Eicosanoids in insect immunity: Bacterial infection stimulates hemocytic phospholipase A(2) activity in tobacco hornworms

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Eicosanoids in Insect Immunity: Bacterial Infection

Stimulates Hemocytic Phospholipase A

2

Activity in

Tobacco Hornworms

Hasan Tunaz,

1,2

Youngjin Park,

3

Kemal Büyükgüzel,

4

Jon C. Bedick,

1

A.R. Nor Aliza,

1

and David W. Stanley

1

_*

Intracellular phospholipase A2 (PLA2) is responsible for releasing arachidonic acid from cellular phospholipids, and is thought to be the first step in eicosanoid biosynthesis. Intracellular PLA2s have been characterized in fat body and hemocytes from tobacco hornworms, Manduca sexta. Here we show that bacterial challenge stimulated increased PLA2 activity in isolated hemocyte preparations, relative to control hemocyte preparations that were challenged with water. The increased activity was detected as early as 15 s post-challenge and lasted for at least 1 h. The increased activity depended on a minimum bacterial challenge dose, and was inhibited in reactions conducted in the presence of oleyoxyethylphos-phorylcholine, a site-specific PLA2 inhibitor. In independent experiments with serum prepared from whole hemolymph, we found no PLA2 activity was secreted into serum during the first 24 h following bacterial infection. We infer that a hemocytic intracellular PLA2 activity is increased immediately an infection is detected. The significance of this enzyme lies in its role in launching the biosynthesis of eicosanoids, which mediate cellular immune reactions to bacterial infection. Arch. Insect Biochem. Physiol. 52:1–6, 2003. © 2003 Wiley-Liss, Inc.

KEYWORDS: eicosanoids; insect immunity; bacterial infection; phospholiphase A2

1_{Insect Biochemical Physiology Laboratory, University of Nebraska, Lincoln}

2_{Faculty of Agriculture, Department of Plant Protection, KahramanMaras Sütcü Ìmam University, KahramanMaras, Turkey} 3_{School of Bioresource Sciences, Andong National University Andong, Korea}

4_{Department of Biology, Karaelmas University, 67100 Ìncivez, Zonguldak, Turkey}

Contract grant sponsor: KahramanMaras Sütcü Ìmam University; Contract grant sponsor: Agricultural Research Division, University of Nebraska; Contract grant number: NEB-17-054.

*Correspondence to: David W. Stanley, Insect Biochemical Physiology Laboratory, 311 Plant Industry Building, University of Nebraska, Lincoln, NE 68583-0816. E-mail: dstanley1@unl.edu

Received 17 April 2002; Accepted 25 June 2002

INTRODUCTION

We have been considering the hypothesis that eicosanoids mediate insect cellular immune reac-tions to bacterial infecreac-tions. Several lines of evi-dence support the hypothesis. First, experimental insects treated with pharmaceutical inhibitors of eicosanoid biosynthesis were severely impaired in their ability to clear bacterial infections from hemolymph circulation (Stanley-Samuelson et al.,

1991), and diminished the nodulation reaction to bacterial challenge in experimental insects (Miller et al., 1994; 1996; 1999; Jurenka et al., 1997; Stanley-Samuelson et al., 1997; Stanley et al., 1999; Tunaz et al., 1999; Bedick et al., 2000). While they support the hypothesis, these results are based on the pharmacological actions of compounds that are understood and intended for use in human and animal medicine. A fundamental assumption of these studies is that the pharmaceuticals exert

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simi-lar eicosanoid biosynthesis-inhibiting actions in mammalian and insect cells.

In a third line of inquiry, we characterized the biochemistry of eicosanoid biosynthesis in im-mune tissues from several insect species, including fat body and hemocytes from tobacco hornworms, Manduca sexta (Stanley-Samuelson and Ogg, 1994; Gadelhak et al., 1995) and fat body from true ar-myworms, Pseudaletia unipuncta (Tunaz et al., 2001). These studies showed that two of the eicosanoid biosynthesis inhibitors used in our biological studies, indomethacin and naproxin, inhibited prostaglandin biosynthesis in a dose-dependent manner.

