Cellular expression of the cytolytic factor in earthworms Eisenia foetida

Immunology Letters 60 (1998) 23 – 29

Cellular expression of the cytolytic factor in earthworms Eisenia foetida Martin Bilej a,*, Pavel Rossmann a, Marek S& inkora a, Radka Hanus' ova´ a,b, Alain Beschin c, Geert Raes c, Patrick De Baetselier c Department of Immunology, Institute of Microbiology, Academy of Sciences of the Czech Republic, Vı´den' ska´ 1083, 142 20 Prague 4, Czech Republic b Faculty of Science, Charles Uni6ersity, Vinic' na´ 7, Prague 2, Czech Republic c Unit of Cellular Immunology, Flemish Interuni6ersity Institute for Biotechnology, Vrije Uni6ersiteit Brussel, Paardenstraat 65, B-1640 St-Genesius-Rode, Belgium a

Received 28 November 1996; received in revised form 24 April 1997; accepted 25 August 1997

Abstract Coelomic fluid of earthworms contains a 42 kDa protein designated CCF-1 (coelomic cytolytic factor 1), which accounts for approximately 40% of cytolytic activity of the entire coelomic fluid. CCF-1 was documented to be present on cells of the mesenchymal lining of the coelomic cavity as well as on free coelomocytes. Both cellular and humoral levels of CCF-1 were significantly increased after parenteral injection of endotoxin. Moreover, CCF-1 seems to be involved in cell mediated cytotoxicity, because cytotoxic activity is blocked in the presence of anti-CCF-1 monoclonal antibody (mAb). © 1998 Elsevier Science B.V. Keywords: Eisenia foetida; Coelomic fluid; Coelomic cytolytic factor 1 (CCF-1); Lipopolysaccharide; TNF; Coelomocytes; Chloragogenous tissue

1. Introduction Proteolytic, hemolytic, antibacterial, and cytolytic properties of invertebrate body fluids have been documented in numerous papers [1]. Molecules with cytokine-like properties are of particular interest. Beck and Habicht [2,3] proposed the hypothesis that cytokines are phylogenetically highly conserved molecules which might have been present in ‘primitive’ ancestral forms in the animal kingdom for millions of years. Functional analogs of inflammatory cytokines have been described in a large variety of invertebrates including echinoderms, molluscs, insects, and tunicates [4– 18]. Nevertheless, the suggested analogy is mainly based on the crossreactivity of anti-cytokine antibodies, on the sensitivity of invertebrate immunocytes to cytokine * Corresponding author. 0165-2478/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 2 4 7 8 ( 9 7 ) 0 0 1 2 7 - 2

action or, vice versa, on the responsiveness of vertebrate immunocytes to invertebrate factors. Recently, a cytolytic protein that displays a certain degree of functional analogy with mammalian tumor necrosis factor (TNF) has been isolated from the coelomic fluid (CF) of Eisenia foetida earthworms (Oligochaeta, Annelida) [19]. The cytolytic protein designated CCF-1 (coelomic cytolytic factor 1) is a 42 kDa molecule that accounts for approximately 40% of the cytolytic activity of the entire CF directed against the TNF-sensitive tumor cell line L929. The mechanism of lysis is, however, different from that mediated by TNF since a neutralizing anti-TNF monoclonal antibody (mAb) does not affect the lysis of L929 cells by CF and conversely anti-CCF-1 mAbs do not influence the cytolytic activity of TNF [19]. CCF-1 is also involved in opsonizing effects and in hemolytic mechanisms that are known to be closely

M. Bilej et al. / Immunology Letters 60 (1998) 23–29

24

connected with the antibacterial properties of CF. From this point of view CCF-1 may belong to the first phase of defence in earthworms. In mammals, TNF plays an important role as an inflammatory pleiotropic cytokine, serum levels of which can be increased by endotoxin [20]. In this work we focused on documenting the cellular expression of CCF-1, the effect of endotoxin on its expression, and the possible role of CCF-1 in cellular defence of earthworms.

2. Materials and methods

2.1. Earthworms and method of stimulation Adult earthworms, E. foetida (Oligochaeta; Annelida), kept at 20°C in compost were used in all experiments. Two days before experimentation the earthworms were transferred to Petri dishes containing cotton wool soaked with isotonic buffer (LBSS; Lumbricus balanced salt solution; [21]). The earthworms were injected with lipopolysaccharide (LPS from Salmonella typhimurium LT2; 50 mg in 10 ml of 3% agar gel per earthworm) 1, 2, 3 or 4 days before collection of the CF or free coelomocytes. Non-stimulated or shamstimulated (agar alone) worms were used as a negative control. Adult earthworms, Lumbricus terrestris, kept at 15°C in soil were used as a source of target cells in cytotoxicity tests (Section 2.6).

