A humoral opsonin from the solitary urochordate Styela clava

Developmentaland ComparativeImmunology,Vol. 17, pp. 29-39, 1993 Printed in the USA. All rights reserved.

0145-305X/93 $6.00 + .00 Copyright © 1993 Pergamon Press Ltd.

A HUMORAL OPSONIN FROM THE SOLITARY UROCHORDATE Styela clava Karen L. Kelly,* E d w i n L. C o o p e r , * and David A. Raftos'l *Department of Anatomy and Cell Biology, School of Medicine, University of California Los Angeles, Los Angeles, CA 90024 and -f-lmmunobiology Unit, School of Biological and Biomedical Sciences, University of Technology, Sydney, P.O. Box 123, Broadway, N.S.W. 2007, Australia

(Submitted October 1991;Accepted April 1992) DAbstract--Opsonins play a key role in invertebrate humoral immune systems. An opsonin for yeast was identified in the plasma of the tunicate, Styela clava. In vitro cultures of hemocytes with homologous plasma-incubated yeast exhibited significantly higher levels of phagocytosis than controls. Studies indicated that the opsonic activity of Styela clava plasma increased the overall capacity for phagocytosis. Opsonization was inhibited by the carbohydrates mannan, N-acetyi-D-galactosamine,and galactose, and by the divalent cation chelator, EDTA. These data suggest that the Styela clava opsonin may share some functional similarities with a C-type lectin. Incubation of yeast with Styela clava and Styela plicata plasma prior to phagocytosis by hemocytes from both species indicated the Styela clava opsonin is species specific. []Keywords--Tunicate; Urochordate; Opsonin; Lectin; Humoral immunity.

Introduction Phagocytosis is the most prevalent cellular immune defense mechanism in the animal kingdom. In many instances, humoral factors are essential for the inhibition of phagocytosis [see (1) for review]. Opsonins, which are generally humoral components, play a key role in phagocytosis by many invertebrates including crayfish (2), snails (3,4), earthworms (5), Address correspondence to Karen L. Kelly, Department of Anatomy and Cell Biology, UCLA, 73-074 CHS, 10833 Le Conte Ave., Los Angeles, CA 90024-1763.

molluscs (6-12), and tunicates (13-15). In crayfish, naturally occurring hemagglutinins are responsible for opsonic activity of crayfish hemolymph toward sheep erythrocytes; these opsonins appear to be species specific (2). Opsonization has also been identified in the earthworm, Eisenia foetida, using synthetic particles as the targets for phagocytosis (5). However, the most thorough characterization of opsonic activity comes from molluscs. The requirement for humoral opsonins has been demonstrated in a number of molluscan species (3,4,6-12). Fryer and Bayne have demonstrated that the opsonization of yeast by the plasma of Biomphalaria glabrata is a strainspecific and time-dependent process (6). Experiments using the bivalve Corbicula fluminea suggest that the opsonin(s) in this species is a divalent cation-dependent, heat-sensitive protein with a M r ranging from 20-66 kDa. This carbohydrate-specific protein has hemagglutinating activity, is divalent cation dependent, and is found both in the plasma and on the surface of circulating hemocytes (11,12). Other molluscan species, such as M e r c e n a r i a m e r c e n a r i a (8) and Mytilus edulis (9), also have agglutinins/ lectins that act as opsonins. In contrast to molluscs, relatively little is known about humoral recognition molecules of tunicates. The nature of immunological recognition in tunicates is of critical importance since they occupy a key evolutionary position on the phylogenetic line progressing toward verte29

30

brates (16). Tunicates are phylogenetically primitive chordates; hence, they are likely to share immunological characteristics with both vertebrates and invertebrates. Naturally occurring hemagglutinins have been identified in the coelomic fluid of the tunicates Styela plicata and Halocynthia hilgendorfi. However, these agglutinins have no apparent opsonic activity toward fixed rabbit erythrocytes (17). Contrary results were obtained in experiments using the colonial tunicate, Botrylloides leachii. In this species, the HA-2 agglutinin/lectin was shown to be opsonic for sheep erythrocytes and other particles bearing the appropriate carbohydrate moieties on their surfaces (13-15). With these few exceptions, it has proven difficult to confirm the opsonic function of humoral recognition molecules from tunicates. However, this study identifies a humoral opsonin in the plasma of the tunicate, Styela clava.

