fc receptor for shark igm

DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY, Vol. 12, pp. 561-571, 1988 I045-305X88 $3.00 + .00 Printed in the USA Copyright (c) 1988 Pergamon Press plc All rights reserved

Fc RECEPTOR FOR SHARK IgM

Laura Haynes, Laphalle Fuller and E. Churchill McKinney Department of Microbiology and Immunology, University of Miami School of

Medicine, Miami

ABSTRACT Fc receptors for shark IgM have been demonstrated on shark leukocytes. Measurement of receptor binding required treatment of leukocytes with Cytochalasin D to inhibit phagocytosis. EA rosetting assays were carried out using human erythrocytes coated with shark anti-human antibody. Binding to shark leukocytes was demonstrated to be specific to shark IgM in that affinity purified shark IgM and purified Fc5g fragments could block rosette formation, but not shark transferrin, bovine serum albumin or fetal bovine serum. The binding was shown to be saturable and reversible, characteristic of receptor-ligand interaction. Further, it was shown that aff ini ty purified, radioiodinated IgM could also bind Cytochalasin D-treated shark leukocytes in a manner analogous to rosetting. We conclude that Fc receptors appeared early in evolution, and that previous difficulties in demonstrating the Fcgt receptor resulted from non-specific binding associated with phagocytosis.

INTRODUCTION

Receptors for the Fc portion of immunoglobulin molecules have been described on a number of lymphomyeloid cells in mammals, including T and B lymphocytes, monocytes/macrophages, null cells and granulocytes (Table 1). On macrophages and granulocytes , these receptors enhance phagocytosis of antibody coated foreign antigens, while on killer cells they are essential for antibody dependent cellular cytotoxici ty (1). Fc receptors also serve to regulate immune responses. T lymphocytes that express Fc receptors for IgM (Fcg receptors) are capable of giving

help to B cells, and those that express Fcy receptors can suppress B cell responses (2). Fc receptors have also been described on avian cells (3,4) but have not been previously demonstrated on cells from primitive vertebrates (5).

In the nurse shark (Ginglymostoma cirratum), ant ibody dependent cel lular cytotoxicity (ADCC) was shown to be associated with a glass adherent population which most likely expressed Fc receptors (6). The current study describes an assay for these receptors which involves formation of EA rosettes by shark leukocytes and human e r y t h r o c y t e s coa ted with a n o n a g g l u t i n a t i n g quan t i ty of shark immunoglobulin. Shark immunoglobulin, from unimmunized animals, is of the IgM

561

562 Fc RECEPTORS FOR IgM Vol. 12, No. 3

class and reacts with a wide range of xenogeneic erythrocytes (7).

Fc~ t r e c e p t o r s were d e t e c t e d on bo th g lass adhe ren t l e u k o c y t e s and g lass nonadheren t l eukocy tes in the shark. Since the adherent popu la t ion was enr iched for g r anu locy te s and monocy te s , and the nonadheren t popu la t ion was enr iched for l ymphocy te s (8) it was not surpr is ing to f ind cei ls bear ing Fc receptors in both of these p o p u l a t i o n s .

TABLE 1,

Cell Types Expressing Fc Receptors

SYSTEM CELL TYPE

FC RECEPTORS*

? Ix e (~ REFERENCE

MURINE/HUMAN MONOCYTE/ MACROPHAGE + + + + (9-13) BLYMPHOCYTE + + + + (14-18) T LYMPHOCYTE + + + ( 19- 24) NK, K CELL + + + (14,25,26) MAST CELL + + ( 27 ) NEUTROPHIL + (9 ,10 ,27) EOSINOPHIL + + (28 - 30)

DOGFISH PBL** ( 5 ) RAY PBL (5) PLAICE PBL (5)

* These receptors may be expressed by the entire population or by a fraction of that population.

** Peripheral blood lymphocytes.

MATERIALS AND METHODS

A n i m a l s . Nurse sharks (Ginglymostoma cirratum) measur ing two to eight feet in length were kept under opt imal condi t ions at the Miami Seaquar ium, Ocean Wor ld in Fort Lauderda le and the Sea World Shark Insti tute on Long Key. Smal ler (less than 4 ft.) sharks were anes the t ized by immers ion in a 1:106 solut ion of t r icane methane su l fonate whi le la rger sharks were immobi lzed in a sling. An imal s were then bled from the caudal vein. Sodium heparin (Sigma, St. Louis, MO) was added to the blood to give a final concentra t ion o f 100 units/ml. Animals are arbi t rar i ly des igna ted 1 and 2 in the data i l lus t ra ted here; however , numbers do not necessar i ly refer to the same animal in different sets of data.

