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Development of assays to evaluate cellular immune functions in the American lobster (Homarus americanus).

ABSTRACT A significant mortality of the American lobster (Homarus americanus) in western Long Island Sound in 1999 seriously impacted the harvest of this species. Whereas the exact cause or causes of the mortality are still not precisely known, dead and dying lobsters in the initial phase of the die-off were diagnosed with infections with paramoeba, a newly recognized disease condition of lobsters. Because the immune system represents the first line of defense against disease-causing agents and is one of the most sensitive systems to environmental stressors, this study is aimed at the development of new assays to quantify the immune system of lobsters. Flow cytometry allowed the discrimination of different populations of hemocytes based on their relative size and complexity. Also using flow cytometry, natural killer (NK) cell-like activity and its stimulation by human recombinant interleukin 2 (IL-2) are described for the first time in lobsters, as is the expression of TLR2, a pattern recognition receptor, on granular hemocytes. Apoptosis is also measured for the first time in lobster hemocytes in higher proportion in nongranular hemocytes than in granular hemocytes. Also shown is the fact that circulating hemocytes fail to proliferate on stimulation, suggesting that they are terminally differentiated and originate from a separate hematopoietic organ. Overall, several new assays are developed to allow the quantitative evaluation of disease-relevant immune functions for future use in health assessment of the American lobster.

KEY WORDS: immunology, lobster, Homarus americanus

INTRODUCTION

A significant mortality of the American lobster (H. americanus H. Milne Edwards, 1837) in western Long Island Sound (LIS) in 1999 seriously impacted the harvest of this species. A paramoeba associated with histopathologic lesions was described in the nervous tissue of lobsters affected by this die-off (Mullen et al. 2004). This paramoeba was found only in dying or dead lobsters from LIS and not in healthy lobsters from outside the Sound (Mullen et al. 2004). Nevertheless, it is not known if this organism is a primary pathogen that could in and of itself have caused a die-off or if it is an opportunistic pathogen that needed to take advantage of multiple environmental stressors to cause a disease in lobsters. Other potentially contributing factors that have been identified include pesticides used in the control of West Nile Virus, low dissolved oxygen and high water temperature, sewage treatment plant discharges, dredge material and associated pollutants and others.

The immune system is one of the most important single factors determining an individual's resistance to pathogens. Interestingly, it is also one of the systems that is most susceptible and sensitive to the effects of xenobiotics (Tryphonas & Feeley 2001). Environmental stressors (chemical or other) that could have affected lobsters in the fall of 1999 might very well have affected the immune system of lobsters, possibly reducing their resistance to pathogens (for example, paramoeba), possibly resulting in a significant contribution to the die-off. Yet, relatively little is known about the functioning of the immune system in lobsters.

The mammalian immune system consists of acquired or adaptive immunity, with its characteristic specificity and memory for specific antigens, and innate immunity, which is not specific for an antigen and does not have memory but is always ready (because it does not require previous exposure to be efficient) (Kuby 1997). Through evolution, the acquired immune system made its appearance with the acquisition of the jaw in fishes, with no evidence to this day of immunoglobulins, T cell receptor or recombination activating gene (RAG) in jawless vertebrates or invertebrates (Peixoto et al. 2000). Invertebrates lack adaptive or acquired immunity. Their defense mechanisms consist only of an innate immune system, defined in mammalian immunology as the first-line host defense that serves to limit infection in early hours after exposure to microorganisms (Hoffmann et al. 1999). The last few years have seen a resurgence of literature on the almost forgotten innate immune system, with invertebrates often used as models for similar, evolutionarily-conserved systems and mechanisms in mammals (Franc et al. 1999, Kopp & Medzhitov 1999, Ulevitch & Tobias 1999).

