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Synthesis of immune proteins in primary cultures of fat body from Hyalophora cecropia
Insect Biochem. Vol. 18, No. 3, pp. 299-312, 1988
0020-1790/88 $3.00 + 0.00 Copyright © 1988 Pergamon Press plc
Printed in Great Britain. All rights reserved
SYNTHESIS OF I M M U N E PROTEINS IN PRIMARY CULTURES OF FAT BODY FROM HYALOPHOR,4 CECROPIA TINA TRENCZEK*
Department of Immunology, The Biomedical Center, Uppsala University, Box 582, 75123 Uppsala, Sweden INGRID FAYE Department of Microbiology, University of Stockholm, 10691 Stockholm, Sweden (Received 21 August 1987; revised and accepted 4 November 1987) Abstract--Fat body from injured or immunized pupae as well as from untreated pupae of Hyalophora cecropia synthesize and release in vitro the immune proteins P4, attacins, lysozymeand cecropins. During an incubation time of 4 days immune protein synthesis declines relative to the other proteins, i.e. protein synthesis returns to a normal pupal pattern. Treatment of fat body in culture with bacterial compounds (LPS, lyophilized cells of Micrococcus lysodeikticus or heat killed Escherichia coli), but also sterile filtered insect Ringer, causes a relative higher rate of synthesis of the immune proteins. At the same time synthesis of some large proteins (75-100 kD) is switched off. Similar results are obtained by adding hemocytes from injured or immunized pupae, whereas hemocytes from untreated pupae show no effect. The induction of protein synthesis with particular emphasis on the immune proteins will be discussed. Key Word Index: Hyalophora cecropia, insect immunity, protein synthesis, in vitro culture
INTRODUCTION Several insect species respond to injections of live bacteria by de novo synthesis of antibacterial proteins which are detectable in the hemolymph (Boman et al., 1974; G t t z and Boman, 1985). However, this response is not specifically induced by bacteria as injection with sterile Ringer solution will cause a transient synthesis of the same antibacterial proteins (Boman et al., 1974; Faye and Wyatt, 1980; Andersson and Steiner, 1986). Ever since the first physiological studies using diapausing pupae of Hyalophora cecropia it has been known that an injury causes increased protein synthesis (Teller and Williams, 1960), a higher level of oxygen consumption (Harvey and Williams, 1961), enhanced RNA synthesis (Barth et al., 1964; Berry et al., 1967) and changed enzyme activities (Stevenson and Wyatt, 1964). Harvey and Williams (1961) assumed that a factor is diffusing in the hemolymph and Cherbas (1973) isolated a factor, "haemokinin", from damaged wing epidermis which could simulate an injury effect in hemocytes. Faye and Wyatt (1980) showed that tissue cultures of fat body from untreated pupae release antibacterial activity into the medium and
synthesize P4 as well as attacins (PS), presumably induced by the injury of dissection. Although we regard dissection of the fat body to be an unavoidable and extensive injury to the insect we have chosen in vitro studies to clarify some aspects of the "injury effect". In this paper we report on the importance and effects of the medium, endogenous factors, and hemocytes on the synthesis of "immune proteins". MATERIALS AND METHODS
Animals and their treatments H. cecropia pupae were reared on a synthetic diet (Riddiford, 1968; Boman et al., 1981) or obtained commercially from North American dealers in the mid-west. For convenience, the pupae and fat body are named according to their storage conditions: SC-pupae were kept at 6-8°C for 2.5 months (short chilled/homebred), LC-pupae were kept at 6-8°C for 5.5 months (long chilled/homebred) and SN pupae were kept about 2 months in diapause at 22°C under short day conditions (short nonchilled/outdoor bred). LCand SC-pupae that were never adjusted to 22°C prior to the dissection and SN-pupae taken directly are defined as "untreated" pupae. Similarly,pupae (or fat body) that were given a single injection of 50pl sterile filtered Ringer (without sucrose, 310mOsm/kg H20) 3 days before dissection and then kept at 22°C are called "injured" pupae. *To whom all correspondence should be addressed. Abbreviations: ACR=anticoagulant Ringer, FCSffetal As a control, untreated pupae were kept 3 days at room calf serum, K-PBS= potassium-phosphate buffered temperature. "Immune" pupae (or fat body) were injected saline, LPS = lipopolysaccharide, PAGE = polyacryl- with 50/zl Ringer containing either 2 or 5mg/ml LPS amide gel electrophoresis, PIPES = piperazine-N,N'-bis- (Escherichia coil D21, a generous gift from H. G. Boman) (2-ethanesulfonic acid), POPOP= 1,4-di[2-(5-phenyl- or 0.2 mg/ml lyophilized and washed cells of Micrococcus lysodeikticus (Sigma, St Louis, Mo.) and then kept 3 days oxazolyl)]-benzene, PPO---diphenyloxazole, SDS= at 22°C. Antibacterial activity from the hemolymph of all sodiumdodecylsulfate, TCA = trichloroacetic acid. 299
300
TINA TRENCZEK and INGRID FAYE
pupae was tested on E. coli D 31 plates according to Faye and Wyatt (1980).
Dissections and in vitro conditions The dissections were done under lepidopteran Ringer modified after Jungreis et al. (1973): 234mg NaC1, 9.5g KC1, 588mg CaC12(2H20), 3.05g MgC12(6H20), 25ml potassium-phosphate buffer (pH 6.5) and 34.23 g sucrose/l, pH 6.5; osmolarity, 440 + 5 mOsm/kg H20. In vitro incubations were performed in three media (Table 1), which differed in either osmolarity or amino acid composition. The three media were tested in parallel with double samples of fat body from the same pupae. Unless otherwise stated, all experiments were done in triplicate. The osmolarity was determined with the osmometer 3W2 (Advanced Instruments Inc., Needham Heights, Mass.). The pupae were washed in 0.5% iodine solution (Jodopax, Ferrosan), 70% ethanol, and sterile water and then quickly bled through a triangle-shaped cut made in the lateral thorax to reduce the influence of possible hemolymph factors. After dissection under Ringer with a few crystals of PTU (phenylthiourea), free fat body was transferred to fresh Table 1. Composition of the media Medium (raM) Substance
I/IP
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NaHCO2 KCI CaCI2 (2H20) MgCI2 (6H20) Mg2SO4 (7H20) KH2PO4
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4 50 3.1 14 1 8
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#-Alanin Arginine-HC1 Asparagine Asparagine acid Cysteine-HCI Glutamine Glutamine acid Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptopbane Tyrosine Valine
5 5 5
5 2 5 5 10 15 2 1 5 2 2 5 10 2 2 1 2
5 6 I 1 0.5 5 6 5 6 4 4 10 2 1 20 12 5 1 1 5
K-fumarate 1 I K-malate 5 5 K-pyruvate 1 1 K-succinate I 1 -Ketoglutarate 2.5 2.5 PIPES 25 25 Vitamins (100 x )a, /~1 10 10 p-Aminobencoic acidd, ~g 2 2 Phenolredd, mg 1 1 Gentamicind, mg 20 20 aModifications of the medium used by Reddy and Wyatt (1967). bAmino acid compositionwas chosen followingthe results of Duchateau et al. (1953). Amino acids were used in t- or in DL- form, vitamins were from Flow (medium MEME). The pH of all three media was adjusted with KOH. ¢Only medium II. aThe indicated amounts were added to 100ml of medium.
Ringer with PTU. Malpighian tubules, nerves, trachea and tissue from the heart complex were carefully removed (15min). Thereafter, the fat body was washed in Ringer without PTU (15 min), medium with PTU (15 min), medium without PTU (45 min). Finally, pieces of about 50 mg fat body were incubated in 500/~1 medium. This procedure was found to be essential since fat body rinsed only briefly in Ringer will "release" a remarkable number of hemocytes that spread all over the incubation vessel. As incubation vessels, multidishes (24/3.4ml) from NUNC (Denmark) were used. All incubations were carried out under gentle shaking at 27°C, in 5% CO 2 and 90% r.h.
Hemocytes Hemocytes were isolated by dropping hemolymph directly into ice-cold anticoagulant Ringer (ACR): 23.4 mg NaCI, 950mg KC1, 292mg EDTA, 198mg citric acid, 798mg tert. Sodium citrate, 2.5ml potassium-phosphate buffer (pH 6.5) and 2.74g sucrose in I00 ml; osmolarity, 455 mOsm/kg H20. After gentle shaking, the cells were centrifuged at 200g for 10min at 4°C (Sorvall RT 6000), washed 3 times in ACR and resuspended in medium before USe. Incorporation studies After 1 and 3 days in culture, fat body was incubated at 27°C for 24 h in 500t~l medium containing 0.5 mM lysine and 10/~Ci [3H]lysine (for the determination of the total protein synthesis) and 50 #Ci [3H]lysine (for characterizing the specific proteins). In both cases the specific activity was 95.8 mCi/mmol lysine. Controls were incubated with 10-3M cycloheximide added already 30 min prior to the incorporation period. The "labeled" fat body was rinsed twice in ice-cold potassium-phosphate buffered saline (K-PBS: 9.5 g KCI, 234 mg NaC1, 212 mg NaH2PO 4, 1.79 g K2HPO4/1, pH 6.5), briefly dried on filter paper, weight and homogenized in 100 #1 K-PBS. After extraction 3 times with 100 #1 K-PBS, pooled supernatants were centrifuged at 10,000 g at 4°C for 5 rain, and the supernatants stored at -20°C. Aliquots of culture supernatants or supernatants from the fat body homogenates were spotted and dried on 1 crn2 Whatman 3 mm fllterpaper, precipitated with 2ml 10% TCA (containing 10mM lysine) at 4°C and afterwards incubated at 80°C, each step for 30min, followed by washing twice with 2 ml 10% TCA, twice with ethanol-ether (1:1) and once with ether. The filters were finally incubated with 25/~1 tissue solubilizer (Serva) at 50°C for 2 h and counted in 2ml scintillation fluid (0.01% POPOP, 0.4% PPO and 0.25% acetic acid in toluene) for 10 min. Polyacrylamide gel electrophoresis (PAGE) and fluorography SDS-PAGE was performed with 7-20% acrylamide slab gels according to Laemmli (1970) but using a 2-fold concentrated buffer in the 20% acrylamide solution. Electrophoresis was carried out at constant current at 20 mA at 8°C overnight. The gels were fixed in 50% methanol and proteins stained with silver after the following protocol. After 2 h fixation the gels were incubated for 1.5 h in a silver solution (0.5 g AgNO 3, 0.9 ml NH 3, 12.5 ml 0.1 M NaOH, 30 ml methanol p.a., 30 ml H20 per gel) protected from light. They were then washed 3 times in water and developed in 50% methanol with 12% sodium thiosulfate. For fluorography the gels were fixed and stained in 0.13% Kenacid Blue (BDH) in 50% methanol and 5% acetic acid (Harding and Scott, 1983). After destaining, the gels were processed according to Skinner and Griswald (1983). Dried gels were exposed on preflashed Kodak-X-Omat AR X-ray films at -70°C. Molecular weight markers were from BioRad (200-45 kD) and BDH (17-2.5 kD). Radioactive markers (t4C-labeled) were from BRL (43-3 kD).