Materials and Methods

Tunicates Styela clava and Styela plicata were purchased from Marinus Inc., Long Beach, CA. Tunicates were maintained at 15°C in an aerated, sandbed-filtered, 180-L aquarium filled with artificial sea water (ASW) (Instant Ocean, Aquarium Systems, Mentor, OH: 3.4% w/v). Approximately 2 mL of Marine Invertebrate Diet (Carolina Biological, Gladstone, OR) were added to the aquarium every 2 days. Tunicates were allowed to adjust to conditions in the aquarium for I week prior to experimentation. Preparation of Hemocyte Suspensions Tunicates were bled by severing the stolon (Styela clara) or body wall (Styela plicata) and collecting 1.5 mL of the ex-

K.L. Kelly, E. L. Cooper, and D. A. Raftos

uding hemolymph in chilled centrifuge tubes (15 mL, sterile, p o l y s t y r e n e ; Fisher Scientific, San Francisco, CA) containing 1.5 mL of sterile-filtered, artificial sea water (ASW) (Instant Ocean; 3.4% w/v). Hemocyte suspensions were allowed to stand for five minutes so that large debris and cell aggregates sank. The upper 2 mL of each suspension were then transferred to fresh tubes. The number of hemocytes in suspension was determined using a hemocytometer and the volume adjusted with ASW to yield 3 × 10 6 cells/mL. Hemocytes from separate tunicates were not pooled for any assay. Hemocytes from individuals were used for both treatments and controls in several experiments.

Target Particles Saccharomyces cerevisiae (baker's yeast, type II, Sigma Chemicals, St. Louis, MO) was prepared as a 0.25% solution (approximately 1 × 108 particles/ mL) in congo red (sterile-filtered, 0.4% w/v in ASW, Sigma Chemicals). This solution was autoclaved at 120°C for 15 min, washed twice (2000 x g, 5 min), resuspended in ASW, and stored at 4°C for a maximum of 1 week. Preparation of Plasma Cell-flee hemolymph (plasma) was obtained by severing the stolon of several S. clava or incising the body wall of S. plicata. The exuding hemolymph was collected in chilled centrifuge tubes in the absence of ASW (50 mL, sterile, polystyrene; Fisher Scientific). Cells and debris were removed by centrifugation (800 x g, 15 min, twice) and the resulting plasma diluted with ASW to 20%, aliquotted into microcentrifuge tubes, and stored frozen (-4°C). Aliquots of plasma were diluted to 10% with the appropriate medium (ASW, EDTA, or carbohydrate

Humoral opsonin from urochordate

solutions) before experimentation. For experiments with autologous plasma, hemolymph was collected in chilled centrifuge tubes and centrifuged (800 x g, 10 min, 4°C). The plasma was then removed and diluted in ASW to 10%. The pellet of hemocytes was resuspended in 3 mL ASW and allowed to stand for 5 min so that large aggregates and debris sank. The upper 2 mL of each suspension were then transferred to fresh tubes. The number of hemocytes in suspension was determined using a hemocytometer and the volume adjusted with ASW to yield 3 x 10 6 cells/mL. Homologous plasma was defined as plasma from individuals of the same species as the hemocytes. Autologous plasma was obtained from the same individual as the hemocytes, whereas heterologous plasma was from individuals of a different species than the hemocytes.

Incubation Supernatants Hemocyte cultures were prepared as described below under the phagocytosis assay. Supernatants from cultures that had been incubated with ASW-preincubated yeast for 60 min were removed and pooled in microcentrifuge tubes. The s u p e r n a t a n t s were then centrifuged (16,000 x g, 6 min) to remove any remaining yeast particles. These supernatants were then added as resuspension media to the yeast for incubation as described in the phagocytosis assay.

EDTA Solutions EDTA (Sigma Chemicals) was prepared at concentrations of 2 mM, 10 mM, 50 mM, and 250 mM in ASW (pH 7.3). The EDTA solutions were each added in a 1:1 ratio to 20.0% plasma to give final c o n c e n t r a t i o n s of 10.0% plasma with 1 mM, 5 mM, 25 mM, or 125 mM EDTA. These solutions were then

31

added as resuspension media to the yeast for incubation as described in the phagocytosis assay.

