mononuclear and against mycelia by polymorphonuclear

Selective Protection against Conidia byMononuclear and against Mycelia by Polymorphonuclear

Phagocytes in Resistance to Aspergillus

OBSERVATIONSONTHESETWOLINES OF DEFENSEIN VIVO AND IN

VITRO WITH HUMANANDMOUSEPHAGOCYTES

ANDREASSCHAFFNER,HERNDONDOUGLAS,and ABRAHAMBRAUDE, Departments ofMedicine and Pathology, University of California,San Diego, California 92103

A B S T R A C T By comparing natural immunity to As-pergillus fumigatus (AF) in vivo with the action ofhuman or mouse phagocytes against AF in vitro, wedelineated two sequential lines of defense against AF.The first line of defense was formed by macrophagesand directed against spores. Macrophages preventedgermination and killed spores in vitro and rapidlyeradicated conidia in vivo, even in neutropenic andathymic mice. The second was the neutrophilic gran-ulocyte (PMN), which protected against the hyphalform of AF. Human and mouse PMNkilled myceliain vitro. Normal, but not neutropenic mice, stoppedhyphal growth, and eradicated mycelia. Either line ofdefense acting alone protected mice from high chal-lenge doses. Natural immunity collapsed only whenboth the reticuloendothelial system and PMNwereimpaired. These findings are in keeping with the clin-ical observation that high doses of cortisone and neu-tropenia are the main risk factors for invasive asper-gillosis. Cortisone inhibited the conidiacidal activityof mouse macrophages in vivo and of human or mousemononuclear phagocytes in vitro. Cortisone damagedthis first line of defense directly and not through theinfluence of T lymphocytes or other systems modifyingmacrophage function as shown in athymic mice andin vitro. In addition, daily high doses of cortisone inmice reduced the mobilization of PMNso that thesecond line of defense was also impaired. Thus, cor-tisone can break down natural resistance on its own.Myelosuppression rendered mice susceptible only whenthe first line of defense was overpowered by high chal-

Received for publication 13 August 1981 and in revisedform 4 November 1981.

lenge doses, by activated spores that cannot be killedby macrophages, or by cortisone suppression of theconidiacidal activity of macrophages.

The host, thus, can call upon two independentphagocytic cell lines that form graded defense systemsagainst aspergillus. These lines of defense function inthe absence of a specific immune response, whichseems superfluous in the control and elimination of thisfungus.

INTRODUCTION

Invasive aspergillosis, due mainly to Aspergillus fu-migatus (AF)1, is a serious problem in immunocom-promised patients, especially those with hematologicneoplasia, renal transplants, and chronic granuloma-tous disease (1-5). The respiratory tract is generallyregarded as the portal of entry for invasive aspergillosisbecause the lung is frequently infected (1, 2), the dis-ease is prevented by filtration of the air in the envi-ronment of patients at risk (6), and the spores are smallenough to reach the alveoli (7). In both the pulmonaryand the disseminated disease of brain, kidneys, liver,and other organs, the fungus invades blood vessels andcauses thrombosis and infarcts (1, 2).

From clinical observations it has been concludedthat neutropenia and high doses of corticosteroids are

' Abbreviations used in this paper: AF, Aspergillus fu-migatus; agar; TSB, trypticase soy broth; HBSS, Hanks' bal-anced salt solution; FCS, fetal calf serum; PS, penicillin-streptomycin; CA; cortisone acetate; HN2, nitrogen mustard;HCS, hydrocortisone; CFU, colony-forming units; PMN,polymorphonuclear granulocyte; A, difference between twomean values; LD50, median lethal dose.

J. Clin. Invest. The American Society for Clinical Investigation, Inc. * 0021-9738/82/03/0617/14 $1.00

Volume 69 March 1982 617-631

617

the two major risk factors for invasive aspergillosis (1,2) and that the combination may act synergisticallyto break down natural resistance (2). The high inci-dence of aspergillosis in chronic granulomatous disease(5, 8) brings out the importance of phagocytes in re-sistance to aspergilli, but does not discriminate be-tween the relative contributions of polymorphonuclearand mononuclear phagocytes because both are defec-tive in oxidative killing in this disorder (9). Despitethis clinical evidence, it has not yet been shown thatphagocytes can kill either spores (10) or mycelia of AFin vitro, but there is evidence that polymorphonucleargranulocytes (PMN) can damage hyphae (11).

Cortisone lowers the normally high resistance of lab-oratory animals to invasive aspergillosis (12-24). Forexample, mice become extremely susceptible to as-pergillosis after large doses of cortisone (13, 14, 23,24), but not after myelosuppression or neutropenia (14,23). Cortisone suppresses the accumulation of phago-cytes at inflammatory sites, reduces bactericidal andcandidacidal activity of monocytes, and impairsexpression of cell-mediated immunity (25-27). Al-though none of these actions of cortisone that mightaffect aspergillosis have been studied systematically,the interesting observation was made that spores ofAspergillus flavus germinated faster in alveolar mac-rophages of cortisone-treated mice (22, 28).

In order to clarify the mechanisms of action of cor-tisone and to identify the role of myeiosuppression andneutropenia in lowering resistance to aspergillus in-fection, we have conducted experiments in a mousemodel of invasive aspergillosis. Our findings, whichalso include observations made during direct compar-isons in vitro of human and mouse phagocytes, allowus to delineate two lines of cellular defense that mustbe overcome by AF in order to establish disseminatedinfection.

METHODS

Organism

A strain of Aspergillus fumigatus (AF) isolated from thesputum of a patient at the University Hospital of San Diegowas used for all experiments. Conidia were scraped with awire loop from 4- to 6-d cultures on Sabouraud dextrose agar(SDA) plates supplemented with 100 ,g/ml of streptomycinand 100 U/ml penicillin G at 370C. Conidia were washedthree times in 0.9% saline, clumps were disrupted with aTeflon pestle and a Tri-R Stir-R homogenizer (Tri-R Instru-ments, Inc., Rockville Center, N. Y.), and the suspensionfiltered through a 12-ml plastic syringe packed with cottongauze. This procedure gave suspensions of 100% conidia (nomycelia) with > 95% single conidia.

Preincubated conidia. In some experiments conidia werepreincubated in broth as follows: 5 X 107 filtered single co-nidia in a volume of 1-5 ml of 0.15 Msaline were added toa 500-ml Erlenmeyer flask containing 200 ml of trypticase

soy broth (TSB) supplemented with penicillin-streptomycin(PS) and incubated overnight at room temperature withoutshaking. After the conidia were collected by centrifugationat 800 g for 15 min, the pellet was washed three times insaline, resuspended, and dispersed into single conidia as de-scribed for plate cultures. Phase contrast microscopy showedthat preincubated conidia were about four times larger indiameter but still round in form and free of germ tubes.

