Damage to Pseudohyphal Forms of Candida albicans by

Neutrophils in the Absence of Serum In Vitro

RICHARDD. DIAMOND, RAYMONDKRZESICKI, and WELLINGTONJAO, InfectiousDisease Division, Departments of Medicine, Michael Reese Hospital andthe University of Chicago-Pritzker School of Medicine, and Departmentof Pathology, Michael Reese Hospital, Chicago, Illinois 60616

A B S T R AC T Large forms of Candida are char-acteristically present in invasive lesions and are oftencleared by host defenses. Therefore, an in vitro systemwas developed to study interactions between leuko-cytes and pseudohyphae. By light, phase contrast,and electron microscopic observations, in the abseneeof serum, neutrophils attached to and spread overthe surfaces of partially ingested pseudohyphae, whichthen appeared damaged. Using a new assay whichmeasured neutrophil-induiced inhibition of uptake of[14C]cytosine by Candida, damage to Candida in theabsence of serum was 53.04+2.96% by neutrophilsfrom 27 normal subjects. With serum, damage toCandida increased because of opsonization by lowlevels of anti-Candida immunoglobulin G in normalsera. Damage to Candida was inhibited by colchicine,cytochalasin B, and 2-deoxyglucose, which interferedwith spreading of neutrophils over the surfaces ofCandida. Dibutyryl cyclic ANIP, theophylline, andisoproterenol also inhibited damage to Candida.Hydrocortisone was inhibitory in levels (10 /.Nl)achievable with pharmacologic doses in man. Light,fluorescence, and electron microscopy indicated thatneutrophils degranulated after contact with Candida.Quantitative studies revealed only a minimal increasein specific release of lysosomal enzymes from azurophilgranules, but much greater release of lysozyme fronmspecific granules. Candida activated neutrophil oxida-tive microbicidal mechanisms, as shown by iodina-tion of Candida by neutrophils, and chemilumiines-cence from neutrophils interacting with Cauidida.

Preliminary findiings were presented in part at the AnnuwlalMeeting of the American Federation for Clinical Research,May 1977. Clin. Res. 25: 374. (Abstr.), and at the AnnualMeeting of the American Society for Microbiology, May 1977.

Dr. Diamond is a recipient of Research Career Develop-ment Award 00055 fromii the National Institute of Allergyand Infectious Disease, Nationial Inistittutes of Health.

Received for publicationt 11 July 1977 acnd in revised formi19 September 1977.

Unilike live Candida, killed Candida did not inducechemiluminescence, were not iodinated, and did notattach to nieutrophils by microscopy. Like Candidapseudohyphae, contact betweeni neutrophils andhyphal forms of Aspergillus and Rhizopus occurredin the absence of serum. This did not occur withCryptococcus neofornmans, an encapsulated yeast, andwas low with Cantdida yeasts. These findings indicatethat neutrophils can recognize and attach to Candidapseudohyphae, then damage the Catndida. This mayrepresent a general reaction between neutrophils andlarge forms of fungi. Though the size of the organismsprecludes complete ingestion, neutrophil oxidativemicrobicidal mechanisms are activated, and preferen-tial release of conitents of specific granules appears tooccur.

INTRODUCTION

Candidiasis is the most commonly observed systemicmycosis in the compromised host, constituting a signifi-cant percentage of all infections and of causes of deathin this population (1-3). Within invasive lesions,pseudohyphal and hyphal forms of Candida are moreprominent than yeasts (4, 5), and must be cleared ifthe host is to survive. Pseudohyphal and hyphalforms of Carndida can l)e cleared in at least somesystemic infectioins in experimental aniimals (6), and inman (1-3, 7, 8). Reproducible quantitative determina-tions of viability of hyphae and pseudohyphae havebeen difficult. Most in vitro studies have concentratedon the ability of leukocytes to ingest and killyeasts. Such studies (9-13), as well as the observedclinical association of neutropenia with disseminatedcandidiasis (14), suggest that neutrophils are importantin host defenses against systemic candidiasis.

The interactionis of leukocytes with filamentousforms of fungi may not necessarily correlate directlywith observations oIn phagocytosis and killing of yeasts(9-12, 15), especially becausie hyphae and pseudohyphace

The Journal of Clinical Investigation Volume 61 Februiary 1978 349-359 349

are too large to be ingested completely. Wedescribe anew leukocyte mechanism where, in the absence ofserum opsonins (including antibody aind complement),human neutrophils partially ingested and damagedCandida albicans pseudohyphae.

METHODS

Candida and other fungi. An isolate of' Candida albicansorginally obtained f'rom a patienit with systemic candidiasiswas maintained in yeast phase on Sahouraud's agar. Forgermination, yeasts were washe(d in phosphate-bufferedsaline and suspended in Eagle's minimnal essenitial medium,which had been supplemiienited with nonessential aminoacids (Grand Island Biological Co., Grand Island, N. Y.) (16,17). After 4 h of incubation at 37°C, anid at 20-30-mimintervals thereafter, ali(luots were renmoved and examined forgerm tube formation b5 phase contrast microscopy. When.95% of cells had formed germ tubes -30 /AI in length,test tubes were stored at 4°C. Canididat used immediatelyor those stored at 4°C for 18 h gave comparable results. Forsome experiments, Candida were killed by heating at 1000C(in a boiling water bath) for 1 h or by exposure to ultra-violet light for 16 h. Samples were plate(d on Sabouraud'sagar and observed for 72 h to verify- that no live organismsremained. Before incubation with leukocytes, live or killedCandida were washed three times and resuspenided in Hanks'balanced salt solution (HBSS,' Grand Islanid Biological Co.).Patient isolates ofAspergillusfu in iga tus and Rhizopus oryzaewere supplied by Dr. John Rippon, University of Chicago,Chicago, Ill., and a small capsule isolate of Cryptococcusneoformans by Dr. John E. Benniett, National Institutes ofHealth, Bethesda, Md. Aspergillus and( Rhizoppus sporeswere germinated and washed as (described above for Cacn-dida. Cryptococci were heat-killed and washed f(llowingthe same procedure as with Canididla.

