Bagby, G. C., McCall, E., Bergstrom, K. A., and Burger, D. A monokineregulates colony-stimulating activity production by vascular endothelial cells.Blood, 62: 663-668, 1983.

16. Yung, Y. P., and Moore, M. A. S. Long term in vitro culture of murine mastcells. III. Discrimination of mast cell growth factor and granulocyte -CSF. J.Immunol., 129: 1256-1261, 1982.

17. Brown, R. D., Yuen, E., Rickard, K. A., Vincent, P. C., Young, G., andKronenberg, H. Plasma lactoferrin in patients with neutropenia. Blut, 52:289-295, 1986.

18. Fletcher, J., and Willars, J. The role of lactoferrin released by phagocytosingneutrophils in the regulation of colony stimulating activity production byhuman mononuclear cells. Blood Cells, 77; 451-458, 1986.

19. Gentile, P., and Broxmeyer, H. E. Suppression of mouse myelopoiesis by

administration of human lactoferrin in vivo and the comparative action ofhuman transferrin. Blood, 61:982-993, 1983.

20. Broxmeyer, H. E., Williams, D. E., I languì.G., Cooper, S. Gentile, P., Shen,R.-N., Ralph, P., Gillis, S., and Bicknell D. C. The opposing actions in vivoon murine myelopoiesis of purified preparations of lactoferrin and the colonystimulating factors. Blood Cells, in press, 1987.

21. Broxmeyer, H. E., Williams, D. E., Cooper, S., Shadduck, R. K., Gillis, S.,Waheed, A., Urdal, D. L., and Bicknell, D. C. The comparative effects invivo of recombinant murine interleukin-3, natural murine colony stimulatingfactor-1 and recombinant murine granulocyte-macrophage colony stimulating factor on myelopoiesis in mice. J. Clin. Invest., 79: 721-730, 1987.

22. Gross, L. Development and serial cell-free passage of a highly potent strainof mouse leukemia virus. Proc. Soc. Exp. Biol. Med., 94: 767-771, 1957.

23. Thompson, S., and Axelrad, A. A. A quantitative spleen colony assay methodfor tumor cells induced by Friend leukemia virus infection in mice. CancerRes., 28: 2105-2114, 1968.

24. Rowe, W. P., Pugh, W. E., and Hartley, J. W. Plaque assay techniques formurine leukemia viruses. Virology, 42: 1136-1139, 1970.

25. Colton, T. Statistics in Medicine, p. 248F. New York: Little, Brown andCompany, 1974.

26. Chen, L. T., Lu, L., and Broxmeyer, H. E. Effects of purified iron-saturatedhuman lactoferrin on spleen morphology in mice infected with Friend viruscomplex. Am. J. Pathol. 126: 285-292, 1987.

27. Zucali, J. R., Dinari-Ilo, C. A., Oblon, D. J., Gross, M. A., Anderson, L.,and Weiner, R. S. Interleukin-1 stimulates fibroblasts to produce granulocyte-macrophage colony stimulating activity and prostaglandin I :...J. Clin. Invest.,77:1857-1863, 1986.

28. Bagby, G. C., Dinari-Ilo. C. A., Wallace, P., Wagner, C., Hefeneider, S., andMcCall, E. Interleukin-1 stimulates granulocyte-macrophage colony stimulating activity release by vascular endothelial cells. J. Clin. Invest., 78; 1316-1323, 1986.

29. Zucali, J. R., Broxmeyer, H. E., Dinari-Ilo. C. A., Gross, M. A., and Weiner,R. S. Regulation of early human hematopoietic (BFU-E and CFU-GEMM)progenitor cells m n'irò by interleukin-1 induced fibroblast conditioned

medium. Blood, 69: 33-37, 1987.30. Nishiya, K., and Horwitz, D. A. Contrasting effects of lactoferrin on human

lymphocyte and monocyte natural killer activity and antibody-dependent cell-mediated cytotoxicity. J. Immunol., 129: 2519-2523, 1982.

31. Ambruso, D. R., and Johnston, R. B. Lactoferrin enhances hydroxyl radicalproduction by human neutrophils, neutrophil paniculate fractions and anenzyme generating system. J. Clin. Invest., 67: 352-360, 1981.

32. Kijlstra, A., and Jeurissen, H. M. Modulation of classical C3 convertase ofcomplement by tear lactoferrin. J. Immunol., 44: 263-270, 1982.

4188

Research. on February 25, 2020. © 1987 American Association for Cancercancerres.aacrjournals.org Downloaded from

1987;47:4184-4188. Cancer Res   Li Lu, Giao Hangoc, Allen Oliff, et al.   Polycythemia-inducing Strain of Friend Virus ComplexProtective Influence of Lactoferrin on Mice Infected with the

  Updated version

  http://cancerres.aacrjournals.org/content/47/15/4184

Access the most recent version of this article at:

   

   

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/47/15/4184To request permission to re-use all or part of this article, use this link

Research. on February 25, 2020. © 1987 American Association for Cancercancerres.aacrjournals.org Downloaded from