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The fate of protein antigen in earthworms: study in vitro
Immunology Letters, 35 (1993) 1 6 0165 2478 / 93 / $ 6.00 ~ 1993 Elsevier Science Publishers B.V. All rights reserved IMLET 01870
The fate of protein antigen in earthworms: study in vitro M a r t i n Bilej, L u d m i l a Tu6kovfi a n d Jaroslav Rejnek Department of Immunology, Institute of Microbiology, Czechoslovak Academy of Sciences, Prague, Czechoslovakia (Received 30 July 1992; accepted 17 September 1992)
I.
Summary
Earthworm coelomocytes digest protein antigen in vitro. Proteolytic activity was detected both in cell-free coelomic fluid and in cell cultures of free coelomocytes which are effectors of earthworm immunodefence mechanisms. Antigen is cleaved either intracellularly or by proteolytic enzymes released by coelomocytes into the medium. Proteolysis was observed both in non-stimulated and antigen-stimulated cultures. Since significantly higher proteolysis was shown in supernatants from cultures of antigen-stimulated coelomocytes, we can assume that the release of proteolytic enzymes was inducible.
senia foetida do respond to parenteral stimulation by protein antigens and that free coelomocytes cultivated in vitro are able to bind and internalize the antigen used for prestimulation in vivo [7,8]. Furthermore, the presence of antigen can influence the proliferation of coelomocytes in vitro [9]. The aim of this report is to elucidate, whether coelomocytes can also process antigen under in vitro conditions (as has been observed in vivo; Rejnek et al., submitted) and whether the antigen is fragmentated intracellularly or extracellularly by enzymes released by coelomocytes. 3.
3.1. 2.
Materials and Methods
Earthworms
Introduction
Numerous humoral factors participate in elimination of non-self substances have been described in annelids, hemagglutinating, hemolytic, antibacterial molecules were reviewed by Valembois et al. [1], and the presence of protein(s) interacting with antigen was reported by Laulan et al. [2] and Tu~kov~ et al. [3,4]. Furthermore, an efficient proteolytic system, that digests vertebrate proteins but not those of related species has also been detected in annelids [5,6]. We have reported that the earthworms Lumbricus terrestris and EiKey words." Lumbricus terrestris; Eiseniafoetida; Coelomocyte; Coelomic fluid; Fate of antigen; In vitro; Proteolysis Correspondence to." M. Bilej, Dept. of Immunology, Institute of Microbiology, Czechoslovak Academy of Sciences, Videfisk~ 1083, 142 20 Prague 4, Czechoslovakia.
Adult earthworms Lumbricus terrestris kept at 15°C in soil, and Eiseniafoetida kept at 20°C in mold were used in all experiments.
3.2.
Harvesting of coelomic fluid and coelomocytes
Coelomic fluid containing coelomocytes was obtained by puncturing the coelomic cavity with a glass micropipette. The suspension pooled from 30 earthworms was centrifuged (100 x g for 10 min), the coelomic fluid removed and stored at -70°C, and the coelomocytes resuspended in isotonic Lumbricus balanced salt solution (LBSS; [10]). Cell suspensions were purified on a Percoll gradient (Pharmacia, Uppsala, Sweden: 1.094/ 1.078 g/ml; 900 x g for 20 min). The cell-rich fractions from the gradient were collected, washed three times in LBSS and resuspended in isoton-
ic RPMI 1640 medium supplemented with glutamine and antibiotics (2% Antibiotic Antimycotic Solution; Sigma, St. Louis). The viability of the cells never dropped below 90%.
3.3. Evaluation of proteolytic activity of coelomic fluid 50 ~tl of coelomic fluid was incubated for various intervals (2, 4, 8, 24, and 48 h) with the mixture of cold ARS-HSA (5 pg/sample) and 125I-labelled ARS-HSA (100 000 cpm) at room temperature. After incubation, 100 #1 of 20% human serum albumin were added to increase the total amount of protein. Proteins were precipitated with 200 pl of 10% trichloracetic acid (TCA) and the samples were centrifuged. The supernatants were collected, the sediments were solubilized with 1 M NaOH, and radioactivity in both fractions was measured using a gamma counter.