$ ~ p a r a t i o n o f L e u k o c v t e s . Separa t ion of shark l eukocy tes has been descr ibed p rev ious ly (8). The exper iments presented here used unf rac t iona ted cel ls ( total b lood l eukocy te s ) and l eukocy te s f rac t iona ted into glass adheren t and glass nonadheren t populations. All media and salt solutions were adjusted to contain 0.2 M NaC1 and 0.35 M urea which is isotonic for shark leukocytes.

F rac t iona t ion o f Shark I~. Whole b lood was co l lec ted from un immunized sharks.

Vol. 12, No. 3 Fc RECEPTORS FOR IgM 563

The serum was recovered, precipitated with 40% ammonium sulfate, and resuspended in phosphate buffered saline isotonic for human ery throcytes . Fol lowing fract ionat ion on DE-52 cellulose (Whatman, Hil lsboro, OR) ion exchange chromatography, this preparation was found to contain the serum protein transferrin, as well as 7s and 19s immunoglobulin.

EA Rosette Assay. Shark leukocytes were tested for receptors for the Fc portion of Ig molecules using an erythrocyte-antibody (EA) rosetting method. Ammonium sulfate precipitated shark serum served as the source of shark immunoglobulin (Sklg). Human erythrocytes (HuRBC) were mixed with a subagglutinating dilution of Sklg and incubated for 30 minutes at room temperature to form the EA complex (HuRBC-Sklg). This preparation was washed twice prior to use. Shark leukocytes (0.5 X 106) were incubated in 1 ml of medium containing 2.01xM Cytochalasin D (Sigma) for 5 minutes. Then 50 X 106 HuRBC-Sklg cells were added in 0.5ml and incubated for 30 minutes at room temperature. The cells were pelleted by centrifugation (50g) and resuspended by mechanical agitation for microscopic examination. A shark leukocyte to which 3 HuRBC-Sklg's were bound constituted a positive rosette. At least 100 leukocytes were scored in each assay. The percent specific binding of Sklg coated HuRBC was then calculated:

% specific binding = (%rosettesHuRBC.Sklg) - (%rosettesHuRBC)

Saturation of the receptor was measured indirectly by incubating HuRBC's with increasing dilutions of ammonium sulfate precipitated shark serum (1:50, 1:100, 1:200, and 1:500). These complexes were then used for the rosetting reaction with shark leukocytes. Inhibition of binding was measured by incubating shark leukocytes with HuRBC-Sklg plus 0.1 ml of affinity purified shark IgM, shark Fc51.t or an unrelated protein for 30 minutes. The number of rosettes was then determined as described. Reversibility of receptor binding was tested by reacting shark leukocytes with HuRBC-Sklg for 30 minutes after which 0.1 ml of ammonium sulfate precipitated shark serum or medium was added. The mixture was incubated for an additional 30 minutes, and the number of rosettes was then determined.

Rabbit Anti-Shark lg. Shark immunoglobulin was purified from serum as previously described (31) and was used to immunized rabbits. Shark immunoglobulin (0.2 mg) was emulsified in Freund's Complete Adjuvant (Difco, Detroit, MI) at a ratio of 1:1 and injected subcutaneously into a rabbit. A booster injection using Freund's Incomplete Adjuvant was given after ten days, and serum was collected two weeks later. The serum was heat inactivated at 56 ° for one hour, and sterile filtered with a Millex-GS 0.221.tm filter unit (Millipore, Bedford, MA). Anti-shark immunoglobul in was purified from whole rabbit serum as previously described (32). Two-fold dilutions of the rabbit antiserum were tested against purified shark Ig, ammonium sulfate precipitated shark serum and whole shark serum in an Ouchterlony double diffusion analysis to confirm the presence of rabbit anti-shark IgG.

Affinity Chromatography. The rabbit anti-shark Ig was coupled to CNBr-activated Sepharose 4B (Pharmacia) according to manufacturer 's instructions. Ammonium sulfate precipitated shark serum was incubated on the anti-Ig coupled gel for 30 minutes at room temperture. The unbound protein was eluted with coupling buffer and collected in 1 ml fractions. When unbound protein was no longer eluted (after approximately 20 ml was run through column), the bound protein was eluted by lowering the pH with addition of -2 ml of a buffer containing 0.1M glycine and 0.5M NaC1, pH 2.5-3.0. The pH of the column was then raised by several column volumes of 0.05M Tris plus 0.5M NaCI, pH 7.0 until all protein was eluted. Determinations of protein content were made by reading the OD280 of each fraction. The fractions containing the greatest amount of SkIg were pooled for further use.


