INFECrION AND IMMUNITY, May 1993, p. 1779-17850019-9567/93/051779-07$02.00/0Copyright C 1993, American Society for Microbiology

Increased C3 Production in Human Monocytes afterStimulation with Candida albicans Is Suppressed byGranulocyte-Macrophage Colony-Stimulating Factor

ANNE KIRSTI MYRVANG H0GASEN* AND TORE G. ABRAHAMSEN

Department ofPediatric Research, Rikshospitalet, The National Hospital, N-0027 Oslo, Norway

Received 28 December 1992/Accepted 20 February 1993

Activation of the complement system is an important part of host resistance against fungal infections. Whenhuman monocytes, cultured for 2 days or more, were treated in vitro with Candida albicans for 24 h, an

enhancement of their biosynthesis of the complement components C3 and factor B was found. However, whenC. albicans was administered to freshly isolated monocytes, a consistent stimulation of factor B biosynthesisoccurred, while the C3 production was increased in about 50%o of the donors. C. albicans also induced therelease of granulocyte-macrophage colony-stimulating factor (GM-CSF) from the cultured cells, apparently inlarger amounts in the donors in whom no stimulation of C3 production was found. An antibody to GM-CSFadministered with the yeast at the initiation of the monocyte culture caused an increase in the C3 production.Furthermore, when monocytes were treated with recombinant human GM-CSF either at the same time as or

4 days prior to the addition of C. albicans, the increase in C3 production was suppressed or neutralized, whilefactor B biosynthesis was unaffected. Taken together, these results indicate that monocytes respond to C.albicans with an increased production of complement factors. This may be an important mechanism both foropsonization of the fungus and for initiation of an inflammatory reaction. At an inflammatory site, thiscomplement response may be suppressed by locally produced GM-CSF.

Candida albicans is an opportunistic pathogen which maycause severe disseminated disease in immunocompromisedpatients (32). The host defense against fungal infections isaccomplished by phagocytes, including monocytes/macro-phages, in addition to cell-mediated and humoral immunemechanisms (36). Opsonization of C. albicans by comple-ment factors and immunoglobulins enhances phagocytosisand intracellular killing of the yeast by the phagocytes (33).Monocytes/macrophages are the main extrahepatic source ofcomplement factors (6, 20, 22, 41), and they provide all thecomponents necessary for local complement activation. Fac-tor B, the initial component of the alternative pathway, isactivated after direct contact with microorganisms and otherforeign agents. C3 is a key component in the complementsystem, as it represents a joining site for the classical andalternative pathways to a common terminal pathway. Thecleavage products obtained during complement activationare potent anaphylatoxins, chemotaxins, and opsonins,which are important in the local inflammatory reaction (19).The monocyte production of complement factors is mod-

ulated by several physiological and foreign substances, suchas interferons (18), immuncomplexes (27), and lipopolysac-charide (LPS) (38, 39). The ingestion of streptococci hasbeen shown to stimulate C3 production in macrophage-likecell lines (16). Phagocytosis may stimulate the monocytes/macrophages to secrete a variety of inflammatory mediators,such as prostaglandins, cytokines, and reactive oxygenintermediates (7, 8).The purpose of this study was to investigate the influence

of C. albicans on monocyte production of the complementfactors C3 and factor B, since optimal opsonization of fungiis important for their engulfment by phagocytes. It is knownthat granulocyte-macrophage colony-stimulating factor (GM-

* Corresponding author. Electronic mail address: a.k.hogasen@rh.uio.no(INTERNET).

CSF) is released from leukocytes in response to C. albicans(3). In addition to its stimulatory effects on myelopoiesis,this cytokine affects several functions of mature granulo-cytes and monocytes/macrophages (28, 31). Since GM-CSFhas been reported to enhance monocyte function againstfungi (34, 37, 40), we evaluated the possible impact of thiscytokine on complement biosynthesis by monocytes treatedwith C. albicans.