Carbohydrate Solutions Mannan, N-acetyl-D-galactosamine, N-acetyI-D-glucosamine, D( + )glucose, o(+)galactose, sucrose, and 13-lactose (Sigma Chemicals) were each prepared as 2.0% solutions in ASW. The carbohydrate solutions were then added in a 1:1 ratio to 20.0% plasma to give final concentrations of 1.0% carbohydrate and 10.0% plasma. This medium was then incubated with agitation for 45 min before being added as a resuspension medium to the yeast for normal incubation as described below. High-percentage concentrations of carbohydrates were employed to test for the presence or absence of inhibition and not the level of inhibition, as fashioned after the experiments performed by Fryer et al. (10).

Absorption of Plasma Aliquots (800 ixl) of yeast suspensions and sheep red blood cells (SRBC) (approximately 1 x 108 cells/mL in ASW, Sigma Chemicals) were washed once (16,000 x g, 6 min), resuspended in 10.0% S. clara plasma in ASW, and incubated at room temperature for 1.5 h with agitation. The suspensions were then centrifuged (16,000 × g, 6 min). Supernatants were used as yeast-incubating media for tbe phagocytosis assay or were further absorbed by repeating the above procedure.

Phagocytosis Assay Preincubation of yeast. Aliquots (800 Ixl) of y e a s t s u s p e n s i o n s were w a s h e d (16,000 x g, 6 min) and resuspended in 800 ~1 of the appropriate medium (ASW,

32

K.L. Kelly, E. L. Cooper, and D. A. Ratios

10% plasma, incubation supernatants, 10% plasma with EDTA, 10% plasma with 1% carbohydrate solutions, yeastabsorbed plasma, SRBC-absorbed plasma). The yeast suspensions were incubated with the media at room temperature with constant agitation for 1.5 h. Suspensions were then washed three times (16,000 x g, 6 min) and resuspended in ASW to a final concentration of 1.0 x 107 particles/mL.

Phagocytosis of preincubated yeast, q-ianicate cell suspensions (200 pJ) were overlaid onto cover glasses (22 × 22 mm, Gold Seal, Fisher Scientific) that were suspended in a humidified container. Cells were allowed to adhere to cover glasses at 15°C for 2 h. Coverslips were then washed four times with 800 ~1 of ASW, and 50 ~xl of preincubated yeast suspensions were added to each coverslip. The cultures were then incubated for up to 60 min (15°C). Excess yeast was then removed by dipping each coverslip in ASW 10 times. The cells were then fixed in 200 pJ of absolute methanol (15 min, 4°C) and washed in distilled water. Coverslips were then inverted onto microscope slides (3" x 1" x 1 mm, Fisherfinest, Fisher Scientific) and sealed.

Quantification of phagocytosis. Hemocytes that had phagocytosed yeast were counted by phase contrast microscopy (1250x magnification, oil immersion, Zeiss). An example of phagocytic cells is shown if Fig. 1. A minimum of 200 cells from at least four fields of view per coverslip were inspected. Data were recorded as the percentage of hemocytes that had phagocytosed yeast particles (% phagocytic cells). In general, the number of yeast cells per phagocytic hemocyte showed the same changes as the percentage of hemocytes ingesting yeast. Data has, therefore, been restricted to percent phagocytic cells. The number of tunicates that have been assayed are repre-

Figure 1. Phase-contrast micrograph of Styela clava hemocytes having phagocytosed yeast particles (dark, oval structures). 500×, oil immersion.

s e n t e d ; a s s a y s were p e r f o r m e d in duplicate.

Statistical Analysis Statistical analyses were performed with the Mystat software package (Systat, Inc., Evanston, IL). The statistical significance of differences between mean values was determined by the Student's t-test (18). Differences were considered to be significant for t probabilities of less than 5.0%.

Results

Kinetics of the Phagocytic Response Plasma-incubated yeast was phagocytosed more rapidly than ASW-incubated yeast controls (Fig. 2). Differences in the percentage of hemocytes phagocytosing plasma-opsonized and control yeast were apparent, but minimal, within 10 min (t = 0.523, p = 0.608). Such differences reached a maximum within 30 min

Humoral opsonin from urochordate

33

50

40

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percent phagocytic cells

_ - - ~1

20.

¢

lO.

---m--.

0~

0

10

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30

time

40

plasma ASW

50

60

(minutes)

Figure 2. Percent phagocytic cells (±SEM, n = 8) in combinations of hemocytes with yeast incubated in ASW, or homologous plasma cultured at 15°C for various periods.

(t = 3.668, p = 0.002). After 60 min, no significant difference between plasmaopsonized and control yeast was evident (t = 0.093, p = 0.928). At this time approximately 50% of the hemocytes had phagocytosed plasma-opsonized or control yeast. Subsequent experiments were analyzed at 30 min.

