Somatostatin and calcitonin modulate gastropod internal defense mechanisms

DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY, Vol. Ii, pp.487-499, 0145-305X87 $3.00 + .00 Printed in the USA. Copyright (c) 1987 Pergamon Press Ltd. All rights reserved.

1987.

SOMATOSTATIN AND CALCITONIN MODULATE GASTROPOD INTERNAL DEFENSE MECHANISMS

Yvonne Grimm-J~rgensen Department of Physiology University of Connecticut Health Center Farmington, CT 06032

ABSTRACT

Immunoreactive somatostatin and calcitonin are stored in selected mammalian leukocytes. Synthetic somatostatin and calcitonin can alter the functional activity of certain mammalian blood cells. These findings suggest that the two enteroneuro-peptides serve as modulators of the mammalian immune system. The aim of the present study is to determine whether the 2 peptides are also found in gastropod blood cells and whether they may participate in the regulation of the gastropod internal defense mechanisms. We found that somatostatln-like and calcitonln-llke substances are located in a distinct population of hemocytes from the pond snail Physella heterostropha and that the amount of immunoreactlve calcitonin in hemocytes increases after an injury to the body wall. Synthetic somatostatin and calcitonin stimulate the attachment of latex beads to in vitro cultured hemocytes. We conclude that the somatostatin-and calcltonln-like substances may serve as modulators of the internal defense system of gastropods.

INTRODUCTION Molluscan hemocytes have many functlons~ such as assisting in wound repair, shell growth and repair~ digestion of particles, as well as the participation in the internal defense of the organism (1). The hemocytes carry out these functions by means of phagocytosing particles and selectively releasing phagocytosed material at appropriate sites, such as a wound or the mantle edge. Because phagocytosis plays such an important role in the proper function of the internal defense system, it is important to understand the mechanisms that regulate the phagocytic activity of hemocytes. It is known that opsinins in the hemolymph plasma can facilitate phagocytosis by molluscan blood cells (2)9 but other mechanisms that stimulate phagocytosis, such as is observed after injury or invasion by foreign par 487

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ticles, have not been excluded. For instance, it is possible that, similar to the blood cells of higher animals, the hemocytes of gastropods can also release factors that stimulate other blood cells or other targets such as fibroblasts. Recent evidence suggests that various neuropeptides are stored in and can modulate the functional activity of certain vertebrate leukocytes. For instance, immunoreactive somatostatin is found in mononuclear leukocytes from man and pig (3) and in in vitro cultured rat basophilic leukemia cells (4). Synthetic somatostatin stimulates attachment and lysis of IgG2~ coated sheep red cells to and by rat peritoneal macrophages (5) and acts as a potent histamine secretagogue in human leukocytes and rat mast cells (6). It also inhibits the proliferation of T-lymphocytes and Molt-4 lymphoblasts (7). Likewise, immunoreactive calcitonin is found in and synthesized by human leukemia cells (8,9,10). Synthetic human calcitonin inhibits cAMP accumulation in monocytes that have been exposed to latex particles (ii). Because cAMP accumulation and phagocytic activity of leucocytes are inversely related (12), it is likely that calcltonin stimulates the phagocytic activity of these cells. We chose to probe for the presence of immunoreactive somatostatin and calcitonin in the hemocytes of the pondsnail Physella heterostropha, and to explore their possible role in the regulation of hemocyte function.

In this report we will provide evidence that i) hemocytes contain immunoreactive material that resembles vertebrate somatostatin and calcitonin, and 2) that synthetic enteroneuro-peptides stimulate the attachment of latex beads to the hemocytes. These results suggest that hemocytes may secrete substances that, analogous to the lymphokines and enteroneuro-peptides of higher organisms, modulate the functional activity of other blood cells.

MATERIALS AND METHODS Animals The freshwater snails Physella heterostropha were bred and maintained in the laboratory at controlled temperature (22vC) and with controlled lighting (14 hr light, I0 hr dark) as previously described (13). The animals (mature, egg-laying specimens) were 3-5 months old with a shell length of 15 ~m when used.

Collection of hemolymph Hemolymph from several animals was collected from the heart via a drill hole in the shell and transferred to a 35 mm Primaria tissue culture dish (Falcon Plastics). The cells were allowed to attach to the Primaria surface for up to 2 hr. The cell-free supernatant was then removed, centrifuged for 30 sec in a microfuge to remove debris, the supernatant removed and frozen for radioimmunoassay. The cells were gently washed with isotonic snail medium (13) and either fixed for 30 min in freshly prepared 0.4 % p-benzoquinone in 1/2 strength phosphate buffered saline (for immunohistochemistry) or removed from the culture dish with 2 N acetic acid (for somatostatin radioimmunoassay) or distilled water (for calcltonin radloimmunoassay).

