YALE JOURNAL OF BIOLOGY AND MEDICINE 75 (2002), pp. 241-245.Copyright © 2003. All nghts reserved.

ORIGINAL CONTRIBUTION

The Effect of Fluconazole Treatment on TumorNecrosis Factor-oc Production in MurineCandidiasis

Ayse Kalkancia and Semra KustimurDepartment of Microbiology and Clinical Microbiology,Gazi University Faculty of Medicine, Ankara, Turkey

That systemic candidiasis increases IN'-cx has been proved in various studies. In our study, weinvestigated iffluconazole too, which is used in treatment, has an additional effect on the increaseofTNF-a. On the sixth day ofthe experimental infection, it was measured that TNF-a levels ofcan-clidiasis was 699 ± 60 pg/ml whereas it was 683 ± 35 pg/ml in the mice that wvere treated with flu-conazole. TNF-a levels were measured by enzyme-linked immunosorbant assay. Due to thefact thatno statistically significant difference wasfound between them p > .05) it was thought thatflucona-zole did not have an additional effect on the increase of TNF-ax in candidiasis.

INTRODUCTION

Tumor necrosis factor-ax (TNF_ct)bincreases as an essential cytokine duringsepsis that is caused by Candida infections.Candida species activate both the humoraland the cellular immune systems. Beingthe main protector of Candida infections,cellular immunity consists ofmacrophages,neutrophils, mononuclear phagocyte systemand natural killer cells, all orchestrated byT cells [1]. After activated macrophagesare stimulated by lipopolysaccharide, theybecome the largest source ofTNF-az. Otherthan activated macrophages, mast cells, poly-morphonuclear leukocytes, keratinocytes,astrocytes, microglia cells, smooth musclecells, and tumor cells also release TNF-oc[2]. It has been demonstrated that similar

to the increase of TNF-a levels duringCandida infections, some antifungal agentsalso increase TNF-ax level as a side effect.Especially during the treatment of ampho-tericin B (Amp B), systemic side effects arefrequently observed. The increase ofTNF-x level, which can be both protective andhanrnful to the organism, can be seen amongthe side effects. Fluconazole is the mostfrequently used azole-derivated agent, and itis an antifungal with minimum side-effects[3]. Whether fluconazole activates TNF-a ornot has been investigated in a small numberof in vitro [4, 5] and in vivo studies [6, 7].

The purpose of our study is to measureTNF-cx level on the experimental model ofmurine candidiasis and to investigate if flu-conazole has an effect on TNF-a level or not.

aTo whom all correspondence should be addressed: Ayse Kalkanci M.D., Ph.D., Emek Mah.75. Sok 120/4 06510, Ankara, Turkey; Tel.: 90-312-214-10-00/6914; Fax: 90-312-213-98-02;E-mail: aysekalkanci@email.com.bAbbreviations:Amp B, amphotericin B; OD, optic density; PBS, phosphate buffered saline;TNF-a, tumor necrosis factor-a

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242 Kalkanci and Kustimur: The effect of fluconazole on TNF-a production

MATERIALS AND METHODS

Experimental animalsFemale, six- to eight-week-old, 20 to 25

g BALB/c mice, maintained fromHifzisihha Serum Farm, Ankara, Turkeywhere the mice were bred under, standardconditions.

OrganismsC. albicans ATCC 26555 (Kukens,

Istanbul, Turkey) reference strain were used.

The preparation ofyeast suspensionsFour or five colonies of Candida albi-

cans, which had been formed in sabourauddextrose agar (SDA, Oxoid) after 48-hrincubation, were suspended inS ml of sterile,pyrogen-free, 0.9 percent NaCl. Organismswere harvested by low-speed centrifugation(1500 xg), washed three times in sterilesaline, counted with a hemocytometer andsuspended to 3 x 108 cell/ml concentra-tion. Mice were challenged by tail veininjection with 0.2 ml of suspension 99 per-cent of which was blastoconidia [8, 9].Experimental groups are shown in Table 1.

Measurement of Serum TNF-aBlood was collected from the heart.

Serum TNF-a levels were quantified by usingenzyme-linked immunosorbant assay kitcalled Biosource CytoscreenT MouseTNF-cx New Format (United States) [10].

Demonstration of Candida infectionBoth of the kidneys were isolated and

incubated to show Candida infection. Bothofthe kidneys homogenized in 10 ml phos-phate-buffered saline were inoculated inequal amounts into SDA plates which con-tain 100 IU penicillin and 10 ,ug strepto-mycine. The same homogenized suspen-sions were also inoculated into blood agarplates in order to eliminate the presence ofbacterial infections. All of the yeast colonieswere counted and documented after a 48-hr incubation period [8].

Statistical analysisAll statistical analysis were calculated

by the help ofthe computer program, SPSS9:0 1. The graph (Figure 1) which shows theoptic density (ODs) values that correspondto the standards ofTNF-a, was drawn withregression analysis method (y = -35.189 +468.621 XOD and R2 = 0.993)

Table 1. Experimental groups.

