Vol. 58, No. 6INFECTION AND IMMUNITY, June 1990, p. 1796-18010019-9567/90/061796-06$02.00/0Copyright © 1990, American Society for Microbiology

Effect of Friend Leukemia Virus Infection on Susceptibility toCandida albicans

MARLENA A. MOORS,' SYLVIA M. JONES,1t KAREN K. KLYCZEK,2 THOMAS J. ROGERS,'HELEN R. BUCKLEY,' AND KENNETH J. BLANK' 3*

Department of Microbiology and Immunology' and Fels Institute for Molecular Biology and Cancer Research,3 TempleUniversity School of Medicine, Philadelphia, Pennsylvania 19140, and Department of Biology, University of Wisconsin-

River Falls, River Falls, Wisconsin 540222

Received 21 December 1989/Accepted 21 March 1990

Previous studies have demonstrated that Friend leukemia virus (FLV) induces a profound immunosuppres-sion in susceptible mice. The studies described in this report indicate that mice infected with FLV have anincreased susceptibility to subsequent infection with the opportunistic pathogen Candida albicans, as measuredby increased numbers of C. albicans CFU in the kidneys of FLV-infected mice relative to uninfected controls.Experiments in which the NB-tropic and N-tropic strains ofFLV were used suggest that virus replication or theresulting virus burden may be important in the observed increased susceptibility to C. albicans. Sinceneutrophils are believed to be important in the response of mice to systemic Candida infections, the effect ofFLV infection on neutrophil candidacidal activity was investigated. The percentage of neutrophils present inunfractionated Proteose Peptone-elicited peritoneal exudates of mice infected with FLV for 14 days wassignificantly lower than in uninfected control mice or mice infected with FLV for 6 or 10 days. Whenneutrophils from FLV-infected and control mice were purified, adjusted to equal concentrations, and tested forin vitro candidacidal activity, neutrophils from mice infected with FLV for 14 days were deficient in their abilityto kill C. albicans relative to normal controls and mice infected with FLV for 6 or 10 days. Addition of normalmouse serum increased killing in all groups but did not restore candidacidal activity of neutrophils from miceinfected with FLV for 14 days to levels of control neutrophils or neutrophils from mice infected for 6 or 10 dayswith the virus. These results suggest a defect in neutrophil function, at the later stages of FLV infection,involving in vitro candidacidal activity. In addition, neutrophils from FLV-infected mice may be deficient in invivo chemotactic activity. These defects in neutrophil function could account, at least in part, for the observedincreased susceptibility of FLV-infected mice to C. albicans.

Infection of susceptible strains of mice with the murineretrovirus complex Friend leukemia virus (FLV) (3) resultsin a rapid development of erythroleukemia accompanied bya profound immunosuppression (16). It has been demon-strated that susceptibility to viral infection and the resultingimmunosuppression are under genetic control (26, 31, 37).This FLV-induced immunosuppression has been shown toaffect B-cell function (12, 37, 40), T-cell function (14, 33, 34,38, 39), macrophage function (9, 30), natural killer cellfunction (20, 35), leukocyte migration (19), and suppressorcell generation (25). A number of studies have shown thatFLV infection suppresses cellular (38) and humoral (11, 12,37, 40) responses to nominal antigens such as purified proteinderivative and sheep erythrocytes. The effect of FLV-inducedimmunosuppression on the immune response to pathogenicorganisms, however, has not been extensively investigated.In the present study, the effect of FLV infection on thesusceptibility of mice to Candida albicans was examined.

C. albicans is a yeast which is part of the normal gastro-intestinal flora of humans and is often observed as anopportunistic infection in immunocompromised hosts (41).Mice are not normal hosts for C. albicans, but they canbecome infected in an experimental model by intravenous(i.v.) inoculation with live Candida yeasts, resulting in adisseminated disease similar to that seen in humans (41). The

* Corresponding author.t Present address: Department of Microbiology, Immunology and

Parasitology, New York State College of Veterinary Medicine,Cornell University, Ithaca, NY 14853.

