Characterization of monoclonal antibodies to hemocytes of Pseudoplusia includens

J. Insect Physiol. Vol. 42, No.

Pergamon

0022-1910(95)00079-8

Characterization of Monoclonal Antibodies Hemocytes of Pseudoplusia includens MICHAEL

R. STRAND,*+

1,pp.21-3 I, 1996

Copyright 0 I996 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0022.1910/96 $15.00 + 0.00

to

JENA A. JOHNSON*

A panel of monoclonal antibodies (MAbs) was generated against hemocytes from unparasitized Pseudoplusia in&dens and P. includens parasitized by the polydnaviruscarrying parasitoid Microplitis demolitor. Of 862 hybridomas generated, 107 lines secreted antibodies to one or more classes of hemocytes. For the current study, 21 lines were cloned, 11 lines were isotyped and three MAbs were characterized. MAb 48F2DS specifically labeled granular cells, MAb 52F3AS labeled plasmatocytes and MAb 55F2G7 only labeled hemocytes inoculated with M. demolitor polydnavirus (MdPDV). Western blot analysis indicated that each MAb recognized specific antigens when cell proteins were separated on SDS-PAGE gels. Using immunofluorescence microscopy, differences were noted in the proportion of granular cells and plasmatocytes present in parasitized hosts and in the proportion of each cell type labeled by MAb 55F2G7. Double-labeling experiments further suggested that the proportion of granular cells infected by MdPDV declined between 24 h and 7 days post-parasitism while the proportion of infected plasmatocytes did not. Hybridomas

Parasite

Wasp

Polydnavirus

Lepidoptera

INTRODUCTION

University should

of Wisconsin-Madison.

Immunity

Nappi and Christensen, 1986; Rizki et al., 1983; Richards et al., 1989). A more promising approach is classification of cells by antigenic differences using monoclonal antibodies (MAbs). Such markers are essential tools in vertebrate immunity, yet only recently have they received any attention in the study of insect hemocytes (Chain et al., 1992; Mullet et al., 1993). Our primary interest is understanding how parasitic wasps like Microplitis demolitor (Hymenoptera: Bracondae) evade encapsulation in permissive hosts such as Pseudoplusia includens (Lepidoptera: Noctuidae). M. demolitor, like many endoparasitic Ichneumonoidea, carries a symbiotic polydnavirus (MdPDV) that the female wasp injects into a host larva at oviposition (Fleming, 1992; Strand and Pech, 1995a). We previously determined that MdPDV is responsible for suppression of the host encapsulation response toward M. demolitor (Strand and Noda, 1991). Most hemocytes in P. includens are infected by MdPDV by 24 h post-parasitism (p.p.) or injection (p.i.) of virus. Viral transcription, in the apparent absence of replication, then continues in certain hemocytes over the 7 day period necessary for the wasp’s progeny to complete dcvclopment (Strand et al., 1992; Strand, 1994). P. includens hemocytes can be separated by phasecontrast microscopy into five subclasses (morphotypes) (Strand and Noda, 1991). Granular cells and plasmatocytes account for 60-80% and 15-30% respectively of the total hemocyte population of P. includens fifth stadium larvae, and are the primary cell types involved in

Hemocytes play a key role in defending insects against pathogens and parasites that gain entry into the hemocoel (Lackie, 1988). Bacteria and other pathogens are usually phagocytized by hemocytes while metazoan parasites and other macrotargets are eliminated by encapsulation, a process in which hemocytes form a multilayered capsule around the intruder (Rowley and Ratcliffe, 198 1). Classification of insect hemocytes has historically been based upon morphology, histological staining properties, and their role in activities such as phagocytosis or wound healing (summarized in Ratcliffe et al., 1985; Gupta, 1986; Lackie, 1988; Ratcliffe, 1993; Strand and Pech, 1995a). Unfortunately, these characteristics often vary with taxa, life stage, and how hemocytes are collected or maintained in culture. As a result, considerable confusion exists in the literature on the naming of hemocytes, ontogeny and differentiation of subpopulations, and the role of different cell types in immunity. Although morphological markers are important for categorizing hemocytes, more specific biochemical markers are clearly needed to advance our understanding of how the cellular immune response of insects is coordinated. Limited success has been achieved in this area using lectins that bind cell surface determinants (Nappi, 1973;

