Cytopathic Effects of a Pathogenic Naegleria fowleri and an Anti-Nfa1 Antibody on Rat Microglial Cells

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ABSTRACT

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Naegleria fowleri, a free-living amoeba, causes fatal primary amoebic

meningoencephalitis (PAME) in experimental animals and humans. On the

pathogenic mechanisms of N. fowleri concerned with host tissue invasion, the

adherence of amoeba to host cells is most important. The nfa1 gene (360 bp)

was previously cloned from a cDNA library of pathogenic N. fowleri by

immunoscreening, and produced a 13.1 kDa recombinant protein (rNfa1) that showed the pseudopodia specific localization by immunocytochemistry. On the basis of an idea that the pseudopodia-specific Nfa1 protein seems to be

involved in the pathogenicity of N. fowleri, the cytopathic activity of N.

fowleri trophozoites co-cultured with rat microglial cells was observed, and

effects of an antiNfa1 antibody as treating on a co-culture system was

elucidated. By a light, scanning and transmission electron microscope, N.

fowleri trophozoites contacted with microglial cells produced vigorous

pseudopodia and foodcups structure. Microglial cells were destroyed by N.

fowleri trophozoites as showing the necrotic and apoptotic cell death in a

timedependent manner. As the results of 51_{Cr release assay,} _{N. fowleri}

showed 17.8, 24.9, 54.6 and 98.0% cytotoxicity against microglial cells at 3, 6,

12 and 24 hr postincubation, respectively. On contrary, N. fowleri treated

with an antiNfa1 antibody (1:100 dilution) showed 15.5, 20.3, 50.7 and 66.9%

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fowleri trophozoites secreted cytokines, TNF-α, IL-1β and IL-6, as the protective immune response.

Key words: Naegleria fowleri, PAME, nfa1 gene, microglial cell, anti-Nfa1

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LIST OF FIGURES

Fig. 1. Life cycle of Naegleria fowleri 2

Fig. 2. Light microscopic findings of primary cultured rat microglial cells 14

Fig. 3. Electron microscopic findings of microglial cells 14

Fig. 4. Electron microscopic findings of Naegleria fowleri

trophozoites 15

Fig. 5. Light microscopic findings of microglial cells co-cultured with

Naegleria fowleri, or with an anti-Nfa1 antibody for 6 hr 17

Fig. 6. SEM findings of microglial cell co-cultured with only

Naegleria fowleri trophozoite, or with an anti-Nfa1 antibody 18

Fig. 7. TEM findings of microglial cell co-cultured

with Naegleria fowleri trophozoite 19

Fig. 8. Cytotoxicity of Naegleria fowleri trophozoites co-cultured

with microglial cells, or with an anti Nfa1 antibody 21

Fig. 9. Amounts of TNF-α secreted from microglial cell

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Fig. 10. Amounts of IL-1β secreted from microglial cells

with Naegleria fowleri trophozoites 24

Fig. 11. Amounts of IL-6 secreted from microglial cells

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LIST OF TABLES

Table 1. The results of the in vitro cytotoxicity of Naegleria fowleri

trophozoites on microglial cells 21

Table 2. Amounts of TNF-α secreted from microglial cells co-cultured

Table 3. Amounts of IL-1β secreted from microglial cells co-cultured

Table 4. Amounts of IL-6 secreted from microglial cells co-cultured

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N. fowleri, a freeliving amoeba found in widespread areas in moist soil,

water and sediment, exists as a virulent pathogen causing fatal primary amoeba meningoencephalitis (PAME) in experimental animal and humans (Visvesvara and Chang, 1974; Brown, 1979; Zubiaur, 1983). The infection results from introduction of water containing amoebae into the nasal cavity and subsequent passage of the organisms to the central nervous system (CNS) via the olfactory apparatus (Carter, 1968, 1970, 1972). Most human

infections with N. fowleri have been associated with swimming in warm

water, but other reports of infection include tap water (Warhurst and Mann, 1980) and hot baths (De Jonchkeere, 1982).

N. fowleri is thermophilic, thriving best in temperatures from 35℃ to

46℃ tolerating temperature of 40 45℃, while N. gruberi is nonpathognic

having an optimal growth temperature of 22 35℃. In its life cycle, N.

fowleri has three stages; trophozoite, cyst and a temporary flagellate stage

(Fig. 1) (John, 1982; Martinez, 1985; Marciano-Cabral, 1988). Alternatively, trophozoites can occur in two forms, amoeboid and flagellate. The amoeboid form is elongated with a broad anterior end tapered posterior end. The trophozoite, measuring 10 20 ㎛, is the infective and reproductive form and characterized by a nucleus with a large karyosome surrounded by a halo and a dense, prominent central nucleus.

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It moves with broadly rounded, granulefree processes that erupt from the surface; granular cytoplasm flows into them. Frequently, protrusions are

formed in quick succession at different points on the surface of the cell so

that its shape is constantly changing. This pseudopodial granular cytoplasm may have ingested erythrocytes. A contractile vacuole, which ruptures by emptying itself and reforms within a few seconds, in visible in the cytoplasm and serves as a valuable aid in diagnosing and recognizing the presence of amoebic trophozoites and also in avoiding the misidentification.

N. fowleri trophozoites are found in cerebrospinal fluid (CSF) and tissue,

and flagella forms are found in CSF. Cysts have a single nucleus almost identical to that seen in the trophozoite. They are generally round, measuring from 7 to 10 ㎛, and there is a thick double wall. The flagellate form is pearshaped, with two flagella at the broad end. These flagellate forms do no divide, but when the flagella are lost, the amoeboid forms resume reduction (Ma et al., 1990).

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PAME occurs most commonly in healthy, young adults, nonimmunocom-pomised children and associated with swimming or bathing in contaminated

warm waters. The incubation period of this disease produced by N. fowleri

may vary from 2 to 3 days to as long as 7 to 15 days. It has an acute onset with severe headache, anorexia, nausea, vomiting, fever (from 38℃ 40℃), ataxia, sign of meningeal irritation, and encephalitis (Ma et al., 1990). The olfactory neuroepithelium is the anatomic site of the primary lesion

in PAME due to N. fowleri. Subtentacular cells of the olfactory

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mechanism by which N. fowleri penetrates CNS. During it's invasion and

migration into the CNS, N. fowleri follows the mesaxonal spaces of the

unmyelinated olfactory nerve. The olfactory nerve terminates in the olfactory bulb, which is located in the in the subarachnoid space bated by the CSF. The olfactory bulbs are lined by the piaarachnoid membranes and are in close contact with the subarachnoid space. This space in richly vascularized and constitutes the route of dissemination of the amoeba to other areas of the CNS (Martinez, 1985).