Media, drugs, and reagentsSabouraud dextrose agar, TSB, and Bacto tryptone were

obtained from Difco Laboratories, Detroit, Mich.; thiogly-colate from BBL Microbiology Systems, Cockeysville, Md.;cortisone acetate (CA) and nitrogen mustard (HN2) fromMerck Sharp & Dohme, West Point, Pa.; hydrocortisonefrom Calbiochem-Behring Corp., American Hoechst Corp.,San Diego, Calif.; Hanks' balanced salt solution (HBSS),RPMI-1640, Ca++- and Mg++-free phosphate-buffered Dul-beccos' modified Eagle medium, heat-inactivated fetal calfserum (FCS), and lyophilized penicillin-streptomycin (PS)were all purchased from Gibco Laboratories, Grand IslandBiological Co., Grand Island, N. Y. Amphotericin B was pur-chased from E. R. Squibb & Sons, Inc., Princeton, N. J., andstarch and Isopaque-Ficoll from Sigma Chemical Co., SaintLouis, Mo.

AnimalsIn most experiments we used 6-8-wk-old female CF1 mice

from Charles River Breeding Laboratories, Inc., Wilming-ton, Mass. 8-10-wk-old nu/nu- and nu/+-BALB. mice werea gift from Dr. N. 0. Kaplan from the Athymic Mouse Fa-cility, Cancer Center and the Department of Chemistry,University of California, San Diego. Four to six mice werekept in each cage. Cages and water bottles of immunosup-pressed animals were changed three times per week and foodand chlorinated drinking water were offered ad lib. Athymicmice were held in a separate room without other animals.Cages, housing material, water, and food were autoclavedbefore use, and cages were covered with sterile filter caps.The nude mouse wasting syndrome was not observed.

Immunosuppressive regimensCortisone. CA was injected s.c. either in one dose of 5

mgper animal in a volume of 0.2 ml of 0.15 MNaCl solution(saline) 48 h before challenge (13, 24) or in a daily dose of2.5 mg in 0.1 ml of saline for 6 consecutive d. Myelosuppres-sion was induced by HN2dissolved in sterile water accordingto the directions of the manufacturer and either used within1 h or frozen at -20°. Its ability to induce neutropenia wasunchanged after 7 d. HN2 was diluted to a concentration of200 Ag/ml in 0.9% of saline immediately before injectioninto the lateral tail vein. Optimal myelosuppression wasachieved by giving mice 40 Mg of HN2/d on 3 consecutived before challenge. Only one mouse died from HN2withoutfungal challenge in one of the control groups of five to sixmice accompanying each experiment. Larger doses of HN2were lethal in the absence of aspergillus infection.

Immunized mice

Mice were immunized with 5 X 105 live conidia, i.v., 28d before study and were treated with 1 mg/kg of ampho-tericin B in 5% dextrose, i.v., on days 3, 5, and 7 after the

618 A. Schaffner, H. Douglas, and A. Braude

conidial challenge. This established an infection lasting <28d. The IgG titer of pooled serum from five mice taken at 28d, the day of challenge, was >1:1,000, and the IgM titer was>1:100 in an enzyme-linked immunosorbent assay (29) usingfragments of boiled mycelia as antigen. Control mice re-ceived amphotericin B but no injections of conidia.

Challenge of miceIv. model. Washed conidia were injected in 0.5 ml of

0.9% saline in the lateral tail vein. All challenge doses werestandardized by adjusting the concentration of conidiaby hematocytometer counts and culturing serial dilutionsin TSB.

Inhalation model. Mice were placed in the inhalationchamber of Piggot and Emmons (30), which was manufac-tured by the glass shop of the University of California, SanDiego. The chamber permitted simultaneous exposure of 10animals to conidia aerosolized by blowing air over a culturegrowing in the chamber on Sabouraud dextrose agar withPS. The challenge dose was determined from the numberof colony-forming units (CFU) recovered from the lungs ofat least five animals per experiment (see below) and wasuniform for each experiment (±0.3 log SD). The exposuretime was three min, and the challenge dose was varied byusing young cultures (3-4 d, 37°C) for low doses and oldercultures (up to 10 d, 370C) for higher doses.

Clearance of fungi from tissuesAt specified times animals were killed with ether, and

individual organs were homogenized with Teflon pestles anda Tri-R Stir-R homogenizer (Tri-R Instruments, Inc.). Serial1:10 dilutions of each homogenate were mixed with Sa-bouraud dextrose agar in pour plates that were read after48 and 72 h at 370C. In studies of clearance of inhaled co-nidia, the right lower lung lobe was not homogenized, butreserved for histology. To calculate the organ distributions,the number of CFU/ml was multiplied by the total volumeof homogenate obtained.

Blood counts, bone marrow smears, andperitoneal exudative responseBlood obtained from the retroorbital venous plexus (31)

with calibrated heparinized glass capillaries was diluted in0.1 MHCI with 0.1% methylene blue, and leucocytes werecounted in a hematocytometer. Differential counts weredone on Wright-stained smears, except those below 1,000/,ul that were made from supravitally stained cells suspendedin HCI with methylene blue. Bone marrow aspirated fromthe femur of killed animals was examined on Giemsa-stainedsmears. Peritoneal cellular exudate was obtained for cellcounts by inserting a pasteur pipette through a small incision,washing with 12 ml of ice-cold Dulbecco's modified Eaglemedium free of CA++ and Mg++, centrifuging the collectedfluid at 300 g for 10 min at 4°C, and resuspending the cellsin 1 ml of Dulbecco's before counting.

HistologyOrgans were fixed immediately after sacrifice of the an-

imals in 10% phosphate-buffered formalin, pH 7.4, embed-ded in paraffin and stained by Giemsa's method.

In vitro studies with phagocytesViability of phagocytes was always >95% as judged by

trypan blue exclusion. Cells were counted in 0.1 MHCI withmethylene blue in a hematocytometer, and differentialcounts were made in duplicate from Giemsa-stained smears.Mouse peritoneal macrophages were either resident mac-rophages obtained without stimulation or exudative mac-rophages removed 5 d after i.p. injection of 1 ml of 2% starchin saline or 4% thioglycolate in water (31). Resident mac-rophages were >99% mononuclear cells and induced mac-rophages >90% mononuclears (<10% PMN). PMN-rich peri-toneal exudate cells from mice were removed 4 h after i.p.injection of 1 ml 10% Bacto-Tryptone in saline and contained>68% PMN. Human mononuclear phagocytes were sepa-rated by a standard technique (31) from the freshly voideddialysis fluid of four patients with end-stage renal diseasewithout peritonitis; they were not receiving steroids. 2.5X 10'-1 X 107 cells with 64-87% mononuclear cells were re-covered from 2 liter of dialysis fluid. Human PMNwereremoved by dextran sedimentation from heparinized blood(32) and used directly at a purity of >68% PMNor afterpurification by the Isopaque-Ficoll technique (32) to a purityof >96%.