mechanism of resistance to the disseminated form of candi-diasis is not entirely clear. Clinical evidence as well asseveral experimental animal studies suggest that resistanceis dependent, at least in part, on neutrophil function (4, 28,29, 43). The role of activated T cells (2, 13, 15, 21, 44) and/oractivated macrophages (5, 43) in resistance to the dissemi-nated form of the disease is less clear. In contrast, resistanceto the chronic mucocutaneous form of the disease appears tobe mediated primarily by T-cell-dependent immunity (24).Our results indicate that mice infected with FLV have an

increased susceptibility to C. albicans, as measured bygreater numbers of Candida CFU in the kidneys of miceinfected with FLV than in mice infected with C. albicansalone. This observation suggests that FLV-infected mice areunable to generate an effective response against systemicCandida infection. In order to investigate a possible mech-anism for the observed increased susceptibility to C. albi-cans induced by FLV, we studied the effect ofFLV infectionon neutrophil function. Our studies demonstrate that defi-ciencies in in vitro neutrophil candidacidal activity as well asin vivo neutrophil chemotaxis in the later stages of FLV-induced disease may contribute to the increased susceptibil-ity of FLV-infected mice to Candida infection. In addition, itappears that virus replication or the resulting increase invirus burden may be an important factor in this increasedsusceptibility to Candida infection.

MATERIALS AND METHODSMice. In these experiments, 5- to 10-week-old female

BALB/cAn mice obtained from the National Cancer Insti-

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tute (Bethesda, Md.) or from the Temple University CentralAnimal Facility (Philadelphia, Pa.) were used. Five micewere used per experimental group for both in vivo and invitro assays.FLV infection. Two polycythemic strains of FLV were

used in these experiments. The NB-tropic strain (NBFLV) ismaintained by passage in susceptible (Fv-lb homozygous)BALB mice (31). The N-tropic strain (NFLV) is maintainedby passage in susceptible (Fv-J' homozygous) DBA/2 mice(31). For in vivo experiments to measure growth of C.albicans in virus-infected mice, virus stocks were preparedas 10% spleen homogenates from pooled spleens of animalsinoculated with FLV 14 days earlier. Homogenates wereclarified by centrifugation to remove cellular debris. Virustiters were determined by the spleen focus assay (3) to be 3.3x 105 focus-forming units (FFU) per ml for NBFV and 3.9 x105 FFU/ml for NFLV. The virus was diluted in ice-coldsaline to yield the indicated FFU per mouse in a 0.2-mlvolume. Mice received virus intraperitoneally (i.p.) 5 daysbefore inoculation of C. albicans. For in vitro assays tomeasure candidacidal activity of neutrophils, neutrophilswere taken from mice which received i.p. injections of 0.5 mlof virus diluted in ice-cold saline to yield the indicated dose.For these experiments, virus stocks of NBFLV were pre-pared as supematants from infected SC-1 cells (22) and hada titer of 8.7 x 103 FFU/ml.

C. albicans. C. albicans B311 (ATCC 32354) was main-tained at 23°C by weekly transfer on Sabouraud dextroseagar (SDA) plates. For all experiments, C. albicans wassubcultured onto a fresh SDA plate 24 h prior to use. For invivo assays, C. albicans was inoculated into 50 ml ofmedium described by Lee et al. (27) and incubated for 18 to24 h at 23°C with shaking. Cells were washed and adjusted to3.75 x 105/ml in sterile saline, and mice were injected i.v. inthe tail with 0.2 ml of the suspension (7.5 x 104 cells permouse) on day 0. The 50% lethal dose for C. albicans inBALB/c mice is 2.5 x 105 cells. For in vitro neutrophilassays, a colony of yeasts was taken aseptically from a 24-hSDA plate, washed once in sterile saline, and adjusted to 5 x103 cells per ml in neutrophil assay medium.Enumeration of C. albicans in the kidneys. Mice were

sacrificed by cervical dislocation on days 1, 7, 10, and 14after i.v. inoculation with 7.5 x 104 live C. albicans yeastcells. Both kidneys from individual mice were removedaseptically and homogenized with a mechanical tissue ho-mogenizer, in a final volume of 10 ml of sterile phosphate-buffered saline (PBS). The homogenate was serially dilutedand inoculated into plates containing warm SDA. The num-ber of viable C. albicans CFU recovered from the kidneys ofindividual mice was determined after incubation of SDAplates for 48 h at 37°C. Results are expressed as the meanCFU of C. albicans recovered from the kidneys of fiveanimals ± the standard error of the mean. Statistical signif-icance was determined by the Student t test.