Leukocytes. Human peripheral venouis blood anticoagu-lated with preservative-free heparin (Connaught Laboratories,Toronto, Ontario, Canada) was obtained from normal volun-teer subjects. Neutrophils were separated by sedimentationwith 3% dextran (average molecular weight 250,000; Phar-macia Fine Chemicals, Div. of Pharmacia, Inc., Piscataway,N. J.) in saline. Remaining erythrocytes were lysed in anammonium chloride-Tris buffer solution (18). Leukocyteswere washed in modified Hanks' solutioni (19) which con-tained no calcium or magnesium, then suspended in HBSS.For some studies, more purified preparations of neutrophilswere obtained by using Hypaque (sodium and megluminediatrizoates; Winthrop Lalboratories, New York) and Ficoll(Pharmacia Fine Chemicals) followed by dextran sedimenta-tion as described by Boyum (20).

Serum. Serum was obtained from each normal volunteerdonor at the same time as blood for leukocytes, and fromtwo subjects with rheumatoid arthritis (rheumatoid factortiters 1:320 and 1:1,280 by latex fixation). Sera were usedfresh, or after storage at -70°C or heating at 56°C for 60 min.

Immunoglobulins. Two commercial preparations of im-munoglobulin G (IgG) were obtained, one >95% pure(Fraction II, Pentex Company, Kanikakee, Ill.) and one >98%pure (Mann Research, Inc., NewYork). IgG was also purifiedto give a single line on immunoelectrophoresis using minormodifications in the method of Stanworth (21). For someexperiments, IgG was aggregated by heating for 20 min at630C.

' Abbreviation used in this paper: HBSS, Hanks' balancedsalt solution.

Detection of anti-Candida irn utntioglobtlins. Tests foranti-Candida immunoglobulins were performed on sera andimmunoglobulin preparations use(d in these studies. Ag-glutinins and precipitins were detected using slight modifica-tions in the techniq(ue of Preisler et al. (22). For detectionof anitibodies bv in direct fluiorescence, live Ca ndida albican .swere incubated with sera, stainied with goat aniti-hunman IgGconjugated with fluorescein isothiocyaniate (MIicrobiologicalAssociates, Inc., Bethesda, Md.), washed ancd examinied forfluorescence (graded from 0 to 4+).

Radioisotope assays for nmetabolic activity of Candida.Suspensions containinig 1 x 106 Canldida albicants pseudo-hyphae and 1 x 106-2 x 10() neutrophils in HBSSwere placedin 15-ml plastic centrifuge tubes (Corninlg Glass Works,Corning, N. Y), and brought to 1 ml total volume withHBSS. Triplicate tubes were placed in a rotator (ScientificIndustries, Inc., Springfield, Mass.) and( incubated at 37°Cfor timed intervals. Additional tubes were addecl to permitremoval of ali(quots for microseopic observations. To end theincubation, 0.25 ml of 2.5% sodiumii deoxycholate was addedto each tube. This incluced ly sis of leukocytes wvithoutdamage to Caindida cells (26). Control tubes contained onlyCandida pseudohyphae, but neutrophils were added justbefore deoxycholate. After lysis of neeutrophils, remainingCandida were centrifuged, then washed twice in distilledwater, and once in yeast nitrogen base broth which had beensupplemented to contain 1% dextrose and 0.15% asparagine.Radioisotopically labeled compounlds, inicluding ['4C]cyto-sine (59-61 mCi/mmol), [3H]mrannose (2 Ci/mmol), and [3H]-glucose (1.1 Ci/mmllol) (Amershanm/Searle Corp., ArlingtonHeights, Ill.) were diluted in supplemented yeast nitrogenbase broth, but glucose supplementationi was omitted in allstudies were [3H]glucose or [3H]mannose were used.The optimum dose, time period, and temperature for incuba-tioIn were determined fur [I4C]cytosine (0.25 ,uCi andl 1-hincubation at 30°C), as well as for [3H]mannose anid [3H]-glucose (2.0 ,Ci anid 2 h at 30(C). Candida were thenseparated and washed free of unlbouind radioisotope by usingan automated, multiple-sample harvester (Otto Hiller Co.,Madison, Wis.). Filters containiing Candida were air-drie(dovernight, then placed in scinitillation vials with 8 ml Aquasol,(New England Nuclear, Boston, Mass.) and counted in aliquid scintillation counter. The neutrophil-induced per-centage of reduction in uptake of isotope by Candida wascalculated from (mean counts per minute in control tubes- mean counts per minute in experimental tubes)/(mean countsper minute in control tubes) x 100. In some studies, in-stead of reductioni of uptake in Candlida, release of radio-isotopes from prelabeled Candida was measured. For meas-urement of radioisotope release, Candida pseudohyphae inyeast nitrogen base broth supplemented as described abovewere incubated at 30(C for 1 h for labeling with 0.25 uCi['4C]cytosine, and 2 h for labeling with [3H]mannose. Afterwashing three times in HBSS, Candida were resuspenide(din HBSSand incubated with neutrophils at 37°C as describedabove. After 2 h, Candida were pelleted by centrifugation at600g. The supernate and pellet were then removed separatelyfor scintillation counting. The total counts per minute insamples were determined from the sums of counts perminute in supernates and pellets of triplicate tubes. Thepercentage of isotope released was then determined by the(counts per minute in the supernatant)/(total counts perminute in supernatant plus pellet) x 100.

