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Effect of lumpsucker (Cyclopterus lumpus L.) C-reactive protein on DNA synthesis in cultured murine and fish leucocytes
Comp. Bioehem, Physiol., Vol. 67B, pp. 127 to 131
0305-0491/80/0801-0127502.00/0
© Pergamon Press Ltd 1980. Printed in Great Britain
EFFECT OF LUMPSUCKER (CYCLOPTERUS LUMPUS L.) C-REACTIVE PROTEIN ON DNA SYNTHESIS IN CULTURED MURINE AND FISH LEUCOCYTES THELMA C. FLETCHER1, A. W. THOMSON2 and B. A. BALDO3 ~N.E.R.C. Institute of Marine Biochemistry, Aberdeen, AB1 3RA, 2Department of Pathology, University of Aberdeen, Aberdeen, AB9 2ZD, Scotland and 3Roche Research Institute of Marine Pharmacology, N.S.W. 2099, Australia (Received 9 January 1980) Abstract--1. C-reactive protein (CRP) isolated from a marine teleost (Cyclopterus lumpus L.) and structurally resembling mammalian CRP was added to cultures of leucocytes obtained from fish peripheral blood or mouse spleens. 2. A statistically significant dose-dependent stimulation by the fish CRP, of PH]thymidine incorporation into normal mouse spleen cells, was observed. The effect was substantially reduced in the presence of phosphorylcholine. 3. Addition of fish CRP to fish leucocytes or to cultures of nu nu mouse spleen cells did not result in significant stimulation. This suggests that although the CRP is not mitogenic for fish leucocytes it exerts a selective stimulatory effect on mouse T cells. 4. A synergistic stimulatory effect between fish CRP and PHA was abolished by phosphorylcholine and suggests that phosphorylcholine residues may contribute to the binding sites on mammalian lymphocytes.
INTRODUCTION C-reactive protein (CRP) in man is an acute phase reactant, levels of which become elevated during inflammatory processes (Abernethy & Avery, 1941; Hedlund, 1947; Claus et al., 1976). It is characterized by Ca2+-dependent binding to phosphorylcholine residues of pneumococcal C-polysaccharide (Volanakis & Kaplan, 1971): a reaction which can initiate activation of the classical complement pathway through C1 (Kaplan & Volanakis, 1974). Although Mortensen (1979) has recently listed the involvement of CRP with a number of immune reactivities in vitro, its biological function in vivo is still unknown. Amongst the lower vertebrates, CRP-like constituents in teleost fish (Baldo & Fletcher, 1973; Fletcher & Baldo, 1976) have been identified, after detailed chemical characterization, as proteins closely resembling human CRP (Pepys et al., 1978; White et al., 1978), Proof of their direct homology with human CRP can, however, only be established when their amino acid sequence has been compared with the CRP of man (Oliveira et al., 1977). The evolutionary persistence of CRP and the conservation of structure would suggest its functional significance. In fish, the only involvement of CRP so far described, is in the mediation of a cutaneous anaphylactic-like reaction in the skin of the plaice (Fletcher & Baldo, 1974). Speculation on the role of CRP in the immune response has led to examination of its effect on cultured lymphocytes (I-Iornung, 1973). The results obtained are conflicting. Thus H o r n u n g & Fritchi (1971) found stimulation of DNA synthesis, whilst Mortensen et al. (1975) obtained no effect. O n the other hand, Hokama et al. (1973) showed depression of [3H]-thymidine incorporation into leucocyte DNA.
The CRP from the lumpsucker has properties resembling mammalian CRP (White et al., 1978) and the availability of purified material provided an opportunity to examine its in vitro effect on the fishes' own leucocytes and for comparison, those of a mammal. MATERIALS AND METHODS Fish Lumpsuckers (Cyclopterus lumpus L.) were caught in salmon nets off the Aberdeen coast during their shoreward breeding migration between March and June. Plaice (Pleuronectes platessa L.) were caught in shallow coastal waters throughout the year. The fish were maintained in aerated sea water tanks in an aquarium at between 11-13°C.
C-reactive protein This was isolated from lumpsucker eggs by the method of White et al. (1978). The material used in the experiments gave a single line on acrylamide electrophoresis and there was no evidence of dissociation into subunits. Reagents Phytohaemagglutinin (PHA-P, Wellcome Reagents Ltd) and E. coil lipopolysaceharide (LPS, Difco) were reconstituted at 1 mg/ml in distilled water and stored at -20°C. Immediately before use the solutions were thawed and appropriate dilutions made with culture medium. Phosphorylcholine chloride Calcittm salt (Sigma Chemical Co Ltd; lot number 63c-1590) was also dissolved in culture medium. Separation of leucocytes from fish blood Blood was collected from the caudal vein into heparinized tubes (Stayne Laboratories Ltd) and allowed to stand
127
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THELMAC. FLETCHER,A. W. THOMSONand B. A. BALDO
for 90 min at 4°C. The leucocyte-rich plasma was layered over Ficoll-Triosil and leucocytes separated by centrifugation at 400g for 30min at 4°C (B~yum, 1976).
