In vitro activation of hemocytes from the American lobster, Homarus americanus, as measured by production of reactive oxygen species.

ABSTRACT Hemocytes from the American lobster (Homarus americanus) were exposed to several receptor-independent and -dependent putative NADPH oxidase stimulators. This stimulation should result in the generation of cytotoxic reactive oxygen species (ROS) that are used by the cells to control infections by destroying ingested microorganisms. Superoxide ([O.sub.2.sup.-]) and hypocholorous acid (HOCl) were quantified by the use of chemiluminescent probes. Only phorbol myristate acetate (PMA), of all the stimuli tested, produced a strong ROS response, which was characterized primarily by the generation of a ~20-fold increase in HOCl. Unstimulated cells produced small [O.sub.2.sup.-] and HOCl peaks. Exposure of the cells to arachidonic acid, zymosan, Bacillus subtilis, conA, laminarin and E. coli lipopolysaccharide failed to stimulate net [O.sub.2.sup.-] generation. However, most of these agents as well as PMA caused the time to peak [O.sub.2.sup.-] production to be significantly reduced. The significance of this apparent kinetic shift (KS) in the [O.sub.2.sup.-] is unknown. The results suggest that, although lobster hemocytes in vitro do not respond to a number of known ROS elicitors, PMA will stimulate a large HOCl response presumably involving the typical pathway of protein kinase C activation, translocation of cytosolic NADPH oxidase components to the site of enzyme assembly and cellular activation.

KEY WORDS: lobster, hemocytes, immunity, Homarus americanus, reactive oxygen species

INTRODUCTION

Circulating hemocytes play a central role in the defense of invertebrates against various microorganisms that gain access to the hemolymph. American lobster (Homarus americanus H. Milne Edwards, 1837) hemolymph contains numerous granulecontaining and agranular cells which are known to be phagocytic and active in the destruction of ingested microbes (Paterson et al. 1976). These hemocytes probably depend on oxygen-independent antimicrobial activities such as lysosomal hydrolases and oxygen-dependent mechanisms such as the production of ROS. In this study, the ability of lobster hemocytes to generate ROS is examined using luminol and lucigenin to quantify myeloperoxidase (MPO)-dependent HOCl production and [O.sub.2.sup.-] generation, respectively (Stevens & Hong 1984, Dahlgren & Stendahl 1983). Phagocytic cells in many species, when properly activated, undergo a burst of respiratory activity followed by the production of ROS catalyzed by the membrane-associated enzyme NADPH oxidase. The initial ROS produced is [O.sub.2.sup.-] that is dismutated to hydrogen peroxide ([H.sub.2][O.sub.2]), which may be converted to the highly cytotoxic molecules HOCl and chloramines in the presence of MPO and chloride ions (Klebanoff 1985). This pathway has been demonstrated in aquatic invertebrates including certain mollusks and crustaceans (Adema et al. 1991, Anderson 1996, Bell & Smith 1993, Bodhipaksha & Weeks-Perkins 1994, Song & Hsieh 1994); however, ROS production is difficult to demonstrate in a few species, such as the clam Mercenaria mercenaria (Anderson 1994).

The production of ROS is initiated by the respiratory burst enzyme, which becomes activated on translocation of several cytosolic proteins to a membrane-bound complex containing flavocytochrome [b.sub.558], the core of the [O.sub.2.sup.-] generating system (Morel et al. 1991). In mammalian macrophages NADPH oxidase can be activated by receptor-dependent stimuli such as the complement fragment C[5.sub.a], the chemotactic tripeptide fMLP and immune complexes. Receptor-independent stimuli include phorbol 12-myristate 13-acetate (PMA) and certain long-chain unsaturated fatty acids (Leusen et al. 1996). These and other potential NADPH activators, based on the literature on ROS stimulation in mammalian and invertebrate phagocytes, were tested using lobster hemocytes.