Germination rate of conidia ingested byperitoneal macrophages in vitroPeritoneal macrophages were washed three times in HBSS,

adjusted to a final concentration of 1 X 106 cell/ml in RPMI-1640 with 10% FCSand 100 U/ml penicillin and 100 gg/mlstreptomycin. Aliquots of 0.15 ml were placed on 18-mmround glass coverslips in 60-mm plastic dishes and incubatedfor 2 h at 37°C. The dishes were washed three times byvigorous rocking with HBSS to remove nonadherent cellsand incubated in RPMI with FCS and PS overnight. Afterthree more washes with HBSS, the macrophages were cul-tured for 48 h longer either with or without 7.5-30 ug/mlof hydrocortisone. These concentrations are below the ex-pected plasma levels of mice given 5 mg of CA, s.c. (35),and comparable to those reached during high-dose steroidtreatment of man (34, 35). Thereafter the cells were washedtwice more with HBSSand infected with conidia suspendedin 5 ml of RPMI with FCS and PS at a concentration of 1-3 X 106/60-mm dish. After 1 h the dishes were vigorouslywashed six to eight times with HBSS until no extracellularconidia were seen under phase contrast microscopy. At spec-ified times, cell cultures were fixed in 95% ethanol andGiemsa-stained. The phagocytic index varied between ex-periments from 3.5 to 13 (n macrophages/n conidia) withzero to four conidia per single cell, whether cells were ex-posed to cortisone or not. Confluence of the macrophagecultures (10-50%) and estimated cell densities were also notinfluenced by HCS. The germination rate was the percentageof conidia forming mycelia among 100 counted per coverslipfrom triplicate wells.

Microassay for killing of spores by mouseperitoneal macrophagesWashed, thioglycolate-induced peritoneal macrophages

were suspended in RPMI-1640 with 10% FCS and PS at aconcentration of 1 X 106 cells/ml. Aliquots of 0.1 ml wereadded to flat-bottomed cluster wells with a capacity of 350Ml (96-well tissue culture clusters, 0.32 cm2, Costar DataPackaging, Cambridge, Mass.). After 2 h of incubation, wells

Natural Resistance to Aspergillus 619

were washed three times with HBSSand incubated overnightwith 100 ,lA of RPMI with FCS and PS, washed again threetimes with HBSS, and then infected with 100 ul of a sus-pension of 25-250 CFU single conidia per ml of mediumcontaining FCS. To control for killing by medium alone,conidia were dispensed in each experiment to an additionalplate that did not contain macrophages. The medium didnot kill the fungus; instead it promoted growth. After 1 hof phagocytosis, the wells containing macrophages werewashed seven times with HBSS to remove extracellularspores. Culture medium was added to alternate wells, and100 ul of distilled water was added to the remainder to lysethe cells and quench the interaction of phagocytes andspores. After 15 min, these control cultures were examinedfor lysis by phase contrast microscopy and 100 ul of doublestrength TSB with PS was added to promote fungal growth.In test wells macrophages and conidia were cultured to-gether for 48 h before their interaction was quenched bylysis as described for the control wells with lysed macro-phages. The plates were inspected daily for fungal coloniesfor 4 d after quenching. Sporulation and spread to otherwells was prevented by placing a drop of 5% aqueous meth-ylene blue in all wells showing growth, which uniformlyappeared 48 h after quenching.

Assay for killing of mycelia by PMNTube assay. From suspensions of 120-250 CFU/ml of

preincubated conidia in RPMI with 10% FCS and PS, ali-quots of 100 gl were dispensed to 15 X 150-mm flat bottomglass tubes and incubated at 37°C until mycelial growth wasseen by phase contrast microscopy (4-6 h). All tubes werecentrifuged at 800 g for 15 min to ensure that mycelia ad-hered to the bottom of the tubes. 1 ml of a suspension 1-3X 106 PMNin RPMI-1640 with 5% autologous, heat-inac-tivated serum, or medium and serum without PMNwasadded. In preliminary experiments it was determined that3 X 10' cells were required to cover the bottom of the tubesfrom the center to the periphery. To establish contact be-tween the leukocytes and the fungus, all tubes were centri-fuged at 150 g for 5 min. In addition to the medium control,the effect of pelleting leucocytes onto mycelia was controlledwith PMNthat were killed by freezing and thawing (31).After 12-h incubation, 2 ml of distilled water was added toall tubes and the mixture vortexed and incubated for 15 minto lyse the PMN. Thereafter, 2 ml of double strength TSBwith PS was added to promote fungal growth. Quenchedcultures were observed for 4 d, but macroscopic growth in-variably appeared as a clearly visible mycelial networkwithin 48 h.

Micro-well assay. A similar assay was performed in flatbottomed 350-,Ml cluster wells. Preincubated conidia weredispensed in a volume of 100 Ml of medium, reincubateduntil mycelial growth was visible, and centrifuged at 800 gfor 15 min. The supernatant was aspirated by vacuumthrough a 23-gauge needle and replaced with a specifiednumber of phagocytes washed three times in 100ulM of RPMI-1640 with 5% FCS, or with medium containing serum alone.The plates were centrifuged at 150 g for 5 min and incubatedfor 12 h before quenching with 100 lA of distilled water andaddition of double strength TSB. The plates were read forgrowth for 4 d. Methylene blue was added to wells thatturned positive to prevent sporulation.

Statistical analysisThe median lethal dose (LD50) for mice challenged with

conidia was calculated by the method of Reed and Muench

(36). The significance of differences observed in the LD50experiments was computed by comparing the frequency ofdeath with 2 X k contingency tables and by using the Bon-ferroni-X2 statistics tables for correction of multiple com-parisons (37). Differences of pooled data from several LD50experiments were calculated at each dose level becausegroup sizes were unequal from pooling. The results of thein vitro killing assay were also analyzed by 2 X k contingencytables with correction for multiple comparisons. Differencesbetweeen mean values were determined by t test.

RESULTS

Effects of immunosuppressive regimens on phago-cytes. HN2 reduced drastically the total blood leu-kocyte and PMNcounts (Fig. 1), and during the PMNnadir, bone marrow cellularity dropped by >70% witha complete absence of the postmitotic pool of the PMNseries. In contrast, the PMNcounts were unchangedfrom 48 h up to 8 d after injection of 5 mg CA, whilethe mononuclear cell counts dropped by 95% for sev-eral days. After daily injections of 2.5 mg of CA, thecounts of mononuclear cells were further reduced to<50 cell/ill again without decreased PMNcounts.

To compare suppression of the cellular inflamma-tory response by CAand HN2, the peritoneal exudativeresponse to heat-killed conidia was studied in normal,CA-, and HN2-treated mice (Fig. 2). This experimentwas repeated with live conidia, and similar results wereobtained. When mice were treated in this experimentwith CA from 2 d before stimulation with spores untilcells were harvested, the total cellular response fell93% below that of normal mice (P < 0.01). PMNfell95% (P < 0.001), a suppression similar to that in HN2-treated mice.