3.4. Evaluation ofproteolytic activity ofcoelomocytes Isolated coelomocytes (10 6) were cultivated for the intervals described above with the mixture of cold ARS-HSA (5 /~g/sample) and 125I-labelled ARS-HSA (100000 cpm). After the incubation the samples were centrifuged. The liquid portion was TCA precipitated and radioactivity of nonprecitable fragments and of precitable proteins were measured using a gamma counter as described above (Fig. 1, steps 1 and 2). Cell sediments were resuspended, and cells were lysed (0.75 M Tris-HCl buffer, pH 8.8, 1% Nonidet P-40, 1 mM PMSF, 1 mM aprotinin, and 2 mM Na2EDTA) and centrifuged. The intracellular content was precipitated with TCA, and the
coelomocytes in medium I centrlfugatlon medium + TCA 1 supernatant 2 sediment cell 1ysls centrlfugation Intracellular 3 supernatant 4 sediment 5 insoluble remnants of cells
radioactivity was measured in supernatant (3), in precipitate (4), and in insoluble cell remnants (5).
3.5.
Estimation o/ proteolytic activity of cell culture supernatants
Isolated coelomocytes were cultivated for 48 h in the presence or absence of non-labelled ARSHSA (5 pg per sample). After cultivation the samples were centrifuged, the medium removed and proteolytic activity assayed as described for coelomic fluid. 4.
Results
Rates of antigen degradation were followed in vitro. Mixtures of cold and labelled antigens were added to L. terrestris and E. foetida coelomic fluids, and the radioactivity was measured in % 100
100
~.~...+. 8C
8O
60
6O
40
4O
20
"+
0 iIPl 0 4 8
I
2O
I
24
48
h content + TCA
Fig. 1. Experimental protocol.
Lumbricus terrestris +-Eisenia foetida Fig. 2. Proteolytic activity in coelomic fluids. Presented as means + SD of relative decrease (%) of I25I-labelled antigen in sediment after TCA precipitation.
TCA supernatants and precipitates after 2, 4, 8, 24, and 48 h incubation at room temperature. Half of the antigen added was digested during the first 24 h, as 56% (in case of E. foetida) and 42% (in case o f L. terrestris) of radioactivity was found in the supernatants. These values increased to 84 and 71% after 48 h (Fig. 2), The coelomocytes obtained from E. foetida and L. terrestris earthworms were cultivated in vitro with the mixture of cold and ~25I-labelled antigen. After 2, 4, 8, 24, and 48 h the cultivation medium was removed and the cells collected, lysed and centrifuged. The radioactivity was measured in insoluble remnants of cells and in the TCA supernatants and precipitates of cultivation media and cell lysates (Figs. 1 and 3a and b). Almost 20% of the antigen added to the cultures enters the L. terrestris coelomocytes (10% in case o f E. foetida coelomocytes) during the first 2 h and 35 and 30% is internalized by 24 h. Between 24
of
and 48 h no significant internalization of antigen was observed. During the period followed the internalized antigen is continuously cleaved, but the cleavage in E. foetida coelomoeytes appears to be slower since after 24 h less than 20% of the internalized antigen was present in the form of TCA non-precipitable fragments while in L. terrestris coelomocytes it was more than 50%. The proteolytic enzymes produced by coelomocytes appear to be released into the culture medium as very efficient digestion of the labelled antigen in culture supernatants was detected (culture supernatants were collected after 48 h of cultivation of cells without or with antigen (section 3.5,; Fig. 4)). The kinetics o f labelled antigen degradation in supernatants obtained from cultures of coelomocytes incubated in the presence o f antigen is comparable with kinetics of cleavage in coelomic fluids. The significantly lower level of proteolytic activities in media obtained from non-sti-
total radioactivity
of
100%-
total radioactivity
b
100%
"1ti1 I1
-
75%
80%
6O%
28%
25%
0% ~l:l O2 4
8
24
4M
0%
-
0;4
24
h
4~0
h ~5
~4
113 ~ 2
~1
Fig. 3. Relative distribution of radioactivity (%) in: (1) Supernatants obtained after TCA-precipitationof culture medium (cleaved antigen in medium); (2) sediments obtained after TCA-precipitationof culture medium (intact antigen in medium); (3) supernatants obtained after TCA-precipitation of cell lysates (cleaved antigen in cells); (4) sediments obtained after TCA-precipitation of cell lysates (intact antigen in cells); (5) insoluble remnants of cells (membrane bound antigen). The numbers of columns correspond to numbers in Fig. 1. Full line divides the relative levelof radioactivityin cells and in the medium.