We also tested the eicosanoid hypothesis directly by determining the influence of bacterial infection on eicosanoid biosynthesis in true armyworms (Jurenka et al., 1999). In this work, we showed that hemolymph concentrations of a specific

eicosanoid, the prostaglandin PGF2a, increased

sub-stantially by 30 min post-infection (PI) in experi-mental larvae compared to saline-treated control larvae. Moreover, the infection-stimulated increase

in PGF2a was inhibited in larvae that had been

pre-treated with phenidone, a dual cyclooxygenase/ lipoxygenase inhibitor.

The first step in the eicosanoid biosynthesis

pathways is catalyzed by phospholipase A2 (PLA2),

which is responsible for hydrolyzing fatty acids from the sn-2 position of cellular phospholipids. We

char-acterized an intracellular PLA2 in tobacco hornworm

fat body (Uscian and Stanley-Samuelson, 1993) and hemocytes (Schleusener and Stanley-Samuelson, 1996). This background served as the basis of an-other direct test of the eicosanoid hypothesis. In this study, we report on the influence of bacterial

infection on PLA2 activity in tobacco hornworm

hemocytes.

MATERIALS AND METHODS

Organisms

Eggs of the tobacco hornworm, M. sexta, were purchased from Carolina Biological Supply. The lar-vae were reared in individual cups on artificial diet

under semi-sterile conditions (Stanley-Samuelson and Ogg, 1994). Fifth instars, age day 2 to 4, were used in all experiments.

The bacterium, Serratia marcescens, was obtained from the culture collection, School of Biological Sciences, University of Nebraska-Lincoln, and grown to stationary phase under standard condi-tions in nutrient broth (Miller et al., 1994). The bacterial cells were collected by centrifugation, washed, then freeze-dried. Freeze-dried material was reconstituted in pyrogen-free water, and injected into test larvae at dosages described in Results.

Chemicals

Radioactive phosphatidylcholine (1-palmitoyl,

2-arachidonyl [arachidonyl-1-14C], 1.9 GBq/mmol)

was purchased from DuPont New England Nuclear (Wilmington, DE). Analytical grade solvents were purchased from Fisher (Fair Lawn, NJ). Arachidonic acid was purchased from Sigma Chemical Co. (St. Louis, MO), and oleyoxyethylphosphorylcholine (OOPC) was purchased from BioMol (Plymouth Meeting, PA).

Hemocyte Preparation

Hornworms were chilled on ice, then surface sterilized by wiping them with 95% ethanol. Hemolymph was collected by pericardial puncture (Horohov and Dunn, 1983; Schleusener and Stanley-Samuelson, 1996). Freely-dripping hemo-lymph, 0.5 ml, was collected into tubes and di-luted 1:1 in Manduca saline buffer (MSB, described in Schleusener and Stanley-Samuelson, 1996). Af-ter gentle mixing, the tubes were centrifuged at ap-proximately 1,250g for 15 min. The supernatant was drained, and the pellets were gently rinsed with incubation buffer, without disturbing the position of the pellets.

The pellets were transferred to Eppendorf tubes, and approximately 150 ml of incubation buffer was added to each tube. Then the hemocytes were ho-mogenized by sonication, eight 0.5-s bursts at 60 W using a VibraCell sonicator (VibraCell, Danbury, CT). The tubes were centrifuged for 10 min at

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1,300g at 4°C, and the supernatants were trans-ferred to 1.5-ml Eppendorf tubes. The supernatants were centrifuged at 11,750g for 10 min at 4°C. The supernatants from the final centrifuge step were used in all experiments. Protein concentrations were determined using the bicinchoninic acid re-agent (Pierce, Rockford, IL) against bovine serum albumin as quantitative standard. Protein concen-trations were determined at 562 nm using a BioTek microtiter plate reader.