Preparation of neutrophils. Mice were injected i.p. with 1ml of 10% Proteose Peptone in sterile saline. Four hourslater the peritoneal cavities were lavaged with 10 ml ofice-cold PBS (Dulbecco PBS without Ca2" and Mg2+) plus 5U of heparin per ml. Peritoneal-exudate cells were centri-fuged for 10 min at 300 x g, and when necessary, erythro-cytes were removed by hypotonic lysis. Cells were resus-pended in PBS plus 5 mM EDTA at 107/ml and layered ontogradients of Lymphoprep (Ficoll-Hypaque, 1.077 g/cm3;Accurate Chemicals, Westbury, N.Y.) in 15-ml polypropyl-ene conical tubes. Gradients were centrifuged at 400 x g for30 min at 23°C. Neutrophil-rich pellets were washed three

times with PBS, and the cells were adjusted to desiredconcentrations in neutrophil assay medium (RPMI 1640supplemented with 1% fetal calf serum, glutamine, penicil-lin, and streptomycin). Cell samples taken before and afterfractionation were cytocentrifuged and stained with Diff-Quik (Baxter, McGaw Park, Ill.). Pellets were found toconsist of 70 to 95% neutrophils.

Neutrophil killing of C. albicans. The assay for neutrophilcandidacidal activity was modified from the protocol re-ported by Djeu et al. (18). Fifty microliters of neutrophilsadjusted to 1.5 x 106, 8 x 105, or 1 x 105/ml was added totriplicate wells of a 96-well microtiter plate. After incubationfor 30 min at 37°C in 5% CO2, contaminating nonadherentcells were removed by gentle washing with warm assaymedium, and 50 [LI of fresh medium with or without 5%normal mouse serum was then added to the wells. Fiftymicroliters of C. albicans yeast cells adjusted to 5 x 103/mlin neutrophil assay medium with or without 5% normalmouse serum was added to the wells with neutrophils. Theseconcentrations of neutrophils and C. albicans yielded effec-tor/target ratios of 300:1, 160:1, and 20:1, respectively.Control wells contained either neutrophils only or C. albi-cans only. Plates were incubated for 18 h at 37°C in 5% C02,after which the wells were washed twice with sterile distilledH20. Twenty-five microliters of D-[5,6-3H]glucose (NET-807, 60 to 90 Ci/mmol; Dupont, NEN Research Products,Boston, Mass.), adjusted to 10 ,Ci/ml in sterile distilledH20, was added to the wells, and the plates were incubatedfor 20 min at 37°C. Fifty microliters of bleach was added toeach well to remove adherent hyphae, the wells were har-vested onto glass fiber filters by using a Ph.D. cell harvester(Cambridge Technology, Inc., Watertown, Mass.), and labelwas counted on a beta counter. This protocol was found toyield results in which the uptake of 3H-glucose was propor-tional to the amount of Candida growth in the wells (data notshown). Results are expressed as percent inhibition of C.albicans growth by neutrophils, which was calculated asfollows: percent inhibition = [1 - (cpm of wells containingC. albicans plus neutrophils/cpm of wells containing C.albicans only)] x 100.

RESULTS

Effect of FLV infection on clearance of C. albicans from thekidneys. Experiments were conducted to determine theeffect of FLV infection on susceptibility of mice to C.albicans. Animals were inoculated with NBFLV 5 daysbefore i.v. challenge with C. albicans to eliminate thepossible effect of transient (1- to 3-day) interferon-mediatedimmunosuppression on C. albicans growth (6, 10). We choseto measure C. albicans growth in the kidneys, since previousstudies have shown that the kidneys are the primary site ofreplication of C. albicans in infected mice (42). For thesestudies, mice were divided into two groups: group 1 receivedNBFLV 5 days prior to challenge with C. albicans, andgroup 2 received C. albicans alone. Mice were sacrificed 1,7, 10, and 14 days after challenge with C. albicans. Resultsof a representative experiment are shown in Table 1. On day1 after inoculation with C. albicans, there was no differencein the numbers of C. albicans CFU recovered from thekidneys of mice in groups 1 and 2, indicating that bothgroups received the same dose of yeast cells. At days 7, 10,and 14 after inoculation with C. albicans, the number ofCFU in the kidneys of mice which had received FLVcontinued to rise. In contrast, mice which received C.albicans alone had significantly fewer CFU in their kidneys