mulated cells (cultivation without antigen) when compared with those obtained from cells cultivated in the presence of antigen (30% difference in case of L. terrestris and 20% in case of E. foetida) suggests that the release of proteolytic enzymes by coelomocytes is inducible. However these results do not show whether the same cells are responsible for the proteolysis of internalized antigen and also for the release of enzymes. 5.
Discussion
Proteolytic degradation of antigen in L. terrestris and E. foetida coelomocyte cultures has been assayed. In E. foetida coelomocytes, 45% of the antigen present in the culture medium was digested during 48 h while in L. terrestris coelomocytes it was more than 80%. Similarly, 21% of the internalized antigen was cleaved during 48 h in E. foetida coelomocytes while in L. terrestris this figure was 50% (Fig. 3a, b). Thus, comparison with the results obtained in vivo (Rejnek et al., submit-
ted) revealed that in E. foetida coelomic fluid antigen was digested more effectively than in L. terrestris coelomic fluids, while the cleavage of internalized antigen was more pronounced both in vivo and in vitro in L. terrestris coelomocytes. One possible explanation is that inhibitors of proteolysis and hemolysis were found in L. terrestris but not in E. foetida coelomic fluids (Tu6kov~i et al., in preparation: M6hrig et al., in preparation; Rejnek et al., submitted). Furthermore, after antigenic stimulation, we observed a more pronounced increase of total coelomic fluid protein concentration in L. terrestris [5], and in this species there was also an increase in proteolytic activity. The fact that marked proteolytic activity was present in coelomocyte culture medium indicated that the proteolytic enzymes formed in coelomocytes especially after antigenic stimulation were also released into the medium. This again supports the view that the synthesis and release of enzymes may be influenced by the antigen. %
%
1O
1O0
0
8O
80
8O
60
60
6O
"'...,
40
20 a 0 i~Jr O48
%
lOO
1O0
80
60 '-
.
40
40
40
20
2O
20
b IIII
I
24 h
I 0 48
0 I I I I 0 4 8
I
f
I
I
24
I
f
48
0
h
without antigen +-with antigen without antigen -t-with antigen Fig. 4. Proteolytic activity of culture media. Presented as means + SD of relative decrease of radioactivity in sediment obtained after TCA-precipitation. Culture media were collected after 48 h of cultivation of L. terrestris or E.foetida coelomocytesin presence ( + ) or absence ( - ) of antigen.