Table 1. Effect of Lumpsucker C-reactive Protein (CRP), PHA and LPS on cultured mouse spleen cells
Preparation of mouse spleen cells
Treatment
Spleens were obtained from either C3H HeMg mice, bred in the Animal Department, University Medical Buildings, Foresterhill, Aberdeen or from congenitally athymic MFI nu nu animals (OLAC 1976 Ltd). Cell suspensions were prepared by chopping the spleens and gently forcing the tissue through 80-mesh stainless steel gauze using icecold Eagle's minimum essential medium (MEM, Wellcome). Cell clumps and debris were allowed to sediment for 10min and then the supernatant cell suspension removed and washed twice with MEM. An aliquot of cells was diluted in 0.83%NH4C1 to determine the concentration of nucleated cells, which was adjusted to 2 x 10 6 cells/ml. Cell culture
Mouse leucocytes were cultured in MEM, supplemented with 10% pooled normal mouse serum, at 37°C in a humidified atmosphere of 5% CO2, in air, Fish cells were incubated in MEM containing 10% pooled plaice or lumpsucker serum as appropriate, at either 9 or 20°C. Aliquots (0.1 ml) containing 2 x 105 cells were dispensed into wells of flat-bottomed microculture plates (3040, Falcon Plastics) and appropriate dilutions of reagent added to make the final culture volume 0.2 ml. The cultures were maintained for 72 hr. All determinations were replicated 3-6 times. Cell viability
Viability of cultured cells was assessed by Trypan Blue dye exclusion (Fallon et al., 1962). Estimation of D N A synthesis
At 24hr prior to harvesting, 20ktl (0.2/~Ci) [methyl-3H]thymidine (Radiochemical Centre, Amersham) was added to each culture. These were harvested on to Gelman type A glassfibre discs, using a semi-automatic multiple cell harvester (Skatron AS, Norway). Discs were dried at 60°C, transferred to scintillation vials containing 5 ml 0.3% PPO (Koch-Light Laboratories Ltd) in toluene and counted in an automatic beta counter (Packard). Stimulation index represents the ratio of counts per minute (cpm) for treated cultures to cpm obtained from control cultures. Statistics
The significance of differences between means was evaluated using Student's t-test.
RESULTS
The influence of lumpsucker C R P on cultured mouse spleen cells is shown in Table 1. The C R P had a dose-dependent stimulatory effect on D N A synthesis in normal mouse spleen cells but no response was obtained using spleens from congenitally athymic animals which only responded to LPS. Since optimal stimulation of thymidine incorporation in normal spleens was found using 100/~g CRP, this dose was selected in further experiments. To examine whether lumpsucker C R P affected the response of mouse lymphocytes to PHA, these agents were added, together, to normal mouse spleen cell cultures. C R P significantly enhanced the mitogenic effect of P H A (Table 2). This response was considerably greater than a simple summative effect of C R P
CRP (0.2/~g) (1.0 pg) (5.0/tg) (25.0pg) (100,ug) PHA (0.2 pg) LPS (10 #g)
Mean stimulation index ___SD Normal nu nu 1.27 _ 0.24 1.67 4- 0.54 2.73 + 1.91 3.13 +_ 1.51" 12.17 _+ 8.39* 2.31 + 0.38* 2,55 _ 0.54*
0.85 + 0.05 0.95 + 0.04 0.75 ___0.05 0.80 _ 0.10 1.05 +_ 0.47 1.00 + 0.17 6.00 + 1.32"
Results are mean values obtained from at least 3 experiments, each done in triplicate. * Indicates values significantly higher than controls using Student's t-test (P < 0.01). and P H A alone. The combined effects of C R P and PHA were abolished by the addition of 100/~g phosphorylcholine. Although phosphorylcholine did not impair the response to PHA alone it significantly reduced the mitogenic response to CRP. It also significantly depressed [3H]thymidine incorporation by unstimulated lymphocytes. These experiments were done 3 months after those shown in Table 1 and while 100#g CRP gave a stimulation index of 12.17 in the earlier experiments, in the later experiments it was reduced to 2.73. The same preparation of CRP was used but it had been stored as a freeze-dried solid in the interim period. Treatment of fish blood leucocyte cultures with lumpsucker C R P resulted in a significant stimulation of plaice but not of lumpsucker cells at 20°C. Leucocytes from both species responded to P H A at this temperature but a mitogenic effect of LPS on lumpsucker cells was not observed (Table 3). C R P and P H A were not mitogenic for plaice cells at 9°C. DISCUSSION The lumpsucker, a marine teleost, has the highest levels of circulating CRP-like protein of any fish that we have examined: 2000#g/ml serum have been found in some males (Fletcher et al., 1977). In the female, large amounts of CRP are found in the eggs, probably bound to phospholipids, since the C R P has binding specificity for the phosphorylcholine residues widely occurring in lecithin and sphingomyelin (Fletcher & Baldo, 1976). The egg and serum C R P are similar to mammalian C R P in their molecular weight and subunit structure (White et al., 1978). Lumpsucker C R P shows antigenic cross-reactivity with plaice CRP, which in turn has been shown to belong to a family of plasma proteins, each with a single type of subunit, arranged in pentameric symmetry and represented by C R P and serum amyloid P component (Osmand et al., 1977; Pepys et al., 1978). Since we felt justified in describing the protein in fish as C R P on the basis of structural organization, it was of interest to use it in an in vitro system which might give information on its biological function. The lymphocyte, central to the mechanism of immune responsiveness in vertebrates, is an obvious choice for examination as a possible target for functional modulation by CRP. Abernethy & Francis