Inhibitors. In some studies, inhibitors of neutrophil func-tion were added during incubations. In others, nieutrophilswere preincubated with inhibitors for 20 min before theaddition of Candida, and inhibitors remained in the mediafor the duration of the incubation with Candida. Unless

350 R. D. Diamond, R. Krzesicki, and W. Jao

otherwise specified, inhibitors were obtained from SigmaChemical Co. (St. Louis, Mo.). Cytochalasin B was dis-solved in dimethyl sulfoxide. Controls contained dimethylsulfoxide without cytochalasin B, diluted comparably inHBSS. Colchicine was dissolved and directly diluted inHBSS, as were cyclic AMP, theophylline, and isoproterenol.For experiments using 2-deoxyglucose, glucose was excludedfrom HBSS, and controls contained 2-deoxyglucose plusglucose at 10 times the concentration of the 2-deoxyglucose.For studies of the effects of steroid hormones, theirprecursors, and metabolites on neutrophil fuinction, the fol-lowing compounds were dissolved in absolute ethanol: hydro-cortisone, testosteronie, estriol, androsterone, progesterone,dehydroisoandrosterone, and 1 7a-hydroxyprogesterone. Con-trol tubes included absolute ethanol diluted comparably tothe above compounds which had been originally dissolvedin ethanol.

Microscopic observatiotns. At times intervals during theincubation of Candida with neutrophils, aliquots were re-moved for continuous observation under phase contrastmicroscopy using a stage incubator (Incustage, Lab-LineInstruments, Inc., Melrose Park, Ill.), and for staining usingWright-Giemsa. Trials of visual methods for determination ofCanidida viability were made after lysis of neutrophilsusing deoxycholate. Dye exclusion using methylene blue wasperformed on pseudohyphae exactly as described by Lehrerand Cline (23), and Giemsa staining as outlined by Lehrer(9). Viability of neutrophils was determined before andafter incubation with Candida using exclusion of 0.1%trypan blue. In selected experiments, neutrophils werelabeled with 5'Cr (24), and release of 5mCr during incuba-tions was measured. Degranulation of neutrophils was ob-served under phase contrast microscopy, and by fluorescencemicroscopy after staining of granules with a 1: 100,000dilution of acridine orange in HBSS according to themethod of Allison (25) and D'Arcy Hart and Young (26).For electron microscopic studies of neutrophil degranulation,the method of Armstrong and D'Arcy Hart (27) was usedwith slight modifications. Neutrophil granules were labeledwith 10( mg/miil ferritin (twice recrystallized cadmium free;Pentex Company) for 2.5 h at 37°C, washed three timesin HBSS, and incubated with Candida in the usual way.

Four electron microscopy, pellets containing Candida andneutrophils were fixed in 2.5% glutaraldehyde solutionovernight. After washing overnight in cacodylate-bufferedsolution, pellets were postfixed in 2% osmium tetroxide for2 h. They were then dehydrated in increasing, graded con-centrationis of ethanol, and embedded in Epon. Thinsections were cut with diamond knives on an automaticPorter-Blum ultramicrotome (DuPont Instruments, SorvallOperations, Newtown, Conn.). After staining with uranylacetate and lead citrate, sections were mounted on carbon-coated copper grids, examined with an RCA 3H electronmicroscope (Special Products Div., RCACorp., Cherry Hill,N. J.) and photographed.

Iodination of Candida by neutrophils. The fixation ofiodide to Candida (trichloroacetic acid-precipitable iodide)by neutrophils attaching to the surface of pseudohyphae wasmeasured by the method of Klebanoff and Clark (28), withthe f'ollowing modifications. In addition to pH 7.4 sodiumphosphate buffer with added potassium, calcium, and mag-nesium salts, for optimum conditions, the reaction mixturesin triplicate or quadruplicate tubes contained 1 x 107neutrophils, 5 x 106 Candida, or other fungi, but no serumunless otherwise stated. The optimum concentration of 125I1Na was found to be 0.5 ,M, containing 0.2 ,uCi 1251, with a1-h incubation time.

Chemiluminescence by neutrophils attached to Candida

pseudohyphae. Chemiluminescence was measured in aBeckman Model LS 230 liquid scintillation counter (BeckmanInstruments, Inc., Electronic Instruments Div., Schiller Park,Ill.) as described by Stjernholm et al. (29). Vials contained1 x 107 Candida in HBSS, or HBSSalone. Unless otherwisenoted, reaction mixtures contained no serum.

