INFECTION AND IMMUNITY, May 1985, p. 458-4630019-9567/85/050458-06$02.00/0Copyright C) 1985, American Society for Microbiology

Vol. 48, No. 2

Neutrophil Chemotactic Responses Induced by Fresh and SwollenRhizopus oryzae Spores and Aspergillus fumigatus Conidia

ALAYN R. WALDORF* AND RICHARD D. DIAMOND

Evans Memorial Department of Clinical Research and the Department of Medicine, University Hospital, BostonUniversity Medical Center, Boston, Massachusetts 02118

Received 14 December 1984/Accepted 11 February 1985

With the induction of germination, Rhizopus oryzae spores and Aspergillus fumigatus conidia activate thecomplement system and induce neutrophil chemotaxis. In contrast, freshly isolated R. oryzae spores did notinduce neutrophil migration into lung tissue of mice after intranasal inoculation. Moreover, in microchemo-taxis assays neither fresh R. oryzae spores nor A. fumigatus conidia activated sera to stimulate humanneutrophil chemotaxis above control migration until at least 107 or 108 spores or conidia per ml of sera were

used. The increased generation of chemotactic factors by swollen spores and conidia was not due to an

increased surface area, as there was decreased neutrophil chemotactic response to Rhizopus or Aspergillushyphae when compared with swollen spores or conidia.

Mucormycosis and aspergillosis are often rapidly fatalopportunistic fungal infections commonly caused by Rhi-zopus oryzae and Aspergillusfumigatus, respectively. Afterinhalation of the ubiquitous spores, germination occurs,resulting in vascular invasion and tissue necrosis. Alveolarmacrophages (the first phagocytic cells to encounter inhaledorganisms [12]) are effective in inhibiting R. oryzae sporegermination but are unable to kill spores (20, 21). A. fum-igatus conidia are not as effectively inhibited from germinat-ing by alveolar macrophages but can be killed by alveolarmacrophages (16, 17, 20).

Neutrophils appear to be important in host defense mech-anisms, as neutrophil-rich exudates surround hyphal ele-ments in experimentally infected animals in which infectionsclear, and neutropenia is often associated with cases ofaspergillosis and mucormycosis in humans (18). Neutrophilsare effective in damaging hyphal forms, but ineffectiveagainst conidia (5-8, 16). Previous investigators have re-ported that Rhizopus hyphae stimulate neutrophil migrationdirectly and activate C5 in serum (3, 14). However, becauseof the rapid growth of these organisms in vivo after germi-nation, the importance of the early mobilization and migra-tion of neutrophils to the site of spore and conidia germina-tion makes it valuable to understand the mechanisms bywhich R. oryzae spores or A. fumigatus conidia influenceneutrophil migration.

In this communication, we report that freshly isolated R.oryzae spores or A. fumigatus conidia are unable to activatethe complement system to induce neutrophil chemotaxisuntil more than 107 spores or 108 conidia per ml of sera areused. Equivalent numbers of hyphae per milliliter of sera arealso needed to stimulate neutrophil chemotaxis. In contrast,spores and conidia that are swollen with the induction ofgermination significantly stimulate neutrophil migration bythe activation of complement. Swollen spores and conidiaalso induce neutrophil migration into lung tissue after intra-nasal inoculation.

* Corresponding author.

MATERIALS AND METHODS

Organisms. As in previous studies (20, 22), isolates of R.oryzae and A. fumigatus originally obtained from patientswere maintained on potatoe dextrose agar (8). R. oryzaesporangiospores (spores) or A. fumigatus phialoconidia (con-idia) were harvested in saline, vortex mixed to break upclumps, and filtered through eight layers of cheesecloth toremove any hyphal fragments (20). Freshly isolated sporesand conidia are referred to as fresh spores or conidia.Some of the spores or conidia, harvested as above, were

incubated to induce pregermination swelling. R. oryzaespores were diluted to 2 x 105/ml in Sabouraud broth andincubated at 37°C for 4 h. Because of slower germinationtimes and clumping of A. fumigatus conidia in other media,these conidia (2 x 105/ml) were incubated in Eagle minimalessential medium (GIBCO Laboratories, Grand Island, N.Y.)supplemented with nonessential amino acids at 37°C for 4 h.By microscopic observation, more than 75% of the incu-bated spores and conidia were swollen to >1.5 times theiroriginal size, whereas <5% had germinated. These sporesand conidia were designated as swollen.Hyphae were obtained by continuing the incubations of R.