MATERIALS AND METHODS

Preparation and culture of human monocytes. Leukocytebuffy coats were obtained from healthy blood donors. Thebuffy coats were diluted 1:2 in Hanks' balanced salt solution(HBSS) without calcium and magnesium (Whittaker Bio-products, Walkersville, Md.) and centrifuged over Ficoll-Hypaque solution (Nycomed Pharma, Oslo, Norway) ac-

cording to the method described by B0yum (5). Themononuclear cells obtained were suspended in X-VIVO 10serum-free medium (Whittaker Bioproducts). The cell con-

centration was adjusted to 4 x 106 cells per ml, and portionsconsisting of 1 ml of cell suspension per well were incubatedin 12-well plates (Costar, Cambridge, Mass.) at 37°C with 5%CO2 for 4 h. Nonadherent cells were then removed by twovigorous washes in warm HBSS without calcium and mag-nesium. The adherent cell population, which represented 20to 25% of the originally plated cells, consisted of 85 to 95%monocytes as determined by differential counts (Diff-Quik;Merz+Dade, Dudingen, Germany) and nonspecific esterasestaining (a-naphthyl acetate kit; Sigma Chemical Co., St.Louis, Mo.). Viability, determined by trypan blue (Sigma)exclusion, was always above 98%. Contaminating cells weremainly lymphocytes. The number of adherent cells was

estimated by counting cells removed with a cell lifter (Cos-tar). All cell culture experiments were performed with du-plicate wells, and the results were averaged. At the end of

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culture (1 to 5 days), cell supernatants were harvested,centrifuged for 10 min at 1,500 x g to remove cell debris,transferred to cryotubes (Micronic, Lelystad, The Nether-lands), and stored at -70°C. More than 90% of the culturedcells were viable. Cycloheximide (5 ,ug/ml; Sigma) wasadded in some experiments to block protein biosynthesis.

C. albicans isolates. The C. albicans isolates used in thisstudy were obtained from adult or newborn patients. Theyeast preparations were stored at -70°C. Prior to study,samples were grown on Sabouraud agar plates at 37°C for 18h, and then the blastoconidia were harvested and suspendedin HBSS. C. albicans was then always killed by heating at60°C in a water bath for 1 h, washed twice in HBSS, andcounted. Endotoxin contamination of the yeast suspensionswas less than 3 pg/ml for the dilutions employed, as assayedby a Limulus amebocyte lysate chromogen test.

Determination of complement factor C3 and factor B.Double-antibody enzyme-linked immunoassays (EIA) forquantification of antigenic human C3 and factor B wereestablished. Microtiter plates (Immuno Plate Maxisorp;Nunc, Roskilde, Denmark) were coated overnight at 4°Cwith mouse anti-human C3c (Quidel, San Diego, Calif.) ormouse anti-human Ba (Quidel). For the C3 assay, 1% bovineserum albumin (Amtec Diagnostics International Inc., Con-roe, Tex.) was added to block unspecific binding to themicrotiter plate; this was followed by addition of cell super-natants, a rabbit anti-human C3c (Behring, Marburg, Ger-many), and a peroxidase-linked donkey anti-rabbit immuno-globulin (Amersham, Buckinghamshire, United Kingdom).For the factor B assay the cell supernatants were added,followed by goat anti-human factor B (Quidel) and a perox-idase-linked mouse anti-goat immunoglobulin (Jackson Inc.,West Grove, Pa.). ABTS [2,2'-azinobis(3-ethylbenzthiazo-line-6-sulfonic acid); Sigma] was used as the substrate, andA405-492 was determined with an EIA reader (Titertek Mul-tiscan Plus; Flow, Bioggio, Switzerland). All incubationsteps were performed at 37°C for 1 h, and the wells werewashed four times with an automatic plate washer (DelfiaPlatewash; Wallac, Turku, Finland) after each incubation.Dilutions of a normal human serum (NHS) pool were used asstandards. The C3 concentration in the NHS was 0.91mg/ml, as measured by nephelometry. One unit of factor Bwas defined as the amount contained in a 1:106 dilution ofNHS. Cell supernatants (diluted 1:3 in the C3 assay and 1:1.1in the factor B assay), NHS, and antibodies were diluted inphosphate-buffered saline-Tween 20 (Sigma) to a final con-centration of 0.2% Tween 20. All complement factor deter-minations were made in triplicate. The minimum detectableamounts of C3 and factor B were 0.4 ng/ml and 1.1 U/ml,respectively.