Release of enzymes from neutrophils. Enzyme assasyswere kindly performed by Dr. Philip Davies and hiscolleagues (Department of Inflammation and Arthritis, MerckInstitute for Research, Rahway, N. J.). /8-Glucuronidasewas assayed by the method of Talalay et al. (30), usingphenolphthalein glucuronate (Sigma Chemical Co.) as sub-strate. N-acetyl-,8-D-glucosaminidase was assayed accordingto the method of Woolen et al. (31), using p-nitrophenyl-N-acetyl-,8-D-glucosaminidase as substrate. Lactate dehydrog-enase was quantitated by assay of the change of opticaldensity at 340 nm of NADHbeing oxidized to NADby theconversion of pyruvate to lactate. Lysozyme was measuredby using Micrococcus lysodeikticus, according to the methodof Parry et al. (32).

Statistical methods. Means and SEM were comparedusing two sample T tests (33).

RESULTS

Microscopic observations of the interaction ofCandida albicans pseudohyphae with neutrophils.Preliminary observations using light, phase contrast,and fluorescence microscopy suggested that neutro-phils attached to pseudohyphae in the absence ofserum, spread over pseudohyphal surfaces, anddegranulated, resulting in altered Candida morphol-ogy. To extend these findings, electron micrographswere prepared from samples obtained at timed inter-vals during incubations of neutrophils with Candidapseudohyphae. Normal Candida morphology was ap-parent at the start of incubations (Fig. 1). By 20 minof incubation, neutrophils had spread over the sur-faces and around Candida, and degranulation of neu-trophils over the Candida surface was evident asshown in studies where neutrophil lysosomes werelabeled by incubation with ferritin. Ferritin wasspecifically released by neutrophils over the surfacesof pseudohyphae (Fig. 2). By 1 h of incubation,damage to the Candida was apparent, as there werestriking changes in the cell wall and internalmorphology (Fig. 3), compared with normal Candida(Fig. 1), even though Candida were not completelyingested by neutrophils. Candida which were not inclose contact with neutrophils had no apparent al-terations in morphology.

To quantitate this apparent damage to Candidapseudohyphae by neutrophils, several staining tech-niques for light microscopy were used. These includedmethylene blue vital staining of Candida, and Giemsastaining of fixed specimens. As expected, these pro-cedures were useful for Candida yeasts; unfortunately,they did not provide reproducible results with Candidapseudohyphae. Similarly, tube dilutions and platecounts were extremely variable and not reproducible,because of clumping of pseudohyphae.

Damage to Candida Pseudohyphae by Neutrophils in the Absence of Serum 351

FIGURE 1 Electron micrograph showing normal Candida albicans pseudohyphal morphologyseen in transverse section surrounded by neutrophils, at the start of an experiment. Cellwall (CW) and cell membrane (M) are shown by arrows, and internal structures areidentifiable. Magnification x35,910.

Radioisotope assays for metabolic activity ofCandida. Because routine methods for quantitatingviability of fungi gave inconsistent results, use ofradiolabeled compounds was investigated as a methodwhich might permit quantitation of viability ofCandida pseudohyphae before and after incubationwith leukocytes. Experiments included the effects ofneutrophils upon both reduction of uptake of radio-isotopes by Candida, as well as release of radio-isotopes from Candida during incubations (Fig. 4).With a short incubation time (1-2 h), percentagerelease of [14C]cytosine or [3H]mannose was smallerthan reduction in uptake. Prolongation of the incuba-tion time resulted in growth of Candida and inconsistent results. Reduction of uptake of ["4C]-cytosine proved a reproducible method, which cor-related with reduction of uptake of [3H]mannose andglucose, as well as microscopic observations. Uptakeof [14C]cytosine by Candida was linear over a 3-logrange, from 1 x 104 to 1 x 107 live organisms. A ratio of

10 neutrophils: 1 Candida and a 1 h incubation timewere optimum. In 40 experiments using neutrophilsfrom 27 subjects under optimum conditions (Fig. 4),controls (Candida, which leukocytes added at the endof incubations) took up 56,654+2,806 cpm (mean+SEM) of [14C]cytosine. There was a 10.9% range ofvariation within triplicates. Results were comparablein experimental tubes where leukocytes and Candidawere incubated together. To determine whether met-abolic damage was reversible, [14C]cytosine up-take was measured over 3 h immediately after lysis ofleukocytes, and Candida were stored at 4°C for 4and 18 h. In four experiments, uptake of ["4C]-cytosine did not increase at 4 and 18 h over baselinevalues (mean -2.8%, range -11.2 to +3.4%), but in-creased 20.3-38.7% (P <0.025) for control Candidawhere leukocytes were added just before deoxycholatelysis.

Opsonization of live Candida albicans pseudo-hyphae. Because interactions between Candida and

352 R. D. Diamond, R. Krzesicki, and W. Jao

Aw,

I

I i.

4-v...