*Department of Entomology, son, WI 53706, U.S.A. tTo whom all correspondence

Antigens

Madi-

be addressed. 21

22

MICHAEL R. STRAND and JENA A. JOHNSON

capsule formation. Both morphotypes exhibit several changes in morphology and spreading behavior when collected from parasitized or virus-injected hosts (Strand, 1994; Strand and Pech, 1995b). Moreover, granular cells and plasmatocytes infected in vitro with MdPDV exhibit alterations similar to that of cells infected in viva, suggesting that suppression of encapsulation is due primarily to direct infection by MdPDV (Strand, 1994; Strand and Pech, 1995b). The purpose of the current study was to develop hemocyte markers for characterizing cell-cell interactions during capsule formation and infection by MdPDV. Here we report on the production and partial characterization of three MAbs to P. in&dens hemocytes. These markers are part of a much larger panel of antibodies developed for characterization of hemocyte lineages, stages of maturation and cell state.

MATERIALS Insect rearing

AND METHODS

and collection

of wasp components

P. in&dens larvae were reared as outlined by Strand ( 1990) in 30 ml plastic cups at 27°C & 1“C and a 16L:8D photoperiod. Moths were fed a 20% sucrose solution. M. demolitor was reared as described by Strand and Wong ( 199 1). The mixture of ovarial proteins and virus in oviducts of female wasps is referred to as calyx fluid while material from the wasp’s poison gland is called venom. Calyx fluid, venom and gradient purified MdPDV were collected by established methods with quantities used to inject P. includens larvae expressed in wasp equivalents (Strand and Wong, 1991; Strand et al., 1992). Previous studies indicated that P. includens hemocytes exhibit similar alterations whether infected with virus using calyx fluid or gradient purified MdPDV (Strand, 1994; Strand and Pech, 1995b). Hemocyte

treatments,

collection

and purijcation

Since no antibodies had been generated to P. includens hemocytes previously, we opted to immunize mice with a mixture of hemocytes in three physiological states: (1) uninfected, inactivated hemocytes; (2) uninfected, activated hemocytes; and (3) hemocytes infected by MdPDV. Inactivated hemocytes were defined as a cell population in which neither granular cells nor plasmatocytes were aggregated or spread, whereas activated hemocytes were defined as a cell population in which granular cells and plasmatocytes had spread on a foreign surface (Ratcliffe et al., 1985). Hemocytes used in immunizations and most assays were collected from 36-48 h old fifth stadium larvae that were unparasitized or had been injected 24 h previously with 0.5 equivalents of calyx fluid plus venom (Strand and Noda, 1991). Approx. 4.5 x 10’ hemocytes can be collected per larva of this stage (Pech et al., 1994); a cell number that yields 8- 12 pg of soluble protein as measured by Bradford assay (Biorad) using bovine

serum albumen (BSA) as a standard. Injection of 0.5 equivalents of calyx fluid results in 90% infection of hemocytes with MdPDV at 24 h pi. (Strand, 1994). For certain assays, hemocytes or cell-free plasma were collected from unparasitized larvae, larvae parasitized by M. demolitor at 12 h of the fourth stadium, larvae injected with venom alone, or larvae injected with purified MdPDV plus venom. Purified granular cells and plasmatocytes infected in vitro with MdPDV were assayed as described by Strand ( 1994). Inactivated hemocytes were obtained as described by Pech et al. (1994). Briefly, P. includens larvae were anesthetized with C02, surface sterilized with 95% ethanol, and bled from a proleg into 500 ~1 of anticoagulant buffer (0.098 M NaOH, 0.186 M NaCl, 0.00 17 M EDTA and 0.041 M citric acid, pH 4.5) (Mead et al., 1986). Hemocytes were transferred to a 1.8 ml microfuge tube containing 1 ml of cold anticoagulant buffer and centrifuged at 250g in a Lourdes clinical centrifuge. The supernatant was discarded while the hemocyte pellet was resuspended in 1 ml of anticoagulant buffer and incubated at 4°C for 30 min. Just before immunization, hemocytes were repelleted, washed with Ex-cell 400 medium (JRH Biosciences, Lenexa, KS), and resuspended on ice in 50 ml of Ex-cell 400 medium. Activated hemocytes were obtained by collecting hemocytes from larvae as described above with the exception that hemocytes were cultured for 5 h in 24-well culture plates (Corning) (Pech et al., 1994). During this time granular cells and plasmatocytes attached and spread on the surface of the plate. Cells were washed from plates by vigorously pipetting the surface with medium, pelleted and resuspended on ice in 50 ~1 of fresh medium. Hemocytes from calyx fluid plus venom injected larvae were collected 24 h p.i. as described for inactivated hemocytes.