oryzae spores for a total of 5 to 6 h or incubating A.fumigatus conidia in Eagle medium overnight at room tem-perature until .90 to 95% of the spores or conidia germi-nated to .30 pLm in length (8). Both fresh and swollen sporesand conidia, as well as hyphae, were washed twice indistilled water and stored at 4°C until used. Before use theywere again washed twice in Gey's balanced salt solution(medium; GIBCO) before interaction with neutrophils orserum.Animal model. Fresh or swollen R. oryzae spores or A.

fumigatus conidia (106 spores or conidia per mouse) orsterile saline was administered intranasally (19) into 4- to6-week-old female pathogen-free mice (Charles River CD-1strain) obtained from Charles River Breeding Laboratories,Inc., Kingston, N.Y. After 0, 4, 18, or 72 h animals weresacrificed and bronchoalveolar lavaged (13, 20, 22). Briefly,aseptic bronchoalveolar lavage was executed by using a1.1-mm (20-gauge) Angiocath (Parke-Davis Co., Sandy,Utah) for tracheal cannulation and secured with a ligature.

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TABLE 1. Percent recovery of different cell populations after intranasal inoculation of mice with fresh Rhizopus spores or Aspergillusconidia% Recovery after inoculation" with:

Time after Sterile saline R. on-ae A.inoculation (h) fumigatus

PMN BAM PMN BAM PMN BAM

0 0.3 (0.3) 97.5 (2.1) NDDb ND ND ND4 0 (0) 96.7 (3.5) 0 (0) 90.3 (5.5) 15.5 (0.7) 82.0 (4.2)18 0.3 (0.3) 93.7 (6.8) 8.7 (8.7) 87.3 (4.9) 56.3 (9.4)(' 31.7 (8.8)'72 0 (0) 94.3 (7.8) 6.0 (1.4) 92.5 (3.5) 11.3 (2.1) 81.0 (5.6)

aInoculated with 106 fresh R. orvzae spores or A. fiumigatlus conidia. PMN, Polymorphonuclear leukocytes; BAM. bronchoalveolar macrophages. Valuesrepresent the mean percentage (± standard deviation) of total cells recovered.

b ND, Not determined.' P < 0.001 and df = 4 by the Student t test for independent means (two-tailed) compared with saline controls.

Each mouse received 10 1.0-ml lavages with modified Hankssolution (15). Any lavage fluid contaminated with blood wasdiscarded. Cytocentrifuge (Shandon Southern Instruments,Sewickley, Pa.) preparations of lavage cells were stainedwith Diff-Quick (American Scientific Products, McGrawPark, Ill.) and nonspecific esterase strains, and differentialcounts were performed. To confirm the results of broncho-alveolar lavage, lung tissue was taken from representativeanimals, processed, and stained with hematoxylin and eosinfor histological evaluation.

Neutrophils and sera for chemotaxis assays. Clotted bloodfor serum and heparinized (10 U/ml) blood were obtainedfrom normal human volunteers. Neutrophils were obtainedas in previous studies (3) by dextran sedimentation, andresidual erythrocytes were removed by hypotonic lysis.Leukocytes were washed and suspended in Gey's mediumwith 2% bovine serum albumin at 5 x 106 cells per ml. Cellsuspensions were >95% viable by trypan blue dye exclu-sion.

Portions (2 ml) of serum were stored at -70°C before use.Various concentrations of fresh or swollen spores or conidia(102 to 108/ml) or hyphae (103 to 107/ml) were incubated withserum at 37°C for 1 h. Spores, conidia, and hyphae wereremoved from the serum by centrifugation (500 x g for 5min), and the serum was heated at 56°C for 30 min toinactivate residual complement. Serum was then diluted to5% final concentration in gelatin-Veronal-buffered saline(calcium, 7 x 10-5 M; magnesium, 5 x 10-4 M). Zymosan (4mg/ml)-activated serum served as the positive controls, andGey's medium and sera incubated in the absence of activatorserved as negative controls. Control sera, diluted as outlinedabove, were included with each filter in the chemotaxisassay.