Although there is a high degree of conservation of innate mechanisms between vertebrates and invertebrates (Du Pasquier 2001), interspecies differences do exist, and there have been recent challenges to the portability of knowledge from one species to another (Warr et al. 2003). In view of the importance of the immune system in resistance to infectious disease, the infectious paramoeba identified in association with the die-off and the paucity of knowledge in lobster cellular immunology, this study investigates several different cellular innate immune functions and characteristics in lobsters, including natural killer cell-like activity, hemocyte proliferation, immunophenotyping and apoptosis.

MATERIALS AND METHODS

Animals and Hemolymph Sampling

Healthy adult lobsters from Maine devoid of obvious clinical signs of disease or external lesions were maintained at 14[degrees]C in artificial sea water (Instant Ocean, Mentor, Ohio) at a salinity of 24 parts per thousand in a 1,400-L tank equipped with a recirculating filtration system and air bubblers. All lobsters were acclimated for a minimum of 1 wk prior to use in experimental procedures, and they rested for a minimum of 1 wk between consecutive hemolymph sampling. Two milliliters of hemolymph was sampled, using a 20-gauge 1.5-inch needle, from the dorsal vasculature of lobsters and 500 [micro]L was immediately transferred to 2.5 mL anticoagulant. Preliminary studies in our laboratory determined that while acid citrate dextrose (ACD) Vacutainer tubes (Becton Dickinson, Franklin Lakes, NJ) prevented coagulation it considerably reduced the pH of the sample. Mixing 500 [micro]L of lobster hemolymph with 2.5 mL 3.8% (0.129M) buffered sodium citrate (pH 6.1) in Vacutainer tubes (Becton Dickinson, Franklin Lakes, New Jersey) adequately prevented coagulation without significantly modifying pH; this was used for all experiments.

Natural Killer (NK) Cell-like Activity

NK-like activity of lobster hemocytes was measured against NK-sensitive K-562 cells (ATCC, Rockville, MD), a human erythroleukemic cell line, using two-color flow cytometry. The method was modified from one reported before (Chang et al. 1993), as previously described (De Guise et al. 1997). The K-562 target cells were washed twice in complete Dubelco modified Eagle medium (DMEM) medium (DMEM supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM minimal nonessential amino acids, 5 units/mL penicillin, 50 mg/mL streptomycin (media and supplements from Gibco Laboratories Life Technologies Inc., Grand Island, New York) and 10% fetal calf serum (HyClone Laboratories Inc. Logan, Utah), and concentration was adjusted to [10.sup.6] cells/mL. One milliliter of the suspension was forcefully added to 30 [micro]L of a 3-mM solution of the lipophilic carbocyanine membrane dye 3,3'-dioctadecyloxarbocyanine perchlorate (DiO, Molecular Probes, Eugene, Oregon), dissolved in dimethyl sulfoxide (DMSO, Sigma Chemicals Co, St. Louis, Missouri). The K-562 cells were then incubated for 20 min at 37[degrees]C in 5% C[O.sub.2], followed by 2 washes in phosphate buffered saline (PBS), and then resuspended in complete DMEM medium at a concentration of 2 x [10.sup.5] cells/mL.

Hemolymph collected in anticoagulant (see earlier) was either used without further dilution, or further diluted with cell-free lobster hemolymph supernatant (obtained by collecting the supernatant from lobster hemolymph after centrifugation at 220g for 10 min) at different ratios (1 in 2, 1 in 4 or 1 in 8), and 250 [micro]L of this effector cell (hemocyte) suspension were mixed with 250 [micro]L complete DMEM and 50 [micro]L of the K-562 target cell suspension. Tubes without effector cells were used to assess spontaneous target cell mortality. The tubes were centrifuged at x750g for 30 sec, and then incubated at 0[degrees]C in 5% C[O.sub.2] for 150 min. The tubes were then placed on wet ice, the contents filtered on glass wool-filled Pasteur pipets (to remove cellular debris and clumps), and propidium iodide was added to obtain a final concentration of 50 [micro]g/mL. The cell suspensions were then analyzed by flow cytometry using a FACScan (Becton Dickinson, Mountain View, California) equipped with the CellQuest software (Becton Dickinson Immunocytometry Systems, San Jose, California). NK cell-like activity was calculated by subtracting the spontaneous mortality of target cells (mortality of control target cells incubated without lobster cells) from that of target cells incubated with lobster cells. Cell counts on hemolymph samples were performed using a hemocytometer so that the results could be expressed as effector:target cell ratios. In some experiments, recombinant human interleukin-2 (IL-2, Research Diagnostics Inc, Flanders, New Jersey) was added to the samples (0.05, 0.1 or 0.2 ng/mL) to assess its influence on NK cell-like activity.