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Woundin~ of animals The animals were anaesthetized in artificial pond water containing 250 mg/l menthol. A small incision was made in the foot using microscissors. The snails were returned to aerated water and maintained for the specified time. Controls were anesthetized and returned to aerated water.

Somatostatin radioimmunoassay The hemolymph plasma was thawed and an equal volume of 4N acetic acid was added. The extract was subjected to a freeze-thaw cycle, centrifuged and the acid soluble material lyophilized. The cell homogenate was also centrifuged and the supernatant lyophilized. The lyophilisates were dissolved in 0.5 ml of radiolmmunoassay buffer and the amount of i~unoreactivity in triplicate aliquots of I00 ~I each determined as described elsewhere (13). The somatostatin antibody shows 100% crossreactlvlty with somatostain-25 and -28 and a 5% crossreactivlty with salmon calcitonln.

Calcitonin radioimmunoassay After removal of debris, the hemolymph plasma and aqueous cell extracts were lyophilized and resuspended in 0.3 ml assay buffer (0.I M Borate, i% BSA). The immunoreactive calcitonin activity of duplicate I00 UI aliquots was determined using the Immunonuclear Radioimmunoassay Kit No. 2500. The calcitonin antibody recognizes various mammalian calcltonins but fails to crossreact with salmon calctonin and somatostatin-14.

Immunohistochemistry The antibodies to somatostatin and human calcltonln were of histochemical quality and were purchased from ~ Accurate Chemical Corp. The a n t ~ l citonin antibody failed to bind to ---I labeled salmon calcitonin and ~-~llabeled somatostatin-14 (tested at a 1:500 dilution). The specificity of the somatostatin antibody was tested by radioimmunoassay at a dilution of 1:6000. It cannot distinguish between somatostatin-14, -25 and -28 and fails to crossreact with calcitonin.

Prior to incubation with the primary antlsera, the fixed cells were incubated at room temperature for 30 min in Dulbecco's phosphate buffered saline (PBS) followed by a 60 min incubation at RT in PBS containing 20% normal swine serum and 0.3% Triton X-100 to occupy possible free FC receptors and to permeabilize the cells. The cells were then incubated at RT with a 1:500 dilution of the primary antibody in PBS containing 0.3% Triton X-IO0 and 0.I % swine serum (PBTS). After 24 hr the antiserum was removed and replaced with fresh antiserum and the incubation continued for 24 hr. The cells were washed with PBTS and incubated for 30 mln in FITC-conjugated swine antirabbit IgG serum (1:200 in PBTS) and washed 3 times with PBS. The cells were then incubated for I0 min in Hoechst 33258 nuclear stain (0.08 Ng/ml PBS), washed 3 times with PBS and coverslipped with glycerol PBS (3:1).

The following controls were carried out.

I) Replacement of the primary

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antibody with non-immune serum. 2) Preabsorption of the primary antibody with synthetic human calcitonin or somatostatin-14. 3) For calcitonin only, removing primary antibody by passing serum through a calcitonin-sepharose affinity column.

Bead attachment assay Hemolymph was collected as described above and applied to a 12 mm circular coverslip that had previously been coated with alcian blue (14) and moistened with 50 ~I isotonic snail medium (75% isotonic snail saline; 25% Leibowitz L-15 medium (Gibco)). The cells were allowed to attach to the polycationic surface for 60 min. The medium was then removed and the cells rinsed with i00 ~i of isotonic snail medium. This medium was then replaced wi~h I00 ~I of isotonic snail medium containing a 1:4000 dilution (= 2.5 x i0 beads/ml) of fluorescein coated latex particles (1.74 ~ diameter, Polysciences) and the peptides at the appropriate concentration. The cells were incubated for 60 min in a moist chamber at RT, rinsed 5 times in isotonic snail medium and fixed in 2 % freshly prepared paraformaldehyde solution. The cells were rinsed 4 times in 1/2 strength phosphate buffered saline (PBS) and stained for I0 min in the Hoechst 33258 nuclear stain. After extensive washing in I/2 strength PBS, the coverslips were mounted onto glass slides in glycerol:PBS (3:1).