In one series of experiments, to determine whether incu-bation with the spores or conidia destroyed serum compo-nents responsible for chemotaxis, zymosan-activated serawere incubated a second time, immediately after removal ofthe zymosan particles, with 2 x 105 fresh or swollen R.oryzae spores or A. fumigatus conidia. Similarly, R. oryzae-and A. fumigatus-activated sera were incubated a secondtime, immediately after removal of the spores or conidia,with zymosan (4 mg/ml). The second incubtion periods wereat 37°C for 30 min. After the incubations, spores, conidia, orzymosan was removed by centrifugation, and the serum washeated to 56°C for 30 min to inactivate residual complement.To characterize chemotactic activity, the serum was heated

at 56°C for 1 h before activation. Additionally, the C3 and C5contents of the serum were determined by using radialimmunodiffusion plates and goat anti-human C3 or C5 anti-sera (Chappel Laboratories, West Chester, Pa.). Anti-C3 or

anti-C5 antibody (containing the equivalent of 110% of theC3 or C5 concentration) was preincubated with a portion ofserum for 1 h at 37°C (14). Spores, conidia, or zymosan wasthen incubated with anti-C3 or anti-C5 antibody-treated andcontrol serum in parallel, and the serum was assayed forchemotactic activity after the removal of spores, conidia, orzymosan.Chemotaxis assays. Neutrophils (ca. 1.8 x 105 cells in 35

p.l) were placed in the top wells of a microchemotaxisassembly (11; Neuro Probe, Cabin John, Md.) above mi-cropore filters (pore size, 3 ,um; 100-,um thick; Satoriuscellulose nitrate filters; Neuro Probe). Stimulants (55 ,ul)placed below the filter included activated sera and controlsera or graded increments (102 to 107 spores or conidia perwell) of fresh or swollen R. oryzae spores or A. fumigatusconidia. After incubation for 25 to 30 min at 37°C plus 5%CO2, the filters were removed, fixed in methanol, stainedwith hematoxylin, dehydrated in increasing concentrationsof ethanol, cleared with xylene, and mounted on glass slideswith Permount (Fisher Scientific, Fair Lawn, N.J.) (4). Theleading front technique of Zigmond and Hirsch (25) wasused, determining the migration front by measuring themicrometers from the top of one filter to the farthest distancetraveled by two cells per field with a x40 objective. Fivefields per well were counted, all were run in duplicate foreach filter, and the results were averaged. Duplicate filterswere run with every experiment with at least two replicatesof each experiment.

Statistics. Means (± standard deviation) of replicate ex-periments were computed. The Student t test of independentmeans (two tailed) was used to compare lung recovery data,and the Student t test for paired samples (two tailed) wasused to compare results for activated and control sera (9).

RESULTSMigrations of neutrophils into lungs after inoculations. To

determine whether intranasal inoculation of R. oryzae sporesor A. fumigatus conidia might result in migration of neu-trophils into the lungs, fresh or swollen spores or conidiawere instilled intranasally into groups (n = 3 in each group)of animals, and bronchoalveolar lavage cells were collectedat various times. After inoculation with fresh R. oryzaespores or A. fumigatus conidia there was a significant (P <0.001, df = 4) increase in the absolute number of neutrophilsrecovered 18 h after inoculation only in mice inoculated withA. fumigatus (Table 1; the total number of cells recoveredper animal 18 h after inoculation with saline was 5.1 x 105 +1.0; the total number of cells recovered per animal 18 h afterinoculation with A. fumigatus conidia was 8.4 x 105 ± 3.0).In contrast, there was an increase in the absolute number of

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460 WALDORF AND DIAMOND

TABLE 2. Percent recovery of different cell populations after intranasal inoculation of mice with swollen Rhizopus spores or Aspergillusconidia% Recovery after inoculation" with:

Time afterinoculation (h) Sterile saline R. orvzae A. fumigatus

PMN BAM PMN BAM PMN BAM

0 0.3 (0.3) 97.5 (2.1) NDb ND ND ND4 0 (0) 96.7 (3.5) 8.5 (3.5) 73.5 (5.0) 9.0 (4.6) 86.3 (4.9)18 0.3 (0.3) 93.7 (6.8) 44.7 (3.2)' 47.3 (5.0)' 66.7 (14.0)c 30.0 (4.9)'72 0 (0) 94.3 (7.8) 14.5 (6.4) 64.0 (5.2) 5.7 (6.5) 74.3 (8.7)

a Inoculated with 106 swollen R. oryzae spores or A. fumigatus conidia. PMN, Polymorphonuclear leukocytes; BAM, bronchoalveolar macrophages. Valuesrepresent the mean percentage (± standard deviation) of total cells recovered.

b ND, Not determined.c P < 0.001 and df = 4 by the Student t test for independent means (two-tailed) compared with saline controls.

neutrophils recovered 18 h after mice were inoculated intra-nasally with either swollen R. oryzae spores or A. fumigatusconidia (P < 0.001, df = 4; Table 2; the average total numberof cells recovered per animal after inoculation with R.oryzae spores and A. fumigatus conidia was 9.1 x i0 ±+ 2.8and 10.7 x 105 + 4.1, respectively). These results wereconfirmed by histological staining of lung tissue from inocu-lated mice. Polymorphonuclear cells were observed in lungtissue at 18 h only after inoculation with fresh A. fumigatusconidia but not with R. oryzae spores. However, afterinoculation with either swollen spores or conidia, inflamma-tory cells were observed in lung interstitial spaces. At 18 h,there were increasing numbers of neutrophils observed inalveoli and interstitial spaces. The results suggest that swol-len Rhizopus spores caused an influx of neutrophils into lungtissue to a greater degree than did fresh (nonswollen) spores.

Generation of chemotactic activity in vitro by fresh R.oryzae spores or A. fumigatus conidia. In an attempt todetermine the optimal concentration of fresh spores orconidia activating serum chemotactic activity, various con-centrations of fresh R. oryzae spores or A. fumigatus conidiawere incubated with sera. Human sera activated by theaddition of fresh spores or conidia stimulated human neu-trophil chemotaxis significantly (P < 0.001, df = 4) aboveunstimulated control migration (distance migrated for con-trol, 53.3 ± 4.1 p.m) only when at least 107 R. oryzae sporesor 108 A. fumigatus conidia per ml of sera were used (Fig. 1;distance migrated for R. oryzae and A. fumigatus activatedsera, 83.9 ± 2.9 and 98.1 ± 0.7 ,um, respectively).Because fresh spores and conidia did not activate sera for

neutrophil chemotaxis until exceptionally high concentra-tions were used, experiments were done to determinewhether incubation with the spores or conidia destroyedserum components responsible for chemotaxis, either beforeor after their activation. R. oryzae spores or A. fumigatusconidia (2 x 105/ml), at concentrations below that whichactivated sera, were incubated in sera either before or afterzymosan was used to activate sera. In three separate exper-iments, zymosan-activated sera stimulated neutrophil migra-tion (distance migrated, 85.2 ± 2.1 ,um). Postincubation ofR.oryzae spores and A. fumigatus conidia in zymosan-acti-vated sera did not significantly alter the migration of neu-trophils to the sera (distance migrated, 82.8 ± 7.4 and 87.5 +6.2 ,um, respectively). Similarly, preincubation of R. oryzaespores or A. fumigatus conidia in sera which was thenactivated by zymosan induced equivalent chemotaxis ofneutrophils to control zymosan-activated sera (distance mi-grated for R. oryzae and A. fumigatus preincubated sera,87.3 ± 5.8 and 83.2 ± 4.7 ,um, respectively). Therefore, thelack of sera activation by fresh spores or conidia, below 107

or 108 spores or conidia per ml, was not due to the destruc-tion of serum components by spores.