Reagents. Recombinant human GM-CSF and a sheeppolyclonal anti-human GM-CSF were generously providedby Schering-Plough Research, Bloomfield, N.J. Endotoxinlevels in these reagents were <0.02 ng/ml for the dilutionsemployed. The GM-CSF EIA kit (Quantikine; R&D SystemsInc.) was purchased from British Bio-technology ProductsLtd., Oxon, United Kingdom. The assay was performedaccording to the manufacturer's protocol, and the minimumdetectable dose of GM-CSF was 1.5 pg/ml.

Statistics. The statistical significance of differences be-tween test groups was analyzed by Student's paired t test.

RESULTS

Effect of heat-killed C. albicans on monocyte production ofC3. Monocytes are known to produce and secrete comple-

15

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day 1 day2 day3 day4 daySFIG. 1. Effect of C. albicans on monocyte production of com-

plement factor C3 when cells were treated with the yeast for 24 h.Supernatants of control cells (grey bars) and monocytes treated withC. albicans (1.5 x 10' blastoconidia per well) (black bars) wereharvested at the indicated times and analyzed by EIA for C3 contentas described in Materials and Methods. The results are expressed asmeans + standard errors of the mean of 12 separate experiments. *,P < 0.05 versus the control.

ment components during in vitro culture, and this synthesiscan be stimulated or suppressed by various agents (19). Wewere able to detect C3 in monocyte supernatants after aculture period of 24 h, and the C3 concentration increasedduring a 5-day cell culture (Fig. 1). This C3 production couldbe modulated by adding C. albicans (1.5 x 106 blastoconidiaper well) to the monocyte cultures. All experiments in thisstudy were performed with heat-killed C. albicans, as de-scribed in Materials and Methods. The yeast was added toongoing cell cultures, and the supernatants were harvestedafter 24 h. In monocytes cultured for 1 day and longer, theaddition of C. albicans resulted in an increase in C3 produc-tion to a level which exceeded the C3 production by un-treated cells (Fig. 1). To examine whether the increased C3content represented newly synthesized protein, monocytescultured for 4 days were treated with C. albicans (1.5 x 106blastoconidia per well) with and without cycloheximide for24 h. The C3 concentrations obtained were 4.9 ng/ml forcontrol cells, 5.2 ng/ml for cells with C. albicans andcycloheximide, and 9.6 ng/ml for cells treated with C.albicans alone (one representative experiment, n = 6). Thisdemonstrated a dependence on protein biosynthesis for theincreased C3 concentration in the monocyte supernatants.

Different amounts of C. albicans were employed to deter-mine a dose-response relationship for yeast-induced C3synthesis. When 0.03 x 106, 0.3 x 10 , and 3.0 x 106blastoconidia per well were added to monocytes cultured for4 days, the C3 concentration (mean + standard error of themean) after 24 h was 9.3 + 1.7 ng/ml, 11.2 + 1.9 ng/ml (P <0.05 compared with untreated cells), and 11.6 + 1.4 ng/ml (P< 0.05 compared with untreated cells), respectively. Thecorresponding C3 concentration for untreated cells was 8.6± 1.9 ng/ml (n = 5). Thus, the minimum dose of C. albicansnecessary to obtain a significantly increased C3 productionwas 0.3 x 106 blastoconidia per well, while 10 times thisamount did not further augment the C3 concentration in themonocyte supernatants.