The cells were examined under a fluorescence microscope with epi-illumination alternately using filters optimized for the Hoechst dye and fluorescein. The cell borders were examined under bright field phase contrast optics. A minimum of 300 cells were examined on each coverslip. Only latex beads that were totally within the cell boundary were considered to be attached.

In a preliminary experiment, the incubation with the fluorescent latex beads was carried out at 0°C in order to determine the artefactual association of the beads with the cells. We found that no cells contained more than 2 latex beads and that 87% of the cells contained no beads at all. This distribution was not affected when the peptides were added to the medium. When the cells were incubated with latex beads at RT, only 65-75% of the cells showed 0 beads, while the rest of the cells contained up to 4 beads.

RESULTS The amounts of immunoreactive calcitonin and somatostatin in the hemolymph plasma and hemocytes were quantitated by radioimmunoassay. Table 1 shows that the hemolymph of 3-5 month old animals contained 1781 pg/ml of calcitonin-like activity. Thirty one percent of the immunoreactivity was found in the hemocyte fraction while the remaining 69% were associated with the plasma fraction. The hemolymph of separate pools of animals contained approximately 25 pg/ml of immunoreactlve somatostatin, and 55% of this immunoreactivity was associated with hemocytes.

The morphology of hemocytes from Physella heterostropha after attachment to the Primaria surface is illustrated in Figure i. As in other pulmonate

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gastropods~ two cell types can be discerned. Large irregularly shaped cells with numerous processes are the predominant cell type in adult animals. These type I cells are capable of phagocytosis and of amoeboid movement and have been named amoebocytes (15). The other cell type (type II cell) is smaller and more rounded than the type I cell and exhibits fewer processes. It does not attach to the substrate as readily as the type I cell and preparations enriched in one or the other cell type can be obtained by differential plating. It is currently believed that the two cell types represent different phenotypes of the same basic cell type (16).

TABLE I Immunoreactive Immunoreactivity

Calcltonin and Somatostatin pg/ml Hemolymph

in Hemolymph

Percent in Cells

N

Calcitonin

1781 ± 320

31.2 ±

1.2

2

Somatostatin

24.7 ± 5.9

55.1 ± 10.5

5

Each value represents the mean ± S.E.M. of individual pooled samples that contain hemolymph from 20 animals. N = number of pooled samples used to calculate the mean.

FIG. 1 Phase contrast photomicrograph of hemocytes that were allowed to settle on a Primaria culture dish. The arrows point to the rounded type II cells. The asterick is on a type I cell.

Immunofluorescence microscopy of hemocytes stained with antisomatostatin (Fig. 2A) or anticalcitonin (Fig. 3C) sera revealed that type II cells react with the antibodies. Specific staining was never observed in the

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larger type I cells. No specific fluorescence was observed when the primary antisera were incubated with non-immune serum or with peptide-absorbed serum (Fig. 2C and 3A).

FIG. 2 Panel A depicts a cluster of type II cells that have been incubated with antisomatostatin serum. Panel B is the same field, but photographed under optimal conditions for the nuclear stain Hoechst 33258. Panel C shows a mixture of type I and type II cells that have been incubated with antisomatostain and an excess of synthetic somatostatin. The corresponding Hoechst stained exposure is depicted in Panel D.

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FIG. 3 Panel A shows two type I cells that have been incubated with normal rabbit serum and FITC-coupled secondary antiserum. Note the faint non-specific fluorescence in these cells. Panel B shows the same cells photographed under optimal conditlons'for visualization of the Hoechst 332158 nuclear stain. Panel C shows a weakly fluorescent type I cell and a type II cell that shows specific fluorescence after incubation with anticalcitonin serum. Panel D represents the Hoechst nuclear stain for the cells show in Panel C.

The concentration of hemocytes in the hemolymph increases after a skin incision or the introduction of foreign particles (17). Injury also results in an increase in the proportion of type II hemocytes. For instance~ in the pond snail Fossarla modicella the percentage of type II cells increases from 21.48 + 0.92% (mean ± SEM, N=5) to 54.03 ± 5.04% (N=6) within 24 hr after an incision of the body wall (Grimm-J6rgensen, unpublished observation). If the immunoreactive peptides are selectively located in the type II hemocytes, the amount of immunoreactive material associated with the cellular elements of the hemolymph should increase after injury. Figure 4 depicts 2 representative experiments carried out on animals from different egg clutches. The total amount of immunoreactive calcitonln in the hemolymph did not change significantly during the first 2 days after injury. Thereafter, it progressively declined. On the other hand, the amount of i m u n o reactive calcitonin that was associated with the hemocytes was elevated 24 and 48 hr after injury~ and then declined to below the control levels by day 4.