Generation of chemotactic activity in vitro by swollen R.oryzae spores or A. fumigatus conidia. To examine the possi-bility that the preincubation of R. oryzae spores or A.fumigatus conidia may induce a change which would acti-vate sera and be chemotactic for neutrophils, spores andconidia were swollen before incubation in sera R. oryzaespores and A. fumigatus conidia were incubated in growthmedia until >75% had swollen but not germinated. Humansera was then activated by the addition of 102 to 108 swollenR. oryzae spores or A. fumigatus conidia. Significant chemo-taxis (P = 0.05, df = 13) above unstimulated control

100-

90 z

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8

z 70-

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40-

10O2 10Q3 1 04 1 Of 06 10Q7o 108Conidia or Spores (number/ml of sera)

FIG. 1. Neutrophil migration determined by leading front assaystimulated by serum which had been activated by incubation with102 to 108 fresh R. oryzae spores (0) or fresh A. fumigatus conidia(O). Each point denotes the mean of six separate experiments, andthe brackets indicate the standard deviation of the mean. Results ofthe buffer control (B), normal sera (nonactivated sera; N), andzymosan-activated sera (Z) are shown. Asterisks indicate the lowestnumber of spores or conidia per milliliter of sera needed to activatesera above normal control levels (P < 0.001 by the paired-sample ttest).

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NEUTROPHIL CHEMOTAXIS GENERATED BY FUNGAL SPORES 461

neutrophils, above control values, to sera activated by R.oryzae hyphae was not observed until >106 R. oryzaehyphae per ml of sera were used. The activation of sera byhyphae for the stimulation of neutrophil chemotaxis wassignificantly less than that by swollen spores at 103 to 105 perml of sera (P < 0.05, df = 6), and at no number tested wasthere greater activation of sera by hyphae than that by freshspores (Fig. 2). Similarly, sera activated by A. fumigatushyphae induced neutrophil chemotaxis (P < 0.001, df = 8),above control values, at a two-log-higher value than did seraactivated by swollen A. fumigatus conidia (Fig. 3). AlthoughR. oryzae and A. fumigatus hyphae have a greater surfacearea than swollen spores or conidia, there was less seraactivation for neutrophil migration by hyphae.Heat inactivation or preincubation of sera with anti-C3 or

anti-C5 antisera. To characterize the R. oryzae or A. fum-igatus activation of sera which stimulated neutrophil migra-tion, sera were pretreated by heating at 56°C for 1 h orpreincubated with goat anti-human C3 or C5 antisera.Zymosan, which generates chemotactic activity through theactivation of the alternative complement pathway, was used

100-

90-

I I I I I I

102 103 104 101 101 107

Spores (number/ml of sera)FIG. 2. Neutrophil migration determined by leading front assay

stimulated by serum which had been activated by incubation with103 to 107 R. oryzae fresh spores (@-O), swollen spores (a---a),or hyphae (0). Each point denotes the mean of at least threeexperiments, and brackets indicate the standard deviation of themean. Results of buffer control (B), normal sera (nonactivated sera;N), and zymosan-activated sera (Z) are shown. The asterisk indi-cates the lowest number of spores per milliliter of sera inducingsignificant chemotaxis (P < 0.001 by the paired-sample t test)compared with an equivalent number of fresh spores per milliliter ofsera.

migration was observed with as few as 102 swollen R. oryzae

spores (distance migrated for control versus swollen R.oryzae-activated sera, 44.5 ± 4.1 versus 63.4 ± 4.6 ,um) andwas significantly (P < 0.001, df = 10) greater than that withfresh R. oryzae spores at concentrations of 105 and 106 (Fig.2).

Similar to the results observed with R. oryzae spores, seraactivated by swollen A. fumigatus conidia induced signifi-cantly (P = 0.05, df = 13) greater migration of neutrophilsabove unstimulated controls with as few as 102 conidia per

ml of sera (distance migrated for control versus swollen A.fumigatus activated sera, 45.6 ± 3.1 versus 58.3 ± 2.8 ,um,respectively). With 103 to 105 and >106 swollen conidia per

ml of sera, there was significantly (P = 0.01 and P > 0.001,respectively; df = 8) greater migration of neutrophils whencompared with fresh A. fumigatus conidia (Fig. 3).To determine whether the increased level of neutrophil

migration in response to sera activated by swollen spores or

conidia was due solely to an increased surface area, hyphaewere run in parallel with fresh and swollen spores andconidia. Significant chemotaxis (P < 0.001, df = 8) of

(a

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80-

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102 103 104 105 106 107 lo'sConidia (number/ml of sera)

FIG. 3. Neutrophil migration determined by leading front assaystimulated by serum which had been activated by incubation with103 to 108 A. fumigatus fresh conidia (E-*), swollen, conidia(E---E), or hyphae (O). Each point denotes the mean of at leastthree experiments, and brackets indicate the standard deviation ofthe mean. Results of buffer control (B), normal sera (nonactivatedsera; N), zymosan-activated sera (Z) are shown. The asteriskindicates the lowest number conidia inducing significant chemotaxis(P < 0.001 by the paired-sample t test), compared with an equivalentnumber of fresh conidia per milliliter of sera.