Insect immunity, in vitro studies
301 RESULTS
Protein determination
The protein content was determined in microtiter plates (NUNC) using 10pl sample and 200pl Coomassie Blue G250 solution according to Bradford (1976) at 600 nm. BSA in the range of 50-500pg/ml was used as standard.
Assay conditions
During our initial efforts to establish optimal assay conditions for in vitro induction experiments we have studied the influence of different media and osmotic pressure on incorporation kinetics in long-time cultures. Pieces of "untreated" and "injured" fat body from SC-pupae were incubated in three different media (Table 1). The media differed in osmolarity (media I and II) and amino acid composition (media II and III). In the first 24 h the cultured fat body released proteins in the range of 1.5-5.5 pg/mg fat body. The amount of protein was about 2 times higher in medium I compared to media II and III. The fat body showed protein synthesis up to 15 days (later time points were not investigated), however, the rate of synthesis was relatively constant only during the first 4 days. Kinetic studies showed that the increase of newly synthesized proteins released into the medium was directly correlated to the increase of synthesis in the fat body (Fig. 1). Pulse chase experiments (not shown) with 4 h radioactive incorporation after 1 and 4 days also showed that the increase of labeled proteins in the medium was linear up to 7 h (later time points not examined). The incorporation rate in "injured" fat body was higher than in "untreated" fat body. Incorporation of [3H]lysine in TCA-precipitable proteins from "untreated" and "injured" SC-fat body during 24 h is shown in Fig. 1 and Fig. 2 / O A , B, respectively. After 1 day in culture, the incorporation in released proteins from "injured" SC-fat body was in all three media about 7- to 14-fold higher than that obtained from "untreated" SC-fat body.
lmmunoprecipitation
Aliquots of culture supematants, containing 15,000cpm of labeled protein, were incubated with 5 pl pre-immune serum (rabbit) and 25 #1 incubation buffer (10mM Tris, 2.SmM EDTA, 0.15M NaC1, 0.1% NP40 and Img/ml BSA; pH7.5) at 4°C overnight. Sheep-anti-rabbit Ig antibodies (5#1) were added and after 15min at 22°C, the precipitates were centrifuged at 10,000g. The supernatants were incubated at 4°C for 2h with 10pl of monospecific rabbit antibody (Ig fraction) against P4, attacins, lysozyme, synthetic cecropin A(1-37) (Merrifield et al., 1982), arylphorin and flavoprotein, respectively. Antibody against arylphorin and flavoprotein was a generous gift from W. Teller. After a second incubation with sheepanti-rabbit Ig antibody (10#1) at 22°C for 15rain the samples were centrifuged at 10,000g and 4°C for 15rain. The immunoprecipitates were washed twice in incubation buffer, and dissolved in 25 #1 sample buffer at 95°C for 5 min prior to SDS-PAGE. Induction experiments in vitro
After 1 day in culture, the fat body was treated with different agents for 2 days. The following additives, in 20 pl medium, were used: 5 mg/ml LPS, 0.2 mg/ml M. lysodeikticgs, heat-treated E. coil (20#1 of a culture in log-phase), 1)4 (11 pg/culture), 20-hydroxyeedysone (25 pg/ml, resulting in 10-6 M 20-hydroxyecdysone per culture; Sigma), washed epidermis-free cuticle, wing epidermis from a wounded pupae, and washed hcmocytes from either untreated, injured or vaccinated pupae.
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Fig. I. Total protein synthesis in SC-fat body cultured in different media. After 1 day (a, c) and 3 days (b, d) in culture fat bodies from "untreated" (open symbols) and "injured" (closed symbols) SC-pupac were incubated with [3H]lysine up to 24 h. At each time point two samples were taken from medium I (Fq, I ) , medium II (A, A) and medium III (O, O). LB 18/~-E
TIBIA TRENCZEKand INOmD FAYE
302
months (LC) were used since such pupae start adult development without delay. As shown in Fig. 2/(~)A the amount of synthesized and released proteins from "untreated" SN-fat body was high and reached levels of"injured" SC-fat body (Fig. 2 / 0 B . There was no significant differences between "untreated" and "injured" SN-fat body (Fig. 2 / O A,B). In both types of cultures we observed a slightly higher synthesis after 3 days. Homebred LC-pupae showed an even higher incorporation of [3H]lysine into released proteins (Fig. 2/(~)Aa). Experiments with "injured" LC-fat body was not carried out as the fat body of most of these pupae had started to disintegrate. Only one "untreated" pupa that did not show signs of disintegrated fat body after it had been adjusted to room temperature, could be investigated (Fig. 2/(~)Ab). The release of labeled proteins into medium III was slightly lower than from "untreated" pupae taken directly from the cold.
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Fig. 2. Synthesis and release of proteins in relation to the different physiological stage of the fat body. Fat body from "untreated" (A) and "injured" (B) pupae were incubated for 24 h with [3H]lysine after 1 day (first column~ and 3 days (second column) in culture. The pupae were: Q) SC-pupae, (~ SN-pupae and (~) LC-pupae (Aa, not adjusted to room temperature; Ab, adjusted to room temperature prior to dissection). The bars indicate the SEM values of 6 determinations (of 2 determinations in 3Ab).
After 3 days, "untreated" SC-fat body in media II and III showed a slight increase of labeled proteins but still the total amount was 8- to 10-fold lower than from "injured" SC-fat body. In medium I, however, protein synthesis in "untreated" SC-fat body reached comparable levels as that found in "injured" SC-fat body (Fig. lb, d). Since the difference in the basic incorporation level between individual pupae was high, the kinetic studies were analyzed by comparing fat body samples from the same pupae (see Fig. 1).
Protein synthesis in relation to developmental stages Chilled pupae in diapause are asynchronous in terms of the onset of adult development when they are transferred to room temperature during the experiment (Hirano and Gilbert, 1967; Rasenick et aL, 1976, 1978). Therefore we repeated the same experiments with two different batches of pupae in diapause. On the one hand, unchilled pupae (SIN) of the same age were chosen to avoid the onset of the adult development by the temperature shift. On the other hand, pupae which had been kept chilled for 5.5
Electrophoretic analysis of proteins For further clarification of the experimental conditions we analyzed the proteins which were synthesized and secreted by different fat body cultured in different media. All proteins released during 24h after 1 and 3 days in vitro were characterized by SDS--PAGE (Fig. 3). In all samples the main polypeptides are the same, namely P4 (48 kD), attacins (neutral and basic form, 20kD), and lysozyme (13.8 kD) as well as several polypeptides in the range of 75-100kD. Two other polypeptides that were released from the fat body (indicated by ~ ) have apparent molecular weights of about 40,000 and 25,000 and were more prominent in the medium as compared with the hemolymph. The newly synthesized proteins which were released from "untreated" LC-fat body in the media were demonstrated via fluorography in Fig. 4. The "immune proteins" as well as arylphorin and the flavoprotein, and two of the proteins in the range of 7~--100kD, were identified by immunoprecipitation combined with SDS-PAGE and fluorography (Fig. 5). In all fat body cultures after l day, irrespectively of their origin, the two heavily labeled bands were P4 and the attacins. The basic and neutral form of the attacins are not distinguishable due to the overexposure. Lysozyme, cecropins and two polypeptides of 220 and 180kD as well as polypeptides of 55 kD and in the range of 38-40 kD were also labeled. No incorporation could be found in the region of 75-100 kD (Fig. 4, lanes 5-8). The pattern of labeled proteins from the first 24 h after dissection showed a weak label in the polypeptides of 75-100 kD (not shown). When after 3 days in vitro fat body samples from the same pupae were incubated with [3H]lysine, P4 and attacin were synthesized as before and the incorporation into lysozyme and the peptides of 220 and 180 kD was higher. Moreover, at this time proteins >300 kD, in the range of 75-100, 60 and 30kD were synthesized. The radioactive incorporation in the proteins of 75-100 kD showed slight variations between individual pupae and the three different media tested. Adjusting the pH of the medium with NaOH instead of KOH did not result in any difference (Fig. 4, lanes 3 and 4). Polypeptides of 6 and 8 kD
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Fig. 3. SDS-PAGE analysis of pupal hemolymph proteins and proteins secreted from fat body in culture: (a) hemolymph samples (1 #1) were from untreated (1), injured (2) and vaccinated (3) pupae; (b) proteins secreted from fat body in the 3 different media tested, medium I (lanes 1, 2), medium II (lanes 3, 4), medium III (lanes 5, 6). Samples were taken after 1 day (lanes 1, 3, 5) and 3 days (lanes 2, 4, 6). References were a crude preparation or P4 (lane 7), and highly purified neutral and basic attacins (lane 8). The "immune proteins" are indicated by their first letter P4 (P4), attacins (A), lysozyme (L) and cecropins (C). The attacins were kindly provided by Anette Carlsson (Department of Immunology, Uppsala University).