MATERIALS AND METHODS

Animals and Hemolymph Collection

Equal numbers of male and female 1 1/4 lb. lobsters (Homarus americanus) were purchased from commercial suppliers in Stonington, Maine and Martha's Vineyard, Massachusetts. They were banded and held in a 325-gallon recirculated aquarium system, each in an individual plastic chamber. The water was pumped constantly through a filtration system, UV sterilizing chambers and a column containing biobeads with established bacterial cultures. The system contained 34 ppt Instant Ocean (Aquarium Systems, Inc. Mentor, OH) at 12[degrees]C; levels of nitrate/nitrite and ammonia were monitored and full or partial water changes made whenever these parameters were outside the acceptable range. The lobsters were fed three times per week with lobster pellet food (Ziegler Bros., Inc., Gardners, PA) or Mytilus edulis. Hemolymph (2 mL) was withdrawn from the dorsal tail region of the lobster into a 10-mL syringe containing 4 mL of ice-cold crustacean anticoagulant (0.45 M NaCl, 0.1 M glucose, 30 mM trisodium citrate, 26 mM citric acid, 10 mM EDTA, pH 4.6) (Smith & Soderhall 1983). This mixture of cells and anticoagulant was held on ice until centrifuged (x300g, 4[degrees]C, 10 min) to remove the hemocytes. The hemocyte pellet was resuspended in 4 mL of marine crustacean saline (MCS) containing 0.58 M NaCl, 13 mM KCl, 13 mM Ca[Cl.sub.2], 26 mM Mg[Cl.sub.2], 0.54 mM [N.sub.2]HP[O.sub.4] and 50 mM Tris-HCl buffer, pH 7.6 (Smith & Soderhall 1983).

Chemiluminescence Assay

The cell numbers were determined using a hemocytometer and adjusted with MCS to 3 x [10.sup.6]/mL; 0.1 mL of this cell suspension was added to wells of a white 96-well microtiter plate. PMA (phorboll2-myristatel3-acetate) and all other potential chemical hemocyte stimuli tested were purchased from Sigma Chemical Co. (St. Louis, MO). Luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) was prepared according to the method of Scott and Klesius (1981) and added to wells at a final concentration of 10 [micro]M. Lucigenin (10,10'-9,9'-dimethyl-biacridinium nitrate) was dissolved in water and added to other wells at a final concentration of 150 [micro]M. A typical chemiluminescence (CL) run would consist of wells containing 3 x [10.sup.5] hemocytes (aliquots of cells from the same lobster), wells containing hemocytes plus noncytolethal (by trypan blue exclusion) dilutions of putative NADPH oxidase stimuli, wells containing cells and 5 [micro]g/mL PMA and luminol or lucigen added to each well. The plates were placed in a Packard Topcount microplate scintillation and luminescence counter (Packard, Meriden, CT) adjusted for single photon counting. The plates were maintained at 26[degrees]C in the dark and each well was automatically read (0.05 min), all wells were repeatedly read at 3.6-min intervals. The plates were counted for 10-15 min to establish a stable background prior to the addition of stimuli, then about 2 h of counting cycles was carried out. The data from PMA treated cells served as positive controls, making it possible to insure that the hemocytes being tested were capable of responding to stimulation. The areas under the curves of the CL peaks produced by unstimulated and treated hemocytes were determined, as were the temporal intervals between addition of stimuli and the observed peaks. For any given treatment, the significance between the total CL peak areas and peak times of untreated and stimulated cells were determined. The following stimuli were tested: PMA (1-10 [micro]g/mL), arachidonic acid (1.5-25.0 [micro]M), zymosan (ratio of hemocyte to zymosan particles of 1:2-1:25), E. coli serotype 111: B4, LPS (6.25-50.0 [micro]g/mL), Bacillus subtilis (ATCC 6051, ratio of hemocytes:bacteria of 1:1-1:25), conA (1.25-1,250 [micro]g/mL) and laminarin (0.025-10.0 mg/mL). B. subtilis was grown overnight in nutrient broth at 30[degrees]C, centrifuged out of the broth and resuspended in MCS.

RESULTS

Production of [O.sub.2.sup.-] After PMA Treatment

Lucigenin CL showed a low-level release of [O.sub.2.sup.-] from untreated hemocytes that peaked 50.7 [+ or -] 14.4 min after introduction to the microplate wells (Fig. 1). It is likely that the cells may become partially activated by contact and adhesion to the wells. The addition of 5 [micro]g PMA/mL resulted in a shortened time to peak activity (23.1 [+ or -] 19.0 min, n = 19, P < 0.0001); however, no increase in net [O.sub.2.sup.-] production was caused by PMA treatment. This was concluded after comparing the areas under the ROS curves for PMA-treated and untreated hemocytes. Multiple PMA treatments failed to elicit additional CL responses or any net [O.sub.2.sup.-] production (data not shown).

