Vol. 42. No. 3INFECTION AND IMMUNITY, Dec. 1983. p. 1109-11150019-9567/83/121109-07$02.00/0Copyright © 1983, American Society for Microbiology

Killing of Aspergillus Spores Depends on the AnatomicalSource of the Macrophage

ANDREAS SCHAFFNER, HERNDON DOUGLAS. ABRAHAM l. BRAUDE, AND CHARLES E. DAVIS*

Departiments of Medicine aind Pathology, Unii ersity of Ccaliforniiia Medical Center-, Sain Diego, Cailifornia92103

Received 16 May 1983/Accepted 16 September 1983

To resolve the controversy over the capacity of macrophages to kill or inhibitgermination of Aspergillius spores, we compared this function in peritoneal andalveolar macrophages. Alveolar macrophages from rabbits killed 82 to 90% andcompletely digested 72 to 82% of spores of Aspergillius fimnigaitius in 30 h. Incontrast, peritoneal macrophages could not even inhibit the germination ofingested spores; more than 85% transformed into mycelia within 24 h. Killing byalveolar macrophages was delayed for 3 to 6 h after phagocytosis and wasindependent of oxidative killing mechanisms and immune activation. The abilityof alveolar macrophages to kill Aspergilllis spores without modulation by Tlymphocytes or the generation of oxygen intermediates points out that conceptsbuilt on studies of peritoneal macrophages may be misleading and underscores theimportance of studying the role of macrophages in immunity with cells from theappropriate anatomical site.

We recently reported that macrophages pro-tect mice from invasive aspergillosis by inhibit-ing germination and then killing the spores ofAspergillius fiunigatmis (12). Because aspergillo-sis results from inhalation of spores (1, 11),alveolar macrophages should form the first lineof defense against that infection. Although alve-olar macrophages readily ingest these ubiquitousspores (8, 12), in vitro studies of the capacity of"'resting" macrophages to kill spores or inhibittheir germination have produced conflicting orambiguous results (7, 16; P. F. Lehman, Abstr.Annu. Meet. Am. Soc. Microbiol. 1982, F6, p.327). Most of these in vitro studies have beenconducted with peritoneal macrophages despitethe fact that the alveolar macrophage is thecritical cell.We began to question the reliability of the

peritoneal macrophage as a model for the anti-microbial activity of the alveolar cell when wefound in our recent study that the in vivo activityof liver and lung macrophages against sporeswas much greater than the in vitro activity ofresident or elicited peritoneal macrophages (1).Accordingly, we decided to compare the in vitroactivity of macrophages from different anatomi-cal sites (peritoneum, blood, and lung) and dif-ferent species (mouse, rabbit and humans)against spores of A. firnigatims, Aspergilliis fla-I'us and Aspergilliis niger. Our results showdramatic differences in the antimicrobial activityof alveolar and peritoneal macrophages, raisethe possibility that the site of differentiation of

monocytes to mature tissue macrophages influ-ences these properties, and underscore the im-portance of studying the role of macrophages inimmunity with cells from the appropriate ana-tomical site.

MATERIALS AND METHODS

Organisms. One strain each of A. fimnigauts. A.flaivus, and A. niger, isolated from patients at theUniversity Hospital San Diego was used throughoutthe study. The fungi were propagated, and single sporesuspensions were prepared as previously described(12).

Cell preparations. Peritoneal and alveolar macro-phages were obtained from 10- to 14-week-old femaleCFI mice (Charles River Breeding Laboratories, Inc.,Wilmington, Mass.) which were sacrificed by cervicaldislocation, and New Zealand white rabbits (fromlocal breeders) which were exsanguinated by cardiacpuncture. Representative numbers of mice and rabbitsfrom each breeder were screened for the presence ofinfectious agents. Mice had no evidence of infectionwith Ectromelia virus, Sendai virus. minute hepatitisvirus, Reovirus 3, the pneumonia virus of mice, orMvcoplasina piliionis. Rabbits were free of coccidiaand active infection with Borcdetellai bronchisepticaand Pasteiurella multocida. Peritoneal cells from micewere harvested by standard techniques as describedpreviously (12); alveolar cells were harvested by wash-ing the lungs five times with 1.5 ml of ice-cold Ca-2and Mg2--free Dulbecco phosphate-buffered saline(GIBCO Laboratories, Grand Island. N.Y.) through ablunted 20-gauge needle inserted into the trachea.Rabbit alveolar macrophages were obtained by thesame technique with a 13-gauge needle and volumes of

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1110 SCHAFFNER ET AL.