HN2i

3 4DAYS

FIGURE 1 Blood leukocyte changes in mice given HN2. Re-sponse to three daily doses of 40 Mg HN2 i.v. Mean±SD fromthree animals. (-): total white cell count; (A): PMN-count;(A): PMN-counts from representative individual animalsfrom subsequent experiments.


B 72 h

E total cells

E neutrophil granulocytes

E mononuclear cells

e 7w 107

0

a

x2eac0

*. s xlO-e 5x1

S0.01E

6

controls 5mg CA HN2 controls 5mg CA H N2

FIGURE 2 Peritoneal cellular inflammatory response to heat-killed conidia. A. Peritoneal cel-lular response 24 h after intraperitoneal challenge with 107 heat-killed conidia in 1 ml of 0.15M NaCl. Mean±SD from six animals per group. All cell counts HN2 vs. controls and CA vs.controls: P < 0.001. B. Response 72 h after injection of spores. 5 mg CA: animals given 5 mgof cortisone acetate s.c. 48 h before challenge. HN2: 3 X 40 Ag HN2 i.v. Mean±SD from fouranimals per group. Mononuclear cells: HN2 vs. control P < 0.001, CA vs. control P < 0.05.PMN: HN2 vs. control P < 0.001, CA vs. control NS.

The number of resident peritoneal macrophages re-covered after immunosuppression without stimulationwith spores was 4.47±0.8 X 106 (mean±SD from sixanimals) in HN2-treated, and 3.95±0.83 X 106 in CA-treated mice compared with 5.4±1.6 X 106 in normalmice, demonstrating that the influx of new phagocytesrather than the pool of resident macrophages is af-fected by CA and HN2.

Comparative effect of HN2 and CA on suscepti-bility to intravenous conidia. One dose of CA 48 hbefore challenge reduced the LD50 of intravenous chal-lenge by 1.7 log below normal mice (P < 0.01) in bothsimultaneous comparisons and four pooled experi-ments (Table I). After continuous immunosuppressionwith 2.5 mg of CA for 6 d (beginning 2 d before chal-lenge), the LD50 was 2.2 logs lower than that of animalstreated only once with CA.

In contrast, neutropenia induced by HN2 loweredthe LD50 by only 0.52 log (0.1 > P > 0.05, Table I).Because HN2and CAsuppressed inflammation equally,we sought another explanation for the differences insusceptibility to aspergillosis. First, we examined thepossibility that CA suppressed the T lymphocytes bychallenging athymic mice with AF spores, but foundthem equally resistant. Moreover, athymic mice wereas susceptible to immunosuppression with CA as CF1mice (Table I). To exclude the possibility that cortisonemight lower resistance by blocking the early humoralimmune response, we tested the susceptibility of micewith antibody titers of >1:100, which developed after

active infection. They were as susceptible as their non-immunized littermates (Table I). Thus, CA lowers re-sistance even in the presence of humoral antibody.

We turned next to mononuclear phagocytes as thecortisone-susceptible defense system. We comparedthe susceptibility of normal, neutropenic, and CA-treated mice to preincubated spores because studiesof Aspergillus flavus infections suggested that sporesbecome resistant to macrophages after preincubationin broth (20). Normal mice were only moderately moresusceptible to preincubated spores than freshly col-lected spores (Table I: A log LD50:0.76, P < 0.01). Thefindings in CA-treated mice were similar (A logLDso:0.85, P < 0.01). Neutropenic mice, however,were much more susceptible to preincubated conidiathan to fresh conidia (A log LD50:1.55, P <0.001).More important, myelo-suppressed mice were farmore susceptible to preincubated spores than normalmice (A log LD50:1.32, P < 0.01) but not to restingspores; in fact the HN2-treated mice became as sus-ceptible to preincubated conidia as CA-treated micewere to resting spores. This ability of preincubated(activated) spores to kill neutropenic mice that resistresting spores indicated that another line of defensecan be breached in the absence of PMNby preincu-bated spores, but not resting spores.

Distribution and clearance of i.v.-injected spores.Further insight into the role of macrophages wasgained by comparing the distribution of spores afterintravenous injection into normal, HN2-treated, CA-


24 h

TABLE ISusceptibility of Mice to i.v. Injections of Spores of AF

Resting spores

P value vs.Groups of mice LD5o at 21 d- control

P<

Normal CF1 8.0 X 105 -tMyelosuppressed HN2A 2.4 X I05 NS5 mg CA" (1 dose) 1.7 X 104 0.012.5 mg CA, daily X 6 1.0 X 102** 0.001nu/+ BALBc 2.0 X 106 _tnu/nu BALBc 5.0 x 106 NSnu/nu BALBC, 5 mg CA 3.2 X 103 0.015 mg CA (1 dose)l 6.2 X 104 -Immune, (reinfected), 5

mg CA¶ 5.3 X 104 NS

Preincubated (activated spores)

Normal CF1 1.4 X H0P -tMyelosuppressed HN2A 8.0 X 103 0.0015 mg CA 4.0 X 103 0.001nu/+ BALBc 4.8 X 10500 -tnu/nu BALBc 3.3 X 105* NS

° LD5o were calculated from the pooled data of two to four ex-periments with >30 6-8-wk-old mice per group in each experiment.t Control for subsequent group(s).§ HN2, three daily doses of nitrogen mustard i.v. before challenge." CA, cortisone acetate.T In this experiment only, the mice were 11 wk old. The LD50 ofuntreated littermates was 2.1 X 106.

Single experiments.

treated, and athymic mice. 2 h after injection of 6X 104 conidia i.v., 73% of the injected spores were re-covered as CFU from the liver (95% of recoveredCFU), lungs (1.3%), kidneys (0.3%), and brain(<0.01%). Except for the spleen, the organ distributionof spores was the same in all groups of mice. Spleenweights were reduced by 72 and 69% (mean of fiveanimals per group) after treatment with HN2 or CAand contained about half the CFU of normal mice(2.95%). Thus, spores preferentially located in organsrich in mononuclear phagocytes and differences insusceptibility among various groups of mice could notbe explained by a failure of clearance into certain or-gans. Control mice and myelosuppressed mice eradi-cated >99% of recoverable CFU in 6 d from the livers(Fig. 3A), kidneys (Fig. 3B), spleens, and lungs (datanot shown). No more than 5 CFUwere ever recoveredfrom the brain of normal or HN2-treated mice, andit was sterile after day 4 in both groups. An unexpectedtendency to clear spores faster from the livers of my-elosuppressed animals could not be explained by a re-

4-

S

a1

0E0

-I

V11

3-

2-

A

Smg CA

2 24 48I

96 144

hours after chalilnge

4 -

0

0I.'E

11

oS

2-I

a1-02

B

Smng CA

normalcontrols

2 24 48 96 144

hours after challenge

FIGURE 3 Imnpaired clearance of AF from the liver and kid-ney of cortisone-treated mice after i.v. challenge. A. CFUper milliliter of organ homogenate recovered from the liverof normal (e), myelosuppressed (0), and CA-treated (oneinjection 5 mg cortisone acetate) mice (A) after injection of6 X 104 conidia i.v. Mean±SD of CFU from three animalsper group. At 92 h and thereafter, individual values are giveninstead of the SD for CA-treated mice. Myelosuppressed vs.normal mice: 24 h A log CFU: 0.61, P < 0.05 (two-tailed ttest). Other values: NS. CA-treated vs. control: 24 h: P < 0.05;48 h: P < 0.025. B. CFU recovered from the kidneys.Mean±SD from three animals. Myelosuppressed animalswith same elimination pattern as normal animals omittedfor clarity. CA vs. control. 24 h: P < 0.05; 48 h: P < 0.01;96h: P<0.025; 144h: P<0.025.

distribution of the fungus, because the CFU fell in allorgans simultaneously (Fig. 3A).