2.2. Har6esting of the CF and free coelomocytes CF containing free coelomocytes was obtained by puncturing the coelomic cavity with a glass micropipette. The suspension pooled from earthworms of each experimental group was centrifuged (100× g for 10 min), the cellfree CF collected, recentrifuged (1000× g for 10 min), and the supernatant stored with 1 mM 4-(2-aminoethyl)-benzenesulfonylfluoride (nontoxic serine proteinase inhibitor; Pefabloc® SC; Boehringer Mannheim Biochemica, Germany) at −70°C. Protein concentration of the CF was assessed using a Bio-Rad DC Protein Assay kit (Bio-Rad Laboratories). Free coelomocytes were obtained by washing the earthworms in LBSS containing 5% ethanol [22]. When CF containing free coelomocytes was expelled through dorsal pores, LBSS was collected and immediately centrifuged (100×g for 10 min). The cell pellet was resuspended in LBSS, washed twice in LBSS, and used for further experiments.

2.3. FACS analysis of free coelomocytes Free coelomocytes were collected as described

above, washed in LBSS, preincubated for 20 min at 4°C in 10% inactivated normal swine serum with 0.1% NaN3 to block nonspecific binding, and incubated with 12C9 mAb ([19]; 10 mg/ml in LBSS) for 2 h at 4°C. After washing in LBSS the second fluorescein-labeled swine antimouse Ig antibody (SEVAC, Prague; 1:500; 1 h at 4°C) was applied. As negative controls, the samples were incubated with either an irrelevant IgG1, mAb and subsequently with the secondary antibody or only with the secondary antibody. Flow cytometric analysis was performed using a FACSort flow cytometer (Becton Dickinson Instrument System, Mountain View, CA) equipped with a 15 mW air-cooled 488 nm argon ion laser. For each measurement a minimum of 10 000 events were collected. The FACScan Research Software was used during the data acquisition at the low sample flow rate setting. High green autofluorescence of bacteria normally present in the coelomic cavity was minimized during the data acquisition using combined gate FSC/ SSC and FSC/FL3 using 7-aminoactinomycin D (7-AAD; Molecular Probes; Eugene, OR; [23]). The multivariant analysis was performed with LYSIS II software.

2.4. Immunohistochemistry Segments of intestinal portions of earthworms (control or 24 h after LPS injection) were fixed in one of the following solutions: 1. paraformaldehyde, 4% in 0.1 M phosphate buffer, pH 7.2; 2 h at 20°C with two 15 min rinses in buffer and one overnight at 4°C; 2. paraformaldehyde, 4% in saline with 2% acetic acid; 1 h at 20°C, three rinses with buffer as above; 3. mixture of methanol (six parts), chloroform (three parts), and glacial acetic acid (one part); 1 h at 20°C, direct transfer into absolute alcohol; 4. Stefanini’s reagent (2 g of paraformaldehyde in 15 ml saturated picric acid in distilled water; heat to 60°C, clear with several drops of 2.5% NaOH, filter and complete to 100 ml with phosphate buffer as above). Fixation 15 min at 20°C, three buffer rinses as above. The blocks were dehydrated in graded ethanol-acetone series and embedded in Histoplast S; 6 mm sections were mounted on Vectabond™-coated slides (Vector, Burlingame, CA). The antigen detection was performed by a three-step immunoenzyme method using a Vectastain ABC kit (Vector, Burlingame, CA). No enzyme pretreatment was used. The procedure involved quenching with normal horse serum (20 min) followed without rinse by incubation with anti-CCF-1 12C9 mAb (50 or 25 mg/ml

M. Bilej et al. / Immunology Letters 60 (1998) 23–29

in PBS/0.4% human serum albumin; overnight at 4°C), incubation with biotinylated horse anti-mouse IgG antibody (1 h at 20°C), incubation with the avidin-biotinperoxidase complex (ABC; 1 h at 20°C), development with 3,3%-diaminobenzidine-HCl (DAB)-H2O2 reagent, and counterstaining with haematoxylin. Series of sections were pretreated by microwave oven heating in citrate buffer (0.01 M, pH 6; 5 min at 600 W). Controls were performed either by (a) preincubation of 12C9 mAb with purified antigen (CCF-1; 1 mg CCF-1 and 10 mg of mAb; 1 h at 20°C); (b) omission of the primary 12C9 mAb; (c) omission of both primary and secondary antibodies (to detect endogenous biotin by ABC); (d) incubation with DAB-H2O2 alone (endogenous peroxidase activity); or (e) H.E. staining (autogeneic pigments simulating peroxidase product).