Immunization

and preparation

of hybridomas

Primary immunization of BALB/c mice was initiated by i.p. injection of 200 ~1 of Ex-cell medium containing 2.0 x 10’ each of inactivated, activated and MdPDVinfected hemocytes (6 x 10’ cells total). Mice were injected again with the same number of hemocytes 3, 7, and 10 weeks later. A prefusion series of three boosts was administered on successive days. The first was administered 4 days before the fusion and the subsequent boosts were administered i.p. To determine which mouse showed the strongest immune reaction to hemocyte antigens, mouse serum was tested using an anti-hemocyte EIA developed for screening of hybridoma supernatants (see below). The spleen of the mouse selected for the fusion was harvested and spleen cells were fused with 1 x 10’ PYNSI/l -Ag40 I myeloma cells (NS- 1; ATCC; Rockville, MD) using polyethylene glycol 1500 (BoehringerMannheim, Indianopolis, IN). Cells were cultured in HAT medium (Dulbecco’s Modified Eagle medium plus 60 PM hypoxanthine, 0.2 PM amethopterin, 20 PM thymidine) plus 20% fetal bovine serum (Gibco, Grand

23

MONOCLONALSTOHEMOCYTES

Island, NY) at 37°C with 7% CO,. Mouse blood ceils (OS%, v/v) (Harlan Bioproducts, Indianapolis, IN) were included in the medium as feeder cells. Hybridomas were prescreened by EIA (Lamonte and Kahn, 1988). Flat bottom polystyrene plates (Linbro-Titertek EIA plates; Flow Laboratories, McLean, VA) were coated with 1 pg/well of hemocyte lysate from unparasitized or calyx fluid plus venom injected P. in&dens in PBS plus 10 mM glutathione. Spent medium from colonies was placed in the wells and incubated overnight at room temperature. Fresh culture medium served as the control. Plates were washed in buffer A (50 mM Tris, 150 mM NaCl, 1 mM MgCl,, 20 PM ZnCl,, 5 mM NaN,, pH 7.4) and blocked with 50% fetal calf serum in buffer B (0.25% gelatin in buffer A) for 10 min. Serum was removed and 100 ~1 of alkaline phosphatase-conjugated goat anti-mouse IgG, (125 ng/ml, heavy and light chains; Kirkegaard and Perry Laboratories, Gaithersburg, MD) in buffer B plus 10% calf serum was added to each well. Plates were incubated at RT for 2 h, washed with buffer B, and 200 ~1 of substrate (4-methylumbelliferyl phosphate; Boehringer Mannheim; 0.5 mg/ml in 1 M 2amino-2-methyl-1-propanol, 25 PM ZnCl,, 1 mM MgCl,, pH 10.3) added to each well. Fluorescence was measured on a Titertek Fluoroskan EIA reader. Supernatants of immunoglobulin-secreting hybridomas were subjected to a secondary screen of immunofluorescence microscopy. In some instances, the EIA primary screen was omitted and culture supematants were tested directly Selected microscopy. immunofluorescence by hybridomas were cloned by limiting dilution in 96-well plates at 0.3 and 0.1 cells/well. Clones were considered statistically valid if fewer than 9 wells/plate had colonies and if only a single colony was observed per well. MAbs were isotyped by antibody capture assay using a commercially available dipstick kit (BRL).

Hemocyte

Immunofluorescence data were correlated with previously established characters for hemocyte classification by purifying specific morphotypes on Percoll cushions (Pech et al., 1994). Cushions were formed in sterile 12 x 75 mm roundbottom polystyrene tubes to a total volume of 4 ml. Granular cells were isolated on 47.5% cushions while plasmatocytes, spherule cells and oenocytoids were separated on 63 or 70% cushions. Hemocytes in the Percollpurified fractions were processed for electron microscopy as described by Strand (1994) with sections examined on a Phillips 410 electron microscope. These data were then compared to hemocytes from the same fractions processed for immunofluoresence or light microscopy. Western

microscopy

Hemocytes from unparasitized, parasitized or injected larvae were collected and 103-lo4 cells/well were aliquoted to 96-well culture plates (Coming). Hemocytes were fixed in 5% formalin for 10 min, rinsed in PBS and permeabilized for 15 min in PBT (PBS plus 0.1% Triton X-100). Cells were blocked for 1 h in 3% bovine serum albumen (BSA) (fraction V, Boehringer Mannheim) in PBT followed by incubation 1: 1 with hybridoma or MAb culture supematant for 1 h. After rinsing 4 x in PBT, hemocytes were incubated with fluorescein isothiocyanate (FITC) or rhodamine (Rh)-conjugated goat antimouse secondary antibody (IgG + IgM; Kirkegaard and Perry) diluted 1:20 in PBS plus 3% BSA. Hemocytes were rinsed 4 x in PBS and for double labeling were incubated with a second primary antibody followed by a FITC or Rh-conjugated secondary antibody as described above. Samples were examined using a Nikon Diaphot fluorescence microscope with Hoffman modulation contrast optics.