In contrast to myelosuppression, CA treatment im-paired clearance of the fungus, and the CFU rose in


several organs. A significant and uniform impairmentoccured in the liver between 24 and 48 h after chal-lenge. Thereafter, CFU varied from >500/ml in in-farcted livers to <10 ml in grossly normal livers (Fig.3A). The fungus always multiplied in the kidneys ofCA-treated mice (Fig. 3B), caused cortical lesions witha scant phagocytic response, and finally destroyed themedulla. In contrast, no lesions or fungal growth wereseen in the kidneys of HN2-treated or normal mice.AF multiplied in the brains of five of nine CA-treatedanimals and in the lungs of one. Elimination of thefungus from the spleen was not impaired by CA, andthe fungus did not multiply in this organ.

Athymic mice and heterozygote littermates elimi-nated the fungus at the same rate from the liver (Fig.4), brain, spleen, and lung. In addition, the eliminationpattern of spores from these organs by nu/nu- and nu/+-BALB, mice was similar to that of CF1-mice, in spiteof the higher challenge dose to athymic mice (2 X 105vs. 6 X 104). This higher dose caused kidney infectionwith gross lesions in several nu/nu- and nu/+-mice.Between days 7 and 17 after challenge, >300 CFU/ml of kidney homogenate were recovered from twoof nine nu/nu-mice and three of nine nu/+-mice; theother mice eliminated the fungus from the kidneys bythe pattern seen in CF1 mice with a lower challengedose. Even with this protracted infection, there wasno difference between athymic mice and phenotypi-cally normal littermates.

DAYS AFTER CHALLENGE

FIGURE 4 Clearance of resting conidia from the liver of nu/nu- (0) and nu/+ (X) BALBC mice. Mean±SD of CFU re-covered per milliliter of liver homogenate from three ani-mals per group after injection of 2 X 105 conidia i.v. Therewas no significant difference in clearance between the twogroups (all A CFU:NS).

Preincubation of spores in broth resulted in amarked change in organ distribution. 2 h after injec-tion of preincubated conidia, more fungus was re-covered from the lungs (25%), kidneys (5.5%), andbrain (0.5%), and less from the liver (67%) and spleen(2%) than in experiments with resting spores. The al-tered distribution is attributed to the increased size ofspores and might explain the increased susceptibilityof normal, CA-treated and, in part, neutropenic mice.In addition to the altered distribution, the clearanceof spores was reduced in myelosuppressed mice andthe fungus was not eradicated as it was by normalmice. This difference was independent of the numberof organisms reaching individual organs (Fig. 5A andB). Thus, myelosuppressed mice that eliminate restingspores at least as well as control mice could not mountan effective defense against preincubated spores.Clearance was delayed in all organs as early as 24 hafter challenge and the fungus multiplied, especiallyin the kidneys and brain. In contrast, normal micesuccessfully controlled the infection in the kidneys andbrain at the lower challenge dose. At the higher chal-lenge dose (2 X 104 preincubated conidia), 75 timesmore CFU were recovered per milliliter of brain ho-mogenate than in experiments with 6 X 104 restingspores. The higher challenge caused limited growthof the fungus in the brain of control animals but sig-nificantly less than in myelosuppressed mice (Fig. 6B)and killed no normal animals by day 14, whereas mye-losuppressed animals died within 6 d after challenge.Thus, normal mice controlled the infection even whenchallenged with 2 X 104 preincubated conidia.

Susceptibility of immunosuppressed mice to in-haled conidia. To find out if i.v. challenge was rep-resentative of natural infection, we exposed mice toconidia in an inhalation chamber. Normal mice wereextremely resistant to inhaled conidia (Table II) andHN2-treated mice died only at high challenge doses.One dose of CA also had little effect on the lethalityof inhaled fungi but continuous immunosuppressionwith 2.5 mgof CA for 6 consecutive d killed all animalsexposed to spores, even at the lowest challenge doseof 2.5 X 105 CFU (Table II). In contrast to normals,myelosuppressed mice given one dose of CA were vul-nerable to challenge with a moderate dose of inhaledconidia. Thus, a challenge with <1.5 X 106 sporeskilled neither normal nor HN2-treated mice and only onemouse given a single dose of CA, but all mice treated withone dose of CA plus HN2 died. Mice treated with thiscombined regimen were as susceptible as those given dailyinjections of CA alone (Table II).

Clearance of inhaled conidia. Normal CF1 miceeliminated conidia at highly consistent rates in threestudies with different batches of mice (Fig. 6A). In-haled conidia were cleared efficiently by severely neu-


A KIDNEYS

2 24 48 72 96

B BRAINIo2 * 10 activat*d

*e 2' 103 conidia i.v.* 0 my9losuppressed

0o normal controls

2 24 46 96

HOURS AFTER CHAUENGE

FIGURE 5 Impaired clearance of preincubated (activated) conidia from the kidney and brainafter i.v. injection of myelosuppressed mice. Mean±SDof CFU/ml of organ homogenate fromtwo experiments with 2 X 104 and 2 X 103 preincubated conidia, respectively; each point rep-resents mean of three animals. A. Clearance from the kidneys: A log CFUwith 2 X 104 conidia:48 h: P < 0.05; 72 h: P < 0.01; with 2 X 103 conidia: 48 h: P < 0.005; 96 h: P < 0.001. B.Clearance from the brain. Lower dose 96 h: P < 0.05; higher dose 24 h: P < 0.025.

tropenic mice as expected from their high resistanceto invasive pulmonary aspergillosis (Fig. 6B). Theclearance curves of both normal and neutropenic micefollow first order kinetics with no acquired enhance-ment of elimination that might suggest a specific im-mune response during the clearance studies. Abundantspores, but no mycelia, were seen in histologic sections

of the right lower lobe up to 96 h after challenge. Be-cause spores are prevented from germination and thusare the only recognizable form of the fungus and be-cause >90% of CFU are cleared from the lungs perday, it follows that spores can be cleared efficientlyby a mechanism that is independent of neutrophils.Daily cultures of homogenates of brain, spleen, liver,

TABLE IISusceptibility of Mice to Inhaled Spores of AF

Challengedose" Immunosuppressive regimen

None HN,$ 5 mg daily HN, + 5CAJ CA" mg CAT

8 x 106 2/18°° 16/18 16/18 15/15 18/182 X 106 0/5 1/5 2/5 - -

1.5 X 106 0/8 0/8 - 8/8 -1 x lo6 0/8 0/8 1/8 8/81t 8/8tt

2.5 X 105 0/8 0/8 0/8 8/8tt 8/8tt

° Each challenge dose of spores represents an independent experiment.I Mice myelosuppressed with 3 X 40 lAg of nitrogen mustard.§ Mice immunosuppressed with a single dose of cortisone acetate."Mice immunosuppressed with daily 2.5 mg of cortisone acetate for 6 d.¶ Mice immunosuppressed with 3 X 40 ,ug of nitrogen mustard and a single dose of 5mg cortisone acetate.o Number dead at 21 d over the number challenged.I P vs. control <0.01.