The final diagnosis of PAME is based on the isolation and culture of freeliving amoeba from CSF or the demonstration of amoebic trophozoites in biopsied brain tissue. Antibodies may be detected in serum; however, serologic tests usually are of no value in the diagnosis of infection with freeliving amoeba (John, 1982). Amphotericin B reportedly cured one case of PAME (Ma et al., 1990).

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To date, the factors which determine susceptibility to Naegleria

infection and subsequent development of PAME have not been definitively established. There is no evidence for hereditary immunologic deficiencies.

Several investigators have suggested that N. fowleri releases cytolytic

substance which account for invasiveness and tissue damage in vivo and for cytopathogenicity in vitro (Culbertson, 1971; Brown, 1979; Chang, 1979; Zubiaur, 1983). Indeed, electron microscopy of brain from mice infected with

N. fowleri revealed areas of extensive demyelinization containing trophozoites

surrounded by a clear halo (Carter, 1968; Culbertson, 1971; Chang, 1974). It

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results in the rapid destruction of brain tissue (Chang, 1979). Acid

phosphatase activity of N. fowleri amoeba in CNS lesions has been detected

(Feldman, 1977). Acid phosphatase has been detected also, in vivo along membranes of the hostparasite interface.

In same free living amoeba, Acanthamoeba, cytopathogenic effects of

Acanthamoeba on host cells require adhesion of amoeba to host cell (Yang et

al., 1997; Shin et al., 2001), phagocytosis, amoebic proteolytic enzymes include serine proteases (Hadas and Mazur, 1991; Mitra et al., 1994, 1995), contactdependent metalloproteases (Khan et al., 2000), elastases (Ferrante and Bates, 1988), cysteine proteases (Hadas, 1993), and cytotoxic proteinases, induced by mannosemediated adhesion (Leher et al., 1998). It has been shown that adhesion is one of the crucial steps for the pathogenicity of amoeba (Yang, 1997) as nonpathogenic amoeba exhibit significant decreased binding to host cells. In addition, Khan et al. (2000) reported that pathogenicity would be a complex process which involves both contact dependent and contactindependent pathways in order to kill host cells quickly and to reduce the degree to which defense can be induced.

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Microglia, the resident macrophages of the CNS (Perri and Gordon, 1989), are generally considered to have a mesodermal origin and to be derived from the macrophage precursors originating in the bone marrow, that infiltrate the brain during fetal development to established a resident population (Cuadros and Navascues, 1989). Resting microglia form a neuronglia network with potential macrophagelike functions (Nakajima and Kohsaka, 1993), including expression of major histocompatibility complex class

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II (MHC class II) (Sedgwick et al., 1993), intercellular adhesion molecule I (ICAMI) (Zielasek et al., 1993), phagocytosis of microorganisms (Chao et al., 1992; Nakajima and Kohsaka, 1993), and free radicals (i.e. nitric oxide (NO) and reactive oxygen intermediates (RO)) (Chao et al., 1993, 1996; Hu et al., 1995). On the other hand, microglia represent a sensor for pathological events in the CNS (Kreutzberg, 1996). During the development of inflammatory processes, microglia migrate, differentiate, proliferate, and display amoeboid shape, and phagocytose degenerating cells at sites of inflammation. This type of cell, also named activated microglia, plays an important role in immune host defense (Chao et al., 1993, 1994). These cells may also contribute to neuronal injury (Hu et al., 1995; Chao et al., 1996).

Microglial cells occur in three morphological forms following cell differentiation, i.e., an amoeboid form during embryogenesis, a ramified shape in the mature normal brain, and a rod shaped morphology around inflammatory lesions in the CNS (Giulian and Baker, 1986; Suzumura et al., 1991). Moreover, they function as phagocytotic cells and produce cytokines such as interleukinl (IL1), IL6, and tumor necrosis factor alpha (TNFα) (Chao et al., 1992; Suzumura et al., 1993). Thus, it has been suggested that

microglial cells play an important role as inflammatory cells or

immunoregulatory cells in the protective immune system of the CNS (Suzumura et al., 1993).

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In the previous study, an antigenic gene, nfa1, was cloned from a cDNA

library of pathogenic N. fowleri by immunoscreenig using the immune and

the infected sera, and produced a recombinant 13.1 kDa protein. In addition, the immunolocalization study of the Nfa1 protein revealed that this protein

was abundant in pseudopodia and around food vacuoles of N. fowleri

trophozoites (Shin et al., 2001; Cho et al., 2003). It was suggested that the

nfa1 gene might be related with a mechanism of pathogenicity of N. fowleri

(Cho et al., 2003; Jeong et al., 2004). Any attempt to study the cytopathic

effects of pathogenic N. fowleri against actual target cells of PAME, that is,

rat microglial cells, has not yet been reported. On the basis of an idea that above pseudopodia-specific Nfa1 protein seems to be involved in the

pathogenicity of N. fowleri concerned with the hosttissue contact, this study

was performed. To determine whether a pathogenic N. fowleri showed the

cytopathic effects against primary cultured rat microglial cells, the

morphological changes of microglail cells co-cultured with N. fowleri

trophozoites was observed by a light, scanning and transmission electron

microscope. And then, the in vitro cytotoxicity of N. fowleri against rat

microglial cells were also observed. By adding an anti-Nfa1 antibody in the coculture system, the effects of an anti-Nfa1 antibody on the cytopathology

and in vitro cytotoxicity of N. fowleri against microglial cells was elucidated.

In addition, the cytokine release from microglial cells in co-culture system was estimated.

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The trophozoites of N. fowleri (Cater NF69 strain, ATCC NO. 30215)

were axenically cultured at 37℃ in Nelson's medium (Willaert, 1971).