50 ml. The peritoneal cavity of each rabbit was washedwith 120 ml of cold Dulbecco phosphate-bufferedsaline through a plastic catheter inserted through asmall midline incision. Human and rabbit mononuclearblood leukocytes were harvested from blood anti-coagulated with heparin (10 U/ml) by the Ficoll-Isopa-que technique (2). Viability of cells was always >95%as judged by trypan blue exclusion. Cells were count-ed in 0.1 N HCI with methylene blue in a hemacytome-ter, and differential counts were done in duplicate onGiemsa-stained smears.

Preparation of sera. Human and rabbit sera wereseparated from blood after coagulation at room tem-perature for 1 h and storage at 4°C for 1 h more withcentrifugation at 140 x g and recentrifugation of thesupernatant at 1,500 x g for 10 min. Serum was thenfiltered (0.45 ,um) and stored at -70°C until used.Rabbit immune serum was obtained from rabbits im-munized with six subcutaneous injections of 108 conid-ia from A. fumigatus in Freund complete adjuvantgiven at intervals of 14 days. This serum gave animmunoglobulin G titer of >1:1000 against conidia asdetermined by an enzyme-linked immunosorbent as-say (12). No antibody was detectable in pooled serafrom two shipments of nonimmunized mice and rab-bits.

Germination rates of spores ingested by macrophagemonolayers. Peritoneal (>60% macrophages) and alve-olar (>90% macrophages) cells were washed once inGey balanced salt solution (GIBCO) and suspended ata concentration of 2 x 106 cells per ml in Medium 199(GIBCO) supplemented with 100 U of penicillin perml, 100 jig of streptomycin per ml (GIBCO), and 10%fetal bovine serum (GIBCO) for mouse cells or 20%rabbit serum for rabbit cells. Mononuclear blood leu-kocytes (25 to 32% monocytes) were suspended at aconcentration of 107 cells per ml in Medium 199 with25% human serum of random blood types. Samples(0.1 ml) of cell suspensions were placed on 15-mmround glass cover slips, prepared as described byNagakawara et al. (10), in 16-mm-diameter plasticcluster wells (Costar Data Packaging, Cambridge,Mass.) and incubated with 0.5 ml of the appropriatemedium. After 2 h of in vitro culture, peritoneal andalveolar cells were challenged with 0.1 ml of 1 x 101 to4 x 105 spores per ml suspended in Medium 199.Blood-derived macrophages were cultured for 2 or 10days in vitro before challenge with the same number ofspores. These times were chosen because, between 2and 10 days, mononuclear phagocytes differentiate toepithelioid and multinucleated giant cells and lose, to agreat extent, their capacity to generate reactive oxy-gen intermediates (2). Medium was changed on days 1,3, 5, 7, and 10.

After phagocytosis for 45 min, the medium wasaspirated through a 20-gauge needle, the wells werewashed four to five times with prewarmed Gey bal-anced salt solution, and 0.5 ml of fresh medium wasreplaced for continued culture. At specified times, themedium was removed and the trays were dried undervacuum at -15 mmHg (-1,999.5 Pa). Dry cover slipswere fixed with methanol and stained with Giemsastain. The phagocytic index (number of macrophagesto number of spores) was always above 4, with zero tothree conidia per macrophage. The germination ratewas expressed as the percentage of spores formingmycelia among 100 counted per cover slip from four to

six wells per time point. Each experiment was accom-panied by a media control in which germination ratesof spores incubated in medium supplemented with theappropriate serum were enumerated under the phasecontrast microscope from triplicate wells. Late in thetransformation of spores (after germination of >90%of the spores), individual fungi could no longer becounted accurately because of mycelial growth. Atthis time, almost all spores had germinated and a valueof 95% was assigned to these wells for computation.Anaerobic conditions were created in anaerobic jars(GasPak; BBL Microbiology Systems, Cockeysville,Md.) by using fresh catalyst and indicator for eachexperiment as proposed by the manufacturer. Thissystem gives within 1 h an atmosphere with <0.02%02 and 4 to 10% C02, according to the manufacturer.Anaerobic conditions were verified for each experi-ment with the indicator strip belonging to the systemand a liquid methylene blue redox indicator (9) assur-ing a redox potential of <-230 mV (4).