blotting

Hemocytes were collected and washed in anticoagulant buffer, resuspended in lysis buffer, and homogenized using a plastic pestle. Cellular debris was removed by centrifugation at 1OOOg for 2 min. Supematants were separated on 10% SDS-polyacrylamide mini gels ( 15 pg of protein/well) under reducing conditions. Proteins were transferred to Nitrobind nitrocellulose paper (Hoeffer, San Francisco, CA) using a semi-dry transfer unit (Hoeffer). Blotted proteins were visualized as outlined by Strand et al. (1994) using spent medium for a specific MAb diluted 1: 10 in buffer and peroxidase-labeled, goat anti-mouse IgG (Kirkegaard and Perry) as the secondary antibody. The secondary antibody was diluted 1:lOOO and 3,3’-diaminobenzidine plus 0.3% NiCl, was used as the visualizing substrate (Harlowe and Lane, 1988).

RESULTS Hemocyte

Immunojluorescence

separation

morphotypes

Previous light microscopic studies indicated the presence of five morphological classes of hemocytes in P. in&dens: ( 1) granular cells that are adhesive, granular and when spread in vitro, assume a circular profile; (2) plasmatocytes that are adhesive, variably granular and when spread, assume a fibroblastic profile; (3) spherule cells that are nonadhesive, phase-bright and contain large cytoplasmic inclusions; (4) oenocytoids that are nonadhesive, oval shaped, and exhibit endogenous phenoloxidase activity; and (5) prohemocytes that are nonadhesive, phase- bright and rounded (Strand and Noda, 1991). With the exception of prohemocytes, hemocytes conforming to these characteristics can be isolated to high purity on Percoll cushions and maintained in vitro (Pech et al., 1994). Prohemocytes are rarely observed and are not referred to in our results. To further classify the cells in our Percoll fractions, hemocytes from each fraction were examined by transmission electron microscopy. The granular cell fraction contained hemocytes that were uniformly rounded, 6-8 pm in diameter with several cytoplasmic granules, abun-

24

MICHAEL

R. STRAND

dant rough endoplasmic reticulum (RER) and variably shaped nuclei [Figs l(A,B)]. Lysosomes and small pseudopodial extensions were also commonly observed. Cells in the plasmatocyte fraction were spheroidal, approx. 12 pm along their longest axis, with well-formed mitochondria and variably shaped nuclei [Figs l(C,D)]. Few cells contained lysosomes or granules but RER was

and JENA A. JOHNSON

abundant, forming cisternae that were often greatly distended and filled with a fibrous material. Spherule cells were round, 5-8 pm dia and contained large granules with a microtubular substructure throughout the cytoplasm [Fig. l(E)]. Oenocytoids were also rounded, 711 pm dia, with a homogeneous cytoplasm, large mitochondria and vacuoles present [Fig. l(F)].

FIGURE 1. Electron micrographs from different Percoll fractions; (A) and (B), lower and higher of P. includens hemocytes the cytoplasm; (C) and magnification of granular cells with granules, lysosomes (L) and Golgi (G) distributed throughout (D), a lower and higher magnification of plasmatocytes with characteristic cisternae (C) and large nuclei (N) readily visible. In (E), a spherule cells is presented with its large cytoplasmic granules (CR) visible while in (F) an oenocytoid is presented containing numerous cytoplasmic vacuoles (V), large mitochondria (M) and a rounded nucleus (N).

MONOCLONALS

Labeling

characteristics

of selected

MAbs

The fusion resulted in 862 hybridomas with 107 lines secreting antibodies to one or more classes of hemocytes as evidenced by EIA and/or immunofluorescence microscopy. Hemocyte types labeled by these lines are summarized in Table 1. Some antibodies labeled the cell surface while others labeled intracellular features. In addition, some antibodies labeled only a subset of the hemocytes in a given Percoll-purified fraction, only cells that were spread or attached to one another, or a subset of hemocytes from larvae injected with calyx fluid plus venom. Of these lines, 21 were cloned and 11 isotyped (Table 1). Results reported here focus on three MAbs designated 48F2D5, 52F3A5, and 55F2G7. MAb 48F2D5 labeled granular cells specifically [Figs

TABLE

1.