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TABLE 1. Enumeration of C. albicans in the kidneysof FLV-infected and control mice

Day postinfection witha: C. albicans recovery (102 ± SEM)b frommice infected with:

C. albicans FLV FLV + C. albicans C. albicans

1 6 12 2c 19 57 12 860 334 1 ±110 15 13,591 4,759d 46 ± 1214 19 10,172 ± 2,918d 0.3 ± 0.1

a BALB/c mice were inoculated with 6.5 x 104 FFU ofNBFLV on day -5and then with 7.5 x 104 C. albicans yeast cells on day 0. Mice were sacrificedon the days indicated, and CFU of viable C. albicans in the kidneys weredetermined as described in Materials and Methods.

b Mean from one of three experiments with kidneys of five mice.c Not significant compared with value for infection with C. albicans alone.d Significant at P < 0.05 compared with value for C. albicans alone.

at days 7 and 10 than did FLV-infected mice, and they hadvirtually cleared the infection by day 14 postchallenge. It isapparent from these experiments that mice inoculated withNBFLV prior to C. albicans infection are less able toeliminate C. albicans or to prevent its replication in thekidney than are uninfected control mice. These resultssuggest that the previously reported NBFLV-induced immu-nosuppression which was found to affect cellular and hu-moral responses to nominal antigens (11, 12, 37, 38, 40) alsoaffects immunity against a pathogenic organism.Comparison of the effects of NBFLV and NFLV on clear-

ance of C. albicans from the kidneys. Since previous studieshave shown that virus replication may be an important factorin the induction of immunosuppression (10, 31), experimentsto determine whether virus replication was required forincreased susceptibility of mice to C. albicans were per-formed. For these studies, the growth of C. albicans in miceinoculated with NBFLV was compared with growth in miceinoculated with NFLV. NFLV replicates 100- to 1,000-foldless efficiently in BALB/c cells than does NBFLV, by virtueof the expression of the Fv-1 b allele by this mouse strain (31).The effect of virus replication on susceptibility to C. albicanswas examined by infecting mice with 1.3 x 103 FFU ofNBFLV or NFLV and challenging them 5 days later with C.albicans. Mice were sacrificed 10 days after challenge withC. albicans, and the number of CFU in the kidneys wasdetermined. The mean number of CFU (+ standard error ofthe mean) recovered from the kidneys of NBFLV-infectedmice ([2,351 + 1,109] x 102; significant atP < 0.05 comparedwith value for infection with C. albicans alone) was signifi-cantly greater than those recovered from control mice whichreceived C. albicans alone ([70 + 19] x 102) and from micewhich received NFLV and C. albicans ([154 ± 43] x 102; notsignificant compared with value for infection with C. albi-cans alone). These results suggest that virus replication orthe resulting increase in virus burden is an important factorin the observed increased susceptibility of FLV-infectedBALB/c mice to C. albicans.

Effect of FLV infection on in vitro neutrophil candidacidalactivity. Since polymorphonuclear leukocytes (PMN, orneutrophils) are believed to be important cells in the elimi-nation of a systemic Candida infection in both mice (4, 43)and humans (28, 29), it seemed possible that FLV infectioncaused an increase in susceptibility to Candida infection byaffecting PMN function. To test this possibility, neutrophilspurified from Proteose Peptone-elicited peritoneal exudatesof FLV-infected and control mice were tested for their