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rII

CALCITONIN IN HEMOLYMPH PLASMA

I

CALCITONIN IN CELLS

o cC:.

40-

367

E 29.5

o

E "r-

"~

30-

52.1 44.8

Q.

i Z ~

23.7 2 7

20-

I bJ > h~

140 10-

UJ n~ (Z) Z

A

B

DAY 0

A

B

DAY I

A

B

DAY 2

A

B

DAY 5

I

A B DAY 4

FIG. 4 Effect of wounding on the amount of calcitonin-llke material in hemolymph. The numbers above each bar indicate the percentage of calcltonin found in the hemocytes. Each bar represents the mean value of 2 separate pools of hemolymph.

Injury is accompanied by increased phagocytic activity of type I hemocytes near the site of injury (17). The hypothesis that the phagocytic activity of these hemocytes is under the control of regulatory peptides was tested. Table II lists a represenative experiment. Both peptides promoted bead attachment in a dose dependent manner. It should be noted that the addition of the peptides not only resulted in an increase in the number of cells with beads, but also favored the attachment of multiple beads to the hemocytes (see also Table III).

Three other peptides, that are structurally not related to calcitonin or somatostatin, were also evaluated. Table III shows that.neither thyrotropin releasing hormone (TRH) nor eledoisin at 10~7M to 10-bM affected the attachment of latex beads. Bombesin slightly stimulated bead attachment at 10-VM concentration.

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TABLE I I Effect

of Somatostatin

and C a l c l t o n l n

on Bead A t t a c h m e n t t o H e m o c y t e s .

Percent of cells

0 Bead attached

1 kad attached

2 Beads attached

>2 Beads attached

Control

71.98 + 3.36

17.46 + 1,13

7.04 ± 1.54

3.51 ~ 1.28

10-8M

63.01 + 2 . 9 8

23.97 + 1.99

7.91 :t 0.67

5 . 1 0 :t 1.04

lO-7M

*56.58 ± 5.45

25.30 ± 2.14

10.31 + 2.16

7.80 ± 1.90

Control

73.23 + 4.42

16.90 ± 1.38

7.19 + 2.16

2.67 ± 1.38

lo-BM

64.63 + 4 . 9 5

21.31 :t 1.51

8.28 + 1.13

5.77 ± 2.74

28.49 :l: 1 . 1 3

12.10 + 1.05

9.40 + 1.02

Treatment

8omatostatln

Calcitonin

in each category

10-7M

*'50.01

-+ 3.16

*Slgniflcantly different from control (l-a - 0.925) analysis of variance. a'#Signiflcantly

different

from c o n t r o l

(l-a

= 0.975) analysis

of variance.

TABLE III Effects of Various Peptides on the Bead Attachment to Hemocytes

Percent of cells in each category Treatment

TRH

E1edoisln

Bombesin

0 Bead attached

1 Bead attached

Multiple beads attached

CONTROL

54.74 -+ 4.33

29.80 -+ 1.96

15.46 -+ 2.58

10-7M

63.06 -+ 5.24

24.33 -+ 0.46

12.61 + 2.67

10-6H

56.34 + 4.05

2 5 . 7 2 -+ 1 . 3 0

1 7 . 9 5 -+ 2 . 7 6

CONTROL

72.86 ± 3.62

19.31 -+ 1.76

7.83 + 2.03

lO-7M

71.42 ± 4 . 4 8

18.33 ± 1.71

9.75 + 2.83

lO-6M

74.96 + 2.96

18.22 + 1.38

6.81 ± 1.60

CONTROL

57.06 ± 3.68

17.13 + 1.36

25.80 ± 3,86

10-7M

54.47 -+ 2.57

22,63 -+ 0.61

22.90 -+ 2.88

10-6H

"45.71 ± 0.54

22.21 ± 0.43

32.09 + 0.83

The values are the means f SEM from 4 individual cover slips (Eledoisln and Bombesin) and 3 individual cover slips (TRH). At least 350 cells were evaluated on each coverslip. *Significantly different from control, l - a = 0.95, analysis of variance.

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DISCUSSION We have presented evidence that a distinct population of hemocytes from the freshwater gastropod Physella heterostropha contain substances that resemble the mammalian calcitonins and somatostatins. Specific inlnunohistochemical staining is limited to the round type II cells. The existence of the i~unoreactive materials in type II cells is also supported by the finding that the amount of immunoreactive calcitonin associated with the hemocytes increased when the number of circulating type II cells was augmented by infliction of a stab wound to the body wall.