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Fig. 4. SDS-PAGE analysis of proteins synthesized and secreted from fat body during in vitro culture. Each fat body sample was incubated with 50 #Ci [3H]lysine in 500 #1 medium for 24 h after 1 day (lanes 5-7) and 3 days (lanes 1-4) in culture. Samples with I0,000 cpm were separated on 7-25% polyacrylamide slab gels and the results visualized by fluorography. Lanes 1 and 5, LC-fat body in medium I; lanes 2 and 6, in medium II: lanes 3 and 7, in medium III; lane 4, in medium III; but adjustment of pH with NaOH instead of KOH. Lane 8, proteins synthesized and secreted from SN-fat bodies after 3 days in medium III; lane 9, corresponding Kenacid Blue stain to lane 8. The "immune proteins" are indicated as in Fig. 3, arylphorin and flavoprotein marked by Ay and F1, respectively.
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Fig. 5. Immunoprecipitation of "immune proteins" labeled with [3H]lysine. Medium samples of 15,000 cpm were incubated with the different antibodies, the precipitates subjected to SDS-PAGE and the dried gels exposed to X-ray films. Lane M: medium sample from "immune" fat body after 1 day in culture. Immunoprecipitates of cecropin (C), lysozyme (L), attacin (A), P4 (P4), arylphorin (Ay) and flavoprotein (F1). Arylphorin and flavoprotein were precipitated from an "immune" fat body after 3 days in culture. Low cross reactivities of the cecropin antibody to the highly labeled P4 and attacins could not be avoided.
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?ig. 6. S D S - P A G E analysis of induction experiments in vitro. 10,000 cpm of TCA-precipitable proteins of culture supernatants were subjected to SDS PAGE and the results visualized 9y fluorography. To "untreated" fat body was added: supernatants from hemocyte cultures that had been treated with Ringer (a), hemolymph (b), M. lysodeikticus (c) or LPS (d); or hemocytes "rom LC-pupae vaccinated with M. lysodeikticus (e) and LPS (f) respectively; or cuticle (g); damaged wing epidermis (h); P4 (i). Control: "untreated" LC-fat body. SN-fat body was incubated ~ith heat killed E. coli (1), M. lysodeikticus (m), LPS (n) or Ringer (o). Controls: "injured" SN-fat body after 3 days (p) and 1 day (q) and "untreated" SN-fat body after 3 days (r) in culture. "Untreated" SN-fat body incubated with hemocytes from LPS-vaccinated (s), injured (t) and untreated (u) pupae.
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Insect immunity, m vitro studies were now synthesized more than the cecropins (ca 4 kD). "Injured" fat body expressed the same protein pattern as the "untreated" fat body. In contrast, SN-fat body, neither "untreated" nor "injured", showed any label in the region of 75-100, 60 or 30 kD after 1 and 3 days in culture (Fig. 4, lane 8; Fig. 6, lanes p-r). This is due to missing synthesis and not caused by inhibition of protein release or by degradation as illustrated by the Kenacid Blue counterstain of the same sample (Fig. 4, lane 9). Studies with "immune" fat body Since previous results from Faye and Wyatt (1980) showed that the antibacterial activity in the fat body cultures from vaccinated pupae declines, we reexamined this question. To avoid any effect of live bacteria we vaccinated the LC-pupae with either LPS or dead cells of M. lysodeikticus. Injections of 2mg/ml LPS (E. coli D21) in vivo resulted after 8 days in a 50% response of that when injected with live bacteria and no difference in lysozyme activity. Freeze dried cells of M. lysodeikticus injected in vivo (0.2mg/ml) gave only a 30% response of antibacterial activity but comparable lysozyme activity as LPS or live bacteria did. Supernatants taken from "immune" fat body after 1, 2 and 3 days in vitro showed a continuously decreasing synthesis of P4, attacins, lysozyme, cecropins and the polypeptides between 38--40 kD, but a continuously increasing synthesis of the proteins of 75-100 and 180 kD, and higher molecular weight (Fig. 7). Studies in vivo For comparison, in vivo protein labeling with [35S]methionine was carried out. SN-pupae were taken to avoid the onset of adult development. Each pupa received 50/~Ci [35S]methionine in 50/Jl Ringer or in 50/~1 Ringer containing 5 mg/ml LPS. Incubation times were 30 rain, 1, 2, 3, 5, 7, 11 and 24 h, respectively. Constant volumes of hemolymph were analyzed using SDS-PAGE. The radioactive protein pattern obtained in the first 24 h was the same for the injured and immune pupae (Fig. 8). Synthesis of P4 was already detectable after 2 h but distinct at 3 h, and attacins could be seen after 5 h. For lysozyme and cecropins [35S]methionine is not the optimal amino acid to label with (see Engstrtm et al., 1985, Hultmark et al., 1982). Therefore they first were visible after 24 h. One protein of about 40 kD showed a high incorporation after 2 h with a maximum at 11 h. Whether the synthesis of this protein was induced de novo or not was not tested. Induction experiments in vitro To elucidate which factor might be involved in the injury/immune response several presumptive inducers were tested in vitro. The qualitative changes in the radioactive protein pattern were analyzed by fluorography (Fig. 6). When medium or medium with P4 was added to "untreated" LC-fat body no striking difference from the control was detected (Fig. 6, lanes i, k). Treatment with 20-hydroxyecdysone caused no change in the pattern of SN-fat body. However, in cultures of
307
LC-fat body 20-hydroxyecdysone stopped the synthesis of proteins of 75-100 kD (not shown). These proteins were also affected when cuticle or epidermis from wounded pupae was added to LC-fat body (Fig. 6, lanes g, h). Moreover, in the latter case the total amount of newly synthesized and released proteins decreased by about 50%. The addition of heat treated E. coli, LPS or M. lysodeikticus to LC-fat body stopped the synthesis of the proteins in the range of 75-100 kD (Fig. 6, lanes l-n). At the same time high amounts of cecropins were newly synthesized. Changes in the synthesis of P4, attacins or lysozyme were not possible to see due to the overexposure on the fluorographies but short time exposure showed a higher incorporation in these proteins too. Effect of hemocytes In some preliminary experiments we investigated whether hemocytes could transfer any signal to the fat body and if so how the fat body would react to it. Harvesting naive, i.e. not spreading hemocytes (see Cherbas, 1973) from H. cecropia was possible both with preinjection of ACR (Leonard et al., 1985) and directly dropping the hemolymph into ice-cold ACR. Preinjection of ACR did not change the protein pattern but resulted in an enormous increase in total protein synthesis. Thus we ommited the preinjection step. Washed hemocytes from either untreated, injured or vaccinated pupae were added to cultures of "untreated" LC- and SN-fat body and the newly synthesized proteins characterized as before. The addition of "injured" or "immune" hemocytes to the fat body resulted in a higher synthesis of cecropins, lysozyme, the 6-8 kD peptides and to some extent of P4 and attacins (Fig. 6, lanes e, f, s, t). That is to be compared with the addition of naive hemocytes which did not have any effect (Fig. 6, lane u). We obtained similar results when adding 20/zl of supernatants from primary cultures of hemocytes, that had been incubated 10h with 20/zl Ringer, sterile filtered hemolymph, LPS or M. lysodeikticus (Fig. 6, lanes a-d). In both tests the protein synthesis of hemocytes in culture alone was negligible if compared with the fat body in vitro.
DISCUSSION Influence of media and developmental stages Observations made by Kurtti et al. (1975) and Abu-Hakima (1981) revealed that differences in medium composition influence the metabolism of cultured cells and organs, and led us to test media conditions for H. cecropia fat body cultures again. Our idea was that a proper medium should only reflect normal stage specific protein synthesis and not induce "immune protein" synthesis. Three media were tested for constant rates of protein synthesis under long term conditions. Furthermore we analyzed the synthesized proteins by SDS--PAGE and fluorography. By the addition of vitamins, organic acids, and PIPES to the medium of Reddy and Wyatt (1967) fat body protein synthesis continued for at least 15 days, a longer time than reported. This was even possible with increasing rates of synthesis after 4 days. Reddy
308
TINA TRENCZEK and INGRID FAYE
and Wyatt (1967) found a decline in the rate of incorporation after 20 h when they incubated wing epidermis in a medium which did not contain these additives. We did not supplement the media with any protein since BSA (Andersson and Steiner, 1986) and FCS (our observation) gave a slightly higher antibacterial response in vivo than did Ringer. A change in osmolarity from 343 mOsm/kg H20 (medium I) to 430 and 440 mOsm/kg H20 (media II and III) had a remarkable effect on fat body from SC-pupae, which were in deep diapause. The initial rate of protein synthesis in media II and III was maintained for 4 days but not in medium I. The low incorporation rates reflect the diapausing status of the fat body and is not due to any effect of the high osmotic pressure such as lysis of the cells which would stop the incorporation totally (Stevenson and Wyatt, 1962; Reddy and Wyatt, 1967). Further evidence for this interpretation is that "injured" SC-fat body or "untreated" and "injured" SN- and LC-fat body, representing other physiological stages, show high rates of protein synthesis. It is not likely that the higher rate of protein synthesis of "untreated" LC- and SN-fat body is only a sign of the "injury effect" of dissection since all types of fat body were prepared in the same way. The differences in protein synthesis between SCpupae and SN-pupae was about 10-fold (Fig. 2/C)A, QA). That reflects the fact that the basic metabolism in diapause is higher at 25°C than at 6°C. The LC-fat body synthesized and released an even higher amount of protein than the SN-fat body. The hemolymph of LC-pupae chilled for 5.5 months, i.e. long enough to start adult development (Hirano and Gilbert, 1967) had an osmolarity 540 + 20 mOsm/kg H20. This is close to values found in pupae at the second day of adult development (Jungreis et al., 1973). These results indicate that SC-fat body after about 4 days in culture might change metabolism, adjusting to conditions found at the very beginning of adult development (higher temperature, lower osmotic pressure). LC-pupae however, had already switched on their internal program for the imminent onset of adult development. Preparative changes of adult development such as increased activity by various oxidative enzyme systems several days before the first morphological signs of development can be detected were reported by Shappirio (1960). A higher metabolism of SN- and LC-pupae than SC-pupae would also explain the only slight difference between "untreated" and "injured" SN- and LC-fat body in vitro. This idea is supported by the following data. The second way of optimizing the experimental conditions was to analyze the pattern of newly synthesized proteins. Fat body samples from the same pupae synthesized the same proteins in all three media (Fig. 4). However, with LC- and SC-fat body in medium III we observed a relative decrease in the synthesis of the "immune proteins" and an increased synthesis of other proteins. The latter are described as subunits of normal serum proteins such as lipophorin [270 and 88 kD (Chino et al., 1969)], larval storage proteins (LSP 1 and LSP 2 with 89 and 85 kD (Tojo et al., 1978)], flavoprotein [83 kD (Telfer, personal communication)], arylphorin [73 kD (Telfer, 1983)] and vitellogenin [180 and 44kD (Kunkel and Pan,
