A Novel Monoclonal Antibody That Binds to Hemocytes from Shrimps and Oysters

Abstract

The monoclonal antibody (MAb) LITO-1 was produced from a stable hybridoma cell line generated by the fusion of NS1 myeloma cells with spleen cells isolated from Balb/c mice immunized with a paraformaldehyde-fixed hemocyte suspension of Litopenaeus vannamei. This MAb reacted with all three hemocyte subtypes, but no reaction was observed with components of plasma. Immunohistochemistry assays demonstrated that LITO-1 was very effective in specifically distinguishing hemocytes infiltrated in several tissues such as striated muscle, brain, and hepatopancreas. Moreover, this antibody was able to recognize hemocytes from two shrimp species, Litopenaeus schmitti and Farfantepenaeus paulensis, as well as hemocytes of the oyster Crassostrea gigas. No reaction was observed against hemocytes from the terrestrial insect Triatoma klugi or with mammalian RAW cells. This novel MAb can be useful in revealing the presence and function of a conservative epitope in hemocytes of marine crustaceans and mollusks.

Introduction

Hemocytes play an essential role in protecting crustaceans, mollusks, insects, and other invertebrates from parasitic and pathogenic infections. The importance of hemocytes in invertebrate defense mechanisms can be inferred from their functions. They participate in a variety of internal protective mechanisms such as phagocytosis, nodule and capsule formation, and secretion of hydrolytic enzymes and cytotoxic molecules, which contribute to the destruction of pathogenic organisms.^(1–3) Usually crustaceans have three morphologically different hemocyte types: hyaline hemocytes (the smallest cells with few or no cytoplasm granules), semi-granular hemocytes cells with small granules in cytoplasm, and granular hemocytes, which contain large cytoplasm granules.^(1,4,5)

The morphological features or staining criteria traditionally used to identify the hemocyte categories present some experimental difficulties and are insufficient to clarify the precise type of hemocyte, as well as being unreliable criteria to correlate with cellular functions. Other complicating factors in the identification of hemocytes are the very rapid ex vivo processes such as degranulation, activation of defense processes, and apoptosis or necrosis leading to changes in morphology of the cells.^(6–8) Thus an accurate means of identifying antigenic markers on the cell surface for hemocyte types would allow identification based on molecular rather than morphological criteria and provide researchers with a useful analytical tool for discriminating and separating hemocyte subpopulations, such as probes for monitoring hemocyte-associated functions.

One of the most important tools in the study of the cell functions in vertebrates is the use of monoclonal antibodies (MAbs).^(9,10) However, their use in the study of the invertebrate immune system has only begun recently. MAb technology was first applied to invertebrate hemocytes by Reinisch and colleagues⁽¹¹⁾ and Yoshino and Granath,⁽¹²⁾ who raised MAbs against molluskan hemocytes of the soft shell clam Mya arenaria and the snail Biomphalaria glabrata, respectively. Subsequently, MAbs have been raised against sets of hemocytes from several invertebrate groups, including ascidians,⁽¹³⁾ annelids,⁽¹⁴⁾ insects,^(15,16) and crustaceans.^(17–20)

Using hybridoma technology,⁽²¹⁾ an MAb against hemocytes of Litopenaeus vannamei, one of the most widely farmed shrimp species in Brazil and around the world has been prepared.⁽²²⁾ This was done in an attempt to generate a highly specific probe that could be used (1) to characterize hemocytes according to antigenic differences, (2) to identify and isolate molecules that might be important in the defense mechanisms, (3) to localize hemocytes in different organs and tissues, and (4) for practical applications, such as separating hemocyte subpopulations through flow cytometric techniques. The MAb obtained, named LITO-1, appears to label most but not all hemocyte subsets in vitro, and was shown to be useful for identifying hemocytes within various tissues. LITO-1 cross-reacts with hemocytes of two other species of penaeids and one species of mollusk, indicating that the molecule recognized by LITO-1 is conserved among the marine invertebrates included in this study. Therefore, MAb LITO-1 could become a useful tool to identify hemocytes from shrimp and oyster, as well as to localize hemocytes infiltrated into different tissues from these marine invertebrates.