^^^ 4

z

00

3-0zz00 2-

N.

vj 1-

00

Normal CF- mice

* mean, n. 3s-

4-

3.

2-

2 24 46 72 96

c

o nu/nu SALBC

* nu/+ BALSc

o myslosuppressd. normal controls

144 2 2 4I 14

144 2 24 48 96 144

D A 6-2.5mg Cortisone* normal controls

192 2 24 4i 72 98

HOURS AFTER CHALLENGE

FIGURE 6 Clearance of inhaled conidia by normal, myelosuppressed, cortisone-treated, andathymic mice. Clearance of conidia from the lungs after exposure of mice in four independentexperiments to aerosolized spores. Mean±CFU/ml of lung homogenate from three mice pergroup at each time point. Myelosuppressed mice: 3 X 40 ,ug of nitrogen mustard i.v. Cortisone-treated mice: 6 X 2.5 mg of cortisone acetate s.c. B. All A log CFU:NS. C. All A log CFU:NS(two tailed t test). D. A log CFU at all time points: P < 0.005.

and kidneys after inhalation of spores disclosed no fun- the predominant form of the fungus in both CA-gus in organs other than the lung. Athymic mice treated and control mice. By 48 h, the differences be-cleared inhaled spores as effectively as phenotypically tween the clearance curves widened, and histologicnormal littermates (Fig. 6C). sections from CA-treated mice showed progressively

Unlike athymic and myelosuppressed mice, those more mycelia, invasion into blood vessels, and conse-

treated with CA exhibited impaired clearance of AF quent pulmonary infarction. Many spores appeared to(Fig. 6D). By 24 h after inhalation, the clearance rate be ingested by mononuclear phagocytes, and trans-of conidia in CA-treated mice was significantly below formation of spores into mycelia was observed in pha-normal. At this phase of clearance, conidia represented gosomes of CA-treated, but not normal or HN2-treated


6-A

S.

4.

3.

2

4czo 1'0

0 z0z

-IU '.

00-a 6-

244 "

6.

I

mice. Cultures disclosed that invasive growth in thelungs was followed by dissemination of the fungus tothe kidneys, brain, and liver in most CA-treated mice.

Interaction of human and murine macrophageswith resting and preincubated spores of AF. Directevidence for the importance of mononuclear phago-cytes as the first line of defense against AF was ob-tained from studies on the interaction of macrophageswith conidia. When spores were injected i.p., theywere readily ingested by macrophages whether micewere immunosuppressed or not. When the peritonealexudate was harvested 24 h after challenge, the sporeswere still predominantly in macrophages. In threedifferent experiments, CA-treatment significantly im-paired the ability of peritoneal macrophages to pre-vent transformation of spores into mycelia in vivo(Table III). One dose of CA increased the germinationrate at 24 h by two- to threefold and daily treatmentby 8- to 10-fold. The faster germination was indepen-dent of the cellular composition of the peritoneal ex-udate that was similar in mice treated with HN2 orcontinuous CA. Because myelosuppressed mice re-tained the capacity to prevent germination, these ex-periments pointed to a functional disturbance inmononuclear phagocytes produced by CA.

Macrophages also slowed transformation of ingestedspores into mycelia in vitro, but less efficiently thanin vivo. Observations on germination rates were notpossible beyond 24 h because progressive growth ofescaping mycelia destroyed and overgrew the mono-layer of phagocytes. Germination rates were highestbetween 15 and 21 h of co-culture but did not reach

TABLE IIIGermination Rate of Conidia Ingested by Peritoneal Mouse

Macrophages In Viwo

Groups of mice Germination

Controls 3.4±1.1Cortisone, single dose, 5 mg 10.3±2.8tCortisone, 4 X 2.5 mg 30.7±4.2§Nitrogen mustard, 3 X 40 Aig 2.3±1.2"Preincubated conidia 28.2±4.9§nu/nu BALBCII 3.0±1.6nu/nu BALBC, 4 X 2.5 mg cortisone¶ 26.2±5.2§

% Conidia transforming into mycelia in peritoneal macrophages24 h after i.p. challenge with 2 X 106 conidia (mean±SD fromduplicate combined experiments with three to five animals pergroup in each experiment).t vs. control P < 0.01.

vs. control P < 0.001.vs. control:NS.

¶ Single experiment.

a definite plateau at 24 h, indicating that preventionof germination is not synonymous with killing ofspores.

Cortisone inhibited the ability of macrophages toprevent germination of spores in vitro also. Germi-nation rates increased significantly by 2.3- to 3.2-foldafter resident or induced peritoneal mouse macro-phages were exposed to cortisone in vitro or in vivo(Table IV). Resident macrophages, whether exposedto CA or not, were less able to prevent germination;and co-cultures with resident cells had to be evaluatedat 12 h because of fungal overgrowth. Direct stimu-lation of the fungi by cortisone was excluded becauseno cortisone was present in the culture medium whencells were infected, and germination was not enhancedwhen cortisone was added to conidia in medium alone(data not shown).

Humanmacrophages were comparably inhibited bycortisone (Table IV). They also lost the ability to pre-vent germination of ingested spores after cortisonetreatment. The mycelia sprouting from spores piercedthe cells and spread rapidly extracellularly.

Macrophages could not prevent transformation ofpreincubated spores into hyphae. By 24 h after inges-tion, very few nongerminated spores could be madeout in macrophages, and the abundant mycelial formsof the fungus could no longer be counted individuallyso that an exact germination rate could not be deter-mined. Wesolved this technical problem by showingthat spores preincubated in tissue culture medium for6 h before a 10-h period of phagocytosis transformedsignificantly faster than resting spores subjected tophagocytosis for 24 h. Mean transformation rates ±SDfrom two independent experiments were 33±3.8% forpreincubated spores and 22±2.2% for resting spores(P < 0.005).