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462 WALDORF AND DIAMOND

as in previous experiments as a positive control. The incu-bation of 107 fresh R. oryzae spores, 106 swollen R. oryzaespores, 108 resting A. fumigatus conidia, 107 swollen A.fumigatus conidia, or zymosan with untreated serum re-sulted in significantly increased neutrophil migration (P <0.001 and df = 5, compared with serum controls; distancemigrated, 80.8 ± 6.9, 72.8 ± 6.2, 71.3 ± 10.3, and 79.9 ± 8.6,and 84.4 ± 14.7 ,um, respectively). The enhanced serumleukotactic activity produced by both types of R. oryzaespores and both types of A. fumigatus conidia and zymosanwas absent after heat treatment (distance migrated, 53.7 +12.4, 53.7 ± 8.1, 50.6 ± 5.3, 38.5 ± 7.8, 44.1 ± 7.2, and 60.7± 12.9 ,um, respectively). The neutrophil migration ob-served after incubation of sera with both types of R. oryzaespores and A. fumigatus conidia or zymosan was neutralizedby pretreatment with anti-C5 sera (distance migrated, 41.7 +11.3, 52.6 ± 4.4, 55.1 ± 6.4, 46.7 ± 14.6, and 57.4 ± 7.8 p.m,respectively) and in most instances was reduced by pretreat-ment with anti-C3 sera (distance migrated, 54.1 ± 6.2, 51.4± 4.4, 52.0 ± 7.2, 55.7 ± 7.7, and 66.0 ± 6.7 ,um, respec-tively). These results suggested that the spores and conidiaactivated the complement pathway.

Neutrophil migration in vitro to spores or conidia. Restingand swollen R. oryzae spores and A. fumigatus conidia weretested to determine whether they stimulated neutrophilmigration. There was no migration of neutrophils abovebase-line, buffer control levels with up to 108 spores orconidia as stimulators (data not shown).

DISCUSSIONNeutrophils appear to have a major role in host defense

against hyphal forms of R. oryzae (1, 2, 8) and A. fumigatus(16). The importance of mobilization and migration of neu-trophils early in the infectious processes to contain theseorganisms makes it especially important to understand themechanisms by which spores and conidia activate defensemechanisms. We found that R. oryzae spores and A. fum-igatus conidia, as stimulators alone (without serum), wereineffective in inducing neutrophil migration in in vitro stud-ies. Moreover, fresh R. oryzae spores and A. fumigatusconidia did not activate serum to induce neutrophil migra-tion until >107 spores and >108 conidia, respectively, wereused.

Confirming in vitro chemotactic studies were our in vivofindings after intranasal inoculation of fresh Rhizopus spores.Animals inoculated intranasally with fresh R. oryzae sporesand lavaged at times after inoculation had no increasednumbers of neutrophils in lavage fluids. Histological sectionsof lung tissue at times after intranasal inoculation with freshspores confirmed lavage results. In contrast to fresh spores,in vivo inoculations of swollen R. oryzae spores into lungtissue caused a neutrophilic infiltrate. Neutrophils wereobserved in bronchoalveolar lavages within 18 h of inocula-tions with swollen R. oryzae spores. Again, these resultswere confirmed by histological sections of lung tissue.

In contrast to the results observed with fresh R. oryzaespores, inoculation of animals with fresh A. fumigatusconidia induced migrations of neutrophils into lung tissue by18 h. The migration of neutrophils into lung tissue of miceinoculated with fresh A.fumigatus conidia coincided with A.fumigatus germination. We have previously shown that A.fumigatus conidia germinate in lung tissue of normal animalsby 18 h after inoculation and are recovered in lung lavages(20). In contrast, R. oryzae spores remain ungerminated innormal animals for up to 14 days, and germination occurs, invivo, only in suppressed (diabetic or cortisone-treated) mice