Fc5t.t Fragments. Fc5IX fragments were prepared using a modification of the

procedure described by Plaut and Tomasi (33). Three mg of affinity purified shark IgM in 50mM Tris/HCL containing 11.5 mM CaC12 pH 8.0, was treated with trypsin

(enzyme:protein, 1:25) at 56 ° for 30 min. The reaction was stopped by the addition of soy bean trypsin inhibitor ( lmg/mg trypsin). The Fc5IX fragments were purified employing a Fast Protein Liquid Chromatography (FPLC) system and a Superose 6 filtration column equilibrated with 50mM potassium phosphate containing 200 mM NaC1, pH 7.4.

Iodination of Skl~. Shark immunoglobulin was radiolabeled with 1251 using the Enzymobead radioiodination reagent (Bio-Rad, Richmond, CA), fol lowing the manufacturer 's instructions. The reaction mixture consisted of 50IXl of 0.2 M phosphate buffer, pH 7.2, 0.08 mg Sklg in 150IXl of azide-free buffer, 50111 of Enzymobead reagent, 1.0 mCi Na125I (New England Nuclear, Boston, MA) and 25111 of 1% 13 -D-glucose. The reagents were mixed and the reaction proceeded at room temperature for 15-25 minutes. The reaction was terminated by centrifugation, and labeled Sklg was separated from unbound iodide by passage through a Sephadex G-50 (Pharmacia) column. The fractions from the column containing 125i_Sklg were determined by counting 10111 from each fraction on a gamma counter. The fractions with the highest activity were pooled and stored at 4 ° .

Binding of 125I-Sklg tO Shark Leukocvtes. 125I-Sklg was used to test for the presence of Fc receptors on shark leukocytes. The entire binding procedure was carried out at 0°C. The appropriate number of cells was resuspended in 100IXl of serum free medium and the required amount of 125I-Sklg was added to each tube. Tubes were incubated for 1 hour and the reaction was terminated by centrifugation at 400g for 10 minutes. The supernatant was removed and the pellet was counted in a gamma counter. Results are expressed as CPM bound.

RESULTS

Receptors for the Fc portion of Sklg were demonstrated on shark leukocytes in an EA rosette assay which employed human erythrocytes (HuRBC) coated with ammonium sulfate precipitated shark immunoglobulin (Sklg) to form HuRBC-Sklg complexes. Table 2 shows that 15.1% of unfractionated leukocytes, 20.6% of the glass adherent population and 12.4% of the glass nonadherent population expressed this receptor. The differences in these percentages were not statistically significant.

TABLE 2

Formation of EA Rosettes by Shark Leukocytes

CELL PQPI, JLATION u n f r a c t i o n a t e d glass adherent glass nonadherent

% SPECIFIC BINDING 15.1 + 2.2* (n=ll) 20.6 + 2.2 (n=10) 12.4 + 1.4 (n=9)

The assay was carried out in the presence of 2.0 IxM Cytochalasin D to inhibit phagocytosis. Shark leukocytes (0.5 X 106 ) were incubated with 50 X 106 HuRBC-Sklg for 30 minutes in 1.5 ml total volume. The cells were pelleted by centrifugation and resuspended by mechanical agitation for microscopic examination. A shark leukocyte to which 3 HuRBC-Sklgs were bound constituted a positive rosette.* S.E.


The specificity of the interaction between shark leukocytes and HuRBC-Sklg was examined using competitive inhibition. Affinity purified SklgM (25IXg), shark transferrin (25IXg), bovine serum albumin (BSA, lmg) or undiluted FBS was added at the initiation of the EA rosette assay to determine if these substances could interfere with rosette formation. Representative experiments are shown in Figure 1A. Only purified Sklg caused a significant decrease in the number of rosettes observed, from 23.4% to 1.4% in shark 1 and from 20.0% to 2.0% in shark 2. In shark 1, 19.4% of the cells formed rosettes when shark transferrin was added; 23.4%, when BSA was added; and 21.4% when FBS was added. Similar results were observed in shark 2, and indicate that the inhibition of binding was specific for shark IgM.

To confirm that an Fc receptor for the IX chain of shark IgM was being detected, Fc5IX fragments were prepared from affinity purified shark IgM. When Fc5IX fragments were added, 75% inhibition of rosette formation was observed, resulting in a reduction from 21% to 5% EA rosettes in shark 78, and from 16% to 4% in animal 79 (Fig. 1B). These data also indicate that the shark 19s, pentameric immunoglobulin is responsible for the majority of rosettes observed.