Hemocyte Proliferation

Hemolymph collected in sodium citrate anticoagulant (see above) was diluted in an equal volume of sterile sea water (Sigma Chemicals Co, St. Louis, Missouri) supplemented with 10% fetal calf serum, 5 units/mL penicillin and 50 mg/mL streptomycin, with or without mitogens. To measure proliferation, lobster hemocytes were stimulated with the mitogens concanavalin A (conA, Sigma Chemicals Co, St. Louis, Missouri, 0.31-2 [micro]g/mL), phytohemagglutinin (PHA, Sigma Chemicals Co, St. Louis, Missouri. 0.31-2 [micro]g/mL) or lipopolysaccharide (LPS, Sigma Chemicals Co, St. Louis, Missouri, 3.1-200 [micro]g/mL), as well as cultured without mitogen (unstimulated control). Cells (in diluted hemolymph) were plated in triplicate in flat-bottomed 96-well plates in a total volume of 200 [micro]L. Hemocyte proliferation was assessed using incorporation of bromodeoxyuridine (BrdU, Roche Diagnostic Corp., Indianapolis, Indiana), a thymidine analogue, into the DNA of proliferating cells, as detected with a Cell Proliferation ELISA BrdU (colorimetric) kit (Roche Diagnostic Corp., Indianapolis, Indiana). Briefly, the cells were cultured for 48 h at 14[degrees]C, then pulsed for 18 h by addition of BrdU to each well. Plates were centrifuged, the supernatant was discarded and the plates were dried. BrdU incorporation was measured using a peroxidase-labeled antiBrdU monoclonal antibody and subsequent colorimetric quantification and an ELISA plate reader (at 690 nm with a reference wavelength of 450 nm) as per manufacturer's recommendations (Boehringer Mannheim, Mannheim, Germany). Results were expressed as optical density (OD) and compared with control unstimulated cells.

Hemocytes Immunophenotyping

Hemocytes were labeled with antibodies against three pattern recognition receptors that are well conserved across mammals and invertebrates: CD14, Toll-Like Receptor (TLR) 2 and TLR4. Hemolymph was drawn in sodium citrate anticoagulant (see above) and kept at 4[degrees]C. Cell counts and assessment of cell viability were performed using a hemocytometer and staining with Trypan blue using standard procedures. Aliquots of hemolymph diluted in sodium citrate from each lobster were incubated (30 min at 4[degrees]C) with 100 [micro]g/mL of lipopolysaccharide (LPS) dissolved in sterile seawater or sterile seawater as negative control. Neutral buffered formalin was then added to a final concentration of 1%, and the cell suspension incubated for 10 min at 4[degrees]C. Aliquots of the cell suspension, containing approximately 1.0-1.5 x [10.sup.6] cells, were centrifuged, and the supernatant discarded. The cell pellets were resuspended in 20 [micro]L of the fluorescent-labeled antibodies to human surface molecules (PE-CD14 (IgG1), FITC-TLR-2 (IgG2a), PE-TLR-4 (IgG2a)) or isotype controls (all from e-Biosciences, San Diego, California), and incubated for 30 min in the dark, at 4[degrees]C. The cells were then washed in 500 [micro]L PBS and pellets were resuspended in 500 [micro]L PBS. Cell-associated fluorescence was measured using a FACScan flow cytometer. Populations of granulocytes were selected based on their relative size (forward scatter, FSC) and complexity (side scatter, SSC), and mean fluorescence of each histogram for this population of cells was calculated using the CellQuest software. A 2-way analysis of variance, using the SigmaStat Windows 1.0 (Jandel Scientific, San Rafael, California) software and P < 0.05 for statistical significance, was used to assess differences between groups (LPS vs. no stimulation, labeling with antibody of interest vs. isotype control) when labeling was observed.