1976)]. "Immune" LC-fat body gave the same results (Fig. 7). Boman and Steiner (1981) reported a similar phenomenon in vivo after 7 days, when injured pupae synthesized these proteins whereas immunized pupae did not. At this time, injured pupae express no antibacterial activity and have returned to normal conditions. Therefore we will call this phenomenon a "recovery effect". The observation that SN-fat body did not show this effect might be explained by the fact that this fat body did not undergo a temperature shift, i.e. it is still under the resting condition of diapause. Surprisingly, these proteins were also affected by adding 20-hydroxyecdysone, cuticle, wing epidermis, bacteria or hemocytes (Fig. 6). Turn-off effects of normal serum proteins are reported for the heatshock situation in Drosophila (Ashburner and Bonner, 1979), in an Aedes cell line (Carvalho and Rebello, 1987), and as culture shock in Xenopus laevis (Wolffe et al., 1984). The latter authors describe a drastic loss of albumin mRNA parallel with the occurrence of heat shock-like proteins after preparing primary cultures of hepatocytes and the acquisition of full responsiveness of these cells to estrogen is first observed after 2 days in culture. Concerning these observations we do not know if the proteins of H. cecropia described above are unlabeled because of diapause conditions or by a switch-off effect caused by injury or infection although the exact stage of a fat body in vitro is known. Based on the good simulation of diapause conditions, the relative constant protein synthesis between one and four days in culture and the better "recovery effect" we chose medium III for further studies. Influence o f different treatments Until now no factors, bacterial or other, are known to selectively stimulate the immune response in insects. It has, however, been reported that different bacterial substances and hemocytes are involved in the inducing mechanisms of the immune response (Dunn et al., 1985, Andersson and Cook, 1979). In the case of H. cecropia the injury of dissection already induces the synthesis of all "immune proteins". Therefore, we were restricted to search for enhancing stimuli. Furthermore, previous discussions about the possibility of ecdysteroids being involved in the injury effect (Barth et al., 1964; Berry et al., 1967) led us to include this hormone in our studies. Based on the incorporation of [3H]lysine into the immune proteins, especially cecropin, in the different experiments we conclude that:
1. Addition of bacterial substances enhanced the "immune protein" synthesis. Medium added as a control had no effect. 2. Hemocytes from vaccinated pupae also increased "immune protein" synthesis as compared to hemocytes from untreated pupae. 3. Hemocytes from injured pupae and epidermis raised the "immune protein" synthesis as compared to naive hemocytes and medium respectively. 4. Other factors (salt concentration, osmolarity) as indicated by the addition of Ringer (without sucrose), but not P4 or cuticle seemed to
I
2
3
4
Ft Ay
P4
A
C
Fig. 7. SDS-PAGE and fluorography of proteins from "immune" fat body. The fat body was incubated for 24 h the 1st (lane I), 2nd (lane 2) and 3rd day (lane 3) in culture. Samples were prepared and analyzed as described in Fig. 4. Lane 4: reference sample from an "untreated" fat body after 3 days in culture. Identified proteins are marked as in Figs 3 and 4,
309
6
5
4
3
2
1
2
3
4
5
6
b Fig. 8. SDS-PAGE and fluorography of hemolymph proteins synthesized in vivo. Pupae were injected with 50/iCi [35S]methionine in 50/H Ringer (a, male) or in 50 ~tl Ringer containing 5 mg/ml LPS (b, female), respectively. No incorporation was detectable up to 1 h (lane 1). Lanes 2~i: hemolymph samples taken after 3, 5, 7, 11 and 24 h, respectively. Attacins (A), P4 (P4) and vitellogenin (Vg).
310
Insect immunity, in vitro studies influence the "immune protein" synthesis in the same way. In vivo the response to injury is lower and has a
shorter duration than that caused by bacterial infections. Kinetic studies in vitro might show similar differences between the substances used in the present experiments. 20-Hydroxyecdysone stopped the synthesis of the 75-100 kD proteins in LC-fat body but no effect was seen in SN-fat body. A possible explanation is that the addition of the hormone simulates events at the beginning of adult development when the titer and synthesis of larval and pupal proteins is known to drop (Telfer and Williams, 1953; Patel, 1971). However, stress phenomena as described above cannot be excluded. The most striking difference to the experiments from Dunn et al. (1985) is the missing "injury effect" in Manduca larvae as judged by the absence of antibacterial activity of culture supernatants of "untreated" fat body. Dunn and coworkers did not treat their cultures with Ringer or non-bacterial substances like damaged insect tissue. In the present experiments testing antibacterial activity with E. coil D31 plates was not sensitive enough to prove a relationship between synthesis of "immune proteins" and appearance of antibacterial activity. This is, however, strongly indicated by the findings of Faye and Wyatt (1980). The described differences could be due to the different developmental stages investigated. Schneiderman (1957) reported that the injury metabolism is only characteristic of diapausing pupae. In this case the few "injury effects" described in Manduca (Hughes et al., 1983) may be due to minor infections or contaminations with bacterial cell wall. On the other hand the "injury effect" as such is not restricted to diapausing pupae of H. cecropia as shown with other lepidoptera (Marek, 1969; Anderson and Cook, 1979) and in diptera (Komano et al., 1980; Takahashi et al., 1986; Keppi et al., 1986). But as long as the experiments are not done under the same conditions, the differences remain unexplained. The data obtained in this study clearly show that no bacterial agents are necessary to induce the synthesis of P4, attacins, lysozyme and cecropins. The findings from Faye and Wyatt (1980) that untreated fat body synthesize proteins in the molecular weight range of P4 and attaeins (P5) were confirmed by immunoprecipitation and extended to also include lysozyme and cecropins. We could not show any qualitative differences of "immune protein" synthesis between "untreated", "injured" and "immunized" fat body during the first days of in vitro culture. The primary inducing agent(s) remain unknown. Two different mechanisms of induction might exist, one relatively unspecific at the very beginning and another being of a more specific nature maintaining, if necessary, the already induced antibacterial defense mechanism. In the latter case the involvement of hemocytes is clearly shown. Hemocytes from vaccinated pupae also enhance the synthesis of "immune proteins", while naive hemocytes show no effect. Since hemocytes from "injured' pupae or Ringer gave comparable results, signals other than through hemocytes-processed bacterial substances must also
311
be involved. Stress-linked metabolic events such as reported by Ziegler et ai. (1979) should not be neglected and detailed quantitative kinetic studies of the "immune protein" synthesis are necessary for further clarification. kindly acknowledges a postdoctoral fellowship from Gunnar Hannsons forskningsstiftelse. This work was also supported by the Swedish Natural Science Research Council (BU 245 to H. G. Boman) and the Swedish Medical Research Council (Grant 13X-3556 to H. Bennich). We wish to thank Dr H. Bennieh for critical reading of the manuscript. Acknowledgements--TT
REFERENCES
Abu-Hakima R. R. ( 1981) Vitellogenin synthesis induced in locust fat body by juvenile hormone analogue in vitro. Experienta 37, 1309-1311. Andersson K. and Steiner H. (1986) Structure and properties of protein P4, the major bacteria-inducible protein in pupae of Hyalophora cecropia. Insect Biochem. 17, 133-140. Andersson R. S. and Cook M. L. (1979) Induction of lysozymelike activity in the hemolymph and hemocytes of an insect Spodoptera eridania. J. Invert. Path. 33, 197-203. Ashburner A. and Bonner J. J. (1979) The induction of gene activity by heat shock. Cell 17, 241-254. Barth R. H., Bunyarad P. P. and Hamilton T. H. (1964) RNA metabolism in pupae of the oak silkworm, Antheraea pernyi: the effect of diapause, development, and injury. Proc. nam. Acad. Sci. U.S.A. 52, 1572-1580. Berry S. J., Krishnakumaran A., Oberlander H. and Schneiderman H. A. (1967) Effects of hormones and injury on RNA synthesis in saturniid moths. J. Insect Physiol. 13, 1511-1537. Boman H. G. and Steiner H. (1981) Humoral immunity in Cecropia pupae. In Current Topics in Microbiology and Immunology (Edited by Henle W. et al.), Vol. 94/95, pp. 75-91. Springer, Berlin. Boman H. G., Nilsson-Faye I., Paul K. and Rasmuson T. Jr (1974) Insect immunity I. Characteristics of inducible cell-free antibacterial reaction in hemolymph of Samia cynthia pupae. Infect. Immun. 10, 136-145. Boman H. G., Boman A. and Pigon A. (1981) Immune and injury responses in Cecropia pupae-RNA isolation and comparison of protein synthesis in vivo and in vitro. Insect Biochem. 11, 33-42. Bradford M. (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye binding. Analyt. Biochem. 72, 248-254. Carvalho M. G. C. and Rebello M. A. (1987) Induction of heat shock proteins during the growth of Aedes albopictus cells. Insect Biochem. 17, 199-206. Cherbas L. (1973) The induction of an injury reaction in cultured hemocytes from saturniid pupae. J. Insect Physiol. 19, 2011-2023. Chino H., Murakami S. and Harashima K. (1969) Diglyceride-carrying lipoproteins in insect hemolymph. Isolation, purification and properties. Biochim. biophys. Acta 176, 1-26. Duchateau G., Florkin M. and Leclercque J. (1953) Concentrations des bases fixes et types de composition de la base totale de l'hemolymphe des insectes. Archs int. Physiol. LXI(4), 518-549. Dunn P. E., Dai W., Kanost M. R. and Geng Ch. (1985) Soluble peptidoglycan fragments stimulate antibacterial protein synthesis by fat body from larvae of Manduca sexta. Devl comp. Immun. 9, 559-569.