Phagocytic Response to Incubation Supernatants L e v e l s of p h a g o c y t o s i s w e r e increased significantly (t = 4.13, p = 0.003) in cultures of hemocytes with supernatant-incubated yeast over ASWincubated yeast controls; 38.0 --- 2.9% of h e m o c y t e s phagocytosed yeast incubated in supernatants from 60-min cultures of hemocytes with ASW-incubated yeast, while yeast incubated in ASW was phagocytosed by 22.4 -4_-_ 1.4% of hemocytes.

Opsonization in the Presence of EDTA EDTA inhibited the opsonization of yeast by plasma (Fig. 3). Concentrations of EDTA greater than 1 mM in opsonizing media decreased phagocytosis significantly (5 mM EDTA, 32.0 --- 4.1% phagocytic hemocytes; t = 2.347, p = 0.029) when c o m p a r e d to y e a s t opsonized in the absence of EDTA (46.7 --3.9% phagocytic hemocytes).

Absorption of Opsonizing Activity Incubation of yeast with plasma that had previously been absorbed with yeast significantly (t = 2.623, p = 0.021) decreased phagocytosis when compared to yeast opsonized in unabsorbed plasma (Fig. 4). H e m o c y t e s (32.3 -+ 5.3%) phagocytosed yeast incubated in onceabsorbed plasma, while yeast incubated in twice-absorbed plasma was phagocy-

34

K.L. Kelly, E. L. Cooper, and D. A. Raftos

60.

50.

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40

30

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i

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plasma-incubated yeast

ASW-lncubatedyeast . . . . . . . .

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Figure 3. Percent phagocytic cells (-+SEM, n = 5) in combinations of hemocytes with yeast incubated in ASW, homologous plasma, or homologous plasma with EDTA (1 mM, 5 mM, 25 mM, 125 mM) at 15°C for 30 min. EDTA and plasma were combined immediately prior to coincubation with yeast.

tosed by 27.7 - 2.1% of hemocytes. The phagocytosis of yeast incubated in twiceabsorbed plasma did not differ significantly from that of ASW-incubated yeast

controls (28.6 -+ 2.5%; t = 0.095, p = 0.927). In contrast, incubating yeast with plasma that had been previously incubated with SRBC did not significantly (t

60-

50

¸

40

percent phagocytic

30

cells 20

10

ASW

plasma"

twice SRBCyeast-/ absorbed absorbed plasma plasma Figure 4. Percent phagocytic cells (-SEM, n = 6) in combinations of hemocytes with yeast incubated in ASW, homologous plasma, once-absorbed homologous plasma, twice-absorbed homologous plasma, or SRBC-absorbed homologous plasma cultured at 15°C for 30 min. once

\

Humoral opsonin from urochordate

35

= 1.249, p = 0.234) alter phagocytosis (51.0 --- 3.9%) when compared to yeast opsonized in unabsorbed plasma (44.6 3.5%).

0.001) higher than the ASW-incubated yeast controls (23.4 --- 1.6%). S. plicata hemocytes presented with yeast that had been opsonized in homologous S. plicata plasma exhibited levels of phagocytosis (54.4 +- 3.2%) that were significantly (t/> 4.391, p ~< 0.001) higher than cultures presented with yeast that had been opsonized in heterologous S. clava plasma (36.0 --- 2.7%) or ASW (22.0 --- 2.6%).

Phagocytic Response to Autologous, Homologous, and Heterologous Plasma No significant difference (t = 0.444, p = 0.664) was evident in the percentages of phagocytic S. clava hemocytes presented with yeast that had been opsonized in autologous or homologous plasma (Fig. 5). However, incubation of yeast in plasma from S. plicata significantly (t = 2.459, p = 0.028) decreased percent phagocytosis by S. clava hemocytes (34.0 --- 2.9%) when compared to homologous controls (41.8 - 3.0%). The level of phagocytosis in these heterologous plasma-incubated yeast cultures was still significantly (t = 6.295, p <

Phagocytic Response to Carbohydrates Incubation of yeast with plasma containing the c a r b o h y d r a t e s m a n n a n , N-acetyl-D-galactosamine, or galactose significantly (t = 3.602, p = 0.004; t = 5.173, p < 0.001; t = 3.307, p = 0.006, respectively) decreased the percentage of phagocytic hemocytes when compared to yeast incubated in plasma alone (Fig. 6). Phagocytosis was reduced to the S. plicata

S. clava 60-

hemocytes

hemocytes

55 50 45" 4035"

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30" 25"

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m

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F i g u r e 5. Percent phagocytic cells (-+SEM, n = 6) in combinations of S. clava hemocytes with yeast

incubated in ASW, homologous plasma, autologous plasma, or heterologous plasma and in combinations of S. plicata hemocytes with yeast incubated in ASW, homologous plasma, or heterologous plasma cultured at 15°C for 30 rain.