PLA

2

Reactions

For each reaction, 0.05 mCi of substrate was transferred to an Eppendorf tube and dried under

N2. Substrate vesicles were formed by adding 80

ml of incubation buffer and vigorously vortexing the tubes.

The PLA2 reactions were started by adding

aliquots of hemocyte preparations to tubes con-taining substrate vesicles and vortexing the tubes for 15 s. Under standard conditions, 250 mg of hemocyte protein was incubated with 250 ml total volume at 28°C in a shaking water bath for 30 min. Reactions were terminated by adding 0.6 ml of acidified extraction solvent (chloroform:methanol, 2:1, amended with 100 ml 1 N HCl). Arachidonic acid (20 nmol in 10 ml chloroform) was added to each tube as a carrier and free fatty acid standard. Each tube was vortexed for 15 s and centrifuged at 735g for 2 min. The lower organic phase was trans-ferred to an Eppendorf tube. Two more extraction steps followed, each using 0.5 ml chloroform. The

collected organic solvent was dried under N2, and

the reaction products were separated by TLC (silica gel G TLC plates, 20 ´ 20 cm, 0.25 mm thick, Sigma Chemical Co., St. Louis, MO), plates developed in petroleum ether:diethyl ether:glacial acetic acid (80:20:1, v/v). Free fatty acid and phospholipid fractions were localized by scanning the plates on a BioScan 200 Imaging Scanner (Bioscan, Inc., Washington, DC). The amounts of radioactivity in each fraction were estimated by adding scintilla-tion cocktail (Ecolite, ICN Biomedicals, Irvine, CA) and counting on a LKB Wallac 1209 Rackbeta Liq-uid Scintillation Counter (Pharmacia, Turku,

Fin-land) at 96% counting efficiency for 14C. Enzyme

activity was calculated from the liquid scintillation data.

Statistical Analysis

Data were analyzed using the analysis of vari-ance in the General Linear Models procedure, and mean comparisons were made using the Least Sig-nificant Difference test (SAS Institute, 1989).

RESULTS

The influence of bacterial challenge dosage on

PLA2 activity in hemocyte preparations at 30 sec

PI is shown in Figure 1. We registered very low

PLA2 activity in hemocyte preparations challenged

with 0 or 25 mg doses, which increased approxi-mately 6-fold in preparations that had been chal-lenged with 50 mg. Larger challenge doses, 100 and 200 mg, did not stimulate further increases in

hemocyte PLA2 activity.

Bacterial challenge stimulated significant

in-creases in hemocytic PLA2 activity relative to

con-trols (Fig. 2). We recorded relatively low PLA2

activity in hemocyte preparations that had been

injected with water. By comparison, PLA2 activity

in experimental hemocyte preparations was sub-stantially higher than controls at 30 sec PI, and it remained significantly higher for the following 4.5

min. The apparent differences in PLA2 activity were

not significant at 15 and 60 m PI.

The increased PLA2 activity recorded from

hemocyte preparations that had been stimulated with bacteria was inhibited in reactions conducted in the presence of OOPC, a site-specific inhibitor

of PLA2 activity. It can be seen in Figure 3 that the

influence of OOPC was expressed in a dose-de-pendent manner.

We considered the possibility that the increased

PLA2 activity recorded in challenged hemocyte

preparations may be secreted from hemocytes into hemolymph. For these experiments, hornworms were first challenged with bacterial preparations. At selected times, post-challenge hemolymph was withdrawn from the hornworms and hemocytes

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were pelleted by gentle centrifugation. PLA2

activ-ity was then assessed in the resulting cell-free

se-rum. Virtually no PLA2 activity was detected in the

hemolymph at 0, 1, 2, and 4 h post-challenge (data not shown).