ability to inhibit the growth of C. albicans in an in vitromicroassay. Neutrophils were elicited into the peritonealcavities of FLV-infected and control mice by inoculationwith Proteose Peptone as described in Materials and Meth-ods. To test in vitro candidacidal activity, equal numbers ofpurified neutrophils from FLV-infected and control micewere cultured with live C. albicans yeast cells in the pres-ence or absence of normal mouse serum. The inhibition of C.albicans hyphal growth after culture with neutrophils for 18h was measured by uptake of [3H]glucose by survivingCandida hyphae. Neutrophils from normal mice inhibitedthe growth of C. albicans in a dose-dependent fashion (Fig.1). Neutrophils from mice infected with FLV for 6 or 10 daysinhibited the growth of C. albicans in a manner indistinguish-able from that of normal controls (Fig. 1A and B). Neutro-phils from mice infected with FLV for 14 days, on the otherhand, were unable to inhibit the growth of C. albicans in thisassay, even at a ratio of 300 neutrophils to 1 Candida yeastcell (Fig. 1C). Addition of normal mouse serum to thecultures (which does not affect the growth of C. albicans inthe absence of neutrophils) enhanced the ability of neutro-phils in all groups to inhibit the in vitro growth of C. albicans(Fig. 1). Addition of normal mouse serum did not, however,restore in vitro candidacidal activity of neutrophils frommice infected with FLV for 14 days to control levels. At allthree effector-to-target ratios, there was a significant de-crease in killing ability of neutrophils from mice infected for14 days with FLV compared with normal controls (Fig. 1C).These results indicate that although neutrophil candidacidalactivity appears to be intact at 6 and 10 days after infectionwith FLV, neutrophils from mice infected for 14 days withFLV are grossly deficient in their ability to kill C. albicans invitro. This deficiency is most dramatic when normal mouseserum is not present in the cultures.

In the course of these studies, we observed that whensamples of unfractionated Proteose Peptone-elicited perito-neal exudate cells from control mice and mice inoculatedwith NBFLV 6, 10, and 14 days earlier were cytocentrifugedand analyzed for neutrophil content by differential staining,the percentage of neutrophils present in the peritoneal exu-dates of mice infected with FLV for 14 days was consistentlysignificantly lower than that in exudates of control mice ormice infected with FLV for 6 days (data not shown) or 10days (Table 2). These results suggest a defect in the ability ofneutrophils from mice infected with FLV for 14 days tomigrate by chemotaxis from the blood into the peritonealcavity in response to a sterile inflammatory agent. In con-trast, in vivo neutrophil chemotactic activity in response toProteose Peptone appears to be intact in mice infected for 6or 10 days with FLV.

DISCUSSION

The studies described in this report were initiated toexamine the effect of infection of mice with FLV on suscep-tibility to the opportunistic fungal pathogen C. albicans.Although the fact that FLV induces immunosuppression hasbeen known for quite some time, the effect of FLV infection(or infection by any other retrovirus) on susceptibility topathogenic organisms has not been extensively investigated.Certain studies have demonstrated a decrease in the anti-body response to Ascaris antigen (48) and Escherichia coli(23). Another retrovirus, LP-BM5, has been demonstrated tocause the abrogation of resistance to severe mousepox inC57BL/6 mice (8). Although a recent study has shown thatinfection of mice with lymphocytic choriomeningitis virus

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EFFECTOR : TARGETFIG. 1. Inhibition of in vitro C. albicans growth by neutrophils

from normal and FLV-infected mice. Mice were injected i.p. with1.3 x 103 FFU of NBFLV or supernatants from uninfected SC-1cells. PMN were obtained 6 (A), 10 (B), and 14 (C) days later andtested for their ability to inhibit the in vitro growth of C. albicans as

described in Materials and Methods. Symbols: O, normal PMN; *,FV PMN; 0, normal PMN plus normal mouse serum; 0, FLV PMNplus normal mouse serum. Results are from three representativeexperiments (days 6, 10, and 14) and are expressed as the mean

percent inhibition of Candida growth from triplicate wells ± thestandard deviation. The effector/target ratio indicates the number ofPMN relative to the number of C. albicans cells added to the wells.

causes increased susceptibility to the opportunistic fungalpathogen Histoplasma capsulatum (47), to our knowledgethe present study is the first in which infection with a

retrovirus has been demonstrated to increase susceptibilityto a fungal infection in a mouse model. It is clear from thisstudy that mice receiving FLV 5 days prior to inoculation

TABLE 2. Percent PMN in Proteose Peptone-elicited peritonealexudates from normal and NBFLV-infected mice

No. of Mouse group 106 cells recovered/ % PMNexpts mouse (mean ± SD)' (mean ± SD)6

3 Normal 8.3 1.5 65 ± 9FLV (day 10) 6.7 ± 0.6 56 ± 4

4 Normal 4.2 2.1 51 ± 7FLV (day 14) 4.5 ± 3.7 17 ± 3

a Peritoneal exudate cells obtained by peritoneal lavage of Proteose Pep-tone-treated normal and NBFLV-infected (1.3 x 103 FFU) mice were countedin a hemacytometer.