25

estimated in wells containing 100 ml medium in place of effector cells. Total lysis was estimated in wells containing 100 ml 10% Triton X-100 detergent solution in place of effector cells. After 5 h of incubation at 22°C the plates were centrifuged (100× g for 5 min), 50 ml of supernates transferred to individual tubes, and the radioactivity measured using a g-counter. Specific lysis was determined as follows: Specific lysis= 100 ×

experimental cpm− spontaneous cpm total cpm− spontaneous cpm

2.7. Statistics The significance of the data was evaluated by the Student’s t-test.

2.5. E6aluation of cytolytic acti6ity Cytolytic activity was quantified using a cell-killing bioassay [24]. L929 fibrosarcoma cells cultivated in D-MEM supplemented with 10% of fetal calf serum, glutamine, and antibiotics were treated with actinomycin D (1 mg/ml) at a cell concentration of 3× 105 cells/ml. A sample of 100 ml of the cell suspension was mixed in 96-well flat-bottomed culture plates with 100 ml of CF serially diluted in D-MEM. After 18 h of cultivation at 37°C, the viability of the cells was assessed by crystal violet uptake: the medium was decanted from the plates and the cells were stained for 10 min with 100 ml of a 0.5% solution of crystal violet dissolved in 22% ethanol containing 8% formaldehyde. Plates were rinsed properly in water, 100 ml of 30% acetic acid was added and dye uptake measured at 620 nm with a Titertek Multiscan MCC/340 ELISA Reader. The concentration of CF proteins exerting 50% of lysis was estimated by linear regression.

2.6. Cytotoxicity assay Target cells collected from L. terrestris earthworms, as described in Section 2.2, were labeled with 111In-chloride (Amersham, UK) using tropolone (2-hydroxy2,4,6-cycloheptatrienone; Sigma, St. Louis, MO) as a carrier allowing 111In to be transported into the cytoplasm [25]. Briefly, 200 ml of 4.4 mM tropolone solution in 20 mM Hepes-buffered saline was mixed with 3.7 MBq of 111In in 100 ml of saline. Then, 15 ml of 111 In-saline-tropolone solution was added to 100 ml of coelomocyte suspension (107/ml in isotonic RPMI 1640 medium). After the incubation (10 min; 22°C) cells were washed twice in RPMI 1640 medium and resuspended in medium at a cell concentration of 105/ml. Aliquots of 100 ml were added to wells of a 96-well plate containing 100 ml of E. foetida effector cells (at cell concentrations 5×105/ml; 106/ml; 2×106/ml). Spontaneous lysis was

3. Results

3.1. Cellular expression of CCF-1 The expression of CCF-1 on the surface of free coelomocytes was estimated by flow cytometry. Since coelomocytes were homogeneously labeled with 12C9 mAb, CCF-1-positive coelomocytes were considered as a single population of cells for FACS measurements and a peak channel analysis was used to evaluate the acquired data. Free coelomocytes express an immunoglobulin-binding protein [26] and thus the relative positivity was estimated as the difference between the peak channel of the experimental sample (12C9 mAb) and the control (i.e. labeled with an irrelevant IgG; Fig. 1). Since free coelomocytes are most probably derived from the mesenchymal lining of the coelomic cavity [27–29] the presence of CCF-1-bearing cells in crossections of earthworms was estimated by immunohistochemistry. In the formaldehyde- and formaldehyde-acetic-acidfixed tissue from earthworms the antigen reactive with 12C9 mAb was visualized in the cells of chloragogenous tissue and in light foamy (presumably neutrophilic) free large coelomocytes (Fig. 2a). In the former location, the staining assumed a diffuse positivity or slightly granular pattern with lesser expression in the area of typhlosolis. In the coelomocytes, the reaction involved both the cell membranes and delicate cytoplasmic strands; the less numerous cells with compact basophilic cytoplasm (i.e. small coelomocytes) were always negative. The intestinal epithelial cells (gastrodermis), cuticula, smooth muscle cells, nerve cords and nephridia were also negative. The staining was much more intense after microwave pretreatment. In the probes fixed with chloroform-methanol and Stefanini’s liquid the specific staining was faint to absent.