Effects of prolonged treatment with C. albicans on monocyteC3 production. Monocyte morphology and function change

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CANDIDA-INDUCED C3 PRODUCTION IS SUPPRESSED BY GM-CSF 1781

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1100 albicans on monocyte C3 production was obtained when theyeast was administered to monocytes cultured for 2 or 3 days

1000 - O (Fig. 2). Monocytes from 10 additional donors were alsoo treated with C. albicans on day 0, but these were not

included in Fig. 2 since we did not have data for all the other900 treatment periods. In three donors, there was no stimulation

o of C3 production (range, 83 to 113% of the control value),o-

while in the remaining seven donors, the C3 concentration800_ was considerably enhanced (range, 172 to 700% of the

control value). From the combined results obtained when

700_ 0 freshly isolated monocytes from 23 different blood donorswere treated with C. albicans, two qualitatively different

0 0 response patterns of C3 production seemed to emerge. In600_ 0 about 50% of the cases, there was no stimulation or even an

inhibition of C3 production, while in the remaining 12 donorso the C3 production was markedly increased. Daily addition of

500 C. albicans from day 0 through 4 gave results similar to thoseobtained for a single addition on day 0 for each donor (data

400 not shown).o GM-CSF concentrations in monocyte supernatants. Weo 0 0 have shown in a previous study that GM-CSF suppresses300_0 basal and LPS-stimulated C3 production in monocytes (21).

°0-6 Consequently, to elucidate the mechanism for the variable000P C3 responses to C. albicans in the cultured monocytes, we

200 - o 0 0 examined the monocyte supernatants for the presence of

0 0 GM-CSF. Results from four blood donors representing dif-° ° ° 855 ferent response patterns to C. albicans on monocyte C3

100 8 o production are presented in Fig. 3. GM-CSF was not de-0 tected in supernatants harvested on day 5 from monocytes

0 _ without C. albicans, while there were usually detectableamounts after treatment with this yeast. The GM-CSF

nt(h): 120 96 72 48 24 concentration was higher when C. albicans was present fromthe initiation of cell culture than when the yeast was added

n of on days 1 and 4 (Fig. 3). Furthermore, there was an inverseans: day 0 day 1 day 2 day 3 day 4 relationship between GM-CSF and C3 concentrations in the

r. 2. Effect of prolonged stimulation of C. albicans on mono- supernatants (Fig. 3). In one donor (Fig. 3A), in whom C.'3 production. C. albicans was added at the times indicated, albicans inhibited the C3 production when added on days 0ng in treatment periods varying from 24 to 120 h, and all and 1 and caused a moderate C3 response when added onLatants were harvested on day 5 for determination of accumu- day 4, the corresponding levels of GM-CSF in the superna-C3 production. Thirteen different blood donors were exam- tants were high. An opposite pattern was found for the donorand each circle represents the mean C3 concentration (per- shown in Fig. 3D, in whom the concentrations of GM-CSF,e of control value) from one monocyte donor. The horizontal were very low or not detectable and the corresponding C3idicate the mean C3 concentrations in supernatants from all 13 concentrations were increased compared with those in un-ments. treated cells, irrespective of when C. albicans was added to

the cultures. In Fig. 3B and C, the GM-CSF concentrationsare relatively similar, at levels between the values in Fig. 3A

time in culture (23). In the previous experiments, the and D. However, the corresponding C3 concentrations differcytes were treated with C. albicans at different stages quite a lot, suggesting different sensitivities to GM-CSF inI culture for a constant period of time. We also wanted these two blood donors. Nevertheless, in both donors the C3amine the effect of an extended C. albicans treatment, production increased and the GM-CSF concentration fellherefore we added the yeast (1.5 x 10' blastoconidia when C. albicans was added on day 1 or later during cellell) at different stages of cell culture from day 0 to 4. All culture (Fig. 3B and C).natants were harvested on day 5 so that the treatment Effects of adding anti-GM-CSF or GM-CSF together withd varied from 24 to 120 h. A significant increase in C3 C. albicans. Having found an inverse relationship betweenction (P < 0.05) was obtained when C. albicans was the concentrations of GM-CSF and C3 in the monocyteiistered to the cultures on day 1 or later (Fig. 2). There supernatants, we wanted to examine whether the addition ofhowever, considerable variation among the 13 different an anti-GM-CSF antibody would further increase the C3Idonors tested, as demonstrated in Fig. 2. This was production by the yeast-treated monocytes. When such anularly striking for freshly isolated monocytes which antibody was added together with C. albicans on day 0, theteen treated with C. albicans for 120 h. No stimulation C3 concentration in supernatants harvested on day 5 in-)btained in 8 of 13 donors, whereas in the remaining 5 creased, with a mean of 130% (range, 17 to 228%) comparedrs the C3 concentration was markedly increased (Fig. with treatment with C. albicans alone (n = 10). The weakest(hen the yeast was added on day 1, 2, 3, or 4, the effect of the antibody, 17 and 32% increases in the C3)rtion of blood donors who did not respond with in- concentration, was obtained with two donors in whom C3ed C3 production gradually decreased with the duration production after treatment with C. albicans from day 0 was1 culture (Fig. 2). The maximal stimulatory effect of C. markedly augmented compared with that in untreated cells