b Cytocentrifuged samples of PMN purified from Proteose Peptone-elicitedperitoneal exudates as described in Materials and Methods were stained withDiff-Quik, and the percent PMN was determined by nuclear morphology. Atotal of 100 to 200 cells were scored per sample.

with C. albicans demonstrate increased susceptibility toinfection with C. albicans, as measured by a 2- to 4-logincrease in the number of C. albicans colonies recoveredfrom the kidneys of NBFLV-infected mice compared withmice infected with C. albicans alone. These results suggestthat FLV modulates the activity of a cell(s) involved ineliminating a systemic Candida infection.The cells involved in mediating resistance to systemic C.

albicans infection have not been clearly defined. The role ofT-cell-mediated immunity in the response to systemic C.albicans infection is particularly controversial. In this re-gard, evidence both for (2, 13, 21) and against (15, 44) a rolefor T cells in systemic Candida infection has been reported.In addition, the extent of involvement of activated macro-phages in resistance is unclear (5, 43). Numerous reports,however, have provided evidence that PMN are responsible,at least in part, for eliminating a systemic C. albicansinfection in both humans (28, 29) and mice (4, 43). For thisreason, in order to investigate the mechanism by which FLVcauses increased susceptibility to infection with C. albicans,we decided initially to examine the effect of FLV on PMNcandidacidal activity.

Results of these experiments suggest a defect in neutrophilfunction at day 14 after infection with FLV. Specifically, wefound that mice infected with FLV for 14 days, but not for 6to 10 days, were deficient in their ability to inhibit the growthof C. albicans in vitro. The presence of normal mouse serumin the cultures, which presumably provides a source ofopsonin, only partially restored the ability of neutrophilsfrom FLV-infected mice to inhibit Candida growth relativeto normal controls. These results indicate that neutrophilsfrom mice infected for 14 days with FLV have a deficiency inin vitro candidacidal activity. The fact that decreased can-didacidal activity is more dramatic in the absence than in thepresence of normal mouse serum suggests that this defi-ciency may involve effects on non-complement-receptor-mediated uptake and killing of C. albicans.

In addition, a deficiency in the in vivo chemotactic func-tion of neutrophils was suggested by the observation thatfewer PMN were present in Proteose Peptone-elicited peri-toneal exudates of mice infected with FLV for 14 days thanin exudates of normal controls. We cannot, however, ruleout the possibility that neutrophils are indeed elicited intothe peritoneal cavities of mice infected with FLV for 14 daysas efficiently as in normal mice but for reasons related to thepathology of the disease are not easily obtained by lavage. Insupport of our observations, however, are reports of defi-ciencies in in vitro chemotactic activity of neutrophils iso-

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lated from individuals infected with the human immunodefi-ciency virus (1).That an effect of virus infection on PMN activity was not

observed until 14 days after infection may reflect the factthat the virus burden did not reach a critical level until thistime. The disease progressed markedly from days 10 to 14,as manifested by a twofold increase in the size of the spleenover this 4-day period (data not shown). It is therefore notsurprising that defects in neutrophil function were observedat 14 days but not at 10 days after virus infection.The importance of virus replication or virus burden in the

increased susceptibility of FLV-infected mice to C. albicansis suggested by experiments which measure C. albicansgrowth in NFLV- and NBFLV-infected mice. UnlikeNBFLV-infected mice, mice infected with NFLV were ableto restrict the growth of C. albicans in the kidneys. Thisresult is consistent with previous studies which have shownthat increased viral burden due to viral replication correlateswith FLV-induced immunosuppression (10, 31).The deficiencies in neutrophil function at day 14 after

infection with virus may provide an explanation for our invivo studies. A deficiency in the ability of neutrophils tomigrate by chemotaxis to the site of infection, combinedwith an inability of neutrophils to kill C. albicans efficientlyonce they reach the site of infection, could explain theincreased numbers of C. albicans CFU in the kidneys ofmice infected with FLV for 15 and 19 days (days 10 and 14after inoculation with C. albicans respectively). Althoughneutrophils are generally thought of as cells involved veryearly in the response against pathogenic organisms, in thecase of a chronic infection it is possible that organisms whichescape initial attack by neutrophils reach the kidney andelicit neutrophil-containing inflammatory infiltrates. In thecase of the mice infected with FLV for 14 days, neutrophilsmight not be recruited efficiently and those which are re-cruited might be unable to efficiently inhibit the growth of C.albicans in the kidney.These experiments do not address the mechanism by