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Engstr6m A., Xanthopoulos K. G., Boman H. G. and Bennich H. (1985) Amino acid and cDNA sequences of lysozyme from Hyalophora cecropia. EMBO Jl 4, 2119-2122. Faye I. and Wyatt G. R. (1980) The synthesis of antibacterial proteins in isolated fat body from Cecropia silkmoth pupae. Experientia 36, 1325-1326. G6tz P. and Boman H. (1985) In Comprehensive Insect Physiology, Biochemistry, and Pharmacology (Edited by Kerkut G. A. and Gilbert L. I.), pp. 453-485. Pergamon Press, Oxford. Harding C. R. and Scott I. R. (1983) Fluorographylimitations on its use for the quantitative detection of 3Hand ~4C-labelled proteins in polyacrylamide gels. Analyt. Biochem. 129, 371-376. Harvey W. R. and Williams C. M. (1961) The injury metabolism of the Cecropia silkworm. I. Biological amplification of the effects of localized injury. J. Insect Physiol. 7, 81-99. Hirano Ch. and Gilbert L. I. (1967) Phosphatidate phosphohydrolase in the fat body of Hyalophora cecropia. J. Insect Physiol. 13, 163-174. Hughes J. A., Hurlbert R. E., Rupp R. A. and Spence K. D. (1983) Bacteria-induced haemolymph proteins of Manduca sexta pupae and larvae. J. Insect Physiol. 29, 625-632. Hultmark D., Engstr6m A., Bennich H., Kapur R. and Boman H. G. (1982) Insect immunity: isolation and structure of Cecropin D and four minor antibacterial components from Cecropia pupae. Eur. J. Biochem. 127, 207-217. Jungreis A. M. and Jatlow P. and Wyatt G. R. (1973) Inorganic ion composition of haemolymph of the Cecropia silkmoth: change with diet and onotgeny. J. Insect Physiol. 19, 223-225. Keppi E., Zachary D., Robertson M., Hoffmann D. and Hoffmann J. A. (1986) Induced antibacterial proteins in the haemolymph of Phormia terranovae (Dipterae). Insect. Biochem. 16, 395-402. Komano H., Mizuno D. and Natori S. (1980) Purification of lectin in the hemolymph of Sarcophaga peregrina larvae on injury. J. biol. Chem. 225, 2919-2924. Kunkel J. G. and Pan M. L. (1976) Selectivity of yolk protein uptake: comparison of vitellogenins of two insects. J. Insect Physiol. 22, 809-818. Kurtti T. J., Chaudhary S. P. S. and Brooks M. A. (1975) Influence of physical factors on the growth of insect cells in vitro. II. Sodium and potassium as osmotic pressure regulators of moth cell growth. In vitro 11, 274-285. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. Leonard C., S6derh/ill K. and Ratcliffe N. A. (1985) Studies on prophenoloxidase and protease activity of Blaberus craniifer hemocytes. Insect Biochem. 15, 803-810. Marek M. (1969) In vitro synthesis of injury protein and a companion protein in organs of prepupa of Galleria mellonella. Comp. Biochem. Physiol. 29, 1231-1237. Merrifield R. B., Vizioli L. D. and Boman H. G. (1982)
Synthesis of the antibacterial pvptide cecropin A (1-33). Biochemistry 21, 5020-5031. Patel N. G. (1971) Protein synthesis during insect development. Insect Biochem. 1, 391-427. Rasenick M. M., Neuburg M. and Berry S. J. (1976) Brain cyclic AMP levels and the initiation of adult development in the Cecropia silkmoth. J. Insect Physiol. 22, 1453-1456. Rasenick M. M., Neuburg M. and Berry S. J. (1978) Cyclic nucleotide activation of the silkmoth brain--cellular localization and further observation on the patterns of activation. J. Insect Physiol. 24, 137-139. Reddy S. R. R. and Wyatt G. R. (1967) Incorporation of uridine and leucine /n vitro by Cecropia silkmoth wing epidermis during diapause and development. J. Insect Physiol. 13, 981-994. Riddiford L. M. 0968) Artificial diet for Cecropia and other Saturniid silkworms. Science 160, 1461-1462. Schneiderman H. A. (1957) Physiological Triggers and Discontinuous Rate Processes (Edited by Bullock T. H.), pp. 46-59. Shappirio D. G. (1960) Oxidative enzymes and the injury metabolism of diapausing Cecropia silkworms. Ann. N.Y. Acad. Sci. 89, 537-548. Skinner M. K. and Griswald M. D. (1983) Fluorographic detection of radioactivity in polyacrylamide gels with 2,5-diphenyloxazole in acetic acid and its comparison with existing procedures. Biochem. J. 209, 281-284. Stevenson E. and Wyatt G. R. (1962) The metabolism of silkmoth tissues. 1. Incorporation of leucine into protein. Archs Biochem. Biophys. 99, 65-71. Stevenson E. and Wyatt G. R. (1964) Glycogen phosphorylase and its activation in silkmoth fat body. Archs Biochem. Biophys. 108, 420-429. Takahashi H., Komano, H. and Natori S. (1986) Expression of the lectin gene in Sarcophaga peregrina during normal development and under conditions where the defense mechanism is activated. J. Insect Physiol. 32, 771-779. Telfer W. (1983) Arylphorin, a new protein from Hyalophora cecropia: comparison with calliphorin and manducin. Insect Biochem. 13, 601-613. Telfer W. and Williams C. M. (1953) Immunological studies of insect metamorphosis. I. Qualitative and quantitative descriptions of the blood antigens of the Cecropia silkworm. J. gen. Physiol. 36, 389-413. Telfer W. H. and Williams C. M. (1960) The effect of diapause, development and injury on the incorporation of radioactive glycine into the blood proteins of the Cecropia silkworm. J. Insect Physiol. 5, 61-72. Tojo S., Betchaku T., Zicchardi V. J. and Wyatt G. R. (1978) Fat body protein granules and storage proteins in the silkmoth, Hyalophora cecropia. J. Cell Biol. 78, 823-838. Wolffe A. P., Glover J. F. and Tara J. R. (1984) Synthesis of heat-shock like proteins in fresh primary cell cultures. Expl Cell Res. 154, 581-590. Ziegler R., Ashida M., Fallen A. M., Wimer L. T., Wyatt S. S. and Wyatt G. R. (1979) Regulation of glycogen phosphorylase in fat body of Cecropia silkmoth pupae. J. comp. Physiol. 131, 321-332.
0020-1790/88 $3.00 + 0.00 Copyright © 1988 Pergamon Press plc
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SYNTHESIS OF I M M U N E PROTEINS IN PRIMARY CULTURES OF FAT BODY FROM HYALOPHOR,4 CECROPIA TINA TRENCZEK*
Department of Immunology, The Biomedical Center, Uppsala University, Box 582, 75123 Uppsala, Sweden INGRID FAYE Department of Microbiology, University of Stockholm, 10691 Stockholm, Sweden (Received 21 August 1987; revised and accepted 4 November 1987) Abstract--Fat body from injured or immunized pupae as well as from untreated pupae of Hyalophora cecropia synthesize and release in vitro the immune proteins P4, attacins, lysozymeand cecropins. During an incubation time of 4 days immune protein synthesis declines relative to the other proteins, i.e. protein synthesis returns to a normal pupal pattern. Treatment of fat body in culture with bacterial compounds (LPS, lyophilized cells of Micrococcus lysodeikticus or heat killed Escherichia coli), but also sterile filtered insect Ringer, causes a relative higher rate of synthesis of the immune proteins. At the same time synthesis of some large proteins (75-100 kD) is switched off. Similar results are obtained by adding hemocytes from injured or immunized pupae, whereas hemocytes from untreated pupae show no effect. The induction of protein synthesis with particular emphasis on the immune proteins will be discussed. Key Word Index: Hyalophora cecropia, insect immunity, protein synthesis, in vitro culture
INTRODUCTION Several insect species respond to injections of live bacteria by de novo synthesis of antibacterial proteins which are detectable in the hemolymph (Boman et al., 1974; G t t z and Boman, 1985). However, this response is not specifically induced by bacteria as injection with sterile Ringer solution will cause a transient synthesis of the same antibacterial proteins (Boman et al., 1974; Faye and Wyatt, 1980; Andersson and Steiner, 1986). Ever since the first physiological studies using diapausing pupae of Hyalophora cecropia it has been known that an injury causes increased protein synthesis (Teller and Williams, 1960), a higher level of oxygen consumption (Harvey and Williams, 1961), enhanced RNA synthesis (Barth et al., 1964; Berry et al., 1967) and changed enzyme activities (Stevenson and Wyatt, 1964). Harvey and Williams (1961) assumed that a factor is diffusing in the hemolymph and Cherbas (1973) isolated a factor, "haemokinin", from damaged wing epidermis which could simulate an injury effect in hemocytes. Faye and Wyatt (1980) showed that tissue cultures of fat body from untreated pupae release antibacterial activity into the medium and
synthesize P4 as well as attacins (PS), presumably induced by the injury of dissection. Although we regard dissection of the fat body to be an unavoidable and extensive injury to the insect we have chosen in vitro studies to clarify some aspects of the "injury effect". In this paper we report on the importance and effects of the medium, endogenous factors, and hemocytes on the synthesis of "immune proteins". MATERIALS AND METHODS
Animals and their treatments H. cecropia pupae were reared on a synthetic diet (Riddiford, 1968; Boman et al., 1981) or obtained commercially from North American dealers in the mid-west. For convenience, the pupae and fat body are named according to their storage conditions: SC-pupae were kept at 6-8°C for 2.5 months (short chilled/homebred), LC-pupae were kept at 6-8°C for 5.5 months (long chilled/homebred) and SN pupae were kept about 2 months in diapause at 22°C under short day conditions (short nonchilled/outdoor bred). LCand SC-pupae that were never adjusted to 22°C prior to the dissection and SN-pupae taken directly are defined as "untreated" pupae. Similarly,pupae (or fat body) that were given a single injection of 50pl sterile filtered Ringer (without sucrose, 310mOsm/kg H20) 3 days before dissection and then kept at 22°C are called "injured" pupae. *To whom all correspondence should be addressed. Abbreviations: ACR=anticoagulant Ringer, FCSffetal As a control, untreated pupae were kept 3 days at room calf serum, K-PBS= potassium-phosphate buffered temperature. "Immune" pupae (or fat body) were injected saline, LPS = lipopolysaccharide, PAGE = polyacryl- with 50/zl Ringer containing either 2 or 5mg/ml LPS amide gel electrophoresis, PIPES = piperazine-N,N'-bis- (Escherichia coil D21, a generous gift from H. G. Boman) (2-ethanesulfonic acid), POPOP= 1,4-di[2-(5-phenyl- or 0.2 mg/ml lyophilized and washed cells of Micrococcus lysodeikticus (Sigma, St Louis, Mo.) and then kept 3 days oxazolyl)]-benzene, PPO---diphenyloxazole, SDS= at 22°C. Antibacterial activity from the hemolymph of all sodiumdodecylsulfate, TCA = trichloroacetic acid. 299
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TINA TRENCZEK and INGRID FAYE
pupae was tested on E. coli D 31 plates according to Faye and Wyatt (1980).