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1782 H0GASEN AND ABRAHAMSEN

C)

C.albicans: - day 0 day 1 day 4

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C.albicans: - day 0 day 1 day 4 C.albicans: - day 0 day 1 day 4FIG. 3. C3 concentrations and corresponding GM-CSF concentrations in monocyte supernatants harvested on day 5. The results

presented are from four single experiments with four different donors, in which C. albicans had been added to the cell cultures on days 0,1, and 4.

(634 and 700% of control values). The relative ineffective-ness of the antibody in these two donors probably reflectsthe small amounts of GM-CSF induced by the yeast in thesedonors. An irrelevant sheep serum had no effect on C3production (data not shown).We also wanted to examine whether exogenous GM-CSF

could suppress the C. albicans-induced C3 production.Therefore, recombinant human GM-CSF (100 ng/ml) wasadded to some of the monocyte cultures on day 0. Insupernatants of monocytes treated with GM-CSF alone,there was a significant decrease in C3 concentration com-pared with that in control cells (Table 1). This has beenshown in a previous study (21). When the yeast (1.5 x 106blastoconidia per well) was added together with GM-CSF at

TABLE 1. Effect on C3 biosynthesis when C. albicans is addedto cells treated with GM-CSFa

C3 concn (ng/ml)GM-CSF(100 ng/ml) No C. albicans C albicans added on day:

added 0 4

- 6.7 ± 1.5 22.9 ± 8.6 11.8 ± 2.7b+ 3.6 ± 1.3c 10.6 ± 4.0 3.9 ± 1.4c

a GM-CSF was added to cell cultures on day 0, and C. albicans wasadministered to GM-CSF-treated cells and untreated cells on days 0 and 4.The supernatants were harvested on day 5 and analyzed for C3 content. Theresults are expressed as means ± standard errors of the mean of eight separateexperiments.

Significantly increased (P < 0.05) compared with cells without C. albi-cans.

c Significantly decreased (P < 0.01) compared with cells without GM-CSF.

the initiation of cell culture, a reduction of C3 productionwas found in all donors when compared with cells treatedwith the yeast alone (Table 1). Furthermore, when C.albicans was added on day 4 to GM-CSF-treated monocytes,no increase in C3 production was found, and the obtained C3concentration was significantly decreased compared withthat in cells with no GM-CSF (Table 1). Thus, this treatmenthad made the monocytes refractory to the stimulatory effectof C. albicans on C3 production.