Effect of fish CRP on leucocytes
129
Table 2. Effect of Lumpsucker C-Reactive Protein (CRP), PHA and phosphorylcholine on cultured normal mouse spleen cells Treatment
Mean stimulation index + SD
PHA PHA + CRP PHA + CRP + phosphorylcholine PHA + phosphorylcholine CRP Phosphorylcholine CRP + phosphorylcholine
4.65 + 1.25 17.00 ___1.97 4.76 + 0.79 4.81 ___1.51 2.73 _ 1.00 0.40 + 0.15 1.25 _ 0.71
The quantities of reagents added to the cultures were: PHA, 0.2 #g; CRP, 100/~g; phosphorylcholine, 100/~g. Results are mean values obtained from 6 cultures. (1937) described a delayed skin reaction to pneumococcal C polysaccharide in patients with circulating CRP and they attributed this to the activation of a particular cell type by the CRP. Hornung & Fritchi (1971) found, in 10 separate experiments, that human CRP (10/~g/ml) stimulated human lymphocytes by 3.5-21 times the control values. There do not appear to be any other reports of lymphocyte stimulation by CRP. Although we were using a different system, with normal mouse spleen cells and fish CRP, we also found a dose-dependent increase in [3H]thymidine uptake. There was no evidence of cytotoxicity, as judged by dye exclusion experiments and maximal stimulation was given by 100 #g CRP/2 x 105 cells. In contrast, Hornung & Fritchi (1971) had found an 80~o loss in cell viability with CRP at 20 #g/ml. There have been reports of phosphorylcholinedependent binding of CRP to human peripheral blood lymphocytes (Hokama et al., 1973; Williams et al., 1978) and that this is selective for T cells (Mortensen et al., 1975). The latter authors found that CRP binding inhibited certain T cell functions as evidenced by impaired rosette formation with sheep erythrocytes and depressed mixed lymphocyte responses. Further evidence for CRP reacting primarily with T cells was presented by Mortensen (1979) when he found that CRP inhibited the antibody plaque-forming response to T-dependent but not T-independent antigens. Mortensen et al. (1975) tested human CRP on normal and nu nu mouse spleen cells and showed that CRP could bind to T cells of a heterologous species and that binding was preferentially to extra-thymic mouse T cells (Mortensen & Gewurz, 1976). It was therefore of interest that our CRP preparations which caused a
significant stimulation of spleen cells from C3H HeMg mice had no effect on cells from athymic animals, although these same cells responded to E. coil lipopolysaccharide. Kindred (1979) has pointed out that nude mouse spleen cells may represent a large proportion of T-lineage cells and not a "pure B cell" population. In the light of the results of Mortensen et al. (1975) and the fact that the fish CRP stimulates a population of cells apparently absent from the nude mouse spleen, we presume these to be "mature" T cells. Normal mouse spleen cells were used to establish whether stimulation was due to interaction of the CRP with membrane phosphorylcholine residues. These probably are involved in the membrane receptor site, since the addition of phosphorylcholine to the culture medium significantly reduced the stimulation index. Of relevance to our results are the findings of Leon & Takahashi (1970) that mouse myeloma proteins, including MOPC 167 with specificity for phosphorylcholine, significantly stimulate the incorporation of thymidine into human lymphocytes. Lecithin represents 43.6~o of the total phospholipids of human blood lymphocytes (Resch & Ferber, 1975) so that sufficient phosphorylcholine residues should be available. Volanakis & Wirtz (1979) have recently shown that CRP does not bind significantly to model membranes of lecithin, unless lysophosphatidylcholine is also incorporated into the bilayer. They suggest that an alteration of the natural orientation of cell membranes is necessary for in vivo binding of CRP. It is possible that changes taking place during the isolation of the leucocytes are sufficient to allow in vitro binding of CRP but Resch & Ferber (1975) did report
Table 3. Effect of Lumpsucker C-Reactive Protein (CRP), PHA and LPS on cultured fish blood leucocytes
Treatment CRP (100 #g) PHA (2pg) LPS (10 pg)
9°C
Mean stimulation index + SD Plaice Lumpsucker 20°C 20°C
1.32 +__0.76 1.29 + 0.97 N.D.t
2.47 + 0.84* 1.57 ___0.21" N.D.