Digestion of spores by rabbit alveolar macrophages.Noningested spores persisted in washed monolayersand produced mycelia so that reliable observationswere impossible beyond 18 h. To remove more nonin-gested spores, phagocytosis was induced in suspen-sion for 30 min by tumbling macrophages and spores at6:1 or 7:1 in Medium 199 with 20% serum. A 10-mlamount of this mixture was then layered over 15 ml ofFicoll-Isopaque (Histopaque; Sigma Chemical Co., St.Louis, Mo.) and centrifuged at 700 x g for 20 min atroom temperature. The spores sedimented through theFicoll-Isopaque, leaving less than 1% extracellularspores among macrophages at the interface. The re-covered macrophages were washed once in Medium199 and plated on cover slips as described above. Theimproved purification permitted observations up to 30h after phagocytosis. Quantification of staining abilitywith basic dyes has been used as a measure for killingCandida albicans (5). We were unable to use thismethod for conidia because spores lost the ability tofix the stain gradually and showed strong native pig-mentation. Instead, we determined complete digestion(i.e., disappearance) of spores from the decrease of thephagocytic index over time. Selective loss of macro-phages with ingested spores to the supernatant wasexcluded by periodic examination of the supernatantand the monolayer. The phagocytic index was enumer-ated by counting 100 macrophages per cover slip fromsix wells per time point and experiment.

Killing of ingested spores by rabbit alveolar macro-phages. Cells were suspended with spores as describedabove at ratios from 2:1 to 20:1 (number of macro-phages to number of spores) and separated fromnoningested spores by a Ficoll-Isopaque gradient. Thisgave a phagocytic index of 2.4 to 27 as determined onGiemsa-stained smears. Cells (1 x 105 to 3 x 106) in0.5 ml of Medium 199 with 20% serum were thendistributed to flat-bottom glass tubes (15 by 150 mm)and incubated at 37°C for predetermined times beforedisrupting the cells by extensive vortexing in 4.5 ml ofdistilled water for 15 min. The number of CFUs wascounted in pour plates from quadruplicate tubes aftermixing 1 ml of each 1:10 dilution of disrupted cells withSabouraud glucose agar (Difco Laboratories, Detroit,Mich.). Results were calculated after 48 h of culture at37°C from plates with a density between 10 and 100colonies per plate.

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FIG. 1. Germination of Aspergillus spores. Comparison of inhibition after ingestion by mouse macrophagesderived from the peritoneum (O) and lung (0) with spores incubated in medium alone (*). Mean ± SD fromduplicate experiments. A, A. fumigatus; B, A. flavus; C, A. niger.

For determination of killing under anaerobic condi-tions, tubes with ingested spores and media controlswere placed into anaerobic jars as described aboveConversion of the methylene blue indicator (Eh<-230 mV) placed into identical tubes occurred with-in 3 h. At this time no significant killing had occurredin parallel aerobic controls.

Statistical analysis. Mean values were compared by ttest.

RESULTSGermination inhibition. Macrophages ob-

tained from the lungs of mice (Fig. 1) and rabbits(Fig. 2) prevented germination of most ingestedspores of all three aspergilli. Peritoneal macro-phages were less efficient than alveolar cells(three to seven times, P < 0.001 for each cellpair and experiment) when compared side byside with cells obtained from the same animals(Fig. 1 and 2). Peritoneal macrophages frommice were unable to suppress A. flavus (Fig. 1B

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and 3A). The kinetics of germination of sporesfrom A. fumigatus and A. flavus ingested byrabbit alveolar or peritoneal macrophages dif-fered markedly. After the lag phase induced byboth kinds of macrophages, only spores ingestedby peritoneal cells germinated as fast as the cell-free media controls. The germination rate ofspores ingested by alveolar cells leveled offwithin the next few hours, demonstrating furtherinhibition only by alveolar cells (Fig. 2). Weshowed that the antimicrobial capacity of theseperitoneal cells was intact for Staphylococcusepidermidis by the technique of van Furth et al.(15). Washed, resident peritoneal macrophages(107) from one of our fungal experiments killedalmost one log1o of a wild strain of pre-opso-nized S. epidermidis (zero time = 2.4 x 106 CFUcompared with 4.5 x 105 at 1 h after phagocyto-sis). These results are equivalent to those of vanFurth et al. (15).Macrophages derived from rabbit blood

18 24 6 12 18 24Hours after Phagocytosis

FIG. 2. Germination of Aspergillus spores. Comparison of inhibition after ingestion by rabbit macrophagesderived from the peritoneum (U), lung (0), and blood by culture of monocytes for 2 days in vitro (0) with sporesincubated in medium alone (*). Mean ± SD from at least duplicate experiments (blood-derived cells, singleexperiments). A, A. fumigatus; B, A. flavus; C, A. niger.