Hemocyte Apoptosis

After hemolymph collection with anticoagulant (see earlier), cells were counted and 1 x [10.sup.6] cells were resuspended in 1 mL Caltag binding buffer (Caltag Laboratories, Burlingame, California) and adjusted with NaCl to be isotonic with the water in which lobsters were maintained to avoid osmotic shocks to the cells. Five [micro]l Caltag Annexin V-FITC conjugate (Caltag Laboratories, Burlingame, California) and 0.5 [micro]g propidium iodide were added to 1 x [10.sup.5] hemocytes (100 [micro]L of the original cell suspension). After a 15-min incubation at 20[degrees]C, the cell suspension was diluted 1:4 with binding buffer. Apoptosis-associated fluorescence (Annexin V-FITC) and necrotic cell-associated fluorescence (propidium iodide) were measured using a FACScan flow cytometer with the CellQuest software. Negative controls were incubated with isotonic seawater instead of propidium iodide or Annexin-V to adjust the quadrants. Apoptotic cells were defined as Annexin-V positive-staining and propidium iodide negative-staining cells.

RESULTS

Natural Killer (NK) Cell-like Activity

In all experiments, the labeling of target cells with DiO allowed their discrimination from lobster cells based on their fluorescence in FL1. Also, the addition of propidium iodide allowed the discrimination of dead cells, which incorporate the dye and become more fluorescent in FL2 from the less fluorescent live cells. The use of dual fluorescence flow cytometry then allowed the quantification of the proportions of live and dead target cells (Fig. 1) for the calculation of NK cell-like activity as described above. As expected for NK cell activity, increasing the effector:target cell ratio resulted in increased specific mortality of target cells (Fig. 2). The addition of increasing concentrations of cell-free lobster hemolymph supernatant resulted in no increased target cell mortality, supporting that the activity was cell-associated. The addition of 0.2 ng/mL of human recombinant IL-2, a physiologicallyrelevant concentration, increased the specific mortality of target cells, irrelevant of the effector:target cell ratio (Fig. 2).

[FIGURES 1-2 OMITTED]

Hemocyte Proliferation

None of the stimulation regimes resulted in proliferation of lobster hemocytes above the baseline level of BrdU incorporation by unstimulated cells (Fig. 3).

[FIGURE 3 OMITTED]

Hemocyte Immunophenotyping

For CD14 and TLR-4, there was no clear cell labeling (i.e., no discernable increase in cell-associated fluorescence on antibody labeling as compared with isotype control antibodies) either in the presence or absence of LPS (data not shown). The fluorescence of cells labeled with antiTLR-2 was increased significantly (approximately double) compared with controls in the most granular hemocytes from every individual lobster (Fig. 4). There was no clear labeling of TLR2 of the other cell populations examined. Stimulation with LPS did not significantly increase antibody labeling of the most granular cells compared with controls (Fig. 4).

[FIGURE 4 OMITTED]

Hemocyte Apoptosis

Two-color flow cytometry allowed the discrimination of hemocytes labeled with Annexin V only, and therefore considered apoptotic, from those labeled with propidium iodide, demonstrating the lack of integrity of their cell membrane and therefore considered necrotic (Fig. 5). It is important to understand that hemocytes labeled with both probes (double positive) do lack intact cell membranes and should be considered necrotic. The different hemocyte populations differed markedly in the proportion of cells undergoing apoptosis and necrosis, with most of the granular cells viable, and over 40% of the less granular cells undergoing either apoptosis or necrosis (Fig. 5).