1.46 -t- 0.39 1.31 ___0.11" 1.23 ___0.80
Results are mean values obtained from 5 (lumpsucker) or 2 (plaice) separate experiments each done in triplicate. * Indicates values significantly higher than controls using Student's t-test (P < 0.01). 1"N.D. indicates not determined. C.B.P. 67/IB--I
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THELMA C. FLETCHER, A. W. THOMSON and B. A. BALDO
that lysolecithin was present at 1 ~ of the total phospholipids of lymphocytes. Meade & Mertin (1978) have reviewed the role of fatty acids in immunity and the concept that it is changes in membrane phospholipids which underlie lymphocyte triggering. It is therefore of interest that CRP may react directly with a membrane component and that it has a synergistic effect with PHA (a T cell mitogen). Phosphorylcholine abolished the synergistic response to PHA and CRP. Mortensen et al. (1975) found a consistent, but statistically insignificant, enhancement of the response to PHA when human CRP was also added to the human lymphocytes. Since Hokama et al. (1973) found CRP to have an inhibitory effect upon PHA-induced mitogenicity, these conflicting results can only be explained by differences in the CRP preparations and biological systems used. Mitogenic responses, perhaps suggestive of lymphoid heterogeneity, have been reported in fishes (Etlinger et al., 1976; Chiimonczyk, 1978). Peripheral blood leucocytes of our marine teleosts were also responsive to mitogens, but only at 20°C. Although the ambient temperature of both plaice and lumpsucker is below 10°C, the lack of response at 9°C is probably indicative of the absence of stimulation in the natural environment. Our results would indicate that in the artificial system of mouse leucocytes with fish CRP there appears to be some specificity for mitogenicity directed towards T cells, with phosphorylcholine involved in the reaction. The possibility cannot be excluded however, that lumpsucker CRP may share determinants with a naturally occurring antigen to which the mice have previously been exposed. The T cell dependence of the mitogenic response to the CRP could also be explained on this basis. In the "natural" situation, the lumpsucker CRP has no mitogenic effects on its own leucocytes. This is not surprising since the individual fish from which the leucocytes were isolated, all had circulating levels of CRP over I0 times in excess of the 100#g used in cultures. We therefore conclude that in fish, CRP is not involved in lymphocyte activation and that its role, if indeed involved in the immune response, is perhaps more indirect, through complement activation or the release of pharmacological mediators (Baldo & Fletcher, 1975).
REFERENCES ABERNETHY T. J. ~ AVERY O. T. (1941) The occurrence
during acute infections of a protein not normally present in the blood. I. d. exp. Med. 73, 173-182. ABERNETHY T. J. ~g FRANCIS T. (1937) Studies on the somatic C polysaccharide of pneumococcus. I. J. exp. Med. 65, 59-73. BALDO B. A. 8/. FLETCHER T. C. (1973) C-reactive proteinlike precipitins in the plaice. Nature, Lond. 246, 145-146. BALDOB. A. & FLETCHERT. C. (1975) Phylogenetic aspects of hypersensitivity: immediate hypersensitivity reactions in flatfish. Adv. exp. Med. Biol. 64, 365-372. BOYUM A. (1976) Isolation of lymphocytes, granulocytes and macrophages. Scand. J. lmmunol. 5, suppl. 5, 9-15. CH1LMONCZYKS. (1978) In vitro stimulation by mitogens of peripheral blood lymphocytes from rainbow trout (Salmo gairdneri). Ann. lmraunol., Paris 129C, 3-12.