Our experiments did not reveal whether the same cells that digest the internalized antigen also release the enzymes into the culture medium, nor whether different cells are responsible for these two effects. The second possibility seems to be more plausible. More effective cleavage was observed in culture media than in the cells and one can hardly expect release of enzymes by cells containing the substrate. The labelled antigen after being added to L. terrestris and E. foetida coelomocyte cultures enters the cell interior (Fig. 3a, b). Total radioactivity of cells increases during the first 24 h and remains nearly constant between 24 and 48 h. On the other hand the amount of radioactivity in the proteolytic products (TCA non-precipitable fragments) remains unchanged during the first 4 (L. terrestris) or 8 (E. foetida) h and increases slightly between 8 and 24 h of cultivation. These differences suggest that the fragments can either be released or enter the cells possibly in dependence on the actual concentration of the fragments in culture medium. The ability of the fragments to enter the cells or to interact with their surfaces has been shown in experiments where small dialyzable proteolytic fragments were used for stimulation of antigen
binding protein formation by coelomocytes in vitro. Similarly as intact antigen these fragments stimulated the response (in preparation). References [1] Valembois, P., Roch, P. and Lassegues, M. (1986) in: Immunity in Invertebrates (M. Brehelin, Ed.), pp. 74~93, Springer-Verlag, Berlin, Heidelberg. [2] Laulan, A., Morel, M., Lestage, J., Delaage, M. and Chateaureynaud-Duprat, P. (1985) Immunology 56, 751. [3] Tu~kovfi, L., Rejnek, J. and Sima P. (1988) Dev. Comp. Immunol. 12, 287. [4] Tu6kov~., L., Rejnek, J., Bilej, M. and Pospi~il, R. (1991) Dev. Comp. Immunol. 15, 263. [5] Tu~kovfi., L., Rejnek, J., Sima, P. and Ond~ejov~t, R. (1986) Dev. Comp. Immunol. 10, 181. [6] M6hrig, W., Eue, I. and Kauschke, E. (1989) Zool. Jb. Physiol. 93, 303. [7] Bilej, M., Tu6kov~i, L., Rejnek, J. and V~tvi6ka, V. (1990) Immunol. Lett. 26, 183. [8] Bilej, M., Rossmann, P., VandenDriesscbe, T., Scheerlinck, J.P., De Baetselier, P., Tu6kov/t, L., V&vi~ka, V. and Rejnek, J. (1991) Immunol. Lett. 29, 241. [9] Bilej, M., Sima, P. and Slipka, J. (1992) Immunol. Lett. 32, 181. [10] Stein, E.A. and Cooper, E.L. (1981) Dev. Comp. Immunol. 5, 415.
The fate of protein antigen in earthworms: study in vitro M a r t i n Bilej, L u d m i l a Tu6kovfi a n d Jaroslav Rejnek Department of Immunology, Institute of Microbiology, Czechoslovak Academy of Sciences, Prague, Czechoslovakia (Received 30 July 1992; accepted 17 September 1992)
I.
Summary
Earthworm coelomocytes digest protein antigen in vitro. Proteolytic activity was detected both in cell-free coelomic fluid and in cell cultures of free coelomocytes which are effectors of earthworm immunodefence mechanisms. Antigen is cleaved either intracellularly or by proteolytic enzymes released by coelomocytes into the medium. Proteolysis was observed both in non-stimulated and antigen-stimulated cultures. Since significantly higher proteolysis was shown in supernatants from cultures of antigen-stimulated coelomocytes, we can assume that the release of proteolytic enzymes was inducible.
senia foetida do respond to parenteral stimulation by protein antigens and that free coelomocytes cultivated in vitro are able to bind and internalize the antigen used for prestimulation in vivo [7,8]. Furthermore, the presence of antigen can influence the proliferation of coelomocytes in vitro [9]. The aim of this report is to elucidate, whether coelomocytes can also process antigen under in vitro conditions (as has been observed in vivo; Rejnek et al., submitted) and whether the antigen is fragmentated intracellularly or extracellularly by enzymes released by coelomocytes. 3.
3.1. 2.