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FIG. 3. (A) Failure of mouse peritoneal macrophages to inhibit germination of spores of A. flai'is. Residentperitoneal macrophages cultured as monolayers for 4 h in vitro before challenge with spores. By 12 h myceliasprout from ingested spores and pierce the macrophages (Giemsa stain, magnification x500). (B) Suppressionand killing of spores by rabbit alveolar macrophages. Rabbit alveolar macrophages 24 h after ingestion of sporesfrom A. fuinigatus efficiently prevent germination and kill spores as shown by digestion. Ingested spores(arrows) gradually lose their staining ability with the basic dye (from right to left) and finally disappear. (Giemsastain, magnification x2,000).

monocytes by in vitro culture for 2 days behavedlike alveolar cells (Fig. 2). There was no differ-ence between the suppressive activity ofthese "young" human blood-derived macro-

phages that resembled monocytes (Fig. 4A) after2 days of in vitro culture and the epithelioid (Fig.4B) and multinucleated giant cells (Fig. 4C) thatdeveloped after 10 days of culture. Both prepa-

rations prevented the transformation of spores

into mycelia as efficiently as mouse or rabbitalveolar macrophages (Fig. 5). Both prepara-tions also prevented transformation under aero-bic conditions (Fig. 5). Rabbit alveolar macro-phages also efficiently reduced germination ofthe three fungi without oxygen (less than 2% ofmedia control at 24 h, data not shown).

Killing of spores by rabbit alveolar macro-phages. Significant killing of A. fumigatus began

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FIG. 4. Suppression of germination of spores from A. flavus by human blood derived macrophages. (A)Macrophages derived from monocytes by culture in vitro for 2 days, 24 h after ingestion of spores (arrows). Anescaping mycelium is attacked by neighboring phagocytes (Giemsa stain, magnification x 125). (B) Epithelioidmacrophages (Giemsa stain, magnification x500) and (C) multinucleated giant cells derived from bloodmonocytes by in vitro culture for 10 days 24 h after ingestion of spores (Giemsa stain, magnification x325).

6 h after phagocytosis (Fig. 6). The rate of killingwas not affected by a wide range of changes inthe phagocytic index (2.4 to 27), the number ofphagocytes added per well (1 x 105 to 3 x 106),or the number of spores per well (1.2 x 103 to 8.6x 105). Killing was equal under aerobic andanaerobic conditions (Fig. 6). A requirement ofantibody or complement for killing was excludedby demonstrating that macrophages killed as

efficiently in medium supplemented with heat-

inactivated fetal calf serum (reduction of 87 +4% of CFU at 30 h; mean + standard deviation[SD]) as with 20% fresh rabbit serum (90 + 3%of CFU at 30 h) or immune rabbit serum (904% CFU at 30 h).

Digestion of spores by rabbit alveolar macro-phages. Removal of noningested spores by aFicoll gradient permitted longer observation pe-riods and determination of the percentage ofspores that were completely digested. The ger-

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FIG. 5. Germination of Aspergilluts spores. Inhibition of germination by human macrophages derived fromcultures of blood monocytes in vitro for 2 days (a) or for 10 days (d). Cells were obtained from the same donor atan interval of 8 days, and germination rates compared side by side. Media controls (*). Mean ± SD fromtriplicate experiments (A. niger, duplicate experiment). (0, C. 0) Germination rates under anaerobic conditions(<-230 mV). A, A. fitnigaitius; B, A. flaius; C, A. niger.

mination rates obtained by the two techniques(infection of a monolayer or infection of cells insuspension before plating them on cover slips)were compared and did not differ (data notshown). At 30 h, 72 to 82% of ingested sporeshad completely disappeared (Fig. 6). Of theremaining spores, 13 ± 3% (mean ± SD fromthree experiments) had transformed into myce-lia. These attracted neighboring macrophages,which in turn, appeared to suppress extracellu-lar mycelial growth. When the monolayers were

cultured under anaerobic conditions, digestionwas reduced by nearly 50% (Fig. 4). To testwhether this effect reflected a reduction in kill-ing or merely a reduction in digestion, we re-

peated the experiment with heat-killed spores(56°C for 120 min). Although killed spores were

digested only more slowly than live spores aero-

bically (34 ± 7% in 30 h), under anaerobicconditions digestion was drastically reduced (8± 9% in 30 h), indicating an important role foroxygen in the digestion of Aspergillius spores inthis system.