B. Preparation of an anti-Nfa1 polyclonal antibody

B.B. Preparation of an anti-Nfa1 polyclonal antibodyPreparation of an anti-Nfa1 polyclonal antibody

B. Preparation of an anti-Nfa1 polyclonal antibody

For the preparation of the sera to a recombinant Nfa1 protein (rNfa1),

the expression of a nfa1 gene were performed by the method mentioned in

the previous paper (Jeong et al., 2004). An 8weekold female BALB/C mouse (Korea Institute of Science and Technology, Daejeon, Korea) was intraperitoneally injected with a mixture of the rNfa1 protein (50 ㎍) and the same volume of complete Freund's adjuvant (Sigma). The mouse was boosted biweekly with the rNfa1 protein (25 ㎍) and a same volume of incomplete Freund's adjuvant (Sigma). After third boosting, rNfa1 protein (50 ㎍) was injected intravenously without the adjuvant. Four days later, an antiNfa1 polyclonal serum was collected from the mouse blood by centrifuging at 2,500 × g for 30 min at 4℃. ELISA was performed with a purified rNfal protein (5 ㎍/ml) with rabbit antimouse (1:10,000 dilution) whole immunoglobulin conjugated with alkaline phosphate (Sigma). Western blotting for a rNfa1 protein was confirmed by the method in a previously paper (Jeong et al., 2004).

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Microglial cells were prepared by the method of Giulian and Baker (Giulian and Baker, 1991) with some modifications. Mainly, the cortex of the brain were obtained from newborn rats (SpragueDawley, purchsed from KIST in Daejeon, Korea) and homogenized by pumping with 21gauge

syringe. The mixture was centrifuged at 300 × g for 5 min and resuspended

in Dulbecco's modified eagle's medium (DMEM; Sigma, St. Louis, MO) with 10% heatinactivated fetal bovine serum and antibiotics. The suspension was put into 75㎤ tissue culture flasks, and the flasks were incubated for 14 days

at 37℃ in an incubator with a humidified atmosphere consisting of 5% CO2.

On 14 days of culture, microglial cells were harvested by vigorously shaking each culture flask, and then filtered with nylon wool to remove any remaining astrocytes and centrifuged at 300 × g for 5 min. The pellet was resuspended in DMEM with 10 % FBS, and the mixture was incubated at 37℃ for 2 hr. The attached microglial cells were harvested and subjected to subsequent experiments. The purity of microglial cells was nearly 95% by indirect immunofluorescence staining with a FITC-conjugated mouse anti-rat CD68 (ED 1 antibody) (Serotec, Bicester, United Kingdom), as shown in figure 2.

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Microglial cells (2×105_{) were co-cultured with} _{N. fowleri} _trophozoites

(2×105_{) or} _{lysate (0.1, 0.5 and 1 mg/ml) in 24well culture plate for 6 hr and}

12 hr. After co-incubation, samples were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer and postfixed with 1% osmium tetroxide1.5% potassium for 1 hr and then were examined with a light microscope.

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Microglial cells (2×105_{) were seeded onto the labtek II chamber slide}

system (Nunc A/S, Roskilde, Belgium) and trophozoites of N. fowleri (2×105₎

were added to the monolayers. Microglial cells and N. fowleri trophozoites

were incubated for 12 hr. After co-incubation, samples were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4), dehydrated with increasing concentrations of ethanol. Samples were vacuumdried and coated by ultrathin layer (300 Å) of gold/pt in an ion sputter (E-1010, Hitachi, Tokyo, Japan). An image analyzer program (Escan 4000, BumiMi Universe Co., Ltd., Ansan, korea) was used to capture the images of cells and modified surfaces. Samples were characterized by scanning electron microscope (S800, Hitachi, Tokyo, Japan).

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N. fowleri trophozoites were incubated with microglial cells in 24well

culture plate at ratio of 1:1 for 3, 6 and 12 hr, and were fixed in modified Karnovsky's fixative solution in cacodylate buffer (pH 7.4) and postfixed in 1% osmium tetroxide1.5% potassium ferrocyanide. The cells were stained en bloc in 0.5% uranyl acetate, dehydrated through a graded ethanol series, and embedded in resin (Polyscience, Warrington, Pa.). Then, the blocks were sectioned with Ultrostain 1H and 2 (Leica, Vienna, Austria). Specimens were observed and photographed with a Zeiss EM 902 A electron microscope (Leo, Oberkohen, Germany).

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A 51_{Cr (chromium) release assay was performed to determine the}

cytopathic effects of alive N. fowleri trophozoites by the methods of a

previously study (Moore et al., 1991), with some modifications. Target

microglial cells were labeled with 100 μCi [Na]2 51CrO4 per 105 cells for 60

min at 37℃. The cells were washed to remove any unbound radioisotope.

Labeled microglial cells (5×104_{) were added to each well of a 96well culture}

plate and co-incubated with effector N. fowleri trophozoites (5×104_{) in the}

absence or presence of an antiNfal polyclnal antibody in 5% CO2 for 3, 6, 12

and 24 hr. In addition, labeled microglial cells (5×104_{) co-cultured with lysates}

(0.1, 0.5 and 1 mg/ml) of N. fowleri as abovementioned procedures.

Spontaneous release from labeled microglial cells was determined by acquiring the counts per min (cpm) in the supernatant fluid in the absence of trophozoites. All assay were performed in triplicate. At the end of the experimental incubation period, plates were centrifuged at 300 × g for 3 min and the supernatant fluid from each wells was harvested. Each of these for maximal release was lysed with 5% (vol/vol) Triton X100 and harvested. Following 3, 6, 12 and 24 hr, supernatant fluid and lysed cells were counted separately in a gamma counter. The percentage of the radioisotope released from target microglial cells was determined as the index of lysis using the following formula:

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The amounts of tumor necrosis factor (TNF)-α, Interleukin (IL)-1β and IL-6 from microglial cells in a co-culture system was measured by enzyme-linked immnunosorbent assay (ELISA) kits (BioSource International, California, USA). The detection limits for these kits were about 10 pg/ml, 10 pg/ml and 50 pg/ml, respectively. The amounts of cytokine produced by microglial cells were estimated by generating a standard curve according to the manufactures instructions.