[FIGURE 1 OMITTED]

Production of HOCl After PMA Treatment

As was the case for [O.sub.2.sup.-] , a minor CL peak was seen in control wells containing hemocytes and luminol. This "resting" cell activity peaked at 24.4 [+ or -] 15.3 min (Fig. 2). The addition of PMA stimulated HOCl production in all hemocyte preparations tested; however, the result was variable (21.0 [+ or -] 17.3-fold increase, n = 44). The difference between HOCl and [O.sub.2.sup.-] production by PMA-treated hemocytes was significant (P < 0.001, unpaired t-test with the Welch correction). The PMA-stimulated CL peak was recorded at 28.3 [+ or -] 17.8 min and showed no significant temporal difference from that of the untreated cells. No additional luminol-dependent CL could be elicited by PMA treatments ~2 h after the initial stimulation (data not shown).

[FIGURE 2 OMITTED]

ROS Responses to Arachidonic Acid

Arachidonic acid is considered to be a receptor-independent NADPH oxidase activator, as is PMA. Concentrations of 1.5-25.0 [micro]M arachidonic acid resulted in no net [O.sub.2.sup.-] or HOCl production, but it did significantly speed up the [O.sub.2.sup.-] response (P < 0.001 for all concentrations tested, Mann-Whitney nonparametric t-test) (Fig. 3). This effect of several putative ROS stimulators on shortening the [O.sub.2.sup.-] peak time was often seen, and will hereafter be referred to as the KS. In the case of arachidonic acid, the KS seemed to be dose-dependent, but this effect was rarely seen for the other agents studied.

[FIGURE 3 OMITTED]

ROS Responses to Receptor-Dependent Stimuli

Phagocytic stimuli such as zymosan, a yeast cell wall preparation rich in [beta]-1,3-glucans, and bacteria (Bacillus subtilis) produced no net [O.sub.2.sup.-] or HOCl stimulation, although both particles were ingested by the hemocytes (Table 1). Zymosan sometimes produced a KS for the [O.sub.2.sup.-] response. Laminarin, a soluble [beta]-1,3-glucans, occasionally stimulated ROS but no significant net [O.sub.2.sup.-] or HOCl production was seen; however, the typical KS for the [O.sub.2.sup.-] peak was seen at 10 mg/mL (P < 0.05, n = 6), but not at higher concentrations. The plant lectin ConA produced no significant increase in net ROS. The [O.sub.2.sup.-] peaks showed a KS at 1.25 [micro]g/mL (P < 0.005, n = 6) and 12.5 [micro]g/mL (P < 0.005, n = 6), but not at higher concentrations. E. coli lipopolysaccharide (LPS) also failed to stimulate net ROS production, but produced a clear KS for the [O.sub.2.sup.-] peaks at all concentrations tested (6.25-50.0 [micro]g/mL, P < 0.0001).

DISCUSSION

Invading microorganisms encounter a large assortment of defense mechanisms including phagocytes that play an important role in their destruction by the production of ROS. ROS are generated by the action of NADPH oxidase, and there are multiple signal transduction pathways involved in its activation. Like its mammalian counterpart the macrophage, the lobster hemocyte produces little ROS in the resting (untreated) state. To fully and effectively respond to microbial infections, an activated NADPH oxidase system in the hemocytes is probably required. Therefore, the efficacy of several putative activators was studied in hemocyte isolates. In no case did any of the treatments cause hemocyte cytolethality, as determined by trypan blue exclusion.

During activation of mammalian phagocytes, cytosolic protein factors (p47-phox, p67-phox and p40-phox) translocate to cytochrome [b.sub.558 ]in the plasma membrane to form the active enzyme NADPH oxidase (Clark et al. 1990). Electrons supplied by NADPH are transferred to oxygen to produce [O.sub.2.sup.-]. The natural in vivo activation of lobster hemocytes would be expected to involve recognition and binding of microorganisms via "non-self" receptors and/or receptors against PAMPs (pathogen-associated molecular patterns) such as bacterial LPS and fungal mannans and [beta]-1, 3-glucans. In this study, a number of receptor-dependent and -independent activators are evaluated; the products of NADPH oxidase are not only important in controlling infectious diseases and in molecular signaling, but also can possibly contribute to inflammatory disorders (hemocyte infiltration) and tissue necrosis when over-produced.