FIGuRE 2 Electron imicrograph taken after 60 min of, incubation of neutrophils withCandida albicans pseudohyphae. In this study, neutrophil granules had been labeled with ferritin,and excess ferritin was washed away. Ferritin (arrows) is located specifically surrounding thepartially ingested pseudohypha, and nowhere else. Magnification x11,800.

neutrophils occurred in the absence of serum, it waspossible that serum might not effect these interactions,or might increase or decrease damage to Candida byneutrophils. Using neutrophils from 17 normal subjects,autologous serum consistently increased the percentagereduction of [14C]cytosine uptake by Candida (P < 0.001bv twotailed two-sample t test) (Fig. 5). This occurredwhether serum was fresh or heated at 560C for 60 min.When neutrophils were preincubated in serum andwashed before exposure to Candida, there was noincrement. However, with preincubation of Candida inserum followed by washing before incubation withneutrophils, there was augmentation of damage toCandida, comparable to that seen when serum waspresent throughout incubations (Fig. 5). Because opsoniz-ation of the organism by heated serum was as effective asfresh serum, it appeared that anti-Candida IgG might beinvolved rather than complement. Indirect fluorescentantibody determinations using fluorescein-conjugated

anti-IgG indicated faint (1-2+/4+) fluorescence onCandida which had been incubated in normal seradiluted 1:1-1:16 in HBSS. Under the same conditions,there was no fluorescence on Candida surfaces usingfluorescein-conjugated antialbumin or antibody to thethird component of complement. However, when serawere tested for antiCandida antibodies using standardtechniques for detection of agglutinins and precipitins,none were found. Purified preparations of human IgGwere used to determine if low levels of anti-Candidaactivity might be present in immunoglobulins in normalsera. Whenthree different preparations of normal humanIgG were added to incubations containing Candida andneutrophils, an increment in opsonization was seen(P < 0.05), similar to that noted when whole serum wasused (Fig. 5). To establish further that low levels of anti-Candida IgG were interacting with neutrophil Fcreceptors, attempts were made to block activity of anti-Candida IgG. Heat aggregation of IgG (P < 0.01) or the

Damage to Candida Pseudohyphae by Neutrophils in the Absence of Serum 353

FIGURE3 Electron micrograph taken after 60 min of incubation of neutrophils withCandida albicans pseudohyphae, showing changes in cell wall (arrows) and loss of dis-cernable internal morphology of Candida, which is between several neutrophils. Magnificationx5,265.

presence of rheumatoid factor (P < 0.025) in serum (34)both eliminated the increments in opsonization normnallyseen with IgG or whole serum, as did absorption of IgGwith Candida.

Unlike serum, removal of divalent cations from themedium completely inhibited interactions of neutrophilswith Candida albicans pseudohyphae. In two experi-ments in HBSS, neutrophils reduced uptake of [14C]-cytosine into Candida by 83%. However, when 10 mMsodium citrate was added to HBSS, or a calcium andmagnesium-free buffer (modified Hanks' solution) wassubstituted for HBSS, there was no reduction in uptakeof [14C]cytosine into Candida.

Effect of inhibitors of neutrophil function ondamage to Candida albicans pseudohyphae. Inhibi-tors were used which were known to be active ininhibiting the function of neutrophils with severalmicroorganisms. The results were calculated as per-cent inhibition of the [14C]cytosine assay. Severalconcentrations of each inhibitor were tested. Con-centrations of inhibitors were eliminated from studies

if they inhibited uptake of [I4C]cytosine in controlincubations of Candida without neutrophils or if theycaused .-5% decrease in viability of neutrophils, asjudged by phase contrast microscopy and by trypanblue exclusion, as well as 5'Cr release in selectedstudies. The most effective concentration of eachinhibitor was then determined.

Colchicine, cytochalasin B, and 2-deoxyglucose in-hibited the nonphagocytic interaction of neutrophilswith Candida pseudohyphae (Table I). Dibutyrylcyclic AMP, and two compounds known to raiseintracellular levels of cyclic AMP, theophylline andisoproterenol, all inhibited damage to Candidaalbicans pseudohyphae by neutrophils. With thesecompounds, there were no apparent morphologicchanges which were attributable to the inhibitors onreview of Giemsa-stained slides of interactions ofneutrophils and Candida. Hydrocortisone, along withother steroid hormones and metabolites of hormonesfor comparison were also tested. Hydrocortisone andestriol had the highest inhibitory activity of all the

354 R. D. Diamond, R. Krzesicki, and W. Jao

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UR R

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UR R

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FIGURE 4 Effect of neutrophils on1 uptake and release ofra(dioisotopes by Candida albicaiis pseudohyphae. Radio-labeled coMpouLnds are noted oni the horizontal axis, withresults for reductioni in uptake of isotopes (UR), and isotoperelease (R). Percentage of uptake or release of isotopesis shown oni the ordiniate. Each poinit represents the meanof oine or more experimenits performed in triplicate on onienormiial individual. Horizontal lines denote means andbrackets SENI for sets of observations. Experiments wereperfornmed usinig optimum numlbers of nieutrophils, aind anioptinmumIl duratioI Of in'cubationls.

compounids which were tested (Table I). Testosteronehald less inhibitory activity, whereas other steroidstested had little or no inhibitory activity.