Binding of C3 to C. albicans in the presence and absence ofGM-CSF. To clarify the reason for the decreased C3 con-centration in the presence of GM-CSF, we examinedwhether this cytokine might cause an enhanced deposition ofC3 on the C. albicans cell surface. This experiment wasperformed by adding C3 from an NHS pool to the serum-freecell culture medium in a concentration range of 4.1 to 111.1

ng/ml. Heat-killed C. albicans (3.0 x 106 blastoconidia perwell) was then added in the presence and absence of GM-CSF (100 ng/ml), and medium with these additives wasincubated at 37°C with 5% CO2 for 18 h. The medium wasthen centrifuged to remove yeast particles and analyzed forfree C3 by EIA as described in Materials and Methods.There was usually a 10 to 20% decrease in the free C3concentration when C. albicans was added to the medium,indicating that some of the C3 was deposited on the yeastand removed from the medium. GM-CSF did not influencethis deposition. For example, in the medium containing C3 at12.4 ng/ml, the addition of C. albicans with and withoutGM-CSF resulted in a C3 concentration of 10.7 + 0.2 ng/ml(mean + standard deviation, n = 2) and 9.7 0.2 ng/ml,respectively. This result suggests that the decreased C3concentration found in cell supernatants with high endoge-

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INFEcr. IMMUN.

CANDIDA-INDUCED C3 PRODUCTION IS SUPPRESSED BY GM-CSF 1783

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n= 10 10 8 4 6 10FIG. 4. Effect of prolonged stimulation of C. albicans on mono-

cyte production of factor B. C. albicans was added at the timesindicated, resulting in treatment periods varying from 24 to 120 h.All supernatants were harvested on day 5 for determination ofaccumulated factor B production by EIA as described in Materialsand Methods. Data are presented as means + standard errors of themean. *, P < 0.05 versus the control, n, number of experiments.

nous or exogenous GM-CSF levels is not caused by in-creased binding of C3 to the yeast cell surface.

Effects of other clinical isolates of C. albicans on C3 pro-duction. To examine whether the ability of C. albicans toincrease the C3 production in cultured monocytes wasrestricted to a particular isolate, we studied eight otherclinical isolates of C. albicans by adding 3.0 x 106 blasto-conidia per well on day 2 of culture to monocytes obtainedfrom one blood donor. In supernatants harvested 72 h later(day 5), the C3 concentrations ranged from 18.6 to 23.8 nglmlafter treatment with the different yeast isolates, while thecorresponding C3 concentration for untreated cells was 8.7ng/ml. Thus, all of the clinical isolates exerted a similarstimulatory effect on monocyte C3 biosynthesis. On theother hand, addition of latex beads in the same way resultedin a C3 concentration of 5.6 ng/ml. This lack of a stimulatoryeffect is in agreement with previous reports (16, 29).

Effect of C. albicans on monocyte factor B production.Factor B is produced by monocytes in culture but usuallycannot be detected as early as C3 (25). C. albicans wasadded at different stages of the monocyte culture, and thesupernatants were harvested 24 h later and analyzed for thepresence of factor B. We found a moderate but significantincrease in the factor B content in supernatants from C.albicans-treated monocytes (1.5 x 10 blastoconidia perwell) cultured for 2 days or longer. The factor B concentra-tions (mean + standard error of the mean) for control cellswere 3.4 + 0.7 U/ml (day 3, n = 7) and 4.2 + 1.1 U/ml (day5, n = 10), and those for cells treated with C. albicans were4.7 0.4 U/ml (day 3, P < 0.05 versus the control) and 6.1

1.2 U/ml (day 5, P < 0.05 versus the control).C. albicans was then added at different times during the

monocyte culture, and all of the supernatants were har-vested on day 5. C. albicans stimulated factor B productionat all stages of cell culture (Fig. 4). An inhibition of the factorB production was not found in any of the donors, in contrastto the results for the C3 production. The increase in factor B

TABLE 2. Effect on factor B biosynthesis when C. albicans isadded to cells treated with GM-CSF

Factor B concn (U/ml)GM-CSF Cabcn de ndy(100 ng/ml) No C. albicans C. albicans added on day:

added 0 4

3.5 ± 1.4 13.8 ± 3.4b 5.2 ± 1.7b+ 2.5 ± 0.7 8.0 ± 1.5c 5.8 ± 2.3c

a GM-CSF was added to cell cultures on day 0, and C. albicans was addedto GM-CSF-treated cells and untreated cells as indicated. The supematantswere harvested on day 5 and analyzed for factor B content. The results areexpressed as means + standard errors of the mean. (n = 8).