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Microglial cells were separated from the brain cortex of SD newborn rats and cultured with DMEM medium. Two weeks later, microglial cells with three morphological types were observed by a light microscope (Fig. 2A). The purified microglial cells were identified by the immunostaining of a cell surface marker, CD68. (Fig. 2B). For micro-organic observations, microglial cells were observed by a SEM and TEM (Fig. 3). Amoeboid form of microglial cell have long cytoplamic projection (Fig. 3A, B) compared with them of round form (Fig. 3C). In particular, microglial cells had short blunted spinous projection from cell surface (Fig. 3A, B). Nucleus was ovoid or contained clumped chromatin and occasional nucleoli (Fig. 3C). Rough endo-plasmic reticulum was abundant and tended to form elongated cisternae. Golgi apparatus was moderately developed (Fig. 3C).

(24)

F FF Fiiii

gggg....2222.... gggghhhhttttLLLiiiiL iiiicccccccooooppppoooosssscmiiiiccccrrrrmmm nnnnggggssssnnnnddddiiiiffffiiii ooooffff maaaarrrrmmyyyyiiiimpppprrrr eeeeddddttttuuuurrrrccccuuuullll rrrraaaatttt iiiiaaaallllooooggggllllmmiiiimmccccrrrrssss....lllllllcccceeeel In panel A. microglial cells showed amoeboid form (a), rod form (r), and ramified form (ra). (x200). In panel B, microglial cells were immunostained with a FITC-conjugated mouse antirat CD68. (x400).

F FF

Fiiiigggg 3333.... EEEElllleeeeccccttttrrrroooonnnn mmmmiiiiccccrrrroooossssccccooooppppiiiicccc ffffiiiinnnnddddiiiinnnnggggssss ooooffff mmmmiiiiccccrrrroooogggglllliiiiaaaallll cccceeeellllllllssss.... Numerous cytoplasmic projections were showed in microglial cell by a SEM (A, B). The nucleus (N) containing scattered chromatin materials was observed by a TEM (C).

(25)

B BB

B.... MMMoooorrrrpppphhhhoooollllooooggggyyyyM ooooffff NN....NN ffffoooowwwwlllleeeerrrriiii cccchhhhaaaarrrraaaacccctttteeeerrrriiiizzzzeeeedddd bbbbyyyy ppppsssseeeeuuuuddddooooppppooooddddiiiiaaaa aaaannnndddd ffffooooooooddddccccuuuuppppssss ssssttttrrrruuuuccccttttuuuurrrreeee

N. fowleri trophozoties were observed by a light, SEM and TEM (Fig.

4). Foodcups structure and pseudopodia were observed during the cultivation

(Fig. 4A, B). The trophozoite of N. fowleri, measuring 1020 ㎛, was

characterized by a nucleus with a large karyosome surrounded by a halo (Fig. 4C).

F FF

Fiiiigggg 4444.... EEElllleeeeccccttttrrrroooonnnn mE mmiiiiccccrrrroooossssccccooooppppiiiicccc ffffiiiinnnnddddiiiinnnnggggssss ooooffffm NNaaaaeeeegggglllleeeerrrriiiiaaaa ffffoooowNN wwwlllleeeerrrriiii ttttrrrroooopppphhhhoooozzzzooooiiiitttteeeessss.... Food-cups structure was shown by a SEM (4A, B). Obvious karyosome in nucleus was observed by a TEM (4C). N, nucleus; V, vacuole; fc, foodcup structure.

(26)

C CC

C.... CCCCyyyyttttooooppppaaaatttthhhhiiiicccc cccchhhhaaaannnnggggeeeessss ooooffff mmmmiiiiccccrrrroooogggglllliiiiaaaallll cccceeeellllllllssss ccccoooo----ccccuuuullllttttuuuurrrreeeedddd wwiiiitttthhhhww NNNN.... ffffoooowwwlllleeeerrrriiiiw ttttrrrroooopppphhhhoooozzzzooooiiiitttteeeessss

1111.... MMMoooorrrrpppphhhhoooollllooooggggiiiicccc cccchhhhaaaannnnggggeeeessss ooooffff mM mmiiiiccccrrrroooogggglllliiiiaaaallll cccceeeellllllllssss ccccoooo----ccccuuuullllttttuuuurrrreeeedddd wm wwiiiitttthhhhw NNNN.... ffffoooowwwwlllleeeerrrriiii ttttrrrroooopppphhhhoooozzzzooooiiiitttteeeessss

By a light microscope, microglial cells co-cultured with N. fowleri

trophozoites for 6 hr showed severe morphological destruction and decreasing

number (Fig. 5C). In comparison, microglial cells co-cultured with N. fowleri

trophzoites and an anti-Nfa1 antibody showed less destruction than above experimental group (Fig. 5D). Many alive microglial cells remained to be attached on the bottom.

By SEM findings, microglial cells co-cultured with N. fowleri

trophozoites was attacked and destroyed by N. fowleri trophozoites.

N. fowleri showed broad and sticky attachment onto microglial cells

(Fig. 6A, B). When an anti-Nfa1 antibody was treated on N. fowleri

co-cultured with microglial cells, the morphology of N. fowleri was changed

into a round form and less sticky attachment than above experiment group (Fig. 6C, D).

By TEM findings, microglial cells co-cultured with N. fowleri

trophozoites were destroyed in a timedependent manner (Fig. 7). N. fowleri

attached to a microglial cell by a vigorous psedopodium was observed at 3 hr (Fig. 7B). As the incubating time was continued, the food-cups structure of

N. fowleri trophozoite was observed in the contact site of a microglial cell,

and simultaneously a amoeba had dead particles of microglial cells in vacuoles (Fig. 7C). A microglial cell was dead by the necrotic process characterized by membrane swelling and burst of the nucleus membrane (Fig. 7D).

(27)

F FF

Fiiiigggg.... 5555.... LLLLiiiigggghhhhtttt mmmiiiiccccrrrroooossssccccooooppppiiiicccc ffffiiiinnnnddddiiiinnnnggggssss ooooffff mm mmiiiiccccrrrroooogggglllliiiiaaaallllm cccceeeellllllllssss ccccoooo----ccccuuuullllttttuuuurrrreeeedddd wwwwiiiitttthhhh N

NN

Naaaaeeeegggglllleeeerrrriiiiaaaa ffffoooowwwwlllleeeerrrriiii ttttrrrroooopppphhhhoooozzzzooooiiiitttteeeessss ((((CCC)))),,,, oooorrrr wC wwwiiiitttthhhh aaaannnn aaaannnnttttiiii----NNNNffffaaaa1111 aaaannnnttttiiiibbbbooooddddyyyy ((((1111::::111100000000 ddddiiiilllluuuuttttiiiioooonnnn)))) ((((DDDD)))) ffffoooorrrr 6666 hhhhrrrr.... Arrows and arrowheads indicated microglial

cells and N. fowleri trophozoites, respectively. A, microglial cells only; B, N.