Main Groups Subgroups Injections Number of Mice

Control Healthy - 16Saline 0.2 mL 0.9% NaCI* 6

Fluconazole 2 mg/kg fluconazole* 6Fluconazole 4 mg/kg fluconazole* 6Fluconazole 10 mg/kg fluconazole* 6

Infection C. albicans 3 x108 yeast/ml** 6

Treatment C. albicans + fluconazole 3 x 108 yeast/ml + 610 mg/kg fluconazole***

Total 7 52

*Injections were applied every 24 hr for two days.TNF-a levels were measured on the 48th hr.**TNF-a levels were measured six days after the only yeast suspension was injected.***On the fourth day of the infection the treatment of fluconazole was started, was appliedevery 24 hr for two days. On the sixth day of the infection TNF-a levels were measured.

Kalkanci and Kustimur: The effect of fluconazole on TNF-a production 243

Table 2. TNF-uc levels of the experimental groups.

ODs TNFixt levels Mean values of(pg/mI) TNF-cx (pg/mi)

2mg/kg 154 36100 11 20±14100 11

Control 4mg/kg 108 15Groups 110 16 15±1.1

105 14

10Omg/kg 128 24148 34 29±4.7135 28

Treatment C. albicans 1453 645Group + Fluconazole 1602 715 683 ±35

(10Omg/kg) 1544 688

Figure 1. TNF-a levels of all groups.I gzN M M:8.. Mg Ft.11-; N E, .1700

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O u;ontrol 2mgikg Mg/kg 1 uMgikg u. albicans, u. aibicansFluconazole

244 Kalkanci and Kustimur: The effect of fluconazole on TNF-a production

All of the TNF-a values were calcu-lated with the help of this equation.Friedman test, Mann-Whitney U test, Kruskal-Wallis Variance Analysis were used with thehelp of the computer programme, SPSS9:0 1. That the p value was smaller than .01 or.05 in the statistical evaluations was con-sidered as a significant difference.

RESULTS

The average of TNF-cz levels of allgroups is shown in the Figure 1. The averageofTNF-a level was measured to be 20 pg/mlin the group given 2 mg/kg fluconazole, 15pg/ml in the group given 4 mg/kg fluconazole,and 29 pg/ml in the group given 10 mg/kgfluconazole. A statistically significant dif-ference was not found among the three groupsthat were given different doses offluconazole(p > .05).

The average of TNF-x level was <3pg/ml in the healthy control group and alsoin the saline control group which was usedas a solvent of fluconazole. There was nota statistically significant difference amongthe healthy control, saline control, and flu-conazole control groups (p > .05).

Candida colonies were identifed fromthe cultures of the kidneys. Mean of thecolonies was 28 ± 1.5 of the cultures. Theaverage of TNF-ac level was measured tobe 683 pg/ml in the treatment group. It was699 pg/mil in the C. albicans infected groupthat was left without treatment. There was nostatistically significant difference betweenthe two groups (p > .05).

DISCUSSIONToday the treatment of Candida infec-

tion is limited to the use ofantifungal agents.After infection occurs antifungal agents canbe used to directly destroy the causativeagent ofthe infection or it can be used as anon-specific profilactic agent against colo-nization before the infection. Although AmpB is considered to be the most favorableantifungal to oppose the life-threatening

infections, recently non-toxic azole anti-fungal agents have begun to be prefered toAmp B due to the latter's side effects.Fluconazole is a preperation that can beused systemically and orally. The efficiencyof fluconazole has decreased because of itswide use since the beginning of 1990s.Despite the resistance problem, flucona-zole still continues to be used all over theworld [3].

In our study we investigated ifvaryingdoses of systemically administrated flu-conazole increased TNF-a or not. As wassaid in the result section, there was not astatistically significant difference among thethree groups to which different doses offluconazole were applied. That there wasnot a statistically significant differencebetween the healthy control group and thethree fluconazole control groups made usbelieve that fluconazole by itself did nothave an augmentative effect on TNF-xlevel. According to our results, it was thoughtthat during the infection, fluconazole didnot have an additional effect on TNF-alevels, independent ofthe effect of C. albi-cans. In an in vitro study on the subject itwas found that there was not a statisticallysignificant difference between TNF-ax levelsof the peritoneal macrophages cultures ofmice incubated with C. albicans plus flu-conazole and the TNF-a levels of the cul-tures incubated solely with C. albicans [4].In the study ofYamaguchi et al., the immuno-modulating activity of antifungal drugswas reviewed. Fluconazole, which has noimmunologic effect, did not induce in vitroTNF-oc production by macrophages,whereas Amp B slightly but substantiallydoes so [5]. Pitzurra et al. have shown thattreatment with fluconazole induced an effecton macrophages similar to that of prome-ters of TNF-oc synthesis and secretion [6].In the study of Baltch et al., it was evaluatedthat the effects ofcytokines, used singly andin combination, on the microbicidal activityof human monocyte-derived macrophagesagainst intracellular C albicans in the pres-ence and absence of fluconazole. Their

Kalkanci and Kustimur: The effect of fluconazole on TNF-o production 245

results demonstrate that in the presence oflow concentrations of fluconazole, selectedcytokines and their combinations signifi-cantly increase the microbicidal activity ofhuman-monocyte derived macrophagesagainst intracelluar C. albicans [7]. In ananother study it was demonstrated that flu-conazole did not increase TNF-ax level incerebrospinal fluid of the patients withcoccidioidal meningitis [11].

As a result, fluconazole, which wasdemonstrated to be ineffective on TNF-alevel, was emphasized once more as a safeantifungal agent.

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