Screening

results

for hybridomas

25

TO HEMOCYTES

2(A,B)]. Using mixed populations of hemocytes, this antibody only labeled cells that had spread into the rounded morphology diagnostic of granular cells. For hemocytes separated on Percoll cushions, >97% of the cells in the granular cell fraction were labeled, while approx. 5% of the cells in the plasmatocyte fraction were labeled. The cells labeled in the plasmatocyte fraction were also likely granular cells since previous studies suggested a similar level of granular cell contamination in the plasmatocyte fraction (Pech et al., 1994). No spherule cells or oenocytoids were labeled. MAb 48F2D5 stained primarily the granules within granular cells resulting in a punctate staining pattern in cells that were spread on culture plates [Fig. 2(B)]. This staining pattern was consistent for granular cells in a mixed population

and MAbs against

hemocytes

of P. in&dens

Hybridomas Hemocyte

type(s)

No. of hybridomas

recognized

All cell types (granular oenocytoids)

cells, plasmatocytes,

spherule

cells,

Two cell types Granular cells and plasmatocytes Plasmatocytes and oenocytoids

18

9 2

Single cell types Granular cells Plasmatocytes Spherule cells Oenocytoids

14 17 3 7

Other*

19

Hemocytes

from parasitized

18

larvae Monoclonal antibodies type(s) recognized

Designation

Hemocyte

41A2G4 43E9AlO 45E7C4 48F2D5 49B1 lC6 50A3C7 51B3A3 52F3A5 53AlAI 55F2G7 59DSB3

Granular cells and plasmatocyes Plasmatocytes from parasitized hosts Granular cells Granular cells Plasmatocytes Granular cells and plasmatocytes Hemocytes from parasitized hosts Plasmatocytes Plasmatocytes Hemocytes from parasitized hosts Hemocytes from parasitized hosts

42C3G6 46DllC6 49B1 lC6 49E4D7 49G3A3 49G3A8 52DlOC12 56G7B 10 56G5Cl 60F7A2

Plasmatocyte subpopulation Homotypically aggregated plasmatocyes Plasmatocytes Oenocytoids Homotypically aggregated plasmatocytes Homotypically aggregated plasmatocytes Oenocytoids Granular cells Hemocytes from parasitized hosts All hemocytes

Isotype

I@’ IgGl IgG3 IgGl IgG2a IgM IgM IgGl IgGl IgGl IgM

*Designates a wide range of immunocytochemical staining characteristics that includes staining subpopulations of a given Percoll fraction or cells in a particular physiological state (e.g. homotypically aggregated, spread on a foreign surface).

26

MICHAEL

R. STRAND

and JENA A. JOHNSON

FIGURE 2. Light (A) and immunofluorescence (B) micrographs of hemocytes labeled with MAb 48F2D5. Note the punctuate staining of granular cells (G). The slight staining of pllasmatocytes (P) visible in (B) is equal to background of samples incubated with secondary antibody alone (data not presented). Light (C) and immunofluorescence (D) micrographs of hemocytes labeled with MAb 52F3A5. Plasmatocytes (P) are labeled strongly by the antibody whereas granular cells (G) are not. All micrographs are at the same scale as indicated in (A).

of hemocytes, prepurified on gradients, or cultured in the presence of plasma: suggesting the antibody recognizes molecules synthesized by the cell rather than material endocytosed from the hemocoel. Unspread (inactivated) granular cells were also labeled by 48F2D5 as were purified granular cells infected by MdPDV in vitro (data not presented). MAb 52F3A5 labeled plasmatocytes [Figs 2(C,D)]. In a mixed population of hemocytes, staining was restricted primarily to cells that assumed a fibroblastic morphology when spread. When hemocytes were separated on Percoll cushions, only cells in the plasmatocyte fraction were labeled. Approx. 20% of cells in the plasmatocyte fraction never spread in vitro (Pech et al., 1994), and these cells were also stained by 52F3A5. Although we have not yet determined why these cells do not spread in vitro, staining by this antibody confirmed that they share antigenie features with plasmatocytes that spread. MAb 52F3A5 appeared to label the cytoskeletal matrix, with some plasmatocytes staining with greater intensity than others. Purified plasmatocytes infected by MdPDV in vitro were also labeled by 52F3A5 (data not presented). Hemocytes from parasitized, calyx fluid plus venom or MdPDV plus venom injected larvae were stained by MAb 55F2G7, whereas hemocytes from unparasitized