Dissections and in vitro conditions The dissections were done under lepidopteran Ringer modified after Jungreis et al. (1973): 234mg NaC1, 9.5g KC1, 588mg CaC12(2H20), 3.05g MgC12(6H20), 25ml potassium-phosphate buffer (pH 6.5) and 34.23 g sucrose/l, pH 6.5; osmolarity, 440 + 5 mOsm/kg H20. In vitro incubations were performed in three media (Table 1), which differed in either osmolarity or amino acid composition. The three media were tested in parallel with double samples of fat body from the same pupae. Unless otherwise stated, all experiments were done in triplicate. The osmolarity was determined with the osmometer 3W2 (Advanced Instruments Inc., Needham Heights, Mass.). The pupae were washed in 0.5% iodine solution (Jodopax, Ferrosan), 70% ethanol, and sterile water and then quickly bled through a triangle-shaped cut made in the lateral thorax to reduce the influence of possible hemolymph factors. After dissection under Ringer with a few crystals of PTU (phenylthiourea), free fat body was transferred to fresh Table 1. Composition of the media Medium (raM) Substance
I/IP
III b
NaHCO2 KCI CaCI2 (2H20) MgCI2 (6H20) Mg2SO4 (7H20) KH2PO4
4 50 4 15 -8
4 50 3.1 14 1 8
D-Glucose Trehalose
5 20
5 20
Sucrose
(50)°
50
#-Alanin Arginine-HC1 Asparagine Asparagine acid Cysteine-HCI Glutamine Glutamine acid Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptopbane Tyrosine Valine
5 5 5
5 2 5 5 10 15 2 1 5 2 2 5 10 2 2 1 2
5 6 I 1 0.5 5 6 5 6 4 4 10 2 1 20 12 5 1 1 5
K-fumarate 1 I K-malate 5 5 K-pyruvate 1 1 K-succinate I 1 -Ketoglutarate 2.5 2.5 PIPES 25 25 Vitamins (100 x )a, /~1 10 10 p-Aminobencoic acidd, ~g 2 2 Phenolredd, mg 1 1 Gentamicind, mg 20 20 aModifications of the medium used by Reddy and Wyatt (1967). bAmino acid compositionwas chosen followingthe results of Duchateau et al. (1953). Amino acids were used in t- or in DL- form, vitamins were from Flow (medium MEME). The pH of all three media was adjusted with KOH. ¢Only medium II. aThe indicated amounts were added to 100ml of medium.
Ringer with PTU. Malpighian tubules, nerves, trachea and tissue from the heart complex were carefully removed (15min). Thereafter, the fat body was washed in Ringer without PTU (15 min), medium with PTU (15 min), medium without PTU (45 min). Finally, pieces of about 50 mg fat body were incubated in 500/~1 medium. This procedure was found to be essential since fat body rinsed only briefly in Ringer will "release" a remarkable number of hemocytes that spread all over the incubation vessel. As incubation vessels, multidishes (24/3.4ml) from NUNC (Denmark) were used. All incubations were carried out under gentle shaking at 27°C, in 5% CO 2 and 90% r.h.
Hemocytes Hemocytes were isolated by dropping hemolymph directly into ice-cold anticoagulant Ringer (ACR): 23.4 mg NaCI, 950mg KC1, 292mg EDTA, 198mg citric acid, 798mg tert. Sodium citrate, 2.5ml potassium-phosphate buffer (pH 6.5) and 2.74g sucrose in I00 ml; osmolarity, 455 mOsm/kg H20. After gentle shaking, the cells were centrifuged at 200g for 10min at 4°C (Sorvall RT 6000), washed 3 times in ACR and resuspended in medium before USe. Incorporation studies After 1 and 3 days in culture, fat body was incubated at 27°C for 24 h in 500t~l medium containing 0.5 mM lysine and 10/~Ci [3H]lysine (for the determination of the total protein synthesis) and 50 #Ci [3H]lysine (for characterizing the specific proteins). In both cases the specific activity was 95.8 mCi/mmol lysine. Controls were incubated with 10-3M cycloheximide added already 30 min prior to the incorporation period. The "labeled" fat body was rinsed twice in ice-cold potassium-phosphate buffered saline (K-PBS: 9.5 g KCI, 234 mg NaC1, 212 mg NaH2PO 4, 1.79 g K2HPO4/1, pH 6.5), briefly dried on filter paper, weight and homogenized in 100 #1 K-PBS. After extraction 3 times with 100 #1 K-PBS, pooled supernatants were centrifuged at 10,000 g at 4°C for 5 rain, and the supernatants stored at -20°C. Aliquots of culture supernatants or supernatants from the fat body homogenates were spotted and dried on 1 crn2 Whatman 3 mm fllterpaper, precipitated with 2ml 10% TCA (containing 10mM lysine) at 4°C and afterwards incubated at 80°C, each step for 30min, followed by washing twice with 2 ml 10% TCA, twice with ethanol-ether (1:1) and once with ether. The filters were finally incubated with 25/~1 tissue solubilizer (Serva) at 50°C for 2 h and counted in 2ml scintillation fluid (0.01% POPOP, 0.4% PPO and 0.25% acetic acid in toluene) for 10 min. Polyacrylamide gel electrophoresis (PAGE) and fluorography SDS-PAGE was performed with 7-20% acrylamide slab gels according to Laemmli (1970) but using a 2-fold concentrated buffer in the 20% acrylamide solution. Electrophoresis was carried out at constant current at 20 mA at 8°C overnight. The gels were fixed in 50% methanol and proteins stained with silver after the following protocol. After 2 h fixation the gels were incubated for 1.5 h in a silver solution (0.5 g AgNO 3, 0.9 ml NH 3, 12.5 ml 0.1 M NaOH, 30 ml methanol p.a., 30 ml H20 per gel) protected from light. They were then washed 3 times in water and developed in 50% methanol with 12% sodium thiosulfate. For fluorography the gels were fixed and stained in 0.13% Kenacid Blue (BDH) in 50% methanol and 5% acetic acid (Harding and Scott, 1983). After destaining, the gels were processed according to Skinner and Griswald (1983). Dried gels were exposed on preflashed Kodak-X-Omat AR X-ray films at -70°C. Molecular weight markers were from BioRad (200-45 kD) and BDH (17-2.5 kD). Radioactive markers (t4C-labeled) were from BRL (43-3 kD).
Insect immunity, in vitro studies
301 RESULTS
Protein determination
The protein content was determined in microtiter plates (NUNC) using 10pl sample and 200pl Coomassie Blue G250 solution according to Bradford (1976) at 600 nm. BSA in the range of 50-500pg/ml was used as standard.
Assay conditions
During our initial efforts to establish optimal assay conditions for in vitro induction experiments we have studied the influence of different media and osmotic pressure on incorporation kinetics in long-time cultures. Pieces of "untreated" and "injured" fat body from SC-pupae were incubated in three different media (Table 1). The media differed in osmolarity (media I and II) and amino acid composition (media II and III). In the first 24 h the cultured fat body released proteins in the range of 1.5-5.5 pg/mg fat body. The amount of protein was about 2 times higher in medium I compared to media II and III. The fat body showed protein synthesis up to 15 days (later time points were not investigated), however, the rate of synthesis was relatively constant only during the first 4 days. Kinetic studies showed that the increase of newly synthesized proteins released into the medium was directly correlated to the increase of synthesis in the fat body (Fig. 1). Pulse chase experiments (not shown) with 4 h radioactive incorporation after 1 and 4 days also showed that the increase of labeled proteins in the medium was linear up to 7 h (later time points not examined). The incorporation rate in "injured" fat body was higher than in "untreated" fat body. Incorporation of [3H]lysine in TCA-precipitable proteins from "untreated" and "injured" SC-fat body during 24 h is shown in Fig. 1 and Fig. 2 / O A , B, respectively. After 1 day in culture, the incorporation in released proteins from "injured" SC-fat body was in all three media about 7- to 14-fold higher than that obtained from "untreated" SC-fat body.
lmmunoprecipitation
Aliquots of culture supematants, containing 15,000cpm of labeled protein, were incubated with 5 pl pre-immune serum (rabbit) and 25 #1 incubation buffer (10mM Tris, 2.SmM EDTA, 0.15M NaC1, 0.1% NP40 and Img/ml BSA; pH7.5) at 4°C overnight. Sheep-anti-rabbit Ig antibodies (5#1) were added and after 15min at 22°C, the precipitates were centrifuged at 10,000g. The supernatants were incubated at 4°C for 2h with 10pl of monospecific rabbit antibody (Ig fraction) against P4, attacins, lysozyme, synthetic cecropin A(1-37) (Merrifield et al., 1982), arylphorin and flavoprotein, respectively. Antibody against arylphorin and flavoprotein was a generous gift from W. Teller. After a second incubation with sheepanti-rabbit Ig antibody (10#1) at 22°C for 15rain the samples were centrifuged at 10,000g and 4°C for 15rain. The immunoprecipitates were washed twice in incubation buffer, and dissolved in 25 #1 sample buffer at 95°C for 5 min prior to SDS-PAGE. Induction experiments in vitro
After 1 day in culture, the fat body was treated with different agents for 2 days. The following additives, in 20 pl medium, were used: 5 mg/ml LPS, 0.2 mg/ml M. lysodeikticgs, heat-treated E. coil (20#1 of a culture in log-phase), 1)4 (11 pg/culture), 20-hydroxyeedysone (25 pg/ml, resulting in 10-6 M 20-hydroxyecdysone per culture; Sigma), washed epidermis-free cuticle, wing epidermis from a wounded pupae, and washed hcmocytes from either untreated, injured or vaccinated pupae.