Materials and Methods
Earthworms
Introduction
Numerous humoral factors participate in elimination of non-self substances have been described in annelids, hemagglutinating, hemolytic, antibacterial molecules were reviewed by Valembois et al. [1], and the presence of protein(s) interacting with antigen was reported by Laulan et al. [2] and Tu~kov~ et al. [3,4]. Furthermore, an efficient proteolytic system, that digests vertebrate proteins but not those of related species has also been detected in annelids [5,6]. We have reported that the earthworms Lumbricus terrestris and EiKey words." Lumbricus terrestris; Eiseniafoetida; Coelomocyte; Coelomic fluid; Fate of antigen; In vitro; Proteolysis Correspondence to." M. Bilej, Dept. of Immunology, Institute of Microbiology, Czechoslovak Academy of Sciences, Videfisk~ 1083, 142 20 Prague 4, Czechoslovakia.
Adult earthworms Lumbricus terrestris kept at 15°C in soil, and Eiseniafoetida kept at 20°C in mold were used in all experiments.
3.2.
Harvesting of coelomic fluid and coelomocytes
Coelomic fluid containing coelomocytes was obtained by puncturing the coelomic cavity with a glass micropipette. The suspension pooled from 30 earthworms was centrifuged (100 x g for 10 min), the coelomic fluid removed and stored at -70°C, and the coelomocytes resuspended in isotonic Lumbricus balanced salt solution (LBSS; [10]). Cell suspensions were purified on a Percoll gradient (Pharmacia, Uppsala, Sweden: 1.094/ 1.078 g/ml; 900 x g for 20 min). The cell-rich fractions from the gradient were collected, washed three times in LBSS and resuspended in isoton-
ic RPMI 1640 medium supplemented with glutamine and antibiotics (2% Antibiotic Antimycotic Solution; Sigma, St. Louis). The viability of the cells never dropped below 90%.
3.3. Evaluation of proteolytic activity of coelomic fluid 50 ~tl of coelomic fluid was incubated for various intervals (2, 4, 8, 24, and 48 h) with the mixture of cold ARS-HSA (5 pg/sample) and 125I-labelled ARS-HSA (100 000 cpm) at room temperature. After incubation, 100 #1 of 20% human serum albumin were added to increase the total amount of protein. Proteins were precipitated with 200 pl of 10% trichloracetic acid (TCA) and the samples were centrifuged. The supernatants were collected, the sediments were solubilized with 1 M NaOH, and radioactivity in both fractions was measured using a gamma counter.
3.4. Evaluation ofproteolytic activity ofcoelomocytes Isolated coelomocytes (10 6) were cultivated for the intervals described above with the mixture of cold ARS-HSA (5 /~g/sample) and 125I-labelled ARS-HSA (100000 cpm). After the incubation the samples were centrifuged. The liquid portion was TCA precipitated and radioactivity of nonprecitable fragments and of precitable proteins were measured using a gamma counter as described above (Fig. 1, steps 1 and 2). Cell sediments were resuspended, and cells were lysed (0.75 M Tris-HCl buffer, pH 8.8, 1% Nonidet P-40, 1 mM PMSF, 1 mM aprotinin, and 2 mM Na2EDTA) and centrifuged. The intracellular content was precipitated with TCA, and the
coelomocytes in medium I centrlfugatlon medium + TCA 1 supernatant 2 sediment cell 1ysls centrlfugation Intracellular 3 supernatant 4 sediment 5 insoluble remnants of cells
radioactivity was measured in supernatant (3), in precipitate (4), and in insoluble cell remnants (5).
3.5.
Estimation o/ proteolytic activity of cell culture supernatants
Isolated coelomocytes were cultivated for 48 h in the presence or absence of non-labelled ARSHSA (5 pg per sample). After cultivation the samples were centrifuged, the medium removed and proteolytic activity assayed as described for coelomic fluid. 4.
Results
Rates of antigen degradation were followed in vitro. Mixtures of cold and labelled antigens were added to L. terrestris and E. foetida coelomic fluids, and the radioactivity was measured in % 100
100
~.~...+. 8C
8O
60
6O
40
4O
20
"+
0 iIPl 0 4 8
I
2O
I
24
48
h content + TCA
Fig. 1. Experimental protocol.