CLAUSD. R., OSMANDA. P. & GEWURZH. (1976) Radioimmunoassay of human C-reactive protein and levels in normal sera. J. Lab. clin. Med. 87, 120-128. ETLINGER H. M., HODGINSH. O. & CHILLERJ. M. (1976) Evolution of the lymphoid system. I. Evidence for lymphocyte heterogeneity in rainbow trout revealed by the organ distribution of mitogenic responses. J. Immun. 116, 1547-1553. FALLONH. J., FREIE., DAVIDSONJ. D., TRIERT. S. & BURK D. (1962) Leukocyte preparations from human blood: evaluation of their morphologic and metabolic state. J. Lab. clin. Med. 59, 779-791. FLETCHERT. C. • BALDO B. A. (1974) Immediate hypersensitivity responses in flatfish. Science, N.Y. 185, 360361. FLETCHERT. C. & BALDOB. A. (1976) C-reactive proteinlike precipitins in lumpsucker (Cyclopterus lumpus L.) gametes. Experientia 32, 1199-1200. FLETCHERT. C., WHITEA. & BALDOB. A. (1977) C-reactive protein-like precipitin and lysozyme in the lumpsucker Cyclopterus lumpus L. during the breeding season. Comp. Biochem. Physiol. 57B, 353-357. HEDLUNDP. (1947) The appearance of acute phase protein in various diseases. Acta reed. scand. (suppl.) 196, 579-601. HOKAMA Y., PAIK Y. P., YANAGIHARAE. & KIMURA L. (1973) Effect of C-reactive protein and blood-group substances on 3H-thymidine incorporation into DNA of leukocyte. J. reticuloendothel. Soc. 13, 111-121. HORNUNG M. O. (1973) Lymphocyte stimulation by C-reactive protein. In Non-specific Factors lnfluencine Host Resistance (Edited by BRAUN W. & UNGARJ.) pp. 354-357. Karger, Basel. HORNUNG M. & FRITCHIS. (1971) Isolation of C-reactive protein and its effect on human lymphocytes in vitro. Nature New Biol. 230, 84-85. KAPLAN M. H. 86 VOLANAKISJ. E. (1974) Interaction of C-reactive protein complexes with the complement system. I. Consumption of human complement associated with the reaction of C-reactive protein with pneumococcal C-polysaccharide and with the choline phosphatides, lecithin and sphingomyelin. J. Immun. 112, 2135-2147. KINDRED B. (1979) Nude mice in immunology. Prog. Aller#y 26, 137-238. LEON M. A. & TAKAHASHIT. (1970) Stimulation of lymphocytes by myeloma proteins. In Proceedings of the Fifth Leukocyte Culture Conference (Edited by HARRIS J. E.) pp. 299-301. Academic Press, New York. MEADEC. J. & MERTINJ. (1978) Fatty acids and immunity. Adv. Lipid Res. 16, 127-165. MORTENSEN R. F. (1979) C-reactive protein (CRP)mediated inhibition of the induction of in vitro antibody formation. I. T-cell dependence of the inhibition. Cell. lmmunol. 44, 270-282. MORTENSENR. F. & GEWURZH. (1976) Effects of C-reactive protein on the lymphoid system. II. Inhibition of mixed lymphocyte reactivity and generation of cytotoxic lymphocytes. J. lmmun. 116, 1244-1250. MORTENSEN R. F., OSMAND A. P. & GEWURZ H. (1975) Effects of C-reactive protein on the lymphoid system. I. Binding of thymus-dependent lymphocytes and alteration of their functions. J. exp. Med. 141,821 839. OLIVEIRA E. B., GOTSCHLICH E. C. & LIU T.-Y. (1977) Primary structure of human C-reactive protein. Proc. HatH. Acad. Sci. U.S.A. 74, 3148-3151. OSMAND m. P., FRIEDENSON B., GEWURZ H., PAINTER R. H.,
HOFMANN T. & SHELTONE. (1977) Characterization of C-reactive protein and the complement subcomponent Clt as homologous proteins displaying cyclic pentameric symmetry (pentraxins). Proc. HatH. Acad. Sci. U.S.A. 74, 739-743. PEPYS M. B., DASHA. C., FLETCHERT. C., RICHARDSONN., MUNN E. A. & FEINSTEINA. (1978) Anologues in other
Effect of fish CRP on leucocytes mammals and in fish of human plasma proteins, C-reactive protein and amyloid P component. Nature Lond. 273, 168-170. RESCHK. & FERBERE. (1975) The role of phospholipids in lymphocyte activation. In Immune Recognition (Edited by ROSENTHALA. S.) pp. 281-312. Academic Press, New York. VOLANAK1S J. E. & KAPLAN M. H. (1971) Specificity of C-reactive protein for choline phosphate residues of pneumococcal C-polysaccharide. Proc. Soc. exp. Biol. Med. 136, 612-614.
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VOLANAKiSJ. E. & WIRTZ K. W. A. (1979) Interaction of C-reactive protein with artificial phosphatidylcholine bilayers. Nature Lond. 281, 155-157. WHITE A., FLETCHERT. C., TOWt~R C. M. & BALDOB. A. (1978) Isolation of a C-reactive protein-like precipitin from the eggs of the lumpsucker (Cyclopterus lumpus L.). Comp. Biochem. Physiol. 61C, 331-336. WILLIAMS R. C., KILPATRICK K. A., KASSABY M. & ABDIN Z. H. (1978) Lymphocytes binding C-reactive protein during acute rheumatic fever. J. clin. Invest. 61, 1384-1393.