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TIBIA TRENCZEKand INOmD FAYE
302
months (LC) were used since such pupae start adult development without delay. As shown in Fig. 2/(~)A the amount of synthesized and released proteins from "untreated" SN-fat body was high and reached levels of"injured" SC-fat body (Fig. 2 / 0 B . There was no significant differences between "untreated" and "injured" SN-fat body (Fig. 2 / O A,B). In both types of cultures we observed a slightly higher synthesis after 3 days. Homebred LC-pupae showed an even higher incorporation of [3H]lysine into released proteins (Fig. 2/(~)Aa). Experiments with "injured" LC-fat body was not carried out as the fat body of most of these pupae had started to disintegrate. Only one "untreated" pupa that did not show signs of disintegrated fat body after it had been adjusted to room temperature, could be investigated (Fig. 2/(~)Ab). The release of labeled proteins into medium III was slightly lower than from "untreated" pupae taken directly from the cold.
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Fig. 2. Synthesis and release of proteins in relation to the different physiological stage of the fat body. Fat body from "untreated" (A) and "injured" (B) pupae were incubated for 24 h with [3H]lysine after 1 day (first column~ and 3 days (second column) in culture. The pupae were: Q) SC-pupae, (~ SN-pupae and (~) LC-pupae (Aa, not adjusted to room temperature; Ab, adjusted to room temperature prior to dissection). The bars indicate the SEM values of 6 determinations (of 2 determinations in 3Ab).
After 3 days, "untreated" SC-fat body in media II and III showed a slight increase of labeled proteins but still the total amount was 8- to 10-fold lower than from "injured" SC-fat body. In medium I, however, protein synthesis in "untreated" SC-fat body reached comparable levels as that found in "injured" SC-fat body (Fig. lb, d). Since the difference in the basic incorporation level between individual pupae was high, the kinetic studies were analyzed by comparing fat body samples from the same pupae (see Fig. 1).
Protein synthesis in relation to developmental stages Chilled pupae in diapause are asynchronous in terms of the onset of adult development when they are transferred to room temperature during the experiment (Hirano and Gilbert, 1967; Rasenick et aL, 1976, 1978). Therefore we repeated the same experiments with two different batches of pupae in diapause. On the one hand, unchilled pupae (SIN) of the same age were chosen to avoid the onset of the adult development by the temperature shift. On the other hand, pupae which had been kept chilled for 5.5
Electrophoretic analysis of proteins For further clarification of the experimental conditions we analyzed the proteins which were synthesized and secreted by different fat body cultured in different media. All proteins released during 24h after 1 and 3 days in vitro were characterized by SDS--PAGE (Fig. 3). In all samples the main polypeptides are the same, namely P4 (48 kD), attacins (neutral and basic form, 20kD), and lysozyme (13.8 kD) as well as several polypeptides in the range of 75-100kD. Two other polypeptides that were released from the fat body (indicated by ~ ) have apparent molecular weights of about 40,000 and 25,000 and were more prominent in the medium as compared with the hemolymph. The newly synthesized proteins which were released from "untreated" LC-fat body in the media were demonstrated via fluorography in Fig. 4. The "immune proteins" as well as arylphorin and the flavoprotein, and two of the proteins in the range of 7~--100kD, were identified by immunoprecipitation combined with SDS-PAGE and fluorography (Fig. 5). In all fat body cultures after l day, irrespectively of their origin, the two heavily labeled bands were P4 and the attacins. The basic and neutral form of the attacins are not distinguishable due to the overexposure. Lysozyme, cecropins and two polypeptides of 220 and 180kD as well as polypeptides of 55 kD and in the range of 38-40 kD were also labeled. No incorporation could be found in the region of 75-100 kD (Fig. 4, lanes 5-8). The pattern of labeled proteins from the first 24 h after dissection showed a weak label in the polypeptides of 75-100 kD (not shown). When after 3 days in vitro fat body samples from the same pupae were incubated with [3H]lysine, P4 and attacin were synthesized as before and the incorporation into lysozyme and the peptides of 220 and 180 kD was higher. Moreover, at this time proteins >300 kD, in the range of 75-100, 60 and 30kD were synthesized. The radioactive incorporation in the proteins of 75-100 kD showed slight variations between individual pupae and the three different media tested. Adjusting the pH of the medium with NaOH instead of KOH did not result in any difference (Fig. 4, lanes 3 and 4). Polypeptides of 6 and 8 kD
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?ig. 6. S D S - P A G E analysis of induction experiments in vitro. 10,000 cpm of TCA-precipitable proteins of culture supernatants were subjected to SDS PAGE and the results visualized 9y fluorography. To "untreated" fat body was added: supernatants from hemocyte cultures that had been treated with Ringer (a), hemolymph (b), M. lysodeikticus (c) or LPS (d); or hemocytes "rom LC-pupae vaccinated with M. lysodeikticus (e) and LPS (f) respectively; or cuticle (g); damaged wing epidermis (h); P4 (i). Control: "untreated" LC-fat body. SN-fat body was incubated ~ith heat killed E. coli (1), M. lysodeikticus (m), LPS (n) or Ringer (o). Controls: "injured" SN-fat body after 3 days (p) and 1 day (q) and "untreated" SN-fat body after 3 days (r) in culture. "Untreated" SN-fat body incubated with hemocytes from LPS-vaccinated (s), injured (t) and untreated (u) pupae.
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Insect immunity, m vitro studies were now synthesized more than the cecropins (ca 4 kD). "Injured" fat body expressed the same protein pattern as the "untreated" fat body. In contrast, SN-fat body, neither "untreated" nor "injured", showed any label in the region of 75-100, 60 or 30 kD after 1 and 3 days in culture (Fig. 4, lane 8; Fig. 6, lanes p-r). This is due to missing synthesis and not caused by inhibition of protein release or by degradation as illustrated by the Kenacid Blue counterstain of the same sample (Fig. 4, lane 9). Studies with "immune" fat body Since previous results from Faye and Wyatt (1980) showed that the antibacterial activity in the fat body cultures from vaccinated pupae declines, we reexamined this question. To avoid any effect of live bacteria we vaccinated the LC-pupae with either LPS or dead cells of M. lysodeikticus. Injections of 2mg/ml LPS (E. coli D21) in vivo resulted after 8 days in a 50% response of that when injected with live bacteria and no difference in lysozyme activity. Freeze dried cells of M. lysodeikticus injected in vivo (0.2mg/ml) gave only a 30% response of antibacterial activity but comparable lysozyme activity as LPS or live bacteria did. Supernatants taken from "immune" fat body after 1, 2 and 3 days in vitro showed a continuously decreasing synthesis of P4, attacins, lysozyme, cecropins and the polypeptides between 38--40 kD, but a continuously increasing synthesis of the proteins of 75-100 and 180 kD, and higher molecular weight (Fig. 7). Studies in vivo For comparison, in vivo protein labeling with [35S]methionine was carried out. SN-pupae were taken to avoid the onset of adult development. Each pupa received 50/~Ci [35S]methionine in 50/Jl Ringer or in 50/~1 Ringer containing 5 mg/ml LPS. Incubation times were 30 rain, 1, 2, 3, 5, 7, 11 and 24 h, respectively. Constant volumes of hemolymph were analyzed using SDS-PAGE. The radioactive protein pattern obtained in the first 24 h was the same for the injured and immune pupae (Fig. 8). Synthesis of P4 was already detectable after 2 h but distinct at 3 h, and attacins could be seen after 5 h. For lysozyme and cecropins [35S]methionine is not the optimal amino acid to label with (see Engstrtm et al., 1985, Hultmark et al., 1982). Therefore they first were visible after 24 h. One protein of about 40 kD showed a high incorporation after 2 h with a maximum at 11 h. Whether the synthesis of this protein was induced de novo or not was not tested. Induction experiments in vitro To elucidate which factor might be involved in the injury/immune response several presumptive inducers were tested in vitro. The qualitative changes in the radioactive protein pattern were analyzed by fluorography (Fig. 6). When medium or medium with P4 was added to "untreated" LC-fat body no striking difference from the control was detected (Fig. 6, lanes i, k). Treatment with 20-hydroxyecdysone caused no change in the pattern of SN-fat body. However, in cultures of
307
LC-fat body 20-hydroxyecdysone stopped the synthesis of proteins of 75-100 kD (not shown). These proteins were also affected when cuticle or epidermis from wounded pupae was added to LC-fat body (Fig. 6, lanes g, h). Moreover, in the latter case the total amount of newly synthesized and released proteins decreased by about 50%. The addition of heat treated E. coli, LPS or M. lysodeikticus to LC-fat body stopped the synthesis of the proteins in the range of 75-100 kD (Fig. 6, lanes l-n). At the same time high amounts of cecropins were newly synthesized. Changes in the synthesis of P4, attacins or lysozyme were not possible to see due to the overexposure on the fluorographies but short time exposure showed a higher incorporation in these proteins too. Effect of hemocytes In some preliminary experiments we investigated whether hemocytes could transfer any signal to the fat body and if so how the fat body would react to it. Harvesting naive, i.e. not spreading hemocytes (see Cherbas, 1973) from H. cecropia was possible both with preinjection of ACR (Leonard et al., 1985) and directly dropping the hemolymph into ice-cold ACR. Preinjection of ACR did not change the protein pattern but resulted in an enormous increase in total protein synthesis. Thus we ommited the preinjection step. Washed hemocytes from either untreated, injured or vaccinated pupae were added to cultures of "untreated" LC- and SN-fat body and the newly synthesized proteins characterized as before. The addition of "injured" or "immune" hemocytes to the fat body resulted in a higher synthesis of cecropins, lysozyme, the 6-8 kD peptides and to some extent of P4 and attacins (Fig. 6, lanes e, f, s, t). That is to be compared with the addition of naive hemocytes which did not have any effect (Fig. 6, lane u). We obtained similar results when adding 20/zl of supernatants from primary cultures of hemocytes, that had been incubated 10h with 20/zl Ringer, sterile filtered hemolymph, LPS or M. lysodeikticus (Fig. 6, lanes a-d). In both tests the protein synthesis of hemocytes in culture alone was negligible if compared with the fat body in vitro.