PMA is a receptor-independent stimulus. The details of PMA activation of NADPH oxidase have been described by Curnutte et al. (1994). PMA activates PKC (protein kinase C) which, in turn, is involved in the rapid phosphorylation of multiple serine residues on p47-phox, promoting its translocation to the site of NADPH oxidase assembly. PMA mimics DAG (diacylglycerol), which is the natural ligand and activator of PKC. In lobster hemocytes, PMA strongly stimulated luminol-dependent CL, indicating the generation of HOCl via the myeloperoxidase pathway (Fig. 2). The stimulated peak HOCl response occurred at about the same time as the small HOCl peak activity characteristic of the untreated cells. Theoretically, the untreated hemocytes should produce no ROS; the low level CL response probably is caused by minor activation associated with handling, as well as attachment to and spreading on the walls of the microplate wells, as suggested by Helmke et al. (1990). Treatment of lobster hemocytes with PMA altered [O.sub.2.sup.-] production kinetics but seemed to have little effect on net [O.sub.2.sup.-] generation. As with HOCl, the untreated cells generated a low and comparatively broad [O.sub.2.sup.-] peak (Fig. 1). The addition of PMA resulted in a more defined [O.sub.2.sup.-] response with a sharper peak occurring more rapidly than seen in the controls. This shorter [O.sub.2.sup.-] peak time, or KS, was a common response to the activators used in this study (Table 1). The physiologic significance of this KS is not known; it might represent a type of accelerated innate immune response, but it does not seem to be connected to cell priming because subsequent stimulation with PMA did not elicit an additional response. Although PMA exposure often resulted in elevated peak [O.sub.2.sup.-] values as compared with untreated hemocytes, the total net [O.sub.2.sup.-] responses were more accurately determined by comparing the areas under the CL curves. Using this calculation it was clear that whereas PMA altered [O.sub.2.sup.-] peak heights and response kinetics, it produced little or no increment in total production.

Long-chain unsaturated fatty acids like arachidonic acids have been shown to be receptor independent activators of NADPH oxidase. Arachidonic acid is involved in activation of rac, a cytosolic protein required for NADPH oxidase activity. In the cytosol, rac is usually present as an inactive complex; arachidonic acid disrupts its binding to Rho-GDI (Abo et al. 1991, Chuang et al. 1993). Rac is involved in phosphorylation of p47-phox and in translocation of p47-phox and p67-phox to the site of NADPH assembly at the membrane. Unlike PMA, arachidonic acid did not stimulate either net HOCl or [O.sub.2.sup.-] production in lobster hemocytes. However, the KS of [O.sub.2.sup.-] generation was seen after exposure to arachidonic acid. In these studies the peak time for the [O.sub.2.sup.-] response was not only shifted to the left, but also was increasingly shortened with increasing arachidonic acid concentrations (Fig. 3).

Attempts were made to activate lobster NADPH oxidase by exposure of hemocytes to receptor-dependent activators related to recognition of bacteria and yeast. Yeast cells and zymosan (a particulate preparation of yeast cell walls) are readily phagocytosed by lobster cells. This response is largely dependent on cellular recognition of [beta]-1, 3-glucans receptors, as demonstrated in crayfish by Barracco et al. (1991). Cellular binding of [beta]-1, 3-glucans has been shown to trigger ROS production in shrimp (Song & Hsieh 1994). In this study, neither zymosan nor laminarin (a soluble [beta]-1, 3-glucan) produced increased net production of [O.sub.2.sup.-], but both compounds could shorten the time to the [O.sub.2.sup.-] peaks. Zymosan apparently was ineffective in enhancing net HOCl generation and laminarin enhanced the HOCl response in a few hemocyte preparations, but neither treatment shifted the time of the HOCl peaks. Natural agglutinating agents (lectins) in invertebrate hemolymph have been shown to act as humoral recognition factors that react with foreign particles and facilitate their eventual phagocytosis (Vasta 1991). The presence of saccharide-recognizing lectins in lobster hemolymph was described by Cornick and Stewart (1973). This activity is akin to the process of opsonization in vertebrates, which involves antibodies as recognition molecules. In mammals, antigen-antibody complexes bind to phagocytes via the [F.sub.c] receptor triggering activation of NADPH oxidase (Kramer et al. 1988). We attempted to use a lobster lectin-zymosan complex to produce a similar result, but could show no significant increase in ROS production. This is consistent with previous results showing that the presence of lectins or other hemolymph proteins are not required for phagocytosis. In some cases plant lectins have been shown to be able to act as opsonins in invertebrate phagocyte systems, and concanavalin A receptors have been described on oyster hemocytes (Sami et al. 1992). ConA is known to bind to human macrophages and stimulate calcium uptake and [O.sub.2.sup.-] generation (Scully et al. 1986). Lobster hemocytes exposed to conA showed no enhanced [O.sub.2.sup.-] generation and only occasional increased HOCl production. Treatment with conA did produce the KS for the [O.sub.2.sup.-] peak, but had no effect on the time of the HOCl peak.