Because multiplication of escaping fungi made itimpossible to test killing by demonstrating a reductionof CFU, we tried to determine if macrophages couldsterilize small conidial inocula in microcultures. In fiveconsecutive experiments, macrophages sterilized a sig-nificant number of wells to which small inocula ofspores were added. Sterilization occurred in a doserange of 2 to 12 CFU per well and was dose dependent.No sterilization was detected when a dose of 22 CFUwas added per well (Table V). In contrast to the find-ings with resting spores, preincubated conidia werenot killed in parallel microculture experiments withthe same cell preparations as predicted by the in-ability of macrophages to prevent their germination(Table V).

Killing of mycelia by human and murine PMN.In view of our in vivo evidence from the susceptibilityand clearance studies that neutrophil granulocytesform the important defense line against AF after ger-


TABLE IVEffect of Cortisone on the Germination Rate of Conidia Ingested by Peritoneal

Macrophages In Vitro-

GerminationCortisone rate exposed Germination rate

Macrophage source exposure cells control cells

Mouse, inducedt in vivo§ 50.3±6%" 21.3±2.9%Mouse, resident in vivo§ 41.0±7.3%" 13.7±3.3%1

in vitroMig HCS/ml

Mouse, inducedt 30 Mg/ml 60.5±5.8%" 20.8±2.1%

7.5 ,g/ml 30.3±3.2%"Mouse, induced" 15 Mug/ml 37.7±3.5%" 4.7±2.1%¶

30,ug/ml 41.7±5.5%"

Human 30 Mg/ml 39±3.6%" 10.3±1.7%

7.5 Mg/ml 50.8±3.4%"Human 15 Mg/ml 60.5±5%" 11.8±2.8%

30 Mg/ml 59.6±2.5%"

a Mean±SDof germination rates at 24 h, pooled data from two independent experiments.I Macrophages harvested 5 d after stimulation with thioglycolate.§ 4 Daily injections of 2.5 mg cortisone acetate before cell harvest."vs. control: P < 0.001; germination rates of media controls were >90% for each ex-periment.¶ Germination rates at 12 h.

a Single experiment.

TABLE VKilling of AF Spores by Mouse Peritoneal Macrophages: Sterilization of Monolayers 48 h after

Ingestion of Resting or Preincubated Spores

Inoculum size(CFU)

Restingspores

1.6±1.12±1.2

3.3±2.55.7±1.5

11.7±1.522.3±4.5

Preincubatedspores

5±27.6±2.5

Number of wells sterilized by:

Mediacontrols

14/561/260/480/480/480/48

0/480/48

Livemacrophages

47/68°26/66-13/4810/4815/48§0/48

1/48"0/24

Quenched (lysed)macrophages

27/68e9/66°1/48-0/48t0/48§0/48

0/48"0/24

* P < 0.001.t P < 0.01.

§ P < 0.05." NS for comparisons between corresponding pairs of wells with live vs. quenched (lysed) macrophages,which served as controls for the test wells.


Exp. No.

123456

45

mination, we examined human and murine PMNfortheir ability to kill mycelia in vitro.

In 12 h human blood leukocytes gently centrifugedon top of mycelial micro-cultures in flat bottom tubessterilized the vast majority of cultures inoculated withup to 26 CFU of conidia. There was no difference inkilling between a crude leukocyte preparation (72%PMN; cells vs. media control P < 0.001) and purifiedPMN(96% PMN, cells vs. media control P < 0.001).Sterilization did not occur when centrifugation wasomitted, the assay was performed in round-bottom tubes,or the leukocytes were killed by freezing or thawing. Ina similar assay performed in micro-wells, we proved thatit was PMNthat killed hyphae by demonstrating that (a)PMNpreparations of >99% purity sterilize microcultures,(b) the killing capacity of cell preparations with low PMNfractions is reduced, and (c) mononuclear cells (monocytesand lymphocytes) in excess of the number present in thePMNpreparations cannot kill hyphae (Table VI). Weobtained similar results with mouse peritoneal leukocytesenriched either for PMNor mononuclear cells (Table VI).

DISCUSSION

On the basis of these findings we delineate for the firsttime two distinct lines of defense against Aspergillusand show that both must be breached before the funguscan establish progressive infection. The following dis-cussion recapitulates and synthesizes our findings insupport of this premise and relates these experimentalresults to human aspergillosis.

Natural resistance directed against spores. Thesestudies show that natural immunity to resting sporesof AF is largely independent of PMN, T lymphocytes,and humoral immunity, and that mononuclear phago-cytes form a very efficient defense system by rapidlykilling the fungus in its conidial stage (Figs. 3, 4, 6).In keeping with the reports that inhaled spores of As-pergillus flavus are seen predominantly in phagosomesof alveolar macrophages (22, 28) and that spores of AFare selectively phagocytized by mononuclear phago-cytes in vitro (10), we found that spores of AF arereadily ingested by macrophages in vitro and in vivoand preferentially removed by organs rich in reticu-loendothelial phagocytes after i.v. injection. Wedem-onstrated both in the peritoneal cavity and in vitrothat macrophages of normal mice prevented germi-nation of spores of AF, the prerequisite inhibitory stepfor killing the fungus in its conidial stage. Finally, weshowed that peritoneal macrophages killed small in-ocula of resting spores in microcultures (Table V).

Cortisone inhibits killing of spores by macro-phages. Cortisone significantly impaired the abilityof macrophages to prevent germination of conidia invitro (Table IV) and in vivo (Table III), and therefore

TABLE VIKilling of Mycelia by Leukocytes: Sterilization of Mycelial

Microcultures after 12 h of Incubation with PMN-richPreparations

Human leukocytes-

FractionCell preparations Inoculum Media control sterilized

No. cells added/well, % PMN CFUconidia

Exp. 1 3 X 105, >99% 5±2 3/32 31/32§Exp. 2 1 X 105, >99% 51±8 0/32 7/3211

Exp. 3 2 X 105, 96% 11±2 0/48 46/48§112 X 105, 72% 11±2 0/48 45/48§2 X 105, 4% 11±2 0/48 24/48§112 x 104, 4% 11±2 0/48 0/48

Mouse peritoneal cell preparationst

Exp. 4 3 X 105, 68% 8±3 0/48 33/48§

Exp. 5 1 X 105, 78% 5±3 3/48 20/48§

Exp. 6 1 X 105, 74% 3±2 9/48 44/48§11 X I05, 7% 3±2 10/44 13/44a@¶

Exp. 7 2 X 105, 73% 47±12 0/48 21/48§12 X 105, 8% 47±12 0/48 0/481T

o Human leukocytes were used either as crude preparations fromdextran sedimented blood or after fractionation by the isopaque-ficoll technique using a single donor per experiment.I PMN-rich exudates were obtained from the peritoneal cavity ofmice 4 h after injection of 10% Bacto tryptone and compared topreparations rich in mononuclear cells harvested 5 d after injectionof 4% thioglycolate (Exp. 6) or 24 h after injection of 10% Bactotryptone (Exp. 7).§ vs. control P < 0.001."I vs. control P < 0.01.¶ P < 0.001.a a vs. control: NS.

to kill the spores. Once transformed into a mycelium,the fungus readily pierces the phagocyte in vitro andin vivo. Thus, failure to prevent germination resultsin impaired clearance of the fungus in vivo (Figs. 3and 6).