0305-0491/80/0801-0127502.00/0
© Pergamon Press Ltd 1980. Printed in Great Britain
EFFECT OF LUMPSUCKER (CYCLOPTERUS LUMPUS L.) C-REACTIVE PROTEIN ON DNA SYNTHESIS IN CULTURED MURINE AND FISH LEUCOCYTES THELMA C. FLETCHER1, A. W. THOMSON2 and B. A. BALDO3 ~N.E.R.C. Institute of Marine Biochemistry, Aberdeen, AB1 3RA, 2Department of Pathology, University of Aberdeen, Aberdeen, AB9 2ZD, Scotland and 3Roche Research Institute of Marine Pharmacology, N.S.W. 2099, Australia (Received 9 January 1980) Abstract--1. C-reactive protein (CRP) isolated from a marine teleost (Cyclopterus lumpus L.) and structurally resembling mammalian CRP was added to cultures of leucocytes obtained from fish peripheral blood or mouse spleens. 2. A statistically significant dose-dependent stimulation by the fish CRP, of PH]thymidine incorporation into normal mouse spleen cells, was observed. The effect was substantially reduced in the presence of phosphorylcholine. 3. Addition of fish CRP to fish leucocytes or to cultures of nu nu mouse spleen cells did not result in significant stimulation. This suggests that although the CRP is not mitogenic for fish leucocytes it exerts a selective stimulatory effect on mouse T cells. 4. A synergistic stimulatory effect between fish CRP and PHA was abolished by phosphorylcholine and suggests that phosphorylcholine residues may contribute to the binding sites on mammalian lymphocytes.
INTRODUCTION C-reactive protein (CRP) in man is an acute phase reactant, levels of which become elevated during inflammatory processes (Abernethy & Avery, 1941; Hedlund, 1947; Claus et al., 1976). It is characterized by Ca2+-dependent binding to phosphorylcholine residues of pneumococcal C-polysaccharide (Volanakis & Kaplan, 1971): a reaction which can initiate activation of the classical complement pathway through C1 (Kaplan & Volanakis, 1974). Although Mortensen (1979) has recently listed the involvement of CRP with a number of immune reactivities in vitro, its biological function in vivo is still unknown. Amongst the lower vertebrates, CRP-like constituents in teleost fish (Baldo & Fletcher, 1973; Fletcher & Baldo, 1976) have been identified, after detailed chemical characterization, as proteins closely resembling human CRP (Pepys et al., 1978; White et al., 1978), Proof of their direct homology with human CRP can, however, only be established when their amino acid sequence has been compared with the CRP of man (Oliveira et al., 1977). The evolutionary persistence of CRP and the conservation of structure would suggest its functional significance. In fish, the only involvement of CRP so far described, is in the mediation of a cutaneous anaphylactic-like reaction in the skin of the plaice (Fletcher & Baldo, 1974). Speculation on the role of CRP in the immune response has led to examination of its effect on cultured lymphocytes (I-Iornung, 1973). The results obtained are conflicting. Thus H o r n u n g & Fritchi (1971) found stimulation of DNA synthesis, whilst Mortensen et al. (1975) obtained no effect. O n the other hand, Hokama et al. (1973) showed depression of [3H]-thymidine incorporation into leucocyte DNA.
The CRP from the lumpsucker has properties resembling mammalian CRP (White et al., 1978) and the availability of purified material provided an opportunity to examine its in vitro effect on the fishes' own leucocytes and for comparison, those of a mammal. MATERIALS AND METHODS Fish Lumpsuckers (Cyclopterus lumpus L.) were caught in salmon nets off the Aberdeen coast during their shoreward breeding migration between March and June. Plaice (Pleuronectes platessa L.) were caught in shallow coastal waters throughout the year. The fish were maintained in aerated sea water tanks in an aquarium at between 11-13°C.
C-reactive protein This was isolated from lumpsucker eggs by the method of White et al. (1978). The material used in the experiments gave a single line on acrylamide electrophoresis and there was no evidence of dissociation into subunits. Reagents Phytohaemagglutinin (PHA-P, Wellcome Reagents Ltd) and E. coil lipopolysaceharide (LPS, Difco) were reconstituted at 1 mg/ml in distilled water and stored at -20°C. Immediately before use the solutions were thawed and appropriate dilutions made with culture medium. Phosphorylcholine chloride Calcittm salt (Sigma Chemical Co Ltd; lot number 63c-1590) was also dissolved in culture medium. Separation of leucocytes from fish blood Blood was collected from the caudal vein into heparinized tubes (Stayne Laboratories Ltd) and allowed to stand
127
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THELMAC. FLETCHER,A. W. THOMSONand B. A. BALDO
for 90 min at 4°C. The leucocyte-rich plasma was layered over Ficoll-Triosil and leucocytes separated by centrifugation at 400g for 30min at 4°C (B~yum, 1976).