When lobster hemocytes were exposed to a Gram-positive bacterium Bacillus subtilis they showed no significant response in terms of increased net [O.sub.2.sup.-] or HOCl production and no detectable KS. Attempts to alter this response by previous opsonization of the bacteria with lobster hemolymph were without result. Receptors for LPS associated with Gram-negative bacteria have been identified on crustacean hemocytes. These receptors can activate immune responses such as the prophenoloxidase system in crayfish (Soderhall & Smith 1986), and can stimulate ROS production in shrimp (Sung et al. 1996). However, lobster hemocytes treated with E. coli LPS failed to show augmented ROS production, but did show a significant reduction in [O.sub.2.sup.-] peak time.

These studies indicate that of several putative NADPH oxidase activators tested, only PMA is effective as indicated by its strong stimulation of luminol-dependent CL. Presumably this response involves PKC activation, p47-phox phosphorylation and NADPH oxidase assembly. The apparent inability of other receptor-dependent and -independent activators to produce a net increase in luminol- or lucigenin-dependent CL may be a result of some inadequacy of the experimental in vitro conditions. However, the hemocytes remained viable during all experimental procedures and seemed to retain their phagocytic capacity. Although little or no increase in total [O.sub.2.sup.-] generation was triggered by any of the treatments, a spike of [O.sub.2.sup.-]-activity was always seen in both untreated and treated hemocytes. Most treatments caused the time interval to this peak to be decreased, sometimes in a dose-dependent manner. The physiologic significance of this reduced [O.sub.2.sup.-] peak time is unknown. A HOCl peak was also seen in untreated cells; however, regardless of their ability to further induce this activity, none of the treatments produced a significant change in the HOCl peak time.

ACKNOWLEDGMENTS

This publication is supported by the National Sea Grant College Program of the US Department of Commerce's National Oceanic and Atmospheric Administration under award #NA 16RG 1354 to the Research Foundation of the State University of New York for New York Sea Grant. The views expressed herein do not necessarily reflect the views of any of those organizations. This is Contribution No. 3800 of the University of Maryland Center for Environmental Science, Chesapeake Biological Laboratory.

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ROBERT S. ANDERSON * AND AMY E. BEAVEN Chesapeake Biological Laboratory, University of Maryland Center for Environmental Science, P.O. Box 38, Solomons, Maryland 20688

* Corresponding author. E-mail: [e-mail]

TABLE 1.
Effects of ROS stimuli on activation of lobster hemocytes.

                                        Net [O.sub.2.sup.-]
                                           Stimulation

Receptor-     PMA
independent     1-10 [micro]g/mL             [+ or -]
stimuli       Arachadonic acid
              1.5-25 [micro]M                    -

Receptor-     Zymosan                            -
dependent       2-25 particles/cell
stimuli       Laminarin                          -
                0.025-10.0 mg/mL
              ConA                               -
                1.25-1250 [micro]g/mL
              Bacillus subtilis                  -
                1-25 bacteria/cell
              E. coli LPS                        -
                6.25-50 [micro]g/mL

              Kinetic     Net HOCl      Kinetic
               Shift     Stimulation     Shift

Receptor-
independent      +           +++           -
stimuli
                 +            -            -

Receptor-     [+ or -]        -            -
dependent
stimuli       [+ or -]     [+ or -]        -

                 +         [+ or -]        -

                 -            -            -

                 +            -            -

Net [O.sub.2.sup.-] or HOCl stimulation means that the total CL (area
under the peak) for the treated hemocytes was significantly greater
than the total CL in the untreated cell peak: this is indicated by +,
or +++ to show major stimulation (~20-fold). Lack of ROS stimulation
is indicated by -; variable responses are marked [+ or -]. A positive
kinetic shift (+) means that the ROS peak time produced by a given
treatment was significantly shortened, as compared to the ROS peak
for untreated cells. In the case of arachadonic acid and ConA, the
Kinetic shift seen for the [O.sub.2.sup.-] peak was dose-dependent; for
laminarin this shift was consistently seen at concentrations at
10 mg/mL, but not at lower levels.