Materials and Methods

Animals

Adult shrimps Litopenaeus vannamei, Farfantepenaeus paulensis, and Litopenaeus schmitti were used in this study. L. vannamei was obtained from the Laboratório de Camarões Marinhos (Departamento de Aquicultura, UFSC), and the indigenous species F. paulensis and L. schmitti were collected in Santo Antônio de Lisboa, in the northwestern region of Santa Catarina Island (27° 30″ S; 48° 33′ 40″ W), southern Brazil. The animals were acclimated into aerated aquaria (25°C; salinity, 32 to 34%) for at least 48 h prior to bleeding. Only apparently healthy animals at the intermolt stage, weighing 20.0 ± 2.5 g, were used in the experiments. Cultivated Pacific oysters Crassostrea gigas were obtained from shellfish farms located in Florianópolis, Santa Catarina State, Brazil, and maintained in the same conditions as described above. Triatoma klugi nymphs III and IV were obtained from the Laboratório de Transmissores de Hematozoários (Departamento de Microbiologia, Imunologia e Parasitologia, UFSC).

Cells

Shrimp hemolymph was withdrawn from the ventral sinus located at the base of the first abdominal segment using a 23-gauge needle on a 3 mL syringe filled with 1.5 mL of cold modified Alsever's solution (MAS) (27 mM sodium citrate, 336 mM NaCl, 115 mM glucose, 9 mM EDTA [pH 7.0]).⁽¹⁷⁾ Hemocytes were separated by centrifugation at 400 g for 5 min at 4°C, and either fixed with 4% paraformaldehyde for 20 min for immunization of mice and for screening of MAbs in microplates or used as primary adherent cultures on slides and analyzed by indirect immunofluorescence assay (IIFA).

Oyster hemolymph was collected from the adductor muscle sinus with a 23-gauge needle on a 3 mL syringe containing 1.5 mL of cold MAS and subsequently centrifuged at 400 g for 5 min at 4°C. The pellets were resuspended in MAS and immediately used for hemocyte monolayer preparation, after which IIFA was performed.

Nymphs III and IV of the insect T. klugi were bled by severing the legs and antennas. Hemolymph was collected and immediately diluted in anticoagulant solution (AS) (30 mM sodium citrate, 62 mM NaCl, 100 mM glucose, 26 mM citric acid, 10 mM EDTA [pH 4.6]).⁽²³⁾ Hemocyte suspension was centrifuged at 400 g for 5 min at 4°C and then resuspended in AS. The hemocytes were used immediately for monolayer preparation, followed by IIFA.

RAW 264.7 cells, a macrophage cell line derived from mouse Mus musculus (ATCC no. TIB-71), were kept at 37°C with 5% CO₂ and cultivated in DMEM-F12 medium (Cultilab, Campinas, Brazil) containing 10% of fetal bovine serum (FBS, Cultilab) and 1% of penicillin-streptomycin-amphotericin B (Sigma-Aldrich, St Louis, MO).

Immunization and preparation of hybridomas

Six- to 8-week-old Balb/c mice were obtained from breeding stocks maintained at the Animal Facility of the Departamento de Microbiologia, Immunologia and Parasitologia, UFSC. All animal procedures were approved by the university's Ethics Committee for Animal Care.

Five Balb/c mice were immunized by subcutaneous injection of 2 × 10⁶ L. vannamei hemocytes fixed in paraformaldehyde followed by three intraperitoneal injections at weekly intervals. Ten days before the last injection, serum titers were estimated by IIFA as described below. Two mice with the highest antibody titers were selected and boosted 3 days before lymphocyte hybridization.

The spleens from the selected mice were dissected and splenocytes were fused with NS1 myeloma cells (ATCC no. TIB-108) at a ratio of 7:1 using 50% polyethyleneglycol-4000 (Sigma-Aldrich) as fusogen. The cells were cultured in RPMI 1640 medium (Cultilab) supplemented with 20% FBS, with hybridomas selected using 0.1 mM hypoxanthine, 0.0004 mM aminopterin, and 0.016 mM thymidine (Sigma-Aldrich). The hybridoma supernatants were screened by IIFA on microplates, and hybridomas giving positive results were subcloned twice by the limited dilution method, and then tested again for their specificity by IIFA on microplates.