[FIGURE 5 OMITTED]

DISCUSSION

Hemocytes, the immune cells of lobsters, are classified according to their morphology into hyaline hemocytes or hyalinocytes, which are generally smaller and contain few granules and granulocytes which are larger and contain a larger number of granules. Granulocytes are further divided into small-granule hemocytes, which have fewer and smaller granules than the more mature large-granule hemocytes (Martin & Hose 1995). Hyalinocytes are believed to be the major cells involved in coagulation, whereas granulocytes are believed to be responsible for most of the phagocytosis (Martin & Hose 1995). Although there are relatively important differences between individuals (as exemplified by the differences between Fig. 4a and 5a), it is likely that the more complex population of cells (the ones with the highest SSC value) are granulocytes, whereas those less complex are hyalinocytes, as was verified in prawns (Yip & Wong 2002) and crayfish (Cardenas et al. 2000). Flow cytometry would then allow the recognition of lobster granulocytes and hyalinocytes, but would not allow the discrimination between small-granule and large-granule hemocytes in our study.

Natural killer cell activity is part of natural immunity. In mammals, it is particularly important in defense against tumor cells and the early phase of viral infections (O'Shea & Ortaldo 1992). The complex mechanisms used to recognize target cells have been elucidated only recently, with receptors that recognize carbohydrates and provide a positive signal for killing and receptors that recognize "self" through intact MHC class I, providing a negative signal that overrides the positive signal (Lanier 1997). In addition, mammalian NK cells are capable of antibody-dependent cellular cytotoxicity (ADCC), through which NK cells recognize (through their FC receptor) antibody-labeled target cells (Kuby 1997). Natural cytotoxicity or NK cell-like activity has been demonstrated in a wide range of species including birds (Sieminski-Brodzina & Mashaly 1991), fish (Greenlee et al. 1991, Shen et al. 2004), earthworms (Cooper et al. 1995), snails (Franceschi et al. 1991) and crayfish (Soderhall et al. 1985) but to the authors' knowledge has never before been demonstrated in lobsters. Whereas NK cell-like activity was attributed to the semigranular and granular cells in crayfish (Soderball et al. 1985) and to the nonadherent round hemocytes in snails (Franceschi et al. 1991), the cell type or mechanism responsible for that function in lobsters in this study was not determined. The enhancement of lobster NK cell-like activity by recombinant human IL-2 represents the first demonstration of a conserved activity for this cytokine in crustaceans, whereas IL-2 was also shown to preserve NK cell-like activity in snails (Franceschi et al. 1991). Whereas tumors have not been described in American lobsters, viral infections do occur in spiny lobsters (Panulirus argus), a related species of lobsters (Shields & Behringer Jr 2004), suggesting the potential for the functional relevance of the NK cell-like activity described here.

Proliferation of lymphocytes is an important initial feature of the acquired immune response to pathogens in vertebrates. Hemocytes collected from the hemolymph failed to proliferate on stimulation with LPS, Con A and PHA, suggesting that circulating hemocytes are terminally differentiated quiescent cells and originate from a separate hematopoietic organ rather than dividing in hemolymph. This is in agreement with findings in crayfish, in which cell proliferation was detected in the hematopoietic tissue but not in circulating hemocytes (Soderhall et al. 2003). Our results are also compatible with the fact that shrimp hemocytes did not survive more than 4 days in culture, suggesting that they did not divide, as compared with ovary and lymphoid cultures that were maintained alive for at least 20 days (Chert & Wang 1999).