which FLV causes decreased PMN function. One possiblemechanism is that FLV has a direct effect on PMN functionas a result of infection of the PMN by the virus. Preliminaryresults from immunofluorescence studies, however, indicatethat PMN from virus-infected mice do not stain with amonoclonal antibody directed against the viral gp70, sug-gesting that PMN do not express viral antigens and thus arenot apparently productively infected (data not shown). An-other possibility is that the virus affects cells which influencePMN function by producing PMN-activating lymphokines.Numerous studies have provided evidence that neutrophilsare activated by lymphokines produced by T cells, includinggamma interferon (17, 36, 45), granulocyte-macrophage col-ony stimulating factor (32, 46), and interleukin-6 (7). SinceFLV has been shown to affect T-cell function (14, 33, 34, 36,38, 39), the increased susceptibility to fungal infection maybe due not to a direct effect on PMN but rather to an effectof virus on T cells which may be needed to activate PMN tokill C. albicans in vivo.

Infection with C. albicans is often seen in individualsinfected with human immunodeficiency virus (41). Since anumber of recent studies have suggested defects in PMNfunction in these individuals (1), investigation of the mech-anism by which FLV infection modulates PMN functionmay provide insight into the association between humanimmunodeficiency virus infection and infection with C.albicans.

ACKNOWLEDGMENTSThis research was supported by Public Health Service grant

A127766 from the National Institutes of Health. We thank GregHarvey for editorial assistance, David M. Mosser and LouisRosenthal for helpful discussions, and Bobbie A. Rice for help withgraphics.

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FRIEND LEUKEMIA VIRUS AND C. ALBICANS INFECTION

virus. J. Immunol. 107:1673-1681.20. Genovesi, E. V., D. Livnat, and J. J. Collins. 1982. Immunother-

apy of murine leukemia. VII. Prevention of Friend leukemiavirus-induced immunosuppression by passive serum therapy.Int. J. Cancer 30:609-624.

21. Giger, D. K., J. E. Domer, S. A. Moser, and J. T. McQuitty.1978. Experimental murine candidiasis: pathological and im-mune responses in T lymphocyte-depleted mice. Infect. Immun.21:729-737.

22. Hartley, J. W., and P. R. Wallace. 1975. Clonal cell lines from aferal mouse embryo which lack host-range restrictions formurine leukemia viruses. Virology 65:128-134.

23. Hirano, S., H. Friedman, and W. S. Ceglowski. 1971. Immuno-suppression by leukemia viruses. VII. Stimulatory effects ofFriend leukemia virus on pre-existing antibody forming cells tosheep erythrocytes and Escherichia coli in non-immunizedmice. J. Immunol. 107:1400-1409.

24. Kirkpatrick, C. H., R. R. Rich, and J. E. Bennett. 1971. Chronicmucocutaneous candidiasis: model building in cellular immu-nity. Ann. Intern. Med. 74:955-978.

25. Kumar, V., T. Caruso, and M. Bennet. 1976. Mechanisms ofgenetic resistance to Friend virus leukemia. III. Susceptibilityof mitogen-responsive lymphocytes mediated by T cells. J. Exp.Med. 143:728-740.

26. Kumar, V., P. Resnick, J. W. Eastcott, and M. Bennet. 1978.Mechanism of genetic resistance to Friend virus leukemia inmice. V. Relevance of Fv-3 gene in the regulation of in vivoimmunosuppression. JNCI 61:1117-1123.

27. Lee, K., H. R. Buckley, and C. Campbell. 1975. A syntheticmedium for the growth of the yeast and mycelial phase ofCandida albicans. Sabouraudia 5:148-152.

28. Lehrer, R. I. 1970. Measurement of candidacidal activity ofspecific leukocyte types in mixed cell populations. I. Normal,myeloperoxidase-deficient and chronic granulomatous diseaseneutrophils. Infect. Immun. 2:42-47.

29. Lehrer, R. I. 1972. Functional aspects of a second mechanism ofcandidacidal activity by human neutrophils. J. Clin. Invest.51:2566-2572.