Release of contetnts of neutrophil granules duringconitact twith Caindida p.seudohyphae. Because de-granulationi was observed microscopically, quantitativestudies were performed. A ratio of neutrophils toCandida of 1:1 gave optimunm release of lysosomalenzymes 8-glucuronidase and N-acetvl-,8-D-glucos-aminidase from neutrophils. There was only a slightbut significant (P < 0.05 by two-sample t test) releaseof lysosomal enzynmes by neutrophils (Table II),though microscopic observations indicated that over90% of neutrophils were attached to Can dida.IHydrocortisone, in the concentration which com-pletely blocked daImage to Catndida as measured bythe [ 14C]cytosine assay, did not inhibit this smallamniount of release of granule enzymes from neutrophils.This was confirmed in fluorescence microscopicstudies. In contrast, lysozyme was released in largeramounts during inctubations with pseudohyphae (P< 0.001) (Table II). Hydrocortisone also did notinhibit lysozyme release.

Lice cs. killed Candida albicans pseudohyphae iniodination of Candida and stimutilationt of chem ilu-minescetnce by tleUtrop/uils. In light microscopicstudies, it was noted that neutrophils did not readilyattach to killed pseudohyphae. Therefore, iodinationof pseudohyphae and chemiluminescence by neutro-phils in response to interaction with pseudohyphae

were evaluated, to determine if these processes wereactivated bv surf'ace atttachment of neutrophils topseutdohyphae in the al)sence of serumn, and whetherlive funlgi were re(quired. In the absence of serumii,neutrophils actively iodiniated live Catndida albicatns(Table I1I). In conitrast, iodinationi was muarkedlyreduced wheni pseudohyphae were used which ha(lbeen killed bv heat or ultraviolet light. Comparableresults were obtainied in measuremiients of clhenmilumni-nescence. Live Canidida stimulated chemiiiluminies-cence. b) neutrophils eveni when serumil was notpresent in incul)ations (Fig. 6). However, killedpseudohyphae did niot stinuitilate chemiiluminiescence.

Ititeractiont of ftutngi othler thani Canidida pseuido-hyphae wcith nieutrophils. In order to determine thespecificity of the interactioni between Catndida pseudo-hyphae and neutrophils, other live funigi were tested.By phase contrast microscopic observation, in the ab-seinee of sertum, oinlx 5-8% of Cantdida yeastswere ingested by neuitrophils, and only -55% of theyeasts were attached to the suirfaces of neuitrophils.The encapsulated yeast, Cryptococcuis neoforncatis,was niot ingested by neuetrophils at all in the ab-

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FIGURE 5 Effect of serum and IgG on opsonization of liveCandida albicanis pseudohyphae. Interactioni of Candidawvith neutrophils is measured by neutrophil-iniduced reduc-tion of uptake of [14C]cvtosine by Catidida, shown on theordinate. Each point represenits the mean of at least twoexperiments, each performed in triplicate. Results are shownusing buffer (HBSS) alone. The solid line connects mean aindSENI values for experiments performed in HBSSwith thoseperformed with 10% heated (A) or fresh serum from 17normal sul)jects. Shown separately, connected to restilts incontrols by dotted lines, are results of preincubation ofCandida albicans (PREINC C.a.) or neutrophils (PREINCWBC) in serum, which was washed away before incubations.Also shown separately are results using two commerical (FRII,M; FR II,P) and one freshly purified (DEAE) preparationsof IgG, as well as heat (A) aggregated IgG, and seracontaining rheumatoid factor (RF+).

Damage to Candida Pseudohyphae by Neutrophils in the Absence of Serum

00

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355

TABLE IEffect of Inhibitors of Neutrophil Functiont onl Reduction of

Cytosine Uptake by Candida albicans Pseudohyphue

Inhibitor (concentration added) Inhibition*

Colchicine (0.1 mM) 62.1

Cytochalasin B (2 gg/ml)4 44.7Cvtochalasiin B (20 ,ug/ml)t 57.6

2-Deoxyglucose (0.2 mrM) 100.(2-Deoxyglucose (0.2 mM) + gluicose (2 mM) 0.0

Dibutyryl cyclic AMP(1 mM) 42.8Theophylline (1 mM) 48.1Isoproterenol (1 mrM) 65.2

Hydrocortisonie (1 uM)§ 34.6Hydrocortisone (101uM) 100.0Estriol (1 ,uM) 69.9Estriol (10 ,M) 100.0Testosterone (101(tM) 48.5Androsterone (10 ,uM) 14.3Progesterone (10 ,uM) 0.0Dehydroisoandrosterone (10 uM) 2.317a-Hydroxyprogesterone (10 ,uM) 10.5

* Calculated from the results of [14C]cytosiine uptake in thepreseniee and absence of inhibitors. Vatlues represent meansof' at least two separate experiments, each performed intriplicate.t Conitrols contained equivalent concenitrationis of dimethyl-sulfoxide.§ Controls for all corticosteroids contained equivalent con-centrattions of ethanol.

secIee of serum, nor was there close surface contactbetween neutrophils and yeasts. In contrast, Rhizopusoryzae and Aspergillus fumigatus became coveredwith neutrophils spreading over the surfaces of

TABLE IISelective Release of Markers of Azurophil and Specific

Granules of Neutrophils During Interactionwith Candida Pseudohyphae

Present in incubation Release*

10 MM N-Acetyl-fl-Netutro- Hydro- f3-Glucuron- D-gluco-philst Candida§ cortisone idase saminidase Lysozyme

Yes No No 5.7+2.4 3.6±0.6 1.0±1.0Yes Yes No 12.6+1.9 11.1±1.1 27.9±1.5Yes No Yes 6.0+1.0 3.6+1.1 0.1+0.1Yes Yes Yes 9.0+4.5 13.6±2.2 28.7±4.6

* Mean+SEM.Each tube contained 5 x 106 neutrophils.