(28)

F FF

Fiiiigggg.... 6666.... SSSESEEEMMMM ffffiiiinnnnddddiiiinnnnggggssss ooooffff mmiiiiccccrrrroooogggglllliiiiaaaallll cccceeeellllllll ccccoooo----ccccuuuullllttttuuuurrrreeeedddd oooonnnnllllyyyy wmm wwwiiiitttthhhh NNNNaaaaeeeegggglllleeeerrrriiiiaaaa ffffoooowwwwlllleeeerrrriiii ttttrrrroooopppphhhhoooozzzzooooiiiitttteeee ((((AAA,,,, BA B)))),,,, aaaannnndddd aaaannnn aaaannnnttttiiii----NBB NNNffffaaaa1111 aaaannnnttttiiiibbbbooooddddyyyy ((((CCCC,,,, DDDD)))).... In panel A

and B, N. fowleri showed broad and sticky attachment onto microglial cells.

In panel C and D, N. fowleri showed weaker attachment and round

(29)

F FF

Fiiiigggg.... 7777. TTTETEEEMMMM ffffiiiinnnnddddiiiinnnnggggssss ooooffff mmiiiiccccrrrroooogggglllliiiiaaaallll cccceeeellllllllssss ccccoooo----ccccuuuullllttttuuuurrrreeeedddd wmm wwwiiiitttthhhh NNNNaaaaeeeegggglllleeeerrrriiiiaaaa ffffoooowwwwlllleeeerrrriiii ttttrrrroooopppphhhhoooozzzzooooiiiitttteeeessss.... Microglia (M) co-cultured with N. fowleri (Nf)

trophozoite for 0, 3, 6 and 12 hr (A, B, C and D), respectively. In panel B, N.

fowleri attached to a microglial cell with a vigorous pseudopodia. In panel C,

the foodcups structure of N. fowleri was shown in a contact region of a

microglial cell (Arrow). In panel D, a microglial cell was more destroyed via

(30)

2222.... IIIInnnn vvvviiiittttrrrroooo ccccyyyyttttoooottttooooxxxxiiiicccciiiittttyyyy ooooffff NN.... ffffoooowNN wwwlllleeeerrrriiii ttttrrrroooopppphhhhoooozzzzooooiiiitttteeeessss oooonnnn mmmmiiiiccccrrrroooogggglllliiiiaaaallll cccceeeellllllllssss

To determine whether N. fowleri trophozoites show the cytotoxicity on

microglial cells, 51_{Cr release assay was carried out. When microglial cells}

were co-cultured with N. fowleri trophozoites, the cytotoxicity of amoeba

on microglial cells was increased from 17.8 to 100 % in a timedependent

manner (Table 1, Fig. 8). N. fowleri co-cultured with microglial cells and

an anti-Nfa1 antibody showed 15.5 ～ 66.9 % cytotoxicity which was more decreased than above experimental groups without antibody (T-test, P<0.05) (Table 1, Fig. 8).

(31)

T T T

Taaaabbbblllleeee 1111....TTThhhheeee rrrreeeessssuuuullllttttTssss ooooffff tttthhhheeee iiiinnnn vvvviiiittttrrrroooo ccccyyyyttttooooxxxxiiiictttyyyy ooooffffccciiiit NaaaaeeeegggglllleeeerrrriiiiNNNaaaa wwwlllleeeerrrriiiiffffoooow ttttrrrroooopppphhhhoooozzzzooooiiiitttteeeessss oooonnnn mmmiiiiccccrrrroooogggglllliiiiaaaallll cccceeeellllllllssssm

a _{% cytotoxicity calculated by the} 51_{Cr amount released from radiolabelled}

microglial cells (mean ± standard variation)

b _{Dilution ratio of an anti-Nfa1 antibody}

F FF

Fiiiigggg.... 8888.... CCCCyyyyttttoooottttooooxxxxiiiicccciiiittttyyyy ooooffff NNNNaaaaeeeegggglllleeeerrrriiiiaaaa ffffoooowwwwlllleeeerrrriiii ttttrrrroooopppphhhhoooozzzzooooiiiitttteeeessss ccccoooo----ccccuuuullllttttuuuurrrreeeedddd wwwiiiitttthhhhw m

mm

miiiiccccrrrroooogggglllliiiiaaaallll cccceeeellllllllssss,,,, oooorrrr wwwwiiiitttthhhh aaaannnn aaaannnnttttiiii----NNNNffffaaaa1111 aaaannnnttttiiiibbbbooooddddyyyy.... M, miroglial cells; A,

N. fowleri trophozoites; Ab, anti-Nfa1 antbody.

0 20 40 60 80 100 120 3 6 12 24 h c y to to x ic it y % M M +A M +A+Ab

(32)

D DD

D.... CCCyyyyttttooookkkkiiiinnnneeeessss rrrreeeelllleeeeaaaasssseeee ffffrrrroooomC mm mm mmmiiiiccccrrrroooogggglllliiiiaaaallll cccceeeellllllllssss ccccoooo----ccccuuuullllttttuuuurrrreeeedddd wwiiiitttthhhhww NNNN.... ffffoooowwwlllleeeerrrriiiiw ttttrrrroooopppphhhhoooozzzzooooiiiitttteeeessss

To determine whether microglial cells show any change in cytokine

release as a result of the protective activity induced by pathogenic N. fowleri

trophozoites, assays for cytokines such as TNF-α, IL-1β and IL-6 were measured with enzyme-linked immunosorbent assay kits. In microglial cells

co-cultured with N. fowleri trophozoites, the amount of TNF-α was increased

at 3 hr and peaked at 6 hr post-incubation, and when an anti-Nfa1 antibody was treated, the increasing was inhibited (Table 2, Fig. 9). The amount of IL-1β was slightly increased at 12 hr post-incubation. No effect of an anti-Nfa1 antibody was shown (Table 3, Fig. 10). The amount of IL-6 was increased from 3 hr to 12 hr post-incubation. No effect of an anti-Nfa1 antibody was shown (Table 4, Fig. 11).