Screening of MAbs through indirect immunofluorescence assay with paraformaldehyde-fixed hemocytes

2 × 10⁴ paraformaldehyde-fixed hemocytes were added per well to a 96-well microplate, which was centrifugated at 400 g for 5 min at 4°C. Cells were fixed with 80% acetone (Merck, Darmstadt, Germany) for 15 min at −20°C, and then rinsed three times with PBS (150 mM NaCl, 1.7 mM Na₂HPO₄H₂O, 9.1 mM Na₂HPO₄ [pH 7.4]) at 5 min intervals. One hundred μL of hybridoma supernatants were added per well and incubated for 1 h at 37°C in a moist chamber. After washing as above, 100 μL of anti-mouse immunoglobulin conjugated with FITC (Sigma-Aldrich) diluted to 1:200 in PBS were incubated at 37°C for 1 h in the dark. The slides were washed with PBS, mounted in glycerine, and examined by fluorescence microscopy. The sera of mice immunized with paraformaldehyde-fixed hemocytes diluted to 1:200 and the supernatant of a hybridoma secreting a monoclonal antibody against rabies virus (LIA) produced in our laboratory were used as positive and negative controls, respectively.

Indirect immunofluorescence assay using adherent primary cultures

In brief, 3 × 10⁵ hemocytes from each animal included in this study were made adherent on separate slides in the presence of 0.2 M CaCl₂ and then left for 2 h. Also, 3 × 10⁵ RAW cells were cultivated onto slides in DMEM-F12 medium and kept at 37°C with 5% CO₂ for 24 h. Adherent cells were fixed with 4% paraformaldehyde for 20 min, washed three times with 0.05% Tween in PBS (PBS-T), and permeabilized for 10 min with 25 mM Tris-HCl, 50 mM NH₄Cl, 0.2% gelatin, and 0.5% Triton X-100 (pH 7.2). Slides were washed three times in PBS at 5 min intervals, and then blocked with 10% skim milk in PBS-T for 1 h. The subsequent steps in the procedure were performed as already described.

Isotyping of the monoclonal antibody

The class and subclass of mouse immunoglobulin produced by hybridomas were determined by sandwich ELISA using SBA Clonotyping System/HRP (Southern Biotech, Birmingham, AL), following the manufacturer's instructions.

Immuno-dot blotting

Hemolymph from L. vannamei was collected in MAS [pH 7.0]; the hemocytes were pelleted and the supernatant was kept at 4°C as plasma. An hemocyte lysate (HL) was prepared by washing hemocytes two times with MAS. Subsequently, 1.5 × 10⁸ cells were suspended in 750 μL of lysis buffer (20 mM Tris-HCl, 15 mM MgCl₂, 10 mM CaCl₂, 350 mM NaCl [pH 7.4]), sonicated twice for 15 s at high frequency (50 − 60 Hz, 240 volts), and kept at −20°C until use. Samples of plasma and HL (3 μL of each) were spotted onto a sheet of Hybond nitrocellulose (NC) paper (GE Healthcare, Freiburg, Germany) and air-dried at room temperature. The NC membranes were blocked overnight with 10% skim milk in PBS-T and then washed three times for 5 min with PBS-T. The membranes were incubated with supernatant of LITO-1 hybridoma for 1 h at room temperature. Sera of mice immunized with paraformaldehyde-fixed hemocytes were diluted to 1:200 and the supernatant of the LIA hybridoma were used as positive and negative controls, respectively. Antibody binding was detected with goat anti-mouse IgG conjugated with peroxidase (Sigma-Aldrich) diluted to 1:1.000 for 1 h at room temperature and washed three times with PBS-T. The reaction color was developed with freshly prepared substrate solution containing 0.15% of DAB (3,3-diaminobenzidine tetrahydrochloride dehydrate) (Sigma-Aldrich) and 0.01% of H₂O₂ (Merck) and halted with distilled water.

Histology and immunohistochemistry

L. vannamei tissues (brain, hepatopancreas, and striated muscle) were fixed overnight in Bouin's solution (58% ethanol, 11% formaldehyde, 7% glacial acetic acid, 0.02 M picric acid). Tissues were rinsed with PBS-IH (500 mM NaCl, 1.5 mM Na₂HPO₄H₂O, 8.3 mM Na₂HPO₄, 2.7 mM KCl [pH 7.4]), dehydrated, and embedded in paraplast. Serial sections (8 μm thickness) were dewaxed and hydrated, and endogenous peroxidase activity was blocked with 5% of H₂O₂ in pure methanol for 10 min. Then, sections were rinsed three times with PBS-IH, blocked with 5% normal goat serum for 1 h, and subsequently incubated at 4°C overnight with supernatant from LITO-1 hybridoma. After washing four times with PBS-IH, binding of the first antibody was detected by incubation with a secondary peroxidase-labeled anti-mouse antibody (Sigma-Aldrich) diluted 1:100 for 3 h at room temperature in the dark. Conjugates were visualized with 0.1 % DAB and 0.3% H₂O₂ in 0.05 M Tris-HCl at room temperature for 2 − 5 min. Standard controls, such as omission and replacement of primary MAb by LIA (a MAb against rabies virus), were carried out. Histological controls were made by staining with hematoxylin and eosin.