DISCUSSION

Discrepancies in reports on the role of macro-phages in resistance to Aspergillius spp. (7, 8, 12,16; 82nd Annu. Meet. Am. Soc. Microbiol.,abstr. F6) prompted this study of the interac-tions between macrophages from differentsources and the three species of Aspergillusimportant for human infections. The results con-

firm our previous observation that alveolar mac-

rophages do not have to be modulated by Tlymphocytes and need no antibody to suppress

and kill spores.These experiments also show differences in

the anticonidial activity of macrophages fromdifferent anatomical sites. Peritoneal cells from

either mice or rabbits are strikingly less activethan their alveolar cells and cannot inhibit thegermination of A. flaI'ius (Fig. 1 and 2). Thesedifferences between alveolar and peritonealmacrophages account for the discrepancy re-ported by us previously (12) between in vitroand in vivo anticonidial activity of macrophagesand the conflicting results of other investigatorson the role of macrophages in resistance toAspergilliis spp. Thus, it is not surprising thatWilliams et al. (16) found that mouse peritonealmacrophages could not kill A. fiingatuis in vitroor that Lehman (82nd Annu. Meet. Am. Soc.Microbiol., abstr. F6) reported that they couldnot suppress germination. Furthermore, unlikethe peritoneal macrophages described in ourearlier paper (12), alveolar macrophages killedspores in vitro as efficiently as lung or livermacrophages in vivo. The greater ability of thealveolar macrophages to kill Aspergilllus sporesis of importance because the lung is the site ofchallenge, and the alveolar macrophage is thecritical cell. Nevertheless, most of the studies ofresistance against Aspergillius spp. have beenconducted with peritoneal macrophages andhave been misleading, even suggesting that mac-rophages must be modulated by T lymphocytesto provide resistance to Aspergillius spores (16).Alveolar macrophages obviously do not requirethese instructions (Fig. 1, 2, and 6).

Other investigators have shown biochemicaldifferences between alveolar and peritonealmacrophages, but these have not been related tofunction (9, 13), and differences in antimicrobialactivity have not been decisive (10). Both perito-neal and alveolar macrophages are derived fromblood monocytes (14) and ultimately from thebone marrow. It is possible therefore, that localfactors present in the lung (but not in the perito-

INFECT. IMMUN.1114 SCHAFFNER ET AL.

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KILLING OF ASPERGILLUS SPORES BY MACROPHAGES 1115

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FIG. 6. Killing and digestion of spores of A. fumni-gatlis by rabbit alveolar macrophages. Macrophageswere infected in suspension, and noningested spores

were removed by a Ficoll gradient and then platedeither in flat-bottom tubes to determine killing byCFUs or on glass cover slips to determine digestion.Mean + SD from five (killing) or three (digestion)experiments. (0) percent reduction of CFU. Killingwas not affected by phagocytic indices (number ofmacrophages to number of spores) between 2.4 and 27.(0) percent reduction of CFU under anaerobic condi-tions (<-230 mV); each experiment was accompaniedby a media control which did not reduce the CFU. (O)percent disappearance (complete digestion) of spores

calculated from the phagocytic index from six wells ateach time point and experiment. (O) percent disap-pearance under anaerobic conditions.

neum) govern maturation and differentiation tocells with strong antimicrobial activity.The lag period of 3 to 6 h between ingestion

and killing (Fig. 6) might be caused by the sporewall which could protect the spore against neu-

trophils. Spores of A. fiumigatius are resistant tohigh concentrations of H202 and to killing byneutrophil granulocytes (6). Mycelia do notshare this resistance and are killed by neutro-phils (12), probably through oxidative mecha-nisms (3). Thus, germination seems to makeaspergilli susceptible to oxidative killing by neu-

trophils. Furthermore, alveolar macrophageskill spores at equal rates under aerobic andanaerobic conditions (Fig. 6), and "differentiat-ed" macrophages that resemble epithelioid cellsand can no longer generate important amountsof reactive oxygen intermediates (10) kill sporesas effectively as young (2 days in culture) macro-

phages with high oxidative capacity. These find-ings exclude a major role for oxidative killingmechanisms in the conidiacidal activity of mac-

rophages and point to other killing systems in

macrophages that are not necessarily shared bypolymorphonuclear leukocytes or even allmononuclear phagocytes.