Table 1. Effect of Lumpsucker C-reactive Protein (CRP), PHA and LPS on cultured mouse spleen cells
Preparation of mouse spleen cells
Treatment
Spleens were obtained from either C3H HeMg mice, bred in the Animal Department, University Medical Buildings, Foresterhill, Aberdeen or from congenitally athymic MFI nu nu animals (OLAC 1976 Ltd). Cell suspensions were prepared by chopping the spleens and gently forcing the tissue through 80-mesh stainless steel gauze using icecold Eagle's minimum essential medium (MEM, Wellcome). Cell clumps and debris were allowed to sediment for 10min and then the supernatant cell suspension removed and washed twice with MEM. An aliquot of cells was diluted in 0.83%NH4C1 to determine the concentration of nucleated cells, which was adjusted to 2 x 10 6 cells/ml. Cell culture
Mouse leucocytes were cultured in MEM, supplemented with 10% pooled normal mouse serum, at 37°C in a humidified atmosphere of 5% CO2, in air, Fish cells were incubated in MEM containing 10% pooled plaice or lumpsucker serum as appropriate, at either 9 or 20°C. Aliquots (0.1 ml) containing 2 x 105 cells were dispensed into wells of flat-bottomed microculture plates (3040, Falcon Plastics) and appropriate dilutions of reagent added to make the final culture volume 0.2 ml. The cultures were maintained for 72 hr. All determinations were replicated 3-6 times. Cell viability
Viability of cultured cells was assessed by Trypan Blue dye exclusion (Fallon et al., 1962). Estimation of D N A synthesis
At 24hr prior to harvesting, 20ktl (0.2/~Ci) [methyl-3H]thymidine (Radiochemical Centre, Amersham) was added to each culture. These were harvested on to Gelman type A glassfibre discs, using a semi-automatic multiple cell harvester (Skatron AS, Norway). Discs were dried at 60°C, transferred to scintillation vials containing 5 ml 0.3% PPO (Koch-Light Laboratories Ltd) in toluene and counted in an automatic beta counter (Packard). Stimulation index represents the ratio of counts per minute (cpm) for treated cultures to cpm obtained from control cultures. Statistics
The significance of differences between means was evaluated using Student's t-test.
RESULTS
The influence of lumpsucker C R P on cultured mouse spleen cells is shown in Table 1. The C R P had a dose-dependent stimulatory effect on D N A synthesis in normal mouse spleen cells but no response was obtained using spleens from congenitally athymic animals which only responded to LPS. Since optimal stimulation of thymidine incorporation in normal spleens was found using 100/~g CRP, this dose was selected in further experiments. To examine whether lumpsucker C R P affected the response of mouse lymphocytes to PHA, these agents were added, together, to normal mouse spleen cell cultures. C R P significantly enhanced the mitogenic effect of P H A (Table 2). This response was considerably greater than a simple summative effect of C R P
CRP (0.2/~g) (1.0 pg) (5.0/tg) (25.0pg) (100,ug) PHA (0.2 pg) LPS (10 #g)
Mean stimulation index ___SD Normal nu nu 1.27 _ 0.24 1.67 4- 0.54 2.73 + 1.91 3.13 +_ 1.51" 12.17 _+ 8.39* 2.31 + 0.38* 2,55 _ 0.54*
0.85 + 0.05 0.95 + 0.04 0.75 ___0.05 0.80 _ 0.10 1.05 +_ 0.47 1.00 + 0.17 6.00 + 1.32"
Results are mean values obtained from at least 3 experiments, each done in triplicate. * Indicates values significantly higher than controls using Student's t-test (P < 0.01). and P H A alone. The combined effects of C R P and PHA were abolished by the addition of 100/~g phosphorylcholine. Although phosphorylcholine did not impair the response to PHA alone it significantly reduced the mitogenic response to CRP. It also significantly depressed [3H]thymidine incorporation by unstimulated lymphocytes. These experiments were done 3 months after those shown in Table 1 and while 100#g CRP gave a stimulation index of 12.17 in the earlier experiments, in the later experiments it was reduced to 2.73. The same preparation of CRP was used but it had been stored as a freeze-dried solid in the interim period. Treatment of fish blood leucocyte cultures with lumpsucker C R P resulted in a significant stimulation of plaice but not of lumpsucker cells at 20°C. Leucocytes from both species responded to P H A at this temperature but a mitogenic effect of LPS on lumpsucker cells was not observed (Table 3). C R P and P H A were not mitogenic for plaice cells at 9°C. DISCUSSION The lumpsucker, a marine teleost, has the highest levels of circulating CRP-like protein of any fish that we have examined: 2000#g/ml serum have been found in some males (Fletcher et al., 1977). In the female, large amounts of CRP are found in the eggs, probably bound to phospholipids, since the C R P has binding specificity for the phosphorylcholine residues widely occurring in lecithin and sphingomyelin (Fletcher & Baldo, 1976). The egg and serum C R P are similar to mammalian C R P in their molecular weight and subunit structure (White et al., 1978). Lumpsucker C R P shows antigenic cross-reactivity with plaice CRP, which in turn has been shown to belong to a family of plasma proteins, each with a single type of subunit, arranged in pentameric symmetry and represented by C R P and serum amyloid P component (Osmand et al., 1977; Pepys et al., 1978). Since we felt justified in describing the protein in fish as C R P on the basis of structural organization, it was of interest to use it in an in vitro system which might give information on its biological function. The lymphocyte, central to the mechanism of immune responsiveness in vertebrates, is an obvious choice for examination as a possible target for functional modulation by CRP. Abernethy & Francis