Results

Screening and characterization of monoclonal antibodies

Hybridoma culture media were screened for hemocyte-specific antibodies using an IIFA with paraformaldehyde-fixed hemocytes. From a total of nine hybridomas obtained, one produced antibodies that were positive against L. vannamei hemocytes. Hybridoma cells producing L. vannamei reactive antibodies were cloned by two limiting dilutions and designated as LITO-1. They produced MAb isotyped as IgG1 heavy chain and kappa light chain. Although LITO-1 showed strong immunoreactivity with different subpopulations of hemocytes, with and without granules, some hemocytes of each subset did not show any binding to this MAb (Fig. 1). Moreover, LITO-1 did not react with any secreted plasma component, as demonstrated by immuno-dot blotting assay (Fig. 2).

FIG. 1.

Immunofluorescence pattern of MAb LITO-1 on circulating Litopenaeus vannamei hemocytes. (A) Hemocytes were adhered to slides and stained with LITO-1 followed by incubation with FITC-conjugated anti-mouse immunoglobulin. (B) Evans' blue counterstaining of the same view field observed by phase contrast microscopy. HH, hyaline hemocytes; HG, semi-granular/granular hemocyte. Scale bars, 30 μm.

FIG. 2.

Reactivity of LITO-1 against hemocyte lysate and plasma from L. vannamei by immuno-dot blotting assay. HL (1) and plasma (2) were spotted on nitrocellulose membrane, labeled with LITO-1 (A), sera of mice immunized with hemocytes as positive control (B), or monoclonal antibody against rabies virus (LIA) (C) as negative control, followed by goat anti-mouse IgG-conjugated with peroxidase. Color development was obtained after addition of DAB and H₂O₂.

Histology and immunohistochemistry assay

MAb LITO-1 was capable of identifying hemocytes infiltrated in different tissues of L. vannamei such as brain, hepatopancreas, and striated muscle (Fig. 3). Immunohistochemical assays showed that in the brain positive reaction to LITO-1 was evident only in the hemal vessel wall and none of the adjacent tissues were reactive (Fig. 3A2). Higher magnification of the brain tissue showed that the LITO-1 reactive cells in the hemal vessel had circular shape and no granules were observed in the cytoplasm, consistent with hyaline hemocyte morphology (Fig. 3A3). In hepatopancreas, the positive reaction to LITO-1 was observed mainly in the connective tissue among tubules (Fig. 3B2), which is known as a region with a very large infiltration of hemocytes. In this connective tissue, hemocytes with many granules in their cytoplasm were observed (Fig. 3B3), consistent with semi-granular and granular hemocytes morphology. Immunohistochemistry of the striated muscle showed a weak reaction of LITO-1 with cells present among muscle fibers (Fig. 3C2). LITO-1 bound to long-shaped cells with abundant small granules in the cytoplasm, resembling semi-granular hemocytes infiltrated in the muscle (Fig. 3C3). No reaction was observed when the immunohistochemical assay was performed, either without a primary antibody or using LIA as the first antibody (data not shown).

FIG. 3.

Immunohistochemistry assays in different tissues of L. vannamei. Sections of brain (A), hepatopancreas (B), and striated muscle (C) are stained with hematoxylin-eosin (1) and labeled with LITO-1 (2) or LITO-1 and hematoxylin (3) in higher magnification. Arrows indicate hemocytes labeled by LITO-1. Scale bar, 260 μm (A1, B1, C1, C2); 180 μm (B1, B2); 10 μm (A3, B3, C3).

Cross-reactivity studies of the LITO-1 against immune cells from other animals

The cross-reactivity of LITO-1 against hemocytes from other two shrimp species was investigated. The MAb produced against hemocytes of L. vannamei (Fig. 4A) showed similar reaction patterns for hemocytes from penaeid L. schmitti and F. paulensis (Fig. 4B and C). Indirect immunofluorescence assay using immune cells from other animals was also performed. LITO-1 reacted strongly with hemocytes of the oyster C. gigas (Fig. 4E), but showed no reaction to hemocytes of the insect T. klugi (Fig. 4D) or macrophages of the immortal lineage RAW from M. musculus (Fig. 4F).

FIG. 4.