DISCUSSION Influence of media and developmental stages Observations made by Kurtti et al. (1975) and Abu-Hakima (1981) revealed that differences in medium composition influence the metabolism of cultured cells and organs, and led us to test media conditions for H. cecropia fat body cultures again. Our idea was that a proper medium should only reflect normal stage specific protein synthesis and not induce "immune protein" synthesis. Three media were tested for constant rates of protein synthesis under long term conditions. Furthermore we analyzed the synthesized proteins by SDS--PAGE and fluorography. By the addition of vitamins, organic acids, and PIPES to the medium of Reddy and Wyatt (1967) fat body protein synthesis continued for at least 15 days, a longer time than reported. This was even possible with increasing rates of synthesis after 4 days. Reddy
308
TINA TRENCZEK and INGRID FAYE
and Wyatt (1967) found a decline in the rate of incorporation after 20 h when they incubated wing epidermis in a medium which did not contain these additives. We did not supplement the media with any protein since BSA (Andersson and Steiner, 1986) and FCS (our observation) gave a slightly higher antibacterial response in vivo than did Ringer. A change in osmolarity from 343 mOsm/kg H20 (medium I) to 430 and 440 mOsm/kg H20 (media II and III) had a remarkable effect on fat body from SC-pupae, which were in deep diapause. The initial rate of protein synthesis in media II and III was maintained for 4 days but not in medium I. The low incorporation rates reflect the diapausing status of the fat body and is not due to any effect of the high osmotic pressure such as lysis of the cells which would stop the incorporation totally (Stevenson and Wyatt, 1962; Reddy and Wyatt, 1967). Further evidence for this interpretation is that "injured" SC-fat body or "untreated" and "injured" SN- and LC-fat body, representing other physiological stages, show high rates of protein synthesis. It is not likely that the higher rate of protein synthesis of "untreated" LC- and SN-fat body is only a sign of the "injury effect" of dissection since all types of fat body were prepared in the same way. The differences in protein synthesis between SCpupae and SN-pupae was about 10-fold (Fig. 2/C)A, QA). That reflects the fact that the basic metabolism in diapause is higher at 25°C than at 6°C. The LC-fat body synthesized and released an even higher amount of protein than the SN-fat body. The hemolymph of LC-pupae chilled for 5.5 months, i.e. long enough to start adult development (Hirano and Gilbert, 1967) had an osmolarity 540 + 20 mOsm/kg H20. This is close to values found in pupae at the second day of adult development (Jungreis et al., 1973). These results indicate that SC-fat body after about 4 days in culture might change metabolism, adjusting to conditions found at the very beginning of adult development (higher temperature, lower osmotic pressure). LC-pupae however, had already switched on their internal program for the imminent onset of adult development. Preparative changes of adult development such as increased activity by various oxidative enzyme systems several days before the first morphological signs of development can be detected were reported by Shappirio (1960). A higher metabolism of SN- and LC-pupae than SC-pupae would also explain the only slight difference between "untreated" and "injured" SN- and LC-fat body in vitro. This idea is supported by the following data. The second way of optimizing the experimental conditions was to analyze the pattern of newly synthesized proteins. Fat body samples from the same pupae synthesized the same proteins in all three media (Fig. 4). However, with LC- and SC-fat body in medium III we observed a relative decrease in the synthesis of the "immune proteins" and an increased synthesis of other proteins. The latter are described as subunits of normal serum proteins such as lipophorin [270 and 88 kD (Chino et al., 1969)], larval storage proteins (LSP 1 and LSP 2 with 89 and 85 kD (Tojo et al., 1978)], flavoprotein [83 kD (Telfer, personal communication)], arylphorin [73 kD (Telfer, 1983)] and vitellogenin [180 and 44kD (Kunkel and Pan,
1976)]. "Immune" LC-fat body gave the same results (Fig. 7). Boman and Steiner (1981) reported a similar phenomenon in vivo after 7 days, when injured pupae synthesized these proteins whereas immunized pupae did not. At this time, injured pupae express no antibacterial activity and have returned to normal conditions. Therefore we will call this phenomenon a "recovery effect". The observation that SN-fat body did not show this effect might be explained by the fact that this fat body did not undergo a temperature shift, i.e. it is still under the resting condition of diapause. Surprisingly, these proteins were also affected by adding 20-hydroxyecdysone, cuticle, wing epidermis, bacteria or hemocytes (Fig. 6). Turn-off effects of normal serum proteins are reported for the heatshock situation in Drosophila (Ashburner and Bonner, 1979), in an Aedes cell line (Carvalho and Rebello, 1987), and as culture shock in Xenopus laevis (Wolffe et al., 1984). The latter authors describe a drastic loss of albumin mRNA parallel with the occurrence of heat shock-like proteins after preparing primary cultures of hepatocytes and the acquisition of full responsiveness of these cells to estrogen is first observed after 2 days in culture. Concerning these observations we do not know if the proteins of H. cecropia described above are unlabeled because of diapause conditions or by a switch-off effect caused by injury or infection although the exact stage of a fat body in vitro is known. Based on the good simulation of diapause conditions, the relative constant protein synthesis between one and four days in culture and the better "recovery effect" we chose medium III for further studies. Influence o f different treatments Until now no factors, bacterial or other, are known to selectively stimulate the immune response in insects. It has, however, been reported that different bacterial substances and hemocytes are involved in the inducing mechanisms of the immune response (Dunn et al., 1985, Andersson and Cook, 1979). In the case of H. cecropia the injury of dissection already induces the synthesis of all "immune proteins". Therefore, we were restricted to search for enhancing stimuli. Furthermore, previous discussions about the possibility of ecdysteroids being involved in the injury effect (Barth et al., 1964; Berry et al., 1967) led us to include this hormone in our studies. Based on the incorporation of [3H]lysine into the immune proteins, especially cecropin, in the different experiments we conclude that:
1. Addition of bacterial substances enhanced the "immune protein" synthesis. Medium added as a control had no effect. 2. Hemocytes from vaccinated pupae also increased "immune protein" synthesis as compared to hemocytes from untreated pupae. 3. Hemocytes from injured pupae and epidermis raised the "immune protein" synthesis as compared to naive hemocytes and medium respectively. 4. Other factors (salt concentration, osmolarity) as indicated by the addition of Ringer (without sucrose), but not P4 or cuticle seemed to
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309
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b Fig. 8. SDS-PAGE and fluorography of hemolymph proteins synthesized in vivo. Pupae were injected with 50/iCi [35S]methionine in 50/H Ringer (a, male) or in 50 ~tl Ringer containing 5 mg/ml LPS (b, female), respectively. No incorporation was detectable up to 1 h (lane 1). Lanes 2~i: hemolymph samples taken after 3, 5, 7, 11 and 24 h, respectively. Attacins (A), P4 (P4) and vitellogenin (Vg).
310
Insect immunity, in vitro studies influence the "immune protein" synthesis in the same way. In vivo the response to injury is lower and has a
shorter duration than that caused by bacterial infections. Kinetic studies in vitro might show similar differences between the substances used in the present experiments. 20-Hydroxyecdysone stopped the synthesis of the 75-100 kD proteins in LC-fat body but no effect was seen in SN-fat body. A possible explanation is that the addition of the hormone simulates events at the beginning of adult development when the titer and synthesis of larval and pupal proteins is known to drop (Telfer and Williams, 1953; Patel, 1971). However, stress phenomena as described above cannot be excluded. The most striking difference to the experiments from Dunn et al. (1985) is the missing "injury effect" in Manduca larvae as judged by the absence of antibacterial activity of culture supernatants of "untreated" fat body. Dunn and coworkers did not treat their cultures with Ringer or non-bacterial substances like damaged insect tissue. In the present experiments testing antibacterial activity with E. coil D31 plates was not sensitive enough to prove a relationship between synthesis of "immune proteins" and appearance of antibacterial activity. This is, however, strongly indicated by the findings of Faye and Wyatt (1980). The described differences could be due to the different developmental stages investigated. Schneiderman (1957) reported that the injury metabolism is only characteristic of diapausing pupae. In this case the few "injury effects" described in Manduca (Hughes et al., 1983) may be due to minor infections or contaminations with bacterial cell wall. On the other hand the "injury effect" as such is not restricted to diapausing pupae of H. cecropia as shown with other lepidoptera (Marek, 1969; Anderson and Cook, 1979) and in diptera (Komano et al., 1980; Takahashi et al., 1986; Keppi et al., 1986). But as long as the experiments are not done under the same conditions, the differences remain unexplained. The data obtained in this study clearly show that no bacterial agents are necessary to induce the synthesis of P4, attacins, lysozyme and cecropins. The findings from Faye and Wyatt (1980) that untreated fat body synthesize proteins in the molecular weight range of P4 and attaeins (P5) were confirmed by immunoprecipitation and extended to also include lysozyme and cecropins. We could not show any qualitative differences of "immune protein" synthesis between "untreated", "injured" and "immunized" fat body during the first days of in vitro culture. The primary inducing agent(s) remain unknown. Two different mechanisms of induction might exist, one relatively unspecific at the very beginning and another being of a more specific nature maintaining, if necessary, the already induced antibacterial defense mechanism. In the latter case the involvement of hemocytes is clearly shown. Hemocytes from vaccinated pupae also enhance the synthesis of "immune proteins", while naive hemocytes show no effect. Since hemocytes from "injured' pupae or Ringer gave comparable results, signals other than through hemocytes-processed bacterial substances must also
311
be involved. Stress-linked metabolic events such as reported by Ziegler et ai. (1979) should not be neglected and detailed quantitative kinetic studies of the "immune protein" synthesis are necessary for further clarification. kindly acknowledges a postdoctoral fellowship from Gunnar Hannsons forskningsstiftelse. This work was also supported by the Swedish Natural Science Research Council (BU 245 to H. G. Boman) and the Swedish Medical Research Council (Grant 13X-3556 to H. Bennich). We wish to thank Dr H. Bennieh for critical reading of the manuscript. Acknowledgements--TT
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