36

K.L. Kelly, E. L. Cooper, and D. A. Raftos

50

40

percent phagocytic

30

cells 20

o~ ~=

E

¢-,

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~

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Figure 6. Percent phagocytic cells (-+SEM, n = 6) in combinations of hemocytes with yeast incubated in ASW, homologous plasma, or homologous plasma with various carbohydrates cultured at 15°C for 30 min (n = 6 for each case). Carbohydrates and plasma were combined 45 min prior to coincubation with yeast.

level of ASW-incubated controls (28.6 _+ 2.5%) by these carbohydrates. Yeast incubated with plasma in the presence of N-acetyl-D-glucosamine, glucose, sucrose, or lactose was not phagocytosed at a significantly different rate (t = 0.146, p = 0.886; t = 0.174, p = 0.865; t = 0.461;p = 0.653; t = 0.235, p -= 0.818, respectively) than yeast incubated with plasma in the absence of added carbohydrates (44.6 - 3.5%).

Discussion By definition, an opsonin is any substance that enhances the phagocytosis of a cell or particle by coating and enabling it to adhere to a phagocyte that bears the appropriate receptor for that substance (19). According to this definition, we have identified an opsonin in the plasma of the tunicate, Styela clava, which binds Saccharomyces cerevisiae. Phagocytosis was significantly greater among

Styela clava hemocytes cultured with homologous plasma-incubated yeast than in cultures with ASW-incubated yeast (Fig. 2). This indicates that a factor(s) in S. clara plasma is able to bind yeast and enhance its uptake by phagocytes. Such a capacity for physical attachment to yeast is supported by the ability to absorb opsonizing activity from plasma by prior incubation with yeast. Such binding originally appeared to increase the rate but not the total capacity for phagocytosis. After the initial 10 min of incubation, where approximately 20% of the cells were phagocytic whether in the presence or absence of plasma, phagocytosis of ASW-incubated (control) yeast increased at a linear rate over the remainder of the 60-min period. In contrast, the percentage of phagocytic cells in cultures containing plasmaincubated yeast increased rapidly within 20 to 30 rain before reaching a maximum of approximately 50%. This would indicate that the function of opsonization is

Humoral opsonin from urochordate

to increase the rate of phagocytosis and not to increase the maximum number of phagocytic cells in culture. However, upon further analysis of supernatants from 60-min control cultures, opsonins were released from the hemocytes during culture. This was indicated by the enhanced level of phagocytosis in hemocytes cultured in the presence of 60-min supernatant-incubated yeast. This release of opsonin would, therefore, explain the slow increase in the number of phagocytic cells over time in control cultures. For this reason, the conclusion must be drawn that the binding of opsonin to yeast cells significantly increases the percentage of phagocytic cells over the base level of approximately 20%. Such a capacity to enhance the level of phagocytosis is critical to antipathogen defense. The opsonin that we have identified appears to have some characteristics that are typical of a C-type lectin (20). C-type lectins, which have been identified throughout the animal kingdom, are characteristically Ca 2÷ dependent, are located extracellularly, and have various carbohydrate specificities, most often including D-galactose. An initial indication that opsonic activity in S. clava conforms to this pattern comes from its susc e p t i b i l i t y to inhibition b y EDTA. EDTA, a divalent cation chelator, decreases opsonization when coincubated with plasma and yeast. This inhibition was dose dependent and suggests that Ca 2+ may be required in the binding of S. clava opsonin to yeast. A further characteristic shared by C-type lectins and the S. clava opsonin is a sensitivity to specific inhibition by carbohydrates, particularly galactose configurations. When the carbohydrates mannan, N-acetyl-D-galactosamine, and galactose were c o i n c u b a t e d with S. clava plasma and yeast, opsonization was decreased to near or below the level found in ASW-incubated yeast cultures. N-acetyl-D-glucosamine, glucose, sucrose, or lactose (Fig. 6) had no such in-