"Pattern recognition receptors" are used by the innate immune system to discern molecular patterns in a variety of pathogens that all differ from self (Janeway 1992). Those molecules are well conserved between vertebrates and invertebrates, and the striking homology of the cytoplasmic domain of Drosophila and mammalian TLRs 1, 2, 4 and 6 suggests that an ancestral (prevertebrate) TLR may have adopted a proinflammatory function 500 million years ago (Du et al. 2000). It has been established that TLR4 recognizes the gram-negative product lipopolysaccharides (LPS), whereas TLR2 recognizes various fungal, gram-positive and mycobacterial components (Ozinsky et al. 2000, Tapping et al. 2000). TLR4 was also recognized as the predominant signaling receptor for LPS in human blood, whereas CD14, which is incapable of signal transduction across cell membrane, enhances TLR-mediated cell activation (Tapping et al. 2000). A molecule antigenically homologue to human TLR2, but not to TLR4 nor CD14, has been recognized for the first time on the surface of lobster hemocytes using monoclonal antibodies to the human molecules. This suggests that lobsters may be well equipped to recognize fungal, gram-positive and mycobacterial components, primarily with their granular hemocytes. On the other hand, the lack of reactivity to antibodies to human TLR4 and CD14 on lobster hemocytes suggest that either lobsters lack those molecules and are likely to fail to recognize gram-negative bacteria carrying LPS, or more likely that lobster TLR4 and CD14 are present on the surface of hemocytes but are evolutionary divergent enough to fail to be detected by antibodies to the human molecules. Molecular studies searching for DNA sequences for TLR4 and CD14 in lobsters could answer this question. It is nevertheless interesting to notice that of the two best characterized lobster bacterial diseases, gaffkemia caused by the gram-positive Aerococcus viridans (Steenbergen et al. 1977, Stewart 1993) and limp lobster disease caused by the gram-negative Vibrio fluvialis-like organism (Tall et al. 2003), one is caused by a gram-negative organism for which we have not detected pattern recognition receptors.

Apoptosis, sometimes referred to as programmed cell death, is a very important regulatory mechanism, particularly for development and the immune system. Among other functions, it represents a relatively rapid way to get rid of activated cells once the stimulus has subsided. Apoptosis is a phylogenetically well-conserved phenomenon, and regulatory genes with functions similar to those in mammals have been identified in Drosophila, confirming the broad diversity of species using this mechanism. We have demonstrated apoptosis in lobster cells for the first time, using flow cytometry and Annexin V, a phospholipid-binding protein that recognizes phosphatidylserine externalized in the early phase of apoptosis. A higher percentage of apoptosis and necrosis in nongranular hemocytes, compared with granular, suggests that nongranular hemocytes are either more prone to apoptosis and necrosis or the unlikely possibility that the experimental labeling procedures selectively alter these cells.

CONCLUSION

Overall, we have used flow cytometry to separate lobster hemocytes into two subpopulations, and to demonstrate for the first time NK cell-like activity and its responsiveness to human IL-2, as well as to demonstrate the presence of TLR2 on lobster granular hemocytes. The lack of proliferation of circulating hemocytes in apparently healthy lobsters was also shown for the first time. Given that lobsters live in an environment where they are constantly challenged with exposure to microorganisms, and with the presence of highly conserved mechanisms for innate immunity across several phyla, these functions, which appear relevant to the most frequent diseases of lobsters, will be important tools in future health assessment efforts.

ACKNOWLEDGMENTS

This research was supported by the Connecticut Sea Grant College Program, Grant No. LR/LR-2, through the United States Department of Commerce, National Oceanic and Atmospheric Administration (NOAA), Award No. NA16RG1364.

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SYLVAIN DE GUISE,* BRENDA MORSEY, JENNIFER MARATEA, MICHAEL GOEDKEN, INGA SIDOR AND JAIMES ATHERTON

Department of Pathobiology and Veterinary Science, University of Connecticut, 61 North Eagleville Road, U-89, Storrs, Connecticut 06269

* Corresponding author. E-mail: sylvain.deguise@uconn.edu
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Author:Atherton, Jaimes
Publication:Journal of Shellfish Research
Geographic Code:1USA
Date:Oct 1, 2005
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