26

M. Bilej et al. / Immunology Letters 60 (1998) 23–29

Fig. 1. Flow cytometric analysis and histogram statistics of the expression of CCF-1 on free coelomocytes detected by indirect immunofluorescence with either 12C9 mAb or an irrelevant control mAb. Representative data of one of three independent experiments. Histogram A: staining with an irrelevant control mAb. Histogram B: staining with 12C9 mAb — control earthworms. Histogram C: staining with 12C9 mAb — LPS-stimulated earthworms, day 1.

Preincubation of 12C9 mAb with CCF-1 caused a decreased yet not a complete loss of staining. Following the tests with the secondary antibody, ABC complex, and DAB-H2O2 reagent both the cells of chloragogenous tissue as well as the free coelomocytes remained negative (Fig. 2b); on the other hand, in all three instances the intravascular blood pigment yielded a fluctuating but mostly strong reaction. The circular, and also to a lesser extent the longitudinal, muscle layer exhibited plentiful deep brown pigment granules scattered exclusively in the dorsal (antineural) segment; the same picture was obtained in routine H.E.-stained sec-

Fig. 2. (a) Membraneous and cytoplasmic positivity of intracoelomic clear foamy cells, probably neutrophilic, large coelomocytes (LC). Bottom, part of the gut (G) Formaldehyde-acetic acid, microwavetreated. 12C9 mAb, horse anti-mouse IgG, ABC, DAB-H2O2; counterstained with hematoxylin; ×470. (b) Control test—negative coelomocytes after the omission of 12C9 mAb ×470.

tions. Nonspecific brown staining was also seen in the intestinal content.

3.2. Effect of endotoxin on CCF-1 expression In vertebrates, endotoxin injection induces secretion of inflammatory cytokines including TNF [20]. In earthworms, we have followed the effect of intracoelomic administration of lipopolysaccharide from Salmonella typhimurium LT2 on both humoral and cellular levels of CCF-1. Humoral levels were estimated by an L929 killing bioassay, cellular expression was estimated by FACS analysis and immunohistology. Earthworms respond to body injury or to stress conditions (e.g. puncturing of the coelomic cavity) by a nonspecific increase in the protein concentration of the coelomic fluid [30]. Thus, coelomic fluid levels of CCF1 were estimated as protein concentration of the coelomic fluid corresponding with 50% of L929 lysis. We have found that within 24 h after LPS injection the protein concentration of the entire CF exerting 50% of L929 lysis considerably decreases compared to non-injected or sham-treated controls, in other words the CCF-1 level specifically increases (Table 1). Then the CCF-1 level drops down to control values. The differences in expression of CCF-1 on free coelomocytes isolated from LPS-injected and control earthworms were analyzed by FACSort and expressed as a difference between peak channel of experimental sample stained with 12C9 mAb and control sample stained with an irrelevant IgG1 mAb (Fig. 1). While the peak channel difference in samples from non-injected earthworms was approximately 25 (median channel difference 34), 24 h after LPS treatment it increased to 130 (median channel difference 161; Table 1).

M. Bilej et al. / Immunology Letters 60 (1998) 23–29

27

Table 1 The effect of endotoxin injection on CCF-1 levels in the CF and on its membrane expression

Controls — day 0 LPS-injected — day LPS-injected — day LPS-injected — day LPS-injected — day

1 2 3 4

CF concentration 50% L929 lysis (mg/ml)a

Peak channel differenceb

Median channel differencec

199.59 21.2 95.59 10.3* 135.29 13.2* 149.89 16.1* 203.19 18.7

24.72 98.18 94.37 921.24* 32.57 95.73 31.22 98.48 24.13 94.91

33.48 97.99 127.63 920.56* 28.38 9 5.62 31.62 9 6.04 33.35 98.61

a

Concentration of the CF exerting 50% of L929 lysis. The difference between the peak channel of coelomocytes stained with 12C9 mAb and that of the control sample stained with an irrelevant mAb. c The difference between the median channel of coelomocytes stained with 12C9 mAb and that of the control sample stained with an irrelevant mAb. Indicated as means 9 S.D. of three independent experiments; * significant at PB0.05 level as compared to controls (day 0). b