Cross-reactivity of LITO-1 against cells from different animal groups identified by immunofluorescence. Hemocytes from shrimps L. vannamei (A), L. schmitti (B), F. paulensis (C), insect T. klugi (D), oyster C. gigas (E), and macrophages from M. musculus (RAW cells) (F) were labeled with LITO-1, followed by FITC-conjugated anti-mouse immunoglobulin antibody. Scale bar, 30 μm.

Discussion

The immune system of crustaceans and other invertebrates is primarily related to their hemolymph and to its circulating cells, the hemocytes. The circulating hemocytes are essential in the protection of the animal against invading microorganisms by participating in recognition, phagocytosis, melanization, and cytotoxicity.^(1–3) Despite the importance of these cells for protection of the hosts, their characterization is mostly based on morphological criteria. In order to generate MAbs that could be used to identify hemocytes, paraformaldehyde-fixed hemocytes from L. vanammei were used as immunogen for development of the MAb LITO-1. Hemocytes were fixed prior to use because it is well known that crustacean hemocytes change shape and degranulate very fast in vitro.^(6–8,17) LITO-1 appears to recognize all three morphological classes of hemocytes, albeit 30% of hemocytes in circulation remain unlabeled. Whether LITO-1-negative cells represent some unknown functional subtype of hemocytes remains to be clarified in further studies. Moreover, MAbs reacting with pan-hemocytic antigens have been reported by other authors, despite the fact that a single hemocyte subpopulation was used to immunize mice.^(18,24,25)

LITO-1 did not react with epitopes present in the plasma, indicating that the antigen recognized by this MAb is not a molecule secreted by the hemocytes. The cells labeled by LITO-1 in brain, hepatopancreas, and muscle from shrimp showed morphological characteristics, such as shape and/or granules in cytoplasm, compatible with those of the crustacean hemocytes. LITO-1 recognized hemocytes infiltrated in different organs, but did not show any cross-reaction with other components of the tissues. In fixed and paraplast embedded material, cells stained by LITO-1 are located in places where hemolymph flows, such as among the muscle fibers, hepatopancreatic tubules, and inside a hemal vessel.^(19,20) The MAb described here can be employed to localize hemocytes in situ, and can be very useful in immunohistochemical research and in facilitating hemocytes distribution studies in shrimp tissues, as performed by some authors.^(19,20,26) Moreover, the immunoreactivity of LITO-1 on sections of tissue indicates that the antigenic determinant recognized by this MAb is not affected by the fixation procedures.

The reactivity of LITO-1 against immune cells of other invertebrates and vertebrates was also investigated. This antibody does label hemocytes from other two Brazilian indigenous shrimps (L. schmitti and F. paulensis) and a mollusk (C. gigas) but does not label hemocytes from the insect T. klugi or RAW cells from the mammal M. musculus, the most phylogenetically distant animal compared in our study. The fact that LITO-1 reacted with the hemocytes of an oyster and different penaeids, but not with the hemocytes of a terrestrial insect or with mammalian macrophages, suggests that significant molecular similarities exist between hemocytes from evolutionarily divergent species of marine invertebrates. This result is remarkable, because according to a phylogenetic approach, it would be expected that molecules are more frequently conserved between animals of the same phylum, such as Crustacea and Insecta, instead of phylogenetically distant animals, such as mollusks and crustaceans. On the other hand, this result may indicate the presence of a conserved molecule from hemocytes in marine invertebrates, but absent in terrestrial arthropods. Indeed, morphological as well as molecular differences between terrestrial and aquatic invertebrate hemocytes have been previously described.^(27,28) Future studies are necessary to clarify this important issue.

Efforts to determine the molecular weight of the antigen recognized by MAb LITO-1 through Western blotting have failed. There was no band detectable with LITO-1 due to probable destruction of the epitope during SDS-PAGE processing. It is also possible that the antigen is a non-protein macromolecule, in which case its identification could be pursued by a combination of affinity chromatography, and mass-spectrometry analysis, aided by a LITO-1 immunoprecipitation assay. In conclusion, this novel antibody is potentially instrumental for the research of hemocyte ontogenesis and distribution. In addition, MAb generated in this study may be a promising tool for further research on crustaceous and molluskan immunity and help to unravel important defense mechanisms of the invertebrate immune system.

Footnotes

Acknowledgments

The authors are grateful to Dr. Oleg Lavrukhin for his editorial contribution, and Dr. Carlos José de Carvalho Pinto for providing T. klugi. CHS was a fellowship recipient from Fundação de Apoio à Pesquisa Científica e Tecnológica do Estado de Santa Catarina.

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