A . B .

25

20 0 z

_z 15

U. ~ 10

a. if) ~ 5

NENE Sklg SkTr BSA FIBS

25

20

15~

• SHARK 1

79

MEDIUM Fcg.

SOLUBLE PROTEIN ADDED

FIG. 1 Effect of soluble protein added to the EA rosette assay. A. Purified shark IgM (25~tg, Sklg), shark transferrin (25ixg, SkTr) bovine serum albumin (1 mg, BSA) or undiluted fetal bovine serum (FBS) was added in a volume of 100IXl at the start of the assay and the % specific binding was determined. B. Purified shark Fc5IX fragments ( l i x g ) o r medium was added in a volume of 100 I11 and the % specific binding was determined. Data from two animals are shown in each panel.

To determine whether the observed binding of EA to shark leukocytes occured with the characteristics of receptor-ligand interaction, experiments were designed to


demonstrate receptor saturation and reversibility of binding. To show that binding of EA to the shark Fc receptor was reversible, shark leukocytes were incubated with HuRBC-Sklg for 30 minutes to allow rosette formation, and then either 100111 of ammonium sulfate precipitated serum or medium was added to the assay. This was then incubated for an additional 30 minutes to permit interchange between free Sklg and Sklg bound to HuRBC. Representative experiments are illustrated in Figure 2. For shark 1, when medium was added to the assay 31.3% of the glass adherent population formed rosettes, but when the precipitated serum was added only 9.0% were detected indicating reversibility of binding. For the glass nonadherent population, this drop was from 13.0% to 4.2%. Similar results were seen with shark 2.

30

Z

Z

~' 20 to n',

t,n 1 0

AD 1 AD 2 NONAD 1 NONAD 2

CELL POPULATION

FIG. 2 Reversibi l i ty of Fc receptor binding. Shark leukocytes were incubated with HuRBC-Sklg to form rosettes. After 30 minutes, either ammonium sulfate precipitated serum or medium was added, and the assay was incubated for an additional 30 minutes. The percent binding was then determined for each population. Results from two animals are shown. AD, glass adherent population; NONAD, glass nonadherent popu la t ion .

Two types of experiments were used to demonstrate saturability of the receptor: [1] an EA rosette assay using limiting amounts of shark antibody, and [2] binding of radiolabelled shark IgM. In the first set of experiments, HuRBC's were modified with dilutions of ammonium sulfate precipitated shark serum and then used in the EA rosette assay. In these experiments, the amount of Fc available for binding is limited by the quantity reacted with the surface of the HuRBC. For all three populations tested, the unfractionated leukocytes, the glass adherent population and the glass nonadherent population, the percentage of rosette forming cells was constant until the serum dilution reached 1:500. At this point the percentage of rosettes dropped significantly, indicating that the receptors were no longer saturated (Figure 3). To eliminate contributions of cell:cell interaction and stearic effects that may interfere in these experiments, direct binding was also measured.


_z

=_. e~

_.q =.

lad Ik 0~

2O

• ADHERENT 0 NONADHERENT

15 -' UNFR ACTIONATED

5

0 ' I I I I I I

0 100 200 300 400 500 600

DILUTION OF A M M O N I U M SULFATE P R E C I P I T A T E

FIG 3. Saturability of the Fc receptor. Shark leukocytes were incubated with HuRBC treated with dilutions of ammonium sulfate precipitated shark serum, and the percent specific binding was determined for each p o p u l a t i o n .

Affinity purified shark IgM was radiolabeled and used to directly demonstrate binding of Sklg and to confirm that EA rosetting was mediated by F c b t - I g M interaction. As shown in Figure 4, increasing concentrations of iodinated Sklg result in increased binding to shark cells until saturation was achieved as indicated by the plateau. Reactivity with glass nonadherent cells is depicted, and is representative of results obtained with other fractions of shark leukocytes.

O T,=

X :E Q.

20

10 ~ rn

[ ]

I I I I I I

0 20 40 60 80 100 120

p.I 1 2 5 I-Shark Ig

FIG. 4 Saturability of the Fc receptor demonstrated by binding of 1 2 5 I - S k l g . Glass nonadherent leukocytes (1 X 104) were incubated with increasing volumes of 125I-Sklg for 1 hour at O °. The unbound Ig was washed off and the pellet was counted in a gamma counter. Results are expressed as CPM bound.