larvae or larvae injected with venom only were not [Figs 3(A,B)]. Hemocytes inoculated in vitro with purified MdPDV or calyx fluid were also stained by 55F2G7 while hemocytes inoculated in virro with venom alone were not [Fig 3(C)]. When hemocytes from calyx fluid plus venom injected larvae ( 12 h p.i.) were separated on Percoll cushions, most cells in each fraction were stained by Mab 55F2G7, indicating that this antibody is not specific for any particular morphotype. MAb 55F2G7 stained the surface of hemocytes in a characteristic ring pattern with dense arrays of microvillae often visible on the cell surface [Fig. 3(B)]. This antibody also showed some heterogeneity in staining with some hemocytes staining very brightly while others stained less so. This heterogeneity was apparent even within different Percoll fractions and therefore did not result from differential staining of morphotypes. To determine whether labeling by 55F2G7 was associated with infection of hemocytes by virus or material adsorbed to the surface of cells in the plasma of parasitized hosts, hemocytes from unparasitized larvae were cultured in vitro for 18 h in 50% cell-free plasma from parasitized larvae (48 h p.p.). Reciprocally, hemocytes from parasitized larvae (48 h p. p.) were cultured in 50% plasma from unparasitized larvae. Previous studies indi-

MONOCLONALS

TO HEMOCYTES

FIGURE 3.. Light (A) and immunfluorescence (B) micrographs of hemocytes (24 h p.p.) labeled with MAb 55F2G7. Note the microvillae on the surface of strongly labeled hemocytes (arrow). lmmunofluorescence micrograph (C) of hemocytes with purified MdPDV (0. I wasp equivalents) in vitro and labeled with MAb 55F2G7 (12 h pi.). Light (D) and immunofluorescence (E) micrographs of hemocytes (7 days p.p.) labeled with MAB 55F2G7. Note the variation in staining intensity of hemocytes and absence of staining of the large teratocyte (T) from M. demoliror. All micrographs are at the same scale as indicated in (A).

cated that the majority of hemocytes in circulation are infected by MdPDV at this time while cell-free plasma at this time period does not contain free virus (Strand, 1994). Cells were then processed for immunofluorescence microscopy using MAb 55F2G7. Using hemocytes and plasma from 10 different larvae (each larva representing one replicate), we found that no hemocytes from unparasitized larvae were stained by 55F2G7 when cultured in plasma from parasitized larvae. However, an average of 82.4 f 7.8% SD of hemocytes from parasitized larvae were stained by 55F2G7 when cultured in plasma from unparasitized larvae. The staining patterns described for each antibody were

consistent and repeatable, between cells and larvae. Hemocytes were never stained when either the primary or secondary antibody was omitted. Antibody

isotypes

and Western

blotting

MAbs 48F2D5, 52F3A5 and 55F2G7 isotyped as IgGls (Table 1). Western blots from 10% SDS-PAGE gels indicated that each antibody reacted with hemocyte proteins under reducing conditions in the presence of 2mercaptoethanol (Fig. 4). MAb 48F2D5 reacted with two proteins of 62 and 50 kDa while MAb 52F3A5 reacted with two proteins of approx. 140 and 120 kDa. MAb 55F2G7 reacted only with cells exposed to MdPDV. The

28

MICHAEL R. STRAND and JENA A. JOHNSON

123

were noted in the proportion of granular cells and plasmatocytes that were double-labeled [Figs 5(B,C)]. Here, our interest was to assess for each timepoint, the proportion of granular cells and plasmatocytes that were also labeled by 55F2G7 relative to cells that were not. As expected, most granular cells or plasmatocytes were labeled only by their respective morphotype-specific MAb prior to 24 h p.p. At 24 h p.p., most granular cells were double-labeled by 48F2D5 and 55F2G7, but thereafter the proportion of double-labeled cells declined [Fig. 5(B)]. In contrast, the proportion of double-labeled plasmatocytes remained relatively constant from 24- 144 h P.P. DISCUSSION

FIGURE 4. Western blot of proteins stained with anti-hemocyte MAbs/IgG horseradish peroxidase. Ten pg of protein/lane from cells was separated on 10% SDS-PAGE gels and labeled with MAb 48F2D5 (lane l), 52F3A5 (lane 2) and 55F2G7 (lane 3). Numbers to the left of lane 1 indicate size in kDa of standards.