ACKNOWLEDGMENTS

This study was supported in part by Public Health ServiceInternational Research Fellowship 1 F05 TW 02935. A.S. is aFellow of the Schweizerische Gesellschaft fur Innere Medizin.

LITERATURE CITED

1. Austwick, P. K. 1966. The role of spores in allergies andmycosis of man and animals, p. 321-327. In M. E. Made-lin (ed.), The fungus spores. Proceedings of the 18thsymposium, Colston research society. Blackwell, Lon-don.

2. Boyum, A. 1976. Isolation of lymphocytes, granulocytesand macrophages. Scand. J. Immunol. 5 (Suppl. 5):9-15.

3. Diamond, R. D., R. Krzesicki, B. Epstein, and W. Jao.1978. Damage to hyphal forms of fungi by human leuko-cytes in vitro. Am. J. Pathol. 91:313-350.

4. Holdeman, C. V., E. P. Cato, and W. S. E. Moore. 1977.Anaerobe laboratory manual, 4th ed. Anaerobe Labora-tory Virginia Polytechnic Institute and State University.Blacksburg, Va.

5. Lehrer, R. I., L. G. Ferrari, J. Patterson-Delafield, and T.Sorell. 1980. Fungicidal activity of rabbit alveolar andperitoneal macrophages against Canididi albicans. Infect.Immun. 28:1001-1008.

6. Lehrer, R. I., and R. G. Jan. 1970. Interaction of Aspergil-lus fumigatus spores with human leukocytes and serum.Infect. Immun. 1:345-350.

7. Merkow, L. L., S. M. Epstein, H. Sideransky, and M.Pardo. 1970. The pathogenesis of experimental pulmonaryaspergillosis. Am. J. Pathol. 62:57-66.

8. Merkow, L. L., M. Pardo, S. M. Epstein, E. Verney, andH. Sideransky. 1978. Lysosomal stability during phagocy-tosis of Aspergillus flavus spores by alveolar macrophagesof cortisone treated mice. Science 160:79-81.

9. Myrvik, Q. N., E. S. Leake, and B. Fariss. 1961. Lyso-zyme content of alveolar and peritoneal macrophagesfrom the rabbit. J. Immunol. 86:133-136.

10. Nagakawara, A., C. F. Nathan, and Z. A. Cohn. 1981.Hydrogen peroxide metabolism in human monocytes dur-ing differentiation in vitro. J. Clin. Invest. 68:1243-1252.

11. Rose, H. D. 1972. Mechanical control of hospital ventila-tion and aspergillus infection. Am. Rev. Respir. Dis.105:306-307.

12. Schaffner, A., H. Douglas, and A. Braude. 1982. Selectiveprotection against conidia by mononuclear and againstmycelia by polymorphonuclear phagocytes in resistanceto aspergillus. J. Clin. Invest. 69:617-631.

13. Simon, L. E., E. D. Robin, J. R. Phillips, J. Acevedo, S. G.Axline, and J. Theodore. 1977. Enzymatic basis for bioen-ergetic differences of alveolar versus peritoneal macro-phages and enzyme regulation by molecular 02. J. Clin.Invest. 59:443-448.

14. van Furth, R., M. M. C. Diesselhoff-denDulk, J. A. Rae-burn, T. L. van Zweith, R. W. Crofton, and A. Blusse vanOud Alblas. 1980. Characteristics, origin and kinetics ofhuman and murine mononuclear phagocytes, p. 279-298.In R. van Furth (ed.), Mononuclear phagocytes. Nijhoff,The Hague.

15. van Furth, R., T. L. van Zwet, and P. C. J. Leijh. 1979. Invitro determination of phagocytosis and intracellular kill-ing by polymorphonuclear and mononuclear phagocytes,p. 32.1-32.19. In D. M. Weir (ed.), Handbook of experi-mental immunology, vol. 2, Blackwell Scientific Publica-tions, Oxford.

16. Williams, D. M., M. H. Weiner, and D. J. Drutz. 1981.Immunologic studies of disseminated infection with As-pergillls fitinigatius in the nude mouse. J. Infect. Dis.143:726-733.

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