DISCUSSION

The presence of Fc receptors on leukocytes has been well documented in mammalian systems, but not on cells from primitive vertebrates. These receptors could not be demonstrated by others in the dogfish (Scyliorhinus canicula), the ray (Raja clavata), the plaice (Pleuronectes platessa) (5), or previously by this laboratory using conventional methodology. In our experience, the main difficulty with assays using particulate reagents was the high phagocytic activity of shark leukocytes. Specific binding via an Fc receptor could not be distinguished from nonspecific binding for phagocytosis. The assay described in this report uses Cytochalasin D to prevent phagocytosis . Cytochalasin D interferes with the formation of actin f i laments in the cytosol, without the complicat ion of inhibiting other cellular functions such as glucose transport as is seen with Cytochalasin B (34-36).

Fc receptors were detected on 20.6% of the cells in the glass adherent population which is enriched for monocytes and granulocytes, and on 12.4% of the glass nonadherent population which is enriched for lymphocytes. The presence of Fc receptors on these cell types is consistent with those reported for mammalian cells (Table 1) except that the receptors detected on shark leukocytes were all Fcl.t. The majority of immunoglobulin employed in this assay contained both 7s and 19s IgM which have heavy chains that are antigenically similar and are not separated by gel electrophoresis (37,38). This study did not distinguish potential differences in the binding of 7s and 19s IgM to the Fclx receptor, and it is possible that the two forms have different affinities for the receptor since avian monomeric IgM has been shown to have a lower affinity for Fcl.t than polymeric IgM (3). In the nurse shark, the concentration of 19s IgM is approximately three times greater than that of the 7s in both the serum and the ammonium sulfate precipitate used to prepare HuRBC-Sklg. In this study, Fc51x was able to interfere with 75% of EA rosettes indicating that the pentameric Fc binds the majority of receptors detected. Thus, variation in the concentration and ratio of these two forms may affect the ability to detect Fcl.t receptors on fish cells.

Since unimmunized sharks synthesize antibody to HuRBC, it could be argued that only cells bearing immunoglobulin receptors reactive with HuRBC participate in rosette formation. This does not occur since the glass nonadherent leukocytes, which contain the majority of SIg bearing cells (8), did not form any rosettes with unmodified HuRBC. Only one to two percent of the unfractionated leukocytes and three to five percent of the glass adherent leukocytes formed rosettes with HuRBC alone. For the data presented here, this non-Fc mediated binding was subtracted from the percentage of rosettes detected with HuRBC-Sklg.

The interaction between shark leukocytes and EA complexes was shown to have the characteristics of a receptor and was, therefore, not due to nonspecific binding. Rosette formation could be specifically inhibited by addition of affinity purified shark IgM or shark Fcl.t fragments to the assay, but not by the addition of shark transferrin, BSA or FBS. The interaction was reversible. Shark leukocytes that had formed rosettes with the EA complexes could release the complexes and bind free Sklg, therefore resulting in fewer rosettes in the final preparation. Finally, the interaction was demonstrated to be saturable using two methods: rosette formation using HuRBC modified with varying quantities of Sklg, and directly, by binding of 125I_Sklg. Thus, shark leukocytes have membrane receptors which bind shark immunoglobulin via the Fc portion of IgM.

The functions of Fc receptor bearing leukocytes in the nurse shark are probably quite similar to those seen in higher vertebrates. Shark granulocytes and macrophages are highly phagocyt ic , and opsoniza t ion of par t iculate antigen probably facil i tates ingestion as seen in homeothermic vertebrates. Further, effectors of ADCC-like activity have been demonstrated in the nurse shark (6). The


morphology of these ADCC effector cells has not been determined, but it is known that they are n o n p h a g o c y t i c and are, therefore, distinct from the spontaneously cytotoxic macrophage described in this system (39). Thus Fc receptors for immunoglobul in appeared early in evolution, and appear to subserve functions similar to those observed in other vertebrates. Previous difficulties in detecting the Fclx receptor result from the large number of circulating phagocytes whose activity interferes with receptor assay, not from lack of the receptors.

ACKNOWLEDGEMENTS

We would like to thank the Miami Seaquarium and the Sea World Shark Institute for the housing and maintenance of animals used in this study. In addition, we would like to thank Mantley Dorsey, Jr and Ted Gerula for assistance in handling and bleeding the animals. This work was supported by the National Science Foundation under grant PCM-8302217.

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Received: June, 1987 Accepted: August, 1987

fc receptor for shark igm

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