DISCUSSION

On the mammalian model, PLA2 is the first step

in eicosanoid biosynthesis (Dennis, 1994, 1997; Balsinde et al., 1999). Understanding the

signifi-cance of PLA2 in eicosanoid biosynthesis is

com-plicated by the large and growing number of PLA2

types. Balsinde et al. (1999) recognize ten groups

of PLA2 on the basis of gene sequence data. In

terms of biological actions, these can be sorted into

three major types, the secretory PLA2s, the

cytoso-lic calcium-dependent PLA2s and the intracellular

calcium-independent ones. Among insects, the

PLA2s associated with venoms are secretory

en-zymes, as are the insect digestive PLA2s (Uscian et

al., 1995; Nor Aliza et al, 1999; Rana et al., 1997, 1998; Rana and Stanley, 1999). These are low-mo-lecular weight enzymes, in the range of 13–18 kDa.

Two intracellular PLA2s have been characterized,

the M. sexta fat body and hemocyte PLA2s. Both of

these are calcium-independent. Although the

mo-lecular weights of these insect PLA2s are unknown,

in mammalian systems these are typically high-molecular weight enzymes, in the range of 65–100 Fig. 1. The influence of bac-terial dosages on intracellular PLA2 activity in hemocytes

pre-pared from tobacco horn-worms, M. sexta. Hemocyte preparations were challenged with the indicated dosage of bacteria, then PLA2 activity was

assessed at 30 sec post-chal-lenge. Each point represents the mean of three replicates and the error bars indicate 1 SEM.

Fig. 2. A time course of intrac-ellular PLA2 activity in

hem-ocytes prepared from tobacco hornworms, M. sexta. Experi-mental hemocyte preparations (solid circles) were challenged with bacteria (100 mg), then PLA2 activity was assessed at the

indicated times. Control hem-ocyte preparations (open circles) were challenged with water, then PLA2 activity was similarly

as-sessed. Each point represents the mean of three replicates and the error bars indicate 1 SEM.

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kDa. These intracellular enzymes are associated with release of arachidonic acid for eicosanoid bio-synthesis.

PLA2s associated with the release of arachidonic

acid from cellular phospholipid pools show a pref-erence for arachidonyl-containing substrate

(Den-nis, 1994). The tobacco hornworm hemocyte PLA2

also showed a preference for arachidonyl-contain-ing substrate (Schleusener and Stanley-Samuelson, 1996). Compared to activity against

arachidonyl-containing substrate, the hemocyte PLA2 against

palmitoyl-containing substrate was reduced by ap-proximately 40%. These experiments were con-ducted with simple microsomal-enriched hemocyte preparations, in which the arachidonyl-preferring

PLA2 was not enriched. The preference for

arach-idonyl-containing phospholipid substrate would most likely be expressed in stronger terms with a partially purified enzyme preparation. Nonetheless, the hemocytes feature a calcium-independent,

arachidonyl-preferring PLA2 that probably serves to

release substrate from cellular phospholipid pools for eicosanoid biosynthesis.

Our new findings strongly support this view.

Increased hemocyte PLA2 activity was stimulated

within seconds of bacterial challenge, and the ac-tivity was inhibited in the presence of OOPC. The

PLA2 activity is an intracellular enzyme, entirely

confined within hemocytes, because no PLA2

ac-tivity was detected in serum prepared from horn-worms that had been challenged with bacterial infection.

The significance of this finding lies in a more detailed understanding of eicosanoid mediation of insect cellular immune reactions to bacterial in-fection. Our data indicate that challenged

hemo-cytes express increased PLA2 activity for at least the

first hour after challenge. We infer that eicosanoids are continually formed through at least the first hour of cellular immune reaction to challenge. This idea is supported by the findings of Jurenka et al.

(1999), who reported increased levels of PGF2a in

true armyworm hemolymph at 30 min post-chal-lenge.

ACKNOWLEDGMENTS

Thanks to Ralph Howard (Manhattan, KS) and Gary Blomquist (Reno, NV) for critical comments on a draft of this paper. This study is a contribu-tion of the University of Nebraska Agricultural Re-search Division, Journal Series No. 13,748. This work was supported in part by a fellowship from KahramanMaras Sütcü Ìmam University to H. Tunaz.

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