Because of the mesenchymal origin of free coelomocytes it was of interest to follow CCF-1 expression in cross-sections of earthworms. In control earthworms the specific positivity was recorded in the cells of chloragogenous tissue and in large coelomocytes. In crosssection of LPS-treated earthworms the staining was much stronger with apparent hypertrophy of chloragogenous cells (Fig. 3a and b).

spontaneous release. Anti-CCF-1 mAb when added to the xenogeneic culture, almost completely blocked the cytotoxicity while an irrelevant IgG mAb did not cause any changes in killing. These results indicate that CCF1 may be involved in natural cytotoxicity.

3.3. Cell-mediated cytotoxicity

The coelomic fluid of earthworms E. foetida contains a 42 kDa cytolytic protein designated CCF-1, that lyses L929 murine fibrosarcoma cells and may be involved in opsonizing and hemolytic events of the CF [19]. In this paper we documented the cellular expression of CCF-1 and tried to elucidate its potential role in cellular defense mechanisms of earthworms. The expression of CCF-1 was determined by FACS. We have taken into account both the non-specific binding of the antibodies used due to the presence of immunoglobulin-binding protein [26] as well as the green autofluorescence of coelomocytes. Therefore, the data are reported as the difference between the peak channel of the experimental sample (12C9 mAb) and the control (i.e. labeled with an irrelevant IgG1 mAb). There is a difference of 25 channels of fluorescence intensity between the peak of cells labeled with an irrelevant IgG mAb and 12C9 and the difference is even more pronounced after LPS injection (Table 1). These

Earthworm coelomocytes are able to kill coelomocytes of other lumbricids in xenogeneic culture [31–33]. The killing seems to be independent of CF components since coelomocytes survive in xenogeneic CF [33,34]. We tested the cytotoxic effect of E. foetida effector cells against L. terrestris target coelomocytes. As shown in Table 2 the killing depends on the effector:target ratio (E:T) and at E:T of 1:5 the values of lysis are close to

4. Discussion

Table 2 The effect of anti-CCF-1 mAb 12C9 on natural cytotoxicity of Eisenia foetida coelomocytes exerted versus Lumbricus terrestris target coelomocytes as compared to an irrelevant mAb E:T ratio

Fig. 3. (a) Survey picture of gut with strong cytoplasmic positivity in chloragogenous cells (CC). Weak to negative reaction in typhlosolis (T). Same processing as in Fig. 2a; × 190. (b) Detail of granular positivity in hypertrophic chloragogenous cells (CC). Epithelial cells of the gut (EC) are negative. Formaldehyde fixed, then processed as in Fig. 2a; × 470.

Control +12C9 +irlgG mAb

1:20

1:10

1:5

14.0 9 1.1 4.1 9 0.5* 13.2 9 0.9

7.9 90.6 3.5 90.3* 7.3 90.7

3.5 90.5 4.090.6 3.5 90.3

Indicated as means 9 S.D. of triplicates of two independent experiments, each point in triplicate, * significant at PB0.05 level.