Significantly increased (P < 0.05) compared with cells without C. albi-cans.

c Significantly increased (P < 0.05) compared with cells treated withGM-CSF without C. albicans.

content was more pronounced after 48 h of treatment with C.albicans than after 24 h, but no additional enhancement wasobtained with treatment longer than 48 h (Fig. 4).

Effect of GM-CSF added with C. albicans on monocytefactor B production. An increase in factor B release wasinduced by C. albicans treatment of monocytes from allblood donors. This indicated that GM-CSF did not influencethe production of factor B in the same way as we haddemonstrated for the C3 biosynthesis. To further evaluatethis, recombinant human GM-CSF was added on day 0 andC. albicans was added on either day 0 or 4 to the monocytecultures. GM-CSF did not suppress basal factor B produc-tion (Table 2), as previously described (21). There was asignificant increase in the factor B concentration when C.albicans was added on day 0 to cells with and withoutGM-CSF (Table 2). This increase appears to be lower forGM-CSF-treated cells than for controls, but this was notconsistent and the difference is not statistically significant.Furthermore, pretreatment with GM-CSF for 4 days did notinfluence the stimulatory effect of C. albicans on factor Bproduction (Table 2).

DISCUSSION

This study showed that the production of both C3 andfactor B by cultured monocytes is increased when the cellsare treated with C. albicans. This cellular response to apathogenic fungus may be important, since opsonization bycomplement components secreted by monocytes/macro-phages has been reported (12). This may cause an augmentedbinding and ingestion of the fungi by phagocytes. Comple-ment activation initiated by microorganisms results in cleav-age of C3. The resulting activation products, such as C3a andC3b/iC3b, have important proinflammatory effects (19). C3ais an anaphylatoxin and chemotaxin and stimulates mono-cyte release of cytokines (13, 17), while C3b/iC3b are opson-ins which bind to fungi and other microorganisms (33) andfacilitate binding to complement receptors on the phago-cytes.We measured the free C3 in the monocyte supematants.

This measurement is probably an underestimate of the totalproduction, because some of the C3 released into the super-natant may bind to the surface of C. albicans both nonco-valently to specific complement receptors and covalently asan opsonin (15, 24). This was demonstrated by the removalof 10 to 20% of the free C3 when C. albicans was added toC3-containing medium. GM-CSF did not influence the dep-osition of C3 on the yeast cell surface. The binding of C3 to

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C. albicans may explain the observed plateau in the C3concentration when the monocytes were treated with in-creasing amounts of the yeast.

Earlier studies of complement biosynthesis after stimula-tion with phagocytic agents have given variable results.Yeasts, latex beads, and immunoglobulin-opsonized sheeperythrocytes did not modify Clq or Cls secretion in mono-cytes cultured for 10 to 14 days (2), while zymosan, latexbeads, and sheep erythrocytes suppressed C2 production infreshly isolated monocytes (30). Zymosan caused an earlysuppression of factor B production in mouse macrophages,which was followed by an enhancement after a treatmentperiod of 48 h (29). In our study, we found a significantincrease in factor B production as early as 24 h after additionof C. albicans to monocytes cultured for 2 days or more.This stimulation of factor B production was even strongerwhen the yeast had been added for a period of 48 h or longer,in agreement with the previous report (29).

Stimulation of C3 biosynthesis has been reported in mousemacrophage-like cell lines after treatment with whole, heat-killed streptococci, while latex beads were ineffective (16).In the current study we found that another pathogen, C.albicans, increased C3 synthesis by human monocytes. Thelevel of contaminating LPS in the yeast preparation (<3pg/ml) is not sufficient to induce C3 synthesis, according toprevious studies in our laboratory (21). The stimulatoryeffect of C. albicans on C3 production was more pronouncedthe longer the monocytes had been in culture prior to theaddition of the yeast. A significant increase in the C3concentration was obtained after a stimulatory period of 24h. However, when freshly isolated monocytes were treatedwith C. albicans, the cells failed to respond with increasedC3 production in almost half of the experiments. Thisindicated that in addition to affecting complement biosynthe-sis, the yeast triggered the release of other mediators whichcould inhibit the synthesis of C3.