Lumbricus terrestris +-Eisenia foetida Fig. 2. Proteolytic activity in coelomic fluids. Presented as means + SD of relative decrease (%) of I25I-labelled antigen in sediment after TCA precipitation.
TCA supernatants and precipitates after 2, 4, 8, 24, and 48 h incubation at room temperature. Half of the antigen added was digested during the first 24 h, as 56% (in case of E. foetida) and 42% (in case o f L. terrestris) of radioactivity was found in the supernatants. These values increased to 84 and 71% after 48 h (Fig. 2), The coelomocytes obtained from E. foetida and L. terrestris earthworms were cultivated in vitro with the mixture of cold and ~25I-labelled antigen. After 2, 4, 8, 24, and 48 h the cultivation medium was removed and the cells collected, lysed and centrifuged. The radioactivity was measured in insoluble remnants of cells and in the TCA supernatants and precipitates of cultivation media and cell lysates (Figs. 1 and 3a and b). Almost 20% of the antigen added to the cultures enters the L. terrestris coelomocytes (10% in case o f E. foetida coelomocytes) during the first 2 h and 35 and 30% is internalized by 24 h. Between 24
of
and 48 h no significant internalization of antigen was observed. During the period followed the internalized antigen is continuously cleaved, but the cleavage in E. foetida coelomoeytes appears to be slower since after 24 h less than 20% of the internalized antigen was present in the form of TCA non-precipitable fragments while in L. terrestris coelomocytes it was more than 50%. The proteolytic enzymes produced by coelomocytes appear to be released into the culture medium as very efficient digestion of the labelled antigen in culture supernatants was detected (culture supernatants were collected after 48 h of cultivation of cells without or with antigen (section 3.5,; Fig. 4)). The kinetics o f labelled antigen degradation in supernatants obtained from cultures of coelomocytes incubated in the presence o f antigen is comparable with kinetics of cleavage in coelomic fluids. The significantly lower level of proteolytic activities in media obtained from non-sti-
total radioactivity
of
100%-
total radioactivity
b
100%
"1ti1 I1
-
75%
80%
6O%
28%
25%
0% ~l:l O2 4
8
24
4M
0%
-
0;4
24
h
4~0
h ~5
~4
113 ~ 2
~1
Fig. 3. Relative distribution of radioactivity (%) in: (1) Supernatants obtained after TCA-precipitationof culture medium (cleaved antigen in medium); (2) sediments obtained after TCA-precipitationof culture medium (intact antigen in medium); (3) supernatants obtained after TCA-precipitation of cell lysates (cleaved antigen in cells); (4) sediments obtained after TCA-precipitation of cell lysates (intact antigen in cells); (5) insoluble remnants of cells (membrane bound antigen). The numbers of columns correspond to numbers in Fig. 1. Full line divides the relative levelof radioactivityin cells and in the medium.
mulated cells (cultivation without antigen) when compared with those obtained from cells cultivated in the presence of antigen (30% difference in case of L. terrestris and 20% in case of E. foetida) suggests that the release of proteolytic enzymes by coelomocytes is inducible. However these results do not show whether the same cells are responsible for the proteolysis of internalized antigen and also for the release of enzymes. 5.
Discussion
Proteolytic degradation of antigen in L. terrestris and E. foetida coelomocyte cultures has been assayed. In E. foetida coelomocytes, 45% of the antigen present in the culture medium was digested during 48 h while in L. terrestris coelomocytes it was more than 80%. Similarly, 21% of the internalized antigen was cleaved during 48 h in E. foetida coelomocytes while in L. terrestris this figure was 50% (Fig. 3a, b). Thus, comparison with the results obtained in vivo (Rejnek et al., submit-
ted) revealed that in E. foetida coelomic fluid antigen was digested more effectively than in L. terrestris coelomic fluids, while the cleavage of internalized antigen was more pronounced both in vivo and in vitro in L. terrestris coelomocytes. One possible explanation is that inhibitors of proteolysis and hemolysis were found in L. terrestris but not in E. foetida coelomic fluids (Tu6kov~i et al., in preparation: M6hrig et al., in preparation; Rejnek et al., submitted). Furthermore, after antigenic stimulation, we observed a more pronounced increase of total coelomic fluid protein concentration in L. terrestris [5], and in this species there was also an increase in proteolytic activity. The fact that marked proteolytic activity was present in coelomocyte culture medium indicated that the proteolytic enzymes formed in coelomocytes especially after antigenic stimulation were also released into the medium. This again supports the view that the synthesis and release of enzymes may be influenced by the antigen. %
%
1O
1O0
0
8O
80
8O
60
60
6O
"'...,
40
20 a 0 i~Jr O48
%
lOO
1O0
80
60 '-
.