5 Each tube contained 5 x 106 Candida.

356 R. D. Diamond, R. Krzesicki, and W. Jao

TABLE IIIEffect of Viability of Candida albicans Pseudohyphae on

Iodination of Candida by Neutrophils inthe Absence of Serum

Inhibition ofCandida viability Iodination* iodination

nmol 1251 fixed per10' neutrophilslh %

Live 39.8Heat killed 9.( 76.8Ultraviolet-light killed 13.2 66.0

* Mean of three experiments, each performed in triplicate.

hyphae. These observations were confirmed and betterquantitated using iodination of these fungi by neutro-phils in the presence and absence of serum (Table IV).Dose-response curves were performed for each or-ganism, and the concentration which gave maximumiodination was used. Like Candida pseudohyphae,Rhizopus and Aspergillus were iodinated in theabsence of serum, and showed an increment in iodina-tion when serum was present. The encapsulatedyeast, Cryptococcus neoformans, was iodinated sig-nificantly only when serum was present. Candidayeasts, even in increased numbers to account fordifferences in surface area, were not iodinated asmuch as Candida pseudohyphae.

DISCUSSION

These experiments have established that neutrophilscan damage and probably kill Candida pseudohyphae.Pseudohyphae were too large to be completely in-gested by neutrophils. Nevertheless, neutrophils at-

50

i 30"IJ

2 20

xI0~

IaJ4063

o

LIVE CANDIDA

UV-KILLED____/_\CANDIDA

wz =~5--f--- _-_

EATN- KALLEDCANDI DA

10 20 30 40rIME (MIN)

50 60 70

FIGURE 6 Effect of viability of Candida albicans pseudo-hyphae on chemiluminescence by neutrophils induced oncontact with Candida in the absence of serum. Results usinglive Candida are compared with those using Candida killedby heat or ultraviolet (UV) light.

TABLE IVlodination of Fungi by Neutrophils in the Presence

and Absence of Autologous Serum

Serum inFungus* (growth phase) incubations Iodination

nmol 1251 fixed per107 neutrophilslh

Candida albicans (pseudohyphae) No 50.6Yes 94.9

Candida albicans (yeasts) No 10.0Yes 21.7

Cryptococcus neoformans (yeasts) No 0.5Yes 14.2

Rhizopus oryzae (hyphae) No 24.9Yes 86.6

Aspergillus fumigatus (hyphae) No 65.4Yes 96.3

* Each tube contained 5 x 106 fungi with 1 x 107 neutrophils,except for Candida yeasts, where 5 x 107 tube were requiredfor maximum iodination.

tached to and spread over the surfaces of pseudo-hyphae. Morphologic changes occurred in the Can-dida, as judged by light, phase contrast, and electronmicroscopic observations. Dramatic changes in theCandida cell wall by electron microscopy weresimilar to those described by others who observedorganisms in tissues of infected mice or human pa-tients (35) or in vitro with leukocytes (36). Thesignificance of these morphologic changes was con-firmed by parallel studies using radioisotopicallylabeled nutrients. These experiments indicated thatmetabolism of Candida was impaired after contactwith neutrophils, and that Candida labeled withradioisotopes of cytosine or mannose released thesecompounds during incubations with neutrophils. Be-cause conventional methods for quantitating viabilityof fungi could not be used with pseudohyphae, itremains possible that metabolic damage to Candidapseudohyphae was reversible. However, the con-comitant striking morphologic changes suggestedfungal cell death. In addition, even in the fungiwere not killed, damage to hyphae which are toolarge to be ingested by phagocytic cells might stillbe an important host defense mechanism. Thiscapability of neutrophils may be especially importantbecause the large pseudohyphal and hyphal forms ofCandida are predominant in lesions in tissues (4, 5)which contain both large and small forms of Can-dida. There have been many studies on interactionsof Candida blastospores (or yeasts) with leukocytes(9-13, 23). However, in addition to morphologicdifferences between forms of Candida, antigens (37)and other properties may not be identical. Our ex-

periments indicated several differences between Caci-dida yeasts and pseudohyphae in interactioni withneutrophils.

First, interactions between Catndida pseudohyphaeand neutrophils required no serum. In the preseniceof serum, there was an incremenit in contact betweenCandida and neutrophils, and damage to Cancdidaincreased. This effect was duplicated by normiialhuman IgG, was blocked by heat aggregationi of IgGor by sera which contained rheumatoid factor (34), anidappeared to be attributable to opsonization of Cani-dida by low levels of anti-Candida IgG which waspresent in normal human sera. This is consistenitwith the observations of Weiner and Yount (38), whofound that antibodies to Catndida mainnan were tini-versally present in sera from adult normal controlsubjects.