37

hibitory effects. Since carbohydrates and plasma were removed before the yeast was added to the hemocyte monolayers, this inhibition does not reflect inhibitory effects of sugars on phagocytic cells. The c a r b o h y d r a t e s must have gained their effect by preventing attachment of plasma opsonin to yeast, perhaps through blocking the binding site of the opsonin. The pattern of carbohydrate inhibition suggests that S. clava opsonin binds yeast via galactosyl residues on the yeast cell wall. Galactosyl sugars, galactose, and N-acetyl-D-galactosamine inhibited opsonization, while glucosyl sugars had no effect. However, there were exceptions to this pattern. Lactose, a disaccharide containing galactose, did not prevent opsonization. Perhaps the specific binding site for the opsonin on this sugar was occupied by another element or the steric configuration of lactose hindered the attachment of opsonic factors. One possible binding site would involve carbon 4 of galactose, which is altered when the two galactose residues form a lactose, and which is also the location of the difference between galactose and glucose. Mannan, a homopolysaccharide of o-mannose, inhibited opsonization. Such a capacity for inhibition in the absence of galactosyl residues might again be explained by steric influences that, in this case, promoted binding. The specificity reflected by S. clava opsonin is also indicated by attempts to absorb the opsonin from plasma by incubation with SRBC. Preincubation of plasma with SRBCs did not lead to a decrease in the percentage of phagocytic cells in culture (Fig. 4), indicating that none or little of the opsonin was absorbed from the plasma in the presence of these cells. In contrast, preincubation with yeast before opsonization led to a significant decrease in the level of phagocytosis. Indeed, two cycles of absorption with yeast decreased phagocytosis to the level of ASW-incubated controls (Fig. 4). Apparently, SRBC do not express

38

K.L. Kelly, E. L. Cooper, and D. A. Raftos

moieties to which S. clava opsonin is specific. Clearly, opsonization by S. clava plasma does not result from indiscriminate labelling of target particles. The S. clava opsonin also exhibits species, but not aUogeneic, specificity. Opsonization with autologous or homologous S. clava plasma produces similar degrees of phagocytosis. However, lower levels of phagocytosis were evident after incubation of yeast with heterologous plasma from the congeneric tunicate S. plicata, even though S. plicata hemocytes avidly phagocytosed yeast preincubated with S. plicata plasma. Such data may reflect speciesspecific opsonin/cellular receptor interactions. The divergence of such molecules between these two congeneric species is, h o w e v e r , not a b s o l u t e . Significant opsonization was still evident in heterologous combinations. In summary, we have identified a plasma opsonin(s) from Styela clava.

Some characteristics of the opsonin are consistent with a C-type lectin, which is divalent cation dependent. The opsonin(s) functions by increasing the rate of phagocytosis, has carbohydrate specificities for mannan, galactose, and Nacetyl-D-galactosamine, specifically binds to yeast, and appears to be species, but not allogeneic, specific. We are currently undertaking a molecular characterization of this opsonic activity.

Acknowledgements--We

thank Rick Fairhurst and Steve Sarafian for their contributions. This study was supported in part by the N a t i o n a l S c i e n c e F o u n d a t i o n (grant # D C B 90 05061) and by BRSG funds from the U C L A Schools of Medicine and Dentistry. David A. Raftos was a Fulbright Postdoctoral Fellow and a recipient of a Frederik B. Bang Scholarship in Marine Invertebrate Immunology administered by The American Association of Immunologists.

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Humoral opsonin from urochordate

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16. Field, K. G.; Olsen, G. J.; Lane, D. J.; Giovannoni, S. J.; Ghiselin, M. T.; Raft, E. C.; Pace, N. R. ; Raft, R. A. Molecular phylogeny of the animal kingdom. Science 239:748-753; 1988. 17. Fuke, M. T.; Sugai, T. Studies on the naturally occurring hemagglutinin in the coelomic fluid of an ascidian. Biol. Bull. 143:140-149; 1972. 18. Sokal, R. R.; Rohlf, E J. Biometry, 2nd ed. New York, NY: W. H. Freeman and Co.; 1981. 19. Clark, W. R. The experimental foundations of modern immunology, 3rd edition. New York, NY: John Wiley and Sons Inc.; 1986. 20. Drickamer, K. Two distinct classes of carbohydrate-recognition domains in animal lectins. J. Biol. Chem. 263:9557-9560; 1988.