(33)

T T T

Taaaabbbblllleeee 2222.... AAmAAmmoooouuuunnnnttttssssm ooooffff TTTTNNFNNFFF----αααα sssseeeeccccrrrreeeetttteeeedddd ffffrrrroooommmm mmmmiiiiccccrrrroooogggglllliiiiaaaallll cccceeeellllllllssss ccccoooo----ccccuuuullllttttuuuurrrreeeedddd wwwwiiiitttthhhh NNaaaaeeeegggglllleeeerrrriiiiaaaa ffffoooowNN wwwlllleeeerrrriiii ttttrrrroooopppphhhhoooozzzzooooiiiitttteeeessss

a _{Values are means ± standard deviations}

b _{The ratio (1:1) is the number of microglial cells to the number of}

trophozoites

c _{Ab, dilution ratio of an anti-Nfa1 antibody is 1:100}

F FF

Fiiiigggg.... 9999.... AAAAmmmmoooouuuunnnnttttssss ooooffff TTTTNNFNNFFF----αααα sssseeeeccccrrrreeeetttteeeedddd ffffrrrroooommmm mmmmiiiiccccrrrroooogggglllliiiiaaaallll cccceeeellllllllssss ccccoooo----ccccuuuullllttttuuuurrrreeeedddd w

ww

wiiiitttthhhh NNNNaaaaeeeegggglllleeeerrrriiiiaaaa ffffoooowwwwlllleeeerrrriiii ttttrrrroooopppphhhhoooozzzzooooiiiitttteeeessss.... M, microglia; A, N. fowleri trophozoites; Ab, anti-Nfa1 antibody.

20 40 60 80 100 120 140 160 0 3 6 12 h p g /m l M M +A M +A+Ab

(34)

T T T

Taaaabbbblllleeee 3333.... AAmAAmmoooouuuunnnnttttssssm ooooffff IIIILLLL----1111ββββ sssseeeeccccrrrreeeetttteeeedddd ffffrrrroooommmm mmiiiiccccrrrroooogggglllliiiiaaaallllmm cccceeeellllllllssss ccccoooo----ccccuuuullllttttuuuurrrreeeedddd wwwwiiiitttthhhh NNaaaaeeeegggglllleeeerrrriiiiaaaa ffffoooowNN wwwlllleeeerrrriiii ttttrrrroooopppphhhhoooozzzzooooiiiitttteeeessss

trophozoites

F FF

Fiiiigggg.... 11110000.... AAAmAmmoooouuuunnnnttttssss ooooffff IIIILm LLL----1111ββ sssseeeeccccrrrreeeetttteeeedddd ffffrrrroooomββ mmm mmmmiiiiccccrrrroooogggglllliiiiaaaallll cccceeeellllllllssss ccccoooo----ccccuuuullllttttuuuurrrreeeedddd w

ww

wiiiitttthhhh NNNNaaaaeeeegggglllleeeerrrriiiiaaaa ffffoooowwwwlllleeeerrrriiii ttttrrrroooopppphhhhoooozzzzooooiiiitttteeeessss.... M, microglia; A, N. fowleri trophozoites; Ab, anti-Nfa1 antibody.

50 60 70 80 90 100 110 0 3 6 12 h p g /m l M M +A M +A+Ab

(35)

T TT

Taaaabbbblllleeee 4444.... AAAAmmmmoooouuuunnnnttttssss ooooffff IIIILLLL----6666 sssseeeeccccrrrreeeetttteeeedddd ffffrrrroooommmm mmmmiiiiccccrrrroooogggglllliiiiaaaallll cccceeeellllllllssss ccccoooo----ccccuuuullllttttuuuurrrreeeedddd w

ww

wiiiitttthhhh NNNNaaaaeeeegggglllleeeerrrriiiiaaaa ffffoooowwwwlllleeeerrrriiii ttttrrrroooopppphhhhoooozzzzooooiiiitttteeeessss

trophozoites

F FF

Fiiiigggg.... 11111111.... AAAAmmmmoooouuuunnnnttttssss ooooffff IIIILLLL----6666 sssseeeeccccrrrreeeetttteeeedddd ffffrrrroooommmm mmmmiiiiccccrrrroooogggglllliiiiaaaallll cccceeeellllllllssss ccccoooo----ccccuuuullllttttuuuurrrreeeedddd w

ww

wiiiitttthhhh NNaaaaeeeegggglllleeeerrrriiiiaaaa ffffoooowNN wwwlllleeeerrrriiii ttttrrrroooopppphhhhoooozzzzooooiiiitttteeeessss. M, microglia; A, N. fowleri phozoites; Ab, anti-Nfa1 antibody.

0 100 200 300 400 500 0 3 6 12 h p g /m l _M M +A M +A+Ab

(36)

IIIIV

V.... D

V

D

DIIIIS

D

SS

SC

C

CU

US

U

SS

SIIIIO

O

ON

N

A prominent feature of fatal human and animal cases of infection by

N. fowleri is invasion of the CNS and development of PAME. As the major

route of invasion for the N. fowleri infection, amoeba reach the nasal cavity

and attach to then, and invade the nasal mucosa and olfactory nerve. Concerned with the host-tissue invasion, the adherence of the amoeba to host

cells is the most important step in the mechanism of pathogenicity of N.

fowleri, and a specific pseudopodia projection, called an amoebastome, is

formed (Marciano-Cabral, 1988). Invasive amoeba able to enter the nervous system digest neuronal tissue and other mammalian cells by usually effective cytolysis and phagocytosis as observed in culture or in sections of infected brain tissue (Marciano-Cabral, 1988). Other cytotoxic toxins and cytolytic proteinases have been proposed that amoeba destroy target cells (John, 1982; Ma et al., 1990).

Microglial cells are resident macrophages within the CNS and exhibit phenotypic and functional features of macrophages localized at peripheral non-neural sites. Microglial cells migrate to sites of injury and infection, Phagocytoze dead cells and invading organisms, and produce a variety of factors including cytokines such as TNF-α, IL-1β and IL-6. Because microglial cells appear to possess characteristics of extraneural tissue macrophages, the may be a major cell type involved in host defense against

N. fowleri in the brain (Suzumura et al., 1993). However, the mechanisms

whereby these cells participate in defense against this infection are not understood.