The observation that exposure to cortisone interfereswith the capacity of pure cultures of human and mousemacrophages in vitro and of mouse peritoneal mac-rophages in vivo to prevent germination indicates thatthe drug acts directly on the mononuclear phagocyteand not through T lymphocytes or other systems thatmodify macrophage function. Near maximum im-pairment of human macrophages by 7.5 ,g of hydro-cortisone per milliliter, a concentration similar toplasma levels achieved by high-dose steroid therapyin humans (34, 35) suggests that conidiacidal activityof human macrophages is also impaired in vivo. This


is supported by reports that the candidacidal activityof human monocytes is similarly impaired in vitro byexposure to 16 Ag/ml of HCSand in vivo by treatmentwith two doses of 50 mg prednisone per d (26, 27).

Our findings extend the observations of Merkow etal. (22, 28) that macrophages prevented germinationof ingested conidia of Aspergillus flavus and that cor-tisone enhances intracellular germination, not only byduplicating this phenomenon with AF, but also bydiscovering three new properties of macrophages: (a)they kill ingested spores (Table V), (b) they protectmice even after ablation of PMN(Tables I and II), and(c) their function is impaired by a direct action ofcortisone (Table IV).

Natural resistance against mycelia. Althoughmyelosuppression is believed to be the major risk factorfor invasive aspergillosis in patients (1, 2), it has onlya minor deleterious effect on the natural resistance oflaboratory animals and only with extremely high chal-lenges of conidia (14, 23). The potent defense againstconidia provided by macrophages in our studies ex-plains these discrepancies. The short term myelo-suppression achievable in laboratory animals does notinvolve the pool of tissue macrophages that has a turn-over rate of several months (38) and thus does notaffect the function of the reticuloendothelial system.

Wehave shown that the roles of myelosuppressionand neutropenia become evident only after conidiaescape from the reticuloendothelial system and startmycelial growth. Overwhelming the macrophages byextremely high doses is not a realistic model of humandisease, and it is not surprising that we could not dem-onstrate a significant difference in the susceptibilityof neutropenic and normal mice when we used thistactic in the i.v. model (Table I). However, as sug-gested for Aspergillus fiavus(20), conidia of AF couldbe rendered resistant to macrophages by preincubationin broth. This simple maneuver revealed an impressivedifference in the susceptibility of neutropenic and con-trol mice to aspergillosis and unmasked the importanceof the neutrophil granulocyte (Table I).

Whenwe tried to induce mycelial growth in normalanimals by challenging them with a high dose of prein-cubated conidia, mycelia were scarce and surroundedby typical PMN-rich abscesses. In contrast, abundantmycelia invaded the tissues and encountered little orno PMNresponse in myelosuppressed or CA-treatedanimals. Cultures of the tissues showed that growthwas limited and that fungi were eliminated by normalmice, whereas multiplication was unopposed in im-munosuppressed animals (Fig. 5). These findings,along with efficient killing of mycelia by PMNin vitro(Table VI), establish granulocytes as the major defenseagainst mycelia.

Relative importance of resistance against spores

provided by macrophages and against mycelia byPMN. The strong resistance remaining after ablationof PMNby cytotoxic drugs demonstrates that mac-rophages can protect animals from high challengedoses of resting spores. When this first line of defenseis overpowered with preincubated spores, normal micecan still resist infection, but neutropenic mice areoverwhelmed. Thus, neutrophils form a second line ofdefense by killing mycelia. Natural resistance collapsesonly when both lines of defense are damaged, as seenafter large doses of CA (Tables I and II), which impairkilling of conidia by macrophages and the mobilizationof PMNaround the fungus, or after combined im-munosuppression with one dose of CA plus myelo-suppression with HN2 (Table II). Thus patients re-ceiving organ transplants, who are at high risk ofaspergillosis, receive both high doses of steroids andcytotoxic drugs and are often neutropenic (39). Pa-tients with chronic granulomatous disease are defec-tive in oxidative killing so that their neutrophils wouldnot be expected to kill mycelia nor their mononuclearcells to kill conidia. Old reviews of aspergillosis (1, 2)stem from a time when steroids were part of standardtherapy of acute myelogenous leukemia. Lately, ste-roids are omitted but these patients continue to haveserious problems with invasive aspergillosis. This clin-ical observation does not necessarily contradict ourfindings that mice immunosuppressed with myelotoxicdrugs alone are quite resistant to a challenge withspores, because aplasia induced by treatment of leu-kemia lasts much longer than that induced in our mice.Aspergillosis is generally seen late in therapeutic apla-sia and is not a complication of neutropenic episodesof short duration, an observation in agreement withour experimental findings. Furthermore, bacterialpneumonia often precedes invasive pulmonary asper-gillosis (2), another factor that might affect resistanceprovided by resident pulmonary macrophages.

The disease induced in mice after immunosuppres-sion with a combined regimen of CAand HN2or highdoses of CA alone, which is characterized by invasivefungal growth in the lungs, penetration of pulmonaryblood vessels, formation of infarcts and disseminationto the kidneys, brain, and liver, correspond well to thedescription of human disease (1, 2).

These observations on the existence of alternativesystems of cellular defense that can reinforce or re-place the other has not been brought into focus before.Joint systems of humoral and cellular defense havebeen described in resistance to gram-negative bacteriaand viruses (40). Against fungi, however, humoral im-munity seems to offer little if any resistance (41), sothat phagocytic cells are the main barrier against theseorganisms. It is intriguing that in the absence of ef-fective antibody, the immune system can still call upon


two lines of defense, and that these are two differentkinds of phagocytic cells. It is also remarkable thateach prong of this double system of cellular resistanceis directed at only one of the two stages of the fungus:the macrophage against the spore and the granulocyteagainst the mycelium.

In contrast to aspergilli, more invasive fungi invadethe host even in the presence of intact phagocytic de-fense systems. These fungi differ from aspergillus bytheir dimorphism that dispenses with mycelial growthin vivo. This conversion of the mycelial phase to theresistant tissue phase has been observed when sporesor mycelia of Coccidioides immitis come under attackby macrophages (42) and PMN(43). Control of infec-tion with dimorphic fungi appears to require a specificcell-mediated immune response that is superfluous formonomorphic aspergilli, which are limited to mycelialgrowth in vivo.

ACKNOWLEDGMENTS

This investigation was supported in part by Public HealthService International Research Fellowship 1 F05 TW02935,by the Schweizerische Gesellschaft fuer Innere Medizin, andU. S. Public Health Service training grant AI 07036. Wethank the Athymic Mouse Facility, Cancer Center and theDepartment of Chemistry, University of California, SanDiego (supported by grant number CA-23052) for theathymic mice.

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mononuclear and against mycelia by polymorphonuclear

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