28

M. Bilej et al. / Immunology Letters 60 (1998) 23–29

data indicate that free coelomocytes bear CCF-1 on their surface and this membrane expression is enhanced after endotoxin treatment. It was not possible to detect distinct CCF-1 positive cell populations within the total coelomocyte population. We were not able to reach such a clear and reliable distinction between small and large coelomocytes because the light scatter signals were more homogeneous than those described by Cossarizza et al. [35]. This difference might be affected by the origin of earthworms, season of their collection, extent of natural stimulation and other factors. The cellular expression of CCF-1 was further analyzed by immunohistology. The immunohistochemical detection of CCF-1 was found to be dependent on the method of fixation. Optimal staining was obtained when paraformaldehyde or paraformaldehyde with acetic acid were used and the staining improved significantly using microwave-treated specimens (i.e. more intense staining and lower background). In samples from control earthworms the positivity was localized in the cells of chloragogenous tissue adjacent to the gut wall and in the translucent free large coelomocytes (Fig. 2a) i.e. in cells with macrophage-like function [35]. Small cells with basophilic cytoplasm were always found to be negative for expression of CCF-1. LPS treatment resulted in the hypertrophy of chloragogenous tissue accompanied with a more pronounced staining (Fig. 3a, b). Isolated positive foci of cells were also present in the parietal lining of the coelomic cavity. The higher granularity of cells of chloragogenous tissue could reflect the proteosynthetic and/or secretory activity. In the case of immunohistology we have included all necessary controls to exclude false positivities (Section 2.4). Following the tests with the secondary antibody, ABC complex, and DAB-H2O2 reagent the intravascular blood pigment, longitudinal muscle layer, and intestinal content yielded strong reactions, whilst the cells of chloragogenous tissue as well as free coelomocytes remained negative (Fig. 2b). Moreover, preincubation of anti-CCF-1 mAb 12C9 with immunoaffinity purified CCF-1 caused decreased staining. These controls indicate that the staining of free coelomocytes and in chloragogenous tissue can be considered as the specific binding of anti-CCF-1 mAb 12C9. The observation that CCF-1 levels are increased after LPS treatment is reminiscent of the mammalian cytokine TNF. Indeed LPS was reported to act as a strong TNF-inducing stimulus in vertebrates [20]. Hence, CCF-1 may represent a primitive pleiotropic cytokine-like factor that is strongly produced during inflammatory processes in the coelomic cavity of earthworms. E. foetida coelomic fluid exerts a toxic effect on a variety of cell types, like chicken fibroblasts, guinea pig polymorphonuclear leucocytes, and insect haemocytes

while it does not affect the viability of cells of some molluscs, nematodes, and protozoans, as well as the coelomocytes of other lumbricids [34]. On the other hand, xenogeneic [31–33,35–37] and even allogenic [38] cell-mediated cytotoxicity was recorded. Detected values of intrafamilial killing described in our report are in agreement with those found by Suzuki and Cooper in a similar experimental design [38]. As shown in Table 2, the percentage of the specific lysis is considerably decreased when the cells are incubated in the presence of anti-CCF-1 mAb. While Cossarizza et al. [35] identified small coelomocytes as effectors killing vertebrate tumor cell lines, our results revealing the inhibitory role of 12C9 mAb indicate that CCF-1-positive i.e. large, coelomocytes might be involved in intrafamilial killing. On the other hand we cannot exclude that CCF-1 is secreted and target cells are killed with its soluble form (CCF-1 levels are too low to be detected in culture supernatants reliably; data not shown). These results, together with data presented in our previous paper [19] documenting the humoral role of CCF-1, strongly suggest that CCF-1 represents a pleiotropic factor important in both humoral and cellular responses of earthworms. Acknowledgements This work was supported by the Science Policy Office of the Kingdom of Belgium and by the grant No. 310/93/0610 from the Grant Agency of the Czech Republic. The authors thank Jana Perlova´ and Magdalena Lis' kova´ for excellent technical assistance and Dr Vladimir Dlabac' for providing LPS and helpful discussion. References [1] P. Roch, in: E.L Cooper (Ed.), Advances in Comparative and Environmental Physiology, vol. 23, Springer, New York, 1996, pp. 115 – 150. [2] G. Beck, G.S. Habicht, Immunol. Today 12 (1991) 180. [3] G. Beck, G.S. Habicht, in: E.L. Cooper (Ed.) Advances in Comparative and Environmental Physiology, vol. 24, Springer, New York, 1996, pp. 29 – 48. [4] G. Beck, G.S. Habicht, Proc. Natl. Acad. Sci. USA 83 (1986) 7429. [5] G. Beck, G.R. Vasta, J.J. Marchalonis, G.S. Habicht, Comp. Biochem. Physiol. 92B (1989) 93. [6] G. Beck, G.S. Habicht, Mol. Immunol. 28 (1991) 577. [7] D.A. Raftos, E.L. Cooper, G.S. Habicht, G. Beck, Proc. Natl. Acad. Sci. USA 88 (1991) 9518. [8] D.A. Raftos, E.L. Cooper, D.L. Stillman, G.S. Habicht, G. Beck, Lymphokine Cytokine Res. 11 (1992) 235. [9] G. Beck, R.F. O’Brien, G.S. Habicht, D.L. Stillman, E.L. Cooper, D.A. Raftos, Cell. Immunol. 146 (1993) 284. [10] T.K. Hughes Jr., E.M. Smith, R. Chin, P. Cadet, J. Sinisterra, M.K. Leung, M.A. Shipp, B. Scharrer, G.B. Stefano, Proc. Natl. Acad. Sci. USA 87 (1990) 4426.