In our study, microglial cells cultured from rat newborn brain were used

(37)

against primary cultured rat microglial cells, the morphological change of

microglial cells co-cultured with N. fowleri trophozoites observed by a light,

SEM, TEM. In addition, the in vitro cytotoxicity of N. fowleri against

microglial cells observed. By adding an anti-Nfa1 antibody in the co-culture system, the efforts an anti-Nfa1 antibody on the cytopathology and in vitro

cytotoxicity of N. fowleri against microglial cells was elucidated.

In previous report, trophozoites of N. fowleri in cultivating system and

in mouse brain tissue infected experimentally with N. fowleri were well

immunostained, as results of immunohistochemistry of the Nfa1 protein (Cho et al., 2003). The immunolocalization study of the Nfa1 protein revealed that

this protein was abundant in pseudopodia and around food vacuoles of N.

fowleri trophozoites (Cho et al., 2003). This suggests that the Nfa1 protein is

required for food ingestion and amoeba movement. On the basis of

pseudopodia-specific Nfa1 protein involved in the pathogenicity of N. fowleri

concerned with the host-tissue invasion, cytotoxicity of N. fowleri against

microglial cells were observed by adding an anti-Nfa1 antibody in the co-culture system to elucidate the antibody effect.

In this study, it was shown that microglial cells co-cultured with N.

fowleri trophozoites and an anti-Nfa1 antibody showed less destruction than

groups without anti-Nfa1 antibody by a light microscope. By a SEM, it was

also shown that an anti-Nfa1 antibody was treated on N. fowleri trophzoites

co-cultured with microglial cells, the morphology of N. fowleri was changed

into a round form and less sticky attachment than groups without anti-Nfa1

antibody. However, by a TEM, microglial cells co-cultured with N. fowleri

trophozoites were destroyed in a time-dependent manner. This observations

suggested that effector N. fowleri trophozoites contacted target microglial,

(38)

swelling and burst in the nucleus. Therefore, the lysis of target microglial cells or ingestion of the target cells via food cups and their subsequent channeling into intracytoplasmic food vacuoles. In particular, Brown (Brown,

1979; Zubiaur and Alonoso, 1983) concluded that N. fowleri injured target

cells by repeated nibbling, a process he termed 'trogocytosis'. In our study, it

was confirmed the observation of Brown that N. fowleri can destroy target

cells with early stages for piecemeal ingestion. In addition, when microglial

cells co-cultured with N. fowleri trophozoites, the high cytotoxicity of amoeba

on microglial cells in the 51_{Cr release assay was increased in a}

time-dependent manner, but when an anti-Nfa1 antibody was added, the cytotoxicity was decreased than groups without antibody. Moreover, in the

previous study, the proliferation and the cytotoxicity of N. fowleri against

CHO target cells were observed by adding an anti-Nfa1 antibody in the co-culture system to elucidate the antibody effect. It was shown that

anti-Nfal antibody inhibited the proliferation of N. fowleri trophozoites in a

dose-dependent manner (Jeong et al., 2004). In addition, less CPE has been shown to occur in the presence of an antiNfa1 antibody which immobilizes

or agglutinates N. fowleri trophozoites.

In previous report about Acathamoeba culbertsoni to induce

granulomatous amoeba encephalitis, pathogenic A. culbertsoni induced the

cytopathic effects in primary cultured rat microglial cells, with the effects characterized by necrosis and apoptosis of microglial cells (Shin et al., 2000). Necrosis is marked by swelling and bursting of cellular membranes and organelles following the effector celltarget cell contact event. In contrast, apoptosis is characterized by shrinkage in which the target cell undergoes sequential condensation of chromatin, membrane blebbing, loss of the nuclear envelope, cellular fragmentation into apoptotic bodies, and uptake by

(39)

phagocytic cells. In this study, necrosis via poreforming lytic molecules and apoptosis, both of which disrupt cell membrane integrity, were found to be

two fundamental mechanisms in the cytolysis of target microglial cells by N.

fowleri.

To determine whether microglial cells show changes in cytokines release

as a result of a CPE induced by N. fowleri, the assay for cytokines such as

TNF-α, IL-1β and IL-6 was performed with culture supernatants. We observed that TNF-α and IL-6 peaked early and then decreased at 24 hr, whereas IL-1β peaked at 12 hr and continued to accumulate in the medium through 24 hr (data not shown). In addition, when microglial cells were

co-cultured with N. fowleri and an anti-Nfal antibody for 3 and 6 hr, the

amount of TNF-α secreted from microglial cells was inhibited about 20.3%, 14.1%, respectively. but the amount of IL-1β and IL-6 was not decreased. It suggested that cytokine regulations were different modes. It is well known that microglial cells produce proinflammatory cytokines, such as TNF-α, IL-1 β, IL-3, IL-6, IL-8, IL-12p40 and IL-15, as well as anti-inflammatory cytokine, such as IL-10, TGF-β, for defense against parasites and brain injury (Marciano-cabral et al., 2000; Benedetto et al., 2001). More extensive

studies on the cytokine responses of microglial cells due to N. fowleri are

necessary.

The results of this study could be considered as the cytopathic effects

induced by pathogenic N. fowleri against microglial cells and may be more

directly prominent to understanding the mechanisms of pathogenic N. fowleri.

Moreover, further studies need to determine whether the Nfa1 protein is related to in vivo amoebic pathogenicity.

(40)

V

V.... C

C

CO

O

ON

O

N

NC

C

CL

LL

LU

U

US

SS

SIIIIO

O

ON

O

N

Whether N. fowleri could induce the cytopathogenicity to primary

cultured rat newborn microglial cells as actual target cells of PAME, and an anti-Nfa1 anibody reduce the cytopathogenicity, microglial cells in co-culture

system were observed by electron microscopies and quantitative method. N.

fowleri trophozoites produced pseudopdia to contact microglial cells followed

by inducing a necrotic and apoptotic process. Moreover, there were dead

particles at larger vacuoles and obvious food-cups structure in N. fowleri.