40
40
40
20
2O
20
b IIII
I
24 h
I 0 48
0 I I I I 0 4 8
I
f
I
I
24
I
f
48
0
h
without antigen +-with antigen without antigen -t-with antigen Fig. 4. Proteolytic activity of culture media. Presented as means + SD of relative decrease of radioactivity in sediment obtained after TCA-precipitation. Culture media were collected after 48 h of cultivation of L. terrestris or E.foetida coelomocytesin presence ( + ) or absence ( - ) of antigen.
Our experiments did not reveal whether the same cells that digest the internalized antigen also release the enzymes into the culture medium, nor whether different cells are responsible for these two effects. The second possibility seems to be more plausible. More effective cleavage was observed in culture media than in the cells and one can hardly expect release of enzymes by cells containing the substrate. The labelled antigen after being added to L. terrestris and E. foetida coelomocyte cultures enters the cell interior (Fig. 3a, b). Total radioactivity of cells increases during the first 24 h and remains nearly constant between 24 and 48 h. On the other hand the amount of radioactivity in the proteolytic products (TCA non-precipitable fragments) remains unchanged during the first 4 (L. terrestris) or 8 (E. foetida) h and increases slightly between 8 and 24 h of cultivation. These differences suggest that the fragments can either be released or enter the cells possibly in dependence on the actual concentration of the fragments in culture medium. The ability of the fragments to enter the cells or to interact with their surfaces has been shown in experiments where small dialyzable proteolytic fragments were used for stimulation of antigen
binding protein formation by coelomocytes in vitro. Similarly as intact antigen these fragments stimulated the response (in preparation). References [1] Valembois, P., Roch, P. and Lassegues, M. (1986) in: Immunity in Invertebrates (M. Brehelin, Ed.), pp. 74~93, Springer-Verlag, Berlin, Heidelberg. [2] Laulan, A., Morel, M., Lestage, J., Delaage, M. and Chateaureynaud-Duprat, P. (1985) Immunology 56, 751. [3] Tu~kovfi, L., Rejnek, J. and Sima P. (1988) Dev. Comp. Immunol. 12, 287. [4] Tu6kov~., L., Rejnek, J., Bilej, M. and Pospi~il, R. (1991) Dev. Comp. Immunol. 15, 263. [5] Tu~kovfi., L., Rejnek, J., Sima, P. and Ond~ejov~t, R. (1986) Dev. Comp. Immunol. 10, 181. [6] M6hrig, W., Eue, I. and Kauschke, E. (1989) Zool. Jb. Physiol. 93, 303. [7] Bilej, M., Tu6kov~i, L., Rejnek, J. and V~tvi6ka, V. (1990) Immunol. Lett. 26, 183. [8] Bilej, M., Rossmann, P., VandenDriesscbe, T., Scheerlinck, J.P., De Baetselier, P., Tu6kov/t, L., V&vi~ka, V. and Rejnek, J. (1991) Immunol. Lett. 29, 241. [9] Bilej, M., Sima, P. and Slipka, J. (1992) Immunol. Lett. 32, 181. [10] Stein, E.A. and Cooper, E.L. (1981) Dev. Comp. Immunol. 5, 415.