antibody bound primarily a protein of cu 78 kDa but also crossreacted with several other proteins of larger and smaller size. Hemocytes

in parasitized

hosts

Because granular cells and plasmatocytes undergo severe alterations after exposure to MdPDV, it is difficult to unambiguously identify cell types in parasitized hosts by morphology. Thus, we characterized the relative abundance of these cell types in parasitized hosts using 48F2D5, 52F3A5 and 55F2G7 as markers (Fig. 5). For each timepoint, a parasitized larva was bled and a 1 ~1 aliquot of hemocytes plus plasma was placed into an individual well of a 96 well culture plate containing Excel1 400 medium. Cells were allowed to settle and immunofluorescence then processed for were microscopy. Figure 5(A) presents the mean percentage of hemocytes labeled with each antibody from 1-144 h p.p. At 0 and 2 h p.p., approx. 78% of hemocytes stained with MAb 48F2D5. The percentage of hemocytes stained by this antibody then markedly declined at 24 h p.p., followed by a slow, progressive increase through 7 days p.p. Approx. 20% of hemocytes were stained by MAb 52F3A5 from 0 to 24 h p.p., but thereafter the percentage of stained cells declined to a low of 7% of the total hemocyte population at 7 days p.p. No hemocytes were stained by MAb 55F2G7 at 1 h p.p., but 16% of cells were positive at 2 h p.p. At 24 h p. p. more than 90% of hemocytes were stained but thereafter the percentage of stained cells declined. Teratocytes from M. demolitor that are present in host hemolymph 24 h-7 days p.p. (Strand and Wong, 1991) were not stained by any of the MAbs [Figs

W,E)l. When hemocytes from parasitized hosts were stained with both a morphotype specific (48F2D5 or 52F3F5) and the MdPDV-specific (55F2G7) MAb, differences

This study describes a set of hybridomas raised against hemocytes of P. in&dens, and the initial characterization of three MAbs that recognize subclasses of hemocytes. The hybridomas and MAbs not discussed in this paper (summarized in Table 1) exhibit a range of specificities which suggest that some will be useful as lineage, maturation or activation markers. Two of the MAbs examined in this study, 48F2D5 and 52F3A5, specifically stained granular cells and plasmatocytes, two cell types considered essential in encapsulation of parasites by Lepidoptera. We conclude that these antibodies are specific for these cell types on the basis of two corroborating lines of evidence. First, each antibody stains cells of different morphology and with different spreading characteristics in vitro. Second, when hemocytes are separated on the basis of buoyant density using Percoll cushions, MAb 48F2D5 primarily stains cells designated by Pech et al. ( 1994) as the granular cell fraction and 52F3A5 stains cells in the plasmatocyte fraction. We found during the current study that the ultrastructure of cells in our Percoll fractions are highly uniform, and, in the case of granular cells, spherule cells and oenocytoids, consistent with other descriptions of these cell types in Lepidoptera (summarized by Akai and Sato, 1973; Ratcliffe et al., 1985; Lackie, 1988). Cells we designate as plasmatocytes in P. in&dens also assume a spread morphology in vitro that is typical of plasmatocytes described from related species (Davies et al., 1987), but ultrastructurally these cells contain arrays of RER and cisternae that are much more extensive than generally reported for plasmatocytes (Akai and Sato, 1973; Gupta, 1986). This is not due to our method of purification because the ultrastructure of plasmatocytes from P. in&dens is no different when observed in mixed populations of hemocytes collected directly from the hemocoel (Strand, 1994). It is also not due to differences in viability since plasmatocytes are >98% viable after purification and are fully capable of participating in capsule formation in vitro (Pech et al., 1994, 1995). MAb 55F2G7 only stained hemocytes exposed to MdPDV. We suggest this antibody is specific for hemocytes infected by virus on the basis that it stains cells inoculated in vivo or in vitro with calyx fluid or gradient

MONOCLONALS

24

2

29

TO HEMOCYTES

96

48

144

Time (hours p. p.)

1

2

24

48

96

144

Time (hours p. p.)

1

2

24

48

96

144

Time (hours p. p.)