Finally, pathogenic N. fowleri trophozoites showed the cytopathic effects on

rat microglial cells, showing the morphological changes and strong in vitro cytotoxicity which was inhibited with an anti-Nfa1 antibody. And then, microglial cells secreted cytokines, TNF-α, IL-1β and IL-6, as the protective immune response.

(41)

B

BIIIIB

BL

B

LL

LIIIIO

O

OG

G

GR

RA

R

A

APPPPH

H

HY

Y

1. Benedetto N, Folgore A, Romano Carratelli C, Galdiero F: Effects of

cytokines and prolactin on the replication of Toxoplasma gondii in murine

microglia. Fur Cytokine Netw 12:348-58, 2001

2. Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye

binding. Anal Biochem 72:24854, 1976

3. Brown T: Observations by immunofluorescence microscopy and electron

microscopy on the cytopathogenicity of Naegleria fowleri in mouse

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4. Carter RF: Primary amoebic menigoencephalitis: clinical, pathological and

epidemiological features of six fatal cases. J Pathol Bacteriol 96:125,

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5. Carter RF: Description of a Naegleria species isolated from two cases of

primary amoebic menigoencephalitis and the experimental pathological

change induced by it. J Pathol 100:21744, 1970

6. Carter RF: Primary amoebic meningoencephalitis. An appraisal of present

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7. Chang SL: Etiological, pathological, epidemiological, and diagnostical

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8. Chang SL: Resistance of pathogenic Naegleria to some common physical

(42)

9. Chang SL: Pathogenesis of pathogenic Naegleria amoeba. Folia

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10. Chao CC, Hu S, Close K, Choi CS, Molitor TW, Novick WJ, Peterson PK: Cytokine release from microglia: differential inhibition by pentoxifylline

and dexamethasone. J Infect Dis 166:847853, 1992

11. Chao CC, Anderson WR, Hu S, Gekker G, Martella A, Peterson PK:

Activated microglia inhibit multiplication of Toxoplasma gondii via a

nitric oxide mechanism. Clin Immunol Immunopathol 67:178-183, 1993

12. Chao CC, Gekker G, Hu S, Peterson PK: Human microglial cell defense

against Toxoplama gondii. The role of cytokines. J Immunol

152:1246-1252, 1994

13. Chao CC, Hu S, Peterson PK: Glias: the not so innocent bystanders.

J Neurovirol 2:234-239, 1996

14. Cho MS, Jeong SY, Park S, Kim KH, Kim HI, Sohn S, Kin HJ, Im KI, Shin HJ: Immunological characterizations of a cloned 13.1-kilodalton

protein from pathogenic Naegleria fowleri. Clin Diagn Lab Immunol

10:954-959, 2003

15. Cuadros MA, Navascues J: The origin and differentiation of microglial

cells during development. (Review) Prog Neurobiol 56:173-189, 1998

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25:231254, 1971

17. De Jonckheere JF: Hospital hydrotherapy pools treated with ultraviolet

light; bad bacteriological quality and presence of thermophilic Naegleria.

(43)

18. Feldman MR: Naegleria fowleri: Fine structural localizaion of acid

phosphatase and heme proteins. Exp Parasitol 41:290306, 1977

19. Ferrante A, Bates EJ: Elastase in the pathogenic freeliving amebae

Naegleria and Acanthamoeba spp. Infect Immun 56:33203321, 1988

20. Giulian D, Baker TJ: Characterization of ameboid microglia isolated from

developing mammalian brain. J Neurosci 6:163178, 1986

21. Hadas E, Mazur T: Proteolytic enzymes of pathogenic and nonpathogenic

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아 아 아 아주주주주대대대대학학학교학교교교 대대학대대학학학원원원 의원 의학의의학학학과과과과 오 오 오 오 영영영 환영 환환환 ((((지지지도지도도도교교교수교수 :::: 신수수 신신 호신 호호호 준준준준)))) 파울러자유아메바 (Naegleria fowleri)는 토양과 담수에서 자유생활을 하는 아메바로써 자연환경에서는 세균을 포식하며, 인체와 실험동물에서 원발성 아메 바성 수막뇌염 (priamry amoebic meningoencephalitis)을 일으키는 것으로 알려 져 있다. 숙주 세포로의 침입과 연관해서 파울러 자유아메바의 병원성 기전에서 표적 세포와의 접촉은 아주 중요하다. 본 연구실에서는 파울러 자유아메바에 대 한 감염혈청 및 면역혈청을 생산하여 cDNA로부터 immunoscreenig을 통해 병원 성 및 항원성과 관련이 있을 것으로 사료되며, 특히 위족(pseudopodia)에 특이성 을 갖는 유전자 nfa1을 클로닝 해놓았다. 그리고 클로닝 된 nfa1유전자로부터 13.1 kDa의 재조합 단백질인 Nfa1을 얻었다. 본 연구의 목적은 위족 특이성을 갖는 nfa1 유전자가 파울러자유아메바의 병원성과 관련성이 있는지를 입증하기 위해서, 일차 배양한 쥐의 microglia와 함 께 파울러자유아메바를 혼합 배양함으로써 아메바 영양형들의 병변효과 및 항 Nfa1 항체의 투여효과를 광학, 주사, 및 투과 전자현미경을 이용해서 관찰하였 다. 파울러자유아메바 영양형들은 활발한 위족과 food-cups 구조를 형성하여 microglia에 접촉하는 것을 확인하였다. 혼합 배양 3 시간 후, 소수의 microglia들 은 자유아메바들에 의해서 파괴가 되었으며, 배양 시간이 길어짐에 따라 microglia들은 심하게 파괴되었다. 생화학적 방법인 51_{Cr 분비 실험결과,} microglial 세포들에 대한 자유아메바의 세포독성은 배양 후 3, 6, 12 그리고 24

(48)

시간에 각각 17.8, 24.9, 54.6 그리고 98.0% 이었으며, 항 Nfa1 항체(1:100)를 투여 하였을 경우, microglia들에 대한 세포독성이 각각 15.5, 20.3, 50.7 그리고 66.9% 로 감소하였다. 한편, 파울러자유아메바와 혼합 배양한 microglia들은 방어적인 면역반응으로 TNF-α, IL-1β 그리고 IL-6등의 사이토카인(cytokine)들을 분비한 다.

핵심이 되는말: 파울러자유아메바, 원발성 아메바성 수막뇌염, nfa1 유전자,