As we do not have sufficient data to determine whether all type II cells contain both antigens, it cannot be resolved whether the 2 immunoreactive substances are associated with separate type II cells or co-exist within the same cell as has been demonstrated in thyroid parafollicular cells (18).

The complete biochemical identities of these somatostatln-llke and calcitonin-like activities are also not known. We have shown earlier that the somatostatin-like material in whole hemolymph is heterogeneous and that none of the in~uunoreactlve entities migrate llke somatostatin-14, -25, or -28 on reverse phase thin layer chromatography or HPLC (13,19 and unpublished finding). Because the antibodies used in this study to probe for calcitoninlike activity do not recognize salmon calcitonin, but crossreact with various mammalian calcitonlns, it is likely that the gastropod material more closely resembles the mammalian calcitonins than one of the fish calcitonins. This finding is not unexpected, since it has been demonstrated that the calcitonin-like material in another invertebrate species, the protochordate Cionia intestinalis, closely resembles human calcitonin (20).

Whether the imunoreactive materials are synthesized by the type II hemocytes or produced elsewhere and selectively accumulated, stored, and possibly degraded by these cells, cannot be discerned from the present studies. Proof that hemocytes are capable of synthesizing the immunoreactive substances will require the demonstration of m-RNAs for these peptides as well as careful in vitro biosynthesis studies.

A stabwound to the body wall resulted in an increase in the amount of immunoreactlve calcltonin in the hemocyte fraction; the amount of immunoreactive calcitonin in the hemolymph plasma remained unchanged. This increase was followed by a significant loss of immunoreactive material in the hemocytes and in the plasma, which lasted for up to 4 days after the injury, the longest time period measured. The reasons for these observations are not readily apparent. The initial increase of imunoreactive calcitonin in the cellular fraction is probably due to the proliferation of and mobilization from storage sites of type II cells that follow an injury. It is possible that llke type I cells, a number of type II cells leave the circulation and accumulate at the site of injury which will result in a reduction of total circulating type I and type II cells and might, in part, explain the observed reduction in imunoreactive calcitonin in the hemolymph. Synthetic somatostatin and calcitonin enhanced the attachment of latex beads to in vitro incubated hemocytes in a dose dependent manner indicating

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tha~ these 2 peptides are capable of modulating hemocyte function. At up to I0 -v M 9 TRH~ a peptide that is found in gastropod hemolymph (21) failed to influence bead attachment to hemocytes as did eledoisin whose presence in gastropods has not yet been probed for. The tac~ykinin bombes~n~ on the other hand~ also stimulated bead attachment at I0 M but not i0 M. It is not known whether bombesin-like substances exist in gastropod tissues; therefore, it is not possible to speculate on the possible physiological meaning of this observation.

The concentration of synthetic hormone required to significantly alter the attachment of beads to the hemocytes exceeds the concentration of immunoreactive somatostatin-like and calcltonln-llke material in the hemolymph plasma. This finding is not necessarily in contradiction with the hypothesis that the endogenous somatostatin-like and calcitonin-llke substances function as modulators of amoebocyte function, since it is possible that the type II cells release their products in the immediate proximity of the target cells which will temporarily raise the local peptide concentration to that required for binding to the target cells.

The presence of immunoreactive calcltonin-llke and somatostatin-like substances in hemocytes, and the observation that the synthetic peptides modulate the function of type I cells, suggest that the type II hemocytes may actively participate in the internal defense of gastropods by means of secreting regulatory substances that influence the functional activity of other blood cells. The existence of such a modulating system is not unique to gastropods and resembles the lymphoklne system of higher animals as well as the various modulatory peptlde systems that have recently been described in mammalian leukocytes (reviewed by (22)).

In conclusion, the present study indicates that somatostatin-and calcitonln-llke substances are found in gastropod blood cells, and that synthetic calcitonln and somatostatin can modulate the functional state of hemocytes. Endogenous somatostatin- and calcitonin-llke peptides may belong to a family of substances that are involved in the modulation of the internal defense and healing mechanisms of gastropods.

ACKNOWLEDGEMENTS The skillful technical assistance of Mary Ducor and Linda Forbes is greatly acknowledged. I also thank Dr. Andrew G.M. Bulloch for his invaluable suggestions and discussions during the course of these studies. I wish to thank Mrs. Joan Jannace and Ms. Denis Gagnon for secretarial assistance. Funds to carry out these studies were provided by a grant from the National Research Foundation.

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Received: August, 1986 Accepted: September, 1986