The mean percentage of hemocytes from parasitized P. includens larvae labeled by MAbs 55F2G7, 48F2D5 and 52F3A5. In (A), data for the total hemocyte population collected from larvae l-144 h (7 days) p.p. is presented. Each datum point is the mean percentage (*SD) of cells from three different larvae labeled by each MAb relative to the total number of cells present. One hundred cells per larva from a randomly selected field of view were scored for each time point. In (B), the mean percentage *SD of cells labeled by 48F2D5 or double-labeled by 48F2D5 and 55F2G7 is presented. In (C), the mean percentage ?SD of cells labeled by 52F3A5 or double-labeled by 52F3A5 and 55F2G7 is presented. For both (B) and (C), the values were determined from the same samples used in (A) normalized to 100% for each time point.

purified MdPDV. In contrast, hemocytes are never stained when inoculated with A4. demolitor venom or cultured in plasma from parasitized larvae. At this time we do not know whether the antigen(s) recognized by this antibody are encoded by MdPDV or are cellular proteins whose expression is induced by viral infection. However, we suggest this antibody recognizes cells containing transcriptionally active MdPDV. This is based upon the results of in situ hybridization studies in which expression of a 1.6 kb MdPDV mRNA was monitored in hemocytes (Strand, 1994; Strand and Pech, 1995b). The proportion of hemocytes expressing this viral transcript (O-7 days p.i. or p.p.) was very similar to the proportion of hemocytes stained by 55F2D5 in the current study. What could not be assessed prior to this study was how the relative abundance of granular cells and plasmatocytes changes after parasitism due to the loss of mark-

ers typically used for identification of these cell types. Both granular cells and plasmatocytes lose their ability to adhere to foreign targets 2-4 h p.i. or p.p. (Strand and Noda, 1991), and granular cells specifically undergo a high level of apoptosis at 24-36 h (Strand and Pech, 1995b). Levels of apoptosis and the proportion of cells that contain transcriptionally active MdPDV then decline after 24 h while the number of cells that spread normally on foreign surfaces increases (Strand and Pech, 1995b; Strand, 1994). The sharp drop in the percentage of cells stained by the granular cell-specific MAb 48F2D5 at 24 p.p. is consistent with the high level of apoptosis occurring at this time. The subsequent rise in the proportion of cells stained by MAb 48F2D5, however, suggests new granular cells continue to be produced in parasitized hosts, possibly from hemopoetic tissues or maturation of

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another cell type into granular cells. In contrast, the proportion of cells stained by the plasmatocyte-specific MAb 52F3A5 progressively declines over 7 days suggesting that plasmatocytes are slowly lost from circulation. Support for these conclusions comes from our double-labeling experiments which clearly show that the percentage of granular cells double-labeled by 48F2D5 and 55F2G7 declines with time, whereas the percentage of doublelabeled plasmatocytes does not. A complication in these interpretations is that the antigens recognized by these MAbs could be distributed differently in hemocytes from parasitized hosts causing us, for example, to underestimate the percentage of plasmatocytes in circulation. To date few monoclonal antibodies have been generated to insect hemocytes. Mullett et al. (1993) produced a large number of hybridomas to Galleria mellonella and Blaberus discoidalis but all stained either granular cells or all hemocytes. Chain et al.( 1992) identified three antibodies that reacted to hemocytes and the basement membrane of Periplaneta americana, and one other antibody that appeared specific for hemocytes that are not adhered to a foreign surface. Related studies (Ball et al., 1987; Nardi and Miklasz, 1989) also report crossreactivity between basement membrane and hemocytes. Trenczek and Bennich ( 1991) report the production of antibodies to granular cells and plasmatocytes from Hyalophora cecropia while Willott et al. (1993) report the production of a large panel of antibodies to Manduca sexta that recognize an array of hemocyte types. The small number of antibodies generated by Chain et al. (1992) and Mullet et al. (1993) to hemocytes of cockroaches contrasts sharply with the larger panel of antibodies reported here and by Willott et al. (1993) to hemocytes of Lepidoptera. This could reflect greater heterogeneity in hemocytes of holometabolous insects or differences in methodology and screening. Regardless, we believe monoclonal antibodies will prove to be essential tools in future studies on how the cellular immune response of insects is coordinated.

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MONOCLONALS TO HEMOCYTES petent hemocytes by monoclonal antibodies. In Molecular Sciences (Ed. Hagedom H. H.), 372 pp. Plenum, New York. Willott E., Trenczek M. and Kanost M. R. (1993) Monoclonal antibodies against Manduca sexta hemocytes. Proc. II. ht. Symp. Molec. Insect Sci. 183.

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express our appreciation to L. Pech and D. Trudeau for their assistance in collecting cells used in the study, L. Kahan for use of certain facilities and R. Bromberg for sectioning the EM samples. This work was supported by NIH grant A132617 and Wisconsin Hatch Project 3200.

Acknowledgements-We