It is well known that phagocytosis affects not only com-plement factor production but also other secretory functionsof the monocytes/macrophages. Zymosan and opsonizedbacteria trigger the release of prostaglandins (14). C. albi-cans has been shown to induce the release of tumor necrosisfactor from monocytes (10), and the release of tumor necro-sis factor, gamma interferon, and GM-CSF from large gran-ular lymphocytes with natural killer function (3, 9). In ourstudy GM-CSF was particularly interesting, since we haveshown in a previous work that GM-CSF suppresses basaland LPS-induced C3 production in human monocytes (21).This cytokine is released from monocytes after stimulationwith various parasites, bacteria, and LPS (1, 4, 26), butinduction by C. albicans has not been reported. We demon-strated the presence of GM-CSF in cell supernatants whenC. albicans was added to freshly isolated cells, with decreas-ing levels of this cytokine when the yeast was added duringcell culture. This is in agreement with a previous reportdocumenting that freshly isolated monocytes produce GM-CSF in response to LPS, whereas cells cultured for 1 dayhave lost this ability (26).

In this study we found several indications that GM-CSFmay inhibit monocyte C3 production induced by C. albicans.First, the GM-CSF concentration was high in the monocytesupernatants, where the C3 content was low. Second, anincreased stimulatory effect on the C3 production was foundwhen an anti-GM-CSF antibody was added with the yeast tofreshly isolated monocytes. Third, exogenous GM-CSF,when administered together with C. albicans, suppressedthe stimulatory effect of the yeast on C3 production in

monocytes. Interestingly, monocytes became insensitive tothe stimulatory effect of the yeast on C3 production afterpretreatment with GM-CSF. Furthermore, GM-CSF causedno increase in C3 binding to the surface of C. albicans. Suchan inhibitory effect of GM-CSF on yeast-induced factor Bproduction was not found. Thus, the regulatory mechanismsfor the biosynthesis of these two complement factors seemto differ.GM-CSF has been shown to enhance monocyte binding

and ingestion of yeasts, probably because of increasedexpression of various receptors important for phagocytosis(11, 35). In such experiments, the supernatant fluid ofcultured cells has been removed before the phagocytosisassay. This means that monocyte-produced complementcomponents are not present, and therefore the suppressingeffect of GM-CSF on monocyte C3 production will not berecognized. In addition, the killing of C. albicans by mono-cytes is increased after treatment with GM-CSF, possiblythrough an augmentation of the respiratory burst (34, 37, 40).These experiments have been performed with serum-supple-mented media containing large amounts of complementcomponents. Under these conditions, the contribution ofcomplement factors produced by monocytes is probablynegligible.At an infectious site, however, with rapid growth of C.

albicans and increased activation of complement, the ex-travascular supply of plasma-derived complement factorsmay be limited. Monocyte production of complement com-ponents may then be particularly important in maintainingadequate local complement concentrations. GM-CSF-in-duced suppression of yeast-stimulated complement produc-tion in monocytes as observed in this study may conse-quently reduce the local opsonization of C. albicans andresult in reduced opsonophagocytosis of the yeast. Whetherthe increased expression of monocyte receptors importantfor phagocytosis observed after treatment with GM-CSF (11,35) may compensate for the decreased production of C3remains to be examined.The suppressive effect of GM-CSF on C3 production may

also lead to a reduced local production of complementactivation products with potent chemotactic and anaphylac-tic properties, which then may reduce the recruitment ofleukocytes to the site of inflammation. GM-CSF may in thisway suppress an inflammatory response caused by thefungus. The consequences of this mechanism for the hostresistance against fungi clearly warrant further investiga-tions.

ACKNOWLEDGMENTSThis work was supported by the Norwegian Cancer Society.We thank I. L. Topaas and M. Wold for technical assistance and

Elisabeth Falstr0m for performing the Limulus amebocyte lysatetests.

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