30. Levy, M. H., and E. F. Wheelock. 1975. Impaired macrophagefunction in Friend virus leukemia: restoration by statolon. J.Immunol. 114:962-965.

31. Lilly, F., and T. Pincus. 1973. Genetic control of murine viralleukemogenesis. Adv. Cancer Res. 17:231-277.

32. Lopez, A. F., N. A. Nicola, A. W. Burgess, D. Metcalf, F. L.Battye, W. A. Sewell, and M. Vadas. 1983. Activation ofgranulocyte cytotoxicity by purified mouse colony-stimulatingfactors. J. Immunol. 131:2983-2988.

33. Lopez-Cepero, M., S. Specter, D. Matteucci, H. Friedman, andM. Bendinelli. 1988. Altered interleukin production duringFriend leukemia virus infection. Proc. Soc. Exp. Biol. Med.188:353-363.

34. Matteucci, D., A. M. Giangregorio, M. Lopez-Cepero, S. Spec-

ter, M. Bendinelli, and H. Friedman. 1989. Interleukins 1 and 2production and responsiveness in the early stages of retroviralinfections in mice. Cell. Immunol. 120:10-20.

35. Moody, D. J., S. Spector, M. Bendinelli, and H. Friedman. 1984.Suppression of natural killer cell activity by Friend murineleukemia virus. J. Natl. Cancer Inst. 72:1349-1356.

36. Morrison, D. J., E. Brummer, and D. A. Stevens. 1989. In vivoactivation of peripheral blood polymorphonuclear neutrophilsby gamma interferon results in enhanced fungal killing. Infect.Immun. 57:2953-2958.

37. Morrison, R. P., J. Nishio, and B. Chesebro. 1986. Influence ofthe murine MHC (H-2) on Friend leukemia virus-induced im-munosuppression. J. Exp. Med. 163:301-304.

38. Mortensen, R. F., W. S. Ceglowski, and H. Friedman. 1973.Leukemia virus-induced immunosuppression. IX. Depressionof delayed hypersensitivity and MIF production after infectionof mice with Friend leukemia virus. J. Immunol. 111:1810-1819.

39. Mortensen, R. F., W. S. Ceglowski, and H. Friedman. 1974.Leukemia virus-induced immunosuppression. X. Depression ofT cell-mediated cytotoxicity after infection of mice with Friendleukemia virus. J. Immunol. 112:2077-2086.

40. Odaka, T., H. Ishii, K. Yamaura, and T. Yamamoto. 1966.Inhibitory effect of Friend leukemia virus infection on antibodyformation to sheep erythrocytes in mice. Jpn. J. Exp. Med.36:277-290.

41. Odds, F. C. 1988. Candida and candidosis, 2nd ed. BailliereTindall W. B. Saunders, London.

42. Rogers, T., and E. Balish. 1976. Experimental Candida albicansinfection in conventional mice and germfree rats. Infect. Im-mun. 14:33-38.

43. Rogers, T. J., and E. Balish. 1977. The role of activatedmacrophages in resistance to experimental renal candidiasis.RES J. Reticuloendothel. Soc. 22:309-318.

44. Rogers, T. J., E. Balish, and D. D. Manning. 1976. The role ofthymus-dependent cell-mediated immunity in resistance to ex-perimental disseminated candidiasis. RES J. Reticuloendothel.Soc. 20:291-297.

45. Shalaby, M. R., B. B. Aggarwal, E. Rinderknect, L. P. Sveder-sky, B. S. Finkle, and M. A. Palladino. 1985. Activation ofhuman polymorphonuclear neutrophil functions by interferongamma and tumor necrosis factors. J. Immunol. 135:2069-2073.

46. Weisbart, R. H., D. W. Golde, S. C. Clark, G. G. Wong, andJ. C. Gasson. 1985. Human granulocyte-macrophage colony-stimulating factor is a neutrophil activator. Nature (London)314:361-363.

47. Wu-Hseih, B., D. H. Howard, and R. Ahmed. 1988. Virus-induced immunosuppression: a murine model of susceptibilityto opportunistic infection. J. Infect. Dis. 158:232-235.

48. Yoo, T. J., and M. Bennet. 1981. Effect of herpes simplex virustype 2 and Friend erythroleukemia virus infection on IgEantibody responses to Ascaris antigen in mice. Int. Arch.Allergy Appl. Immun. 65:235-238.

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