Effect of fish CRP on leucocytes
129
Table 2. Effect of Lumpsucker C-Reactive Protein (CRP), PHA and phosphorylcholine on cultured normal mouse spleen cells Treatment
Mean stimulation index + SD
PHA PHA + CRP PHA + CRP + phosphorylcholine PHA + phosphorylcholine CRP Phosphorylcholine CRP + phosphorylcholine
4.65 + 1.25 17.00 ___1.97 4.76 + 0.79 4.81 ___1.51 2.73 _ 1.00 0.40 + 0.15 1.25 _ 0.71
The quantities of reagents added to the cultures were: PHA, 0.2 #g; CRP, 100/~g; phosphorylcholine, 100/~g. Results are mean values obtained from 6 cultures. (1937) described a delayed skin reaction to pneumococcal C polysaccharide in patients with circulating CRP and they attributed this to the activation of a particular cell type by the CRP. Hornung & Fritchi (1971) found, in 10 separate experiments, that human CRP (10/~g/ml) stimulated human lymphocytes by 3.5-21 times the control values. There do not appear to be any other reports of lymphocyte stimulation by CRP. Although we were using a different system, with normal mouse spleen cells and fish CRP, we also found a dose-dependent increase in [3H]thymidine uptake. There was no evidence of cytotoxicity, as judged by dye exclusion experiments and maximal stimulation was given by 100 #g CRP/2 x 105 cells. In contrast, Hornung & Fritchi (1971) had found an 80~o loss in cell viability with CRP at 20 #g/ml. There have been reports of phosphorylcholinedependent binding of CRP to human peripheral blood lymphocytes (Hokama et al., 1973; Williams et al., 1978) and that this is selective for T cells (Mortensen et al., 1975). The latter authors found that CRP binding inhibited certain T cell functions as evidenced by impaired rosette formation with sheep erythrocytes and depressed mixed lymphocyte responses. Further evidence for CRP reacting primarily with T cells was presented by Mortensen (1979) when he found that CRP inhibited the antibody plaque-forming response to T-dependent but not T-independent antigens. Mortensen et al. (1975) tested human CRP on normal and nu nu mouse spleen cells and showed that CRP could bind to T cells of a heterologous species and that binding was preferentially to extra-thymic mouse T cells (Mortensen & Gewurz, 1976). It was therefore of interest that our CRP preparations which caused a
significant stimulation of spleen cells from C3H HeMg mice had no effect on cells from athymic animals, although these same cells responded to E. coil lipopolysaccharide. Kindred (1979) has pointed out that nude mouse spleen cells may represent a large proportion of T-lineage cells and not a "pure B cell" population. In the light of the results of Mortensen et al. (1975) and the fact that the fish CRP stimulates a population of cells apparently absent from the nude mouse spleen, we presume these to be "mature" T cells. Normal mouse spleen cells were used to establish whether stimulation was due to interaction of the CRP with membrane phosphorylcholine residues. These probably are involved in the membrane receptor site, since the addition of phosphorylcholine to the culture medium significantly reduced the stimulation index. Of relevance to our results are the findings of Leon & Takahashi (1970) that mouse myeloma proteins, including MOPC 167 with specificity for phosphorylcholine, significantly stimulate the incorporation of thymidine into human lymphocytes. Lecithin represents 43.6~o of the total phospholipids of human blood lymphocytes (Resch & Ferber, 1975) so that sufficient phosphorylcholine residues should be available. Volanakis & Wirtz (1979) have recently shown that CRP does not bind significantly to model membranes of lecithin, unless lysophosphatidylcholine is also incorporated into the bilayer. They suggest that an alteration of the natural orientation of cell membranes is necessary for in vivo binding of CRP. It is possible that changes taking place during the isolation of the leucocytes are sufficient to allow in vitro binding of CRP but Resch & Ferber (1975) did report
Table 3. Effect of Lumpsucker C-Reactive Protein (CRP), PHA and LPS on cultured fish blood leucocytes
Treatment CRP (100 #g) PHA (2pg) LPS (10 pg)
9°C
Mean stimulation index + SD Plaice Lumpsucker 20°C 20°C
1.32 +__0.76 1.29 + 0.97 N.D.t
2.47 + 0.84* 1.57 ___0.21" N.D.
1.46 -t- 0.39 1.31 ___0.11" 1.23 ___0.80
Results are mean values obtained from 5 (lumpsucker) or 2 (plaice) separate experiments each done in triplicate. * Indicates values significantly higher than controls using Student's t-test (P < 0.01). 1"N.D. indicates not determined. C.B.P. 67/IB--I
130
THELMA C. FLETCHER, A. W. THOMSON and B. A. BALDO
that lysolecithin was present at 1