(20-22). Moreover, some of the A. fumigatus conidia, butnot the R. oryzae spores, are killed by alveolar macrophageswithin 18 h of in vivo incubation, and the number of viableconidia within the lungs of normal animals is reduced within12 h (20, 22). Because some of the fresh A. fumigatus conidiagerminated in the normal mice, some were killed by alveolarmacrophages, and some were removed from the lungs, itwas not possible to follow beyond several hours the influ-ence of fresh (nongerminating) conidia on neutrophil migra-tion and be assured that the results observed were notaffected by the emergence of swollen conidia or hyphae, thekilling of conidia, or the removal from the lung of viableconidia. Because inoculation with either fresh or swollen A.fumigatus conidia induced neutrophil migration at 18 h afterinoculation, this suggests that both fresh and swollen A.fumigatus conidia induced neutrophil migration, perhaps bydifferent mechanisms.

In this study, we investigated the ability of swollen sporesand conidia to activate sera. Swollen spores of both R.oryzae and A. fumigatus generated, in serum, chemotacticfactors for neutrophils above those generated by freshspores or conidia. The activation of sera by swollen sporesand conidia was not due to an increased surface area alone,since hyphae, which have an even greater surface area thanswollen spores, had a reduced serum-activating ability.Previous studies have shown that hyphae of R. oryzae andother Mucorales species can generate chemotactic factorsfor neutrophils directly and by activation of sera in adose-dependent fashion (3, 14). Our results confirm this.However, we found that unlike hyphae, the swollen sporesand conidia were unable to generate a chemotactic factor forneutrophils directly.The absence of a chemotactic response to sera which had

been heated before exposure to spores or conidia andneutralization of the response by anti-C5 antibodies (withonly a reduced response by anti-C3 antibodies) suggestedthat complement was activated. Thus, swollen, but notresting, spores or conidia have the capacity to activate thecomplement pathway. It appears therefore that the normalhost responds with a neutrophilic inflammatory response,stimulated by complement activation, only after the initia-tion of the spore or conidial germination process (i.e.,swelling). As the germination process continues and hyphaeare formed, the stimulation of the inflammatory response ofthe host by the activation of complement returns to levelscomparable to that induced by fresh conidia. However,hyphae then generate a chemotactic factor for neutrophilsdirectly.The clearance of nonswollen spores or conidia may be

similar to the clearance of damaged host cells, neitherinflammatory infiltrates nor immunological responses beingessential (e.g., complement, antibody). Because of the ubiq-uity of fungal spores and conidia, clearance probably takesplace frequently, and it can be argued that like the clearanceof host cells, it is important for the recognition processinvolved to be subtle and nonspecific, since it would beundesirable to use mechanisms that entail unnecessary in-flammatory reactions or specific immune responses (23).Studies have shown that in this nonimmune phagocytosis,the increased hydrophobicity that surfaces usually show isprobably a key factor that enhances their affinity for mem-branes of phagocytic cells and allows recognition by suchcells (23, 24). Fungal spores and conidia are extremelyhydrophobic, and this hydrophobic character decreases withspore swelling and germination (10).

Neutrophils are able to damage and kill fungal hyphae (8)

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NEUTROPHIL CHEMOTAXIS GENERATED BY FUNGAL SPORES 463

but are ineffective against Aspergillus conidia (16). Theresults reported here indicate that only the induction ofgermination was there a corresponding increased activationof serum-induced neutrophil chemotaxis via the complementpathway. In mucormycosis and aspergillosis, where hyphaerapidly penetrate tissue and blood vessels, such activation ofcomplement may provide the early mechanism for attractinginflammatory cells effective in host defense, at the criticaltime when tissue invasion begins.

ACKNOWLEDGMENTSThis work was supported by Public Health Service grants AI-21474

and AI-15338 from the National Institute of Allergy and InfectiousDiseases and training grant HL-07501 from the National Lung andBlood Institute.

LITERATURE CITED1. Bauer, H., J. F. Flanagan, and W. H. Sheldon. 1956. The effects

of metabolic alterations on experimental Rhizopus oryzae (mu-cormycosis) infection. Yale J. Biol. Med. 29:23-32.

2. Bauer, H., J. F. Flanagan, and W. H. Sheldon. 1955. Experi-mental mucormycosis in rabbits with alloxan diabetes. Yale J.Biol Med. 28:29-36.

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