M. Bilej et al. / Immunology Letters 60 (1998) 23–29 [11] T.K. Hughes Jr., E.M. Smith, J.A. Barnett, R. Charles, G.B. Stefano, Dev. Comp. Immunol. 15 (1991) 117. [12] T.K. Hughes Jr., E.M. Smith, M.K. Leung, G.B. Stefano, Ann. New York Acad. Sci. 650 (1992) 74. [13] E. Ottaviani, A. Franchini, C. Franceschi, Biochem. Biophys. Res. Commun. 195 (1993) 984. [14] O. Ouwe-Missi-Oukem-Boyer, E. Porchet, A. Capron, C. Dissous, Dev. Comp. Immunol. 18 (1994) 211. [15] E. Ottaviani, A. Franchini, S. Cassanelli, S. Genedani, Biol. Cell 85 (1995) 87. [16] E. Ottaviani, E. Caselgrandi, C. Franceschi, Biochem. Biophys. Res. Commun. 207 (1995) 288. [17] E. Legac, G.L. Vaugier, F. Bousquet, M. Bajelan, M. Leclerc, Scand. J. Immunol. 44 (1996) 375. [18] B. Scharrer, L. Paemen, E.M. Smith, T.K. Hughes, Y. Lui, M. Pope, G.B. Stefano, Cell Tissue Res. 283 (1996) 93. [19] M. Bilej, L. Brys, A. Beschin, R. Lucas, E. Vercauteren, R. Hanus' ova´, P. De Baetselier, Immunol. Lett. 45 (1995) 123. [20] E.A. Carswell, L.J. Old, R.L. Kassel, S. Green, N. Fiore, B. Willamson, Proc. Natl. Acad. Sci. USA 72 (1975) 3666. [21] E. Stein, E.L. Cooper, Dev. Comp. Immunol. 5 (1981) 415. [22] G.S. Eyambe, A.J. Goven, L.C. Fitzpatrick, B.J. Venables, E.L. Cooper, Lab. Anim. 25 (1991) 61. [23] K. Tobin, E.F. Winton, R.A. Bray, Cytometry 13 (1992) 60. [24] M.R. Ruff, G.E. Gifford, Lymphokines 2 (1981) 236.

. .

29

[25] J.W. Kupiec-Weglinski, J.M. Austyn, P.J. Morris, J. Exp. Med. 167 (1988) 632. [26] J. Rejnek, L. Tuc' kova´, P. S& ima, J. Kostka, Dev. Comp. Immunol. 10 (1986) 467. [27] E.L. Cooper, in: R.K. Wright, E.L. Cooper (Eds.), Phylogeny of Thymus and Bone Marrow-Bursa Cells, Elsevier, Amsterdam, 1976, pp. 9 – 18. [28] M. Bilej, P. S& ima, P. Slı´pka, Immunol. Lett. 32 (1992) 181. [29] E. Fischer, Comp. Biochem. Physiol. 106A (1993) 449. [30] L. Tuc' kova´, J. Rejnek, P. S& ima, Dev. Comp. Immunol. 12 (1988) 287. [31] E.L. Cooper, in: J. R& eha´c' ek, D. Blas' kovic' , W.F. Hink (Eds.), Proc. III Int. Colloquium Invertebrate Tissue Culture, Publishing House of the Slovak Academy of Sciences, Bratislava, 1973, pp. 381 – 404. [32] P. Valembois, P. Roch, D. Boiledieu, in: M.J. Manning (Ed.), Phylogeny of Immunological Memmory, Elsevier, Amsterdam, 1980, pp. 47 – 55. [33] M.M. Suzuki, E.L. Cooper, Immunol. Lett. 44 (1995) 45. [34] E. Kauschke, W. Mohrig, J. Comp. Physiol. B157 (1987) 77. [35] A. Cossarizza, E.L. Cooper, M.M. Suzuki, S. Salvioli, M. Capri, G. Gri, D. Quaglino, S. Franceschi, Exp. Cell. Res. 224 (1996) 174. [36] M.M. Suzuki, E.L. Cooper, Zoolog. Sci. 12 (1995) 443. [37] E.L. Cooper, A. Cossarizza, M.M. Suzuki, S. Salvioli, M. Capri, D. Quaglino, S. Franceschi, Cell. Immunol. 166 (1995) 113. [38] M.M. Suzuki, E.L. Cooper, Nat. Immun. 14 (1995) 11.