Despite the absence of serum, interactioins of neutro-phils with Candida pseudohyphae appeared to activateleukocyte microbicidal mechanisms, as evidencedl bystudies of iodination and chemiluminesceince. lodina-tion of microorganisms generally occurs after phago-cytosis, with interaction of myeloperoxidase and hydro-gen peroxide within phagocy;tic vacuoles (39). Thelight emission in chemiluminescence of neutrophilshas been correlated with the production of oxidizingagents by phagocytosing neutrophils (40). For micro-organisms, this also requires either the presence ofserum opsonins, or opsonization of organisms byincubation with serum prior to incubation withneutrophils (41). In contrast, Candida pseudohyphaerequired neither complete ingestion nor serumiiopsonins to be iodinated by neutrophils or induceneutrophils to chemiluminesce.

lodination (28) and chemiluminescenice (41) haveboth been used as sensitive screening techni(quesfor abnormalities in the phagocytic process. Similarly,the occurrence of iodination and chemiluminescenceduring incubations of neutrophils with Canididapseudohyphae provided useful procedures for (uana-titation of surface interactionis between funlgi andclleukoevtes, which correlated well with light micro-scopic studies. These studies suggested that stir-face factors are present on live but not killed funllgiwhich permit recognition by leukocytes. These fhactorswere not limited to Candida pseudohyphae, as Rhizo-pus oryzae and Aspergillus fu migatus were iodiniatedin the absence of serum, andcl becaimie attachedl toneutrophils on microscopic examinationi. In contrast,iodination of Catndida yeasts was less than iodiniationof pseudohyphae, and Cryptococcuis nteoformanlts,where the yeast cell wall is covered bv a capsuile,was iodinated significantly only wheni serumil waspresent. Consequently, it appears possible that thecapacity of fungi to interact with leukocvtes in theabsence of serumil may reflect a general property ofcell walls of large forms of funLgi.

Damage to Candida Pseudohyphae by Neutrophils in the Absence of Seruim 357

Further insights were gained into interactions be-tween Candida pseudohyphae through the use of sub-stances which are known to inhibit neutrophil func-tion. Several inhibitors of neutrophil phagocytic andother functions also inhibited nonphagocytic damage toCandida pseudohyphae. Damage to pseudohyphae byneutrophils was inhibited by colchicine, cytochalasinB, and 2-deoxyglucose. Colchicine is known to inhibitfunction of neutrophil microtubules, and may inhibitphagocytosis under some conditions as well (42). Cyto-chalasin B inhibits neutrophil microfilaments andblocks phagocytosis, but also causes exocytosis oflysosomal hydrolases from neutrophils, and inhibitshexose transport (43). The compound 2-deoxyglucoseinhibits aerobic and anaerobic cellular metabolism aswell as phagocytosis, though inhibition of phagocyto-sis may occur by a separate mechanism (44). Dibutyrylcyclic AMP, theophylline, and isoproterenol, all ofwhich elevate intracellular levels of 3'5' cyclic AMPin neutrophils, inhibited damage to Candida pseudo-hyphae without clearly interfering with surface contactor spreading over surfaces of Canidida. Elevatedlevels of cyclic AMP have been shown to inhibitseveral functions of neutrophils, such as phagocytosis,release of lysosomal enzymes, and killing of micro-organisms, including Candida yeasts (45). Becausemicroscopic observations indicated that neutrophilsdegranulated during contact with pseudohyphae, theeffects of hydrocortisone were then studied. Besidesthe well-known association between corticosteroidtherapy and invasive candidiasis (4, 5), cortisone mayinhibit release of lysosomal enzymes from neutrophils(46). Hydrocortisone inhibited damage to Candidapseudohyphae by neutrophils at levels (10-1 ,uM)which may be reached in vivo after pharmacologicdoses. Nevertheless, there was no visible or quantita-tive inhibition of degranulation of neutrophils byhydrocortisone. Hydrocortisone appeared to inhibitdamage to pseudohyphae by inhibiting a neutrophilinechanism other than degranulation.

Despite the visual evidence for degranulation ofneutrophils during contact with pseudohyphae, therewas only minimal quantitative release of two lysosomalenzymes from neutrophils. In contrast, lysozyme wasreleased in much larger amounts. Within neutrophils,lysosomal enzymes are located in the azurophilgranules, while lysozyme is present in the specificgranules, which appear to discharge first (47). Con-tact between neutrophils and pseudohyphae may pro-vide a less complete stimtulus for degranulation thanphagocytosis, so that contents of specific granulesare released preferentially.

Further experiments will be required to delineatewhich mechanism is primarily responsible for met-abolic and morphologic damage to Candida pseudo-hyphae by neutrophils. In addition, neutrophilsdirectly recognize Candida pseudohyphae and other

358 R. D. Diamond, R. Krzesicki, and W. Jao

fungi and activate cellular microbicidal mechanisml-sin the absence of serum opsonins by a mechanismwhich must be characterized, although the interactioniis enhanced bv serum. Although the biologic im-portance of this new neutrophil mechanism remains tobe determined, these experiments have established theutility of new methodology which permits quantita-tive studies of the interactions between neutrophilsand large forms of pathogenic fuingi.

ACKNOWLEDGMENTS

We wish to thank Dr. Robert A. Clark for helpful advice,Dr. Philip Davies for helpful discussion and for performingmeasurements of lysosomal enzymes, and Drs. FrederickAuletta, Arthur Schneider, and Clarence Gantt for supply-ing some of the reagents used in these studies. HildaSanders, Martha Newbill, and Jean Makowski providedinvaluable technical assistance with the electroni microscopicstudies.

This work was supported by grant 12145 from the NationalInstitute of Allergy and Infectious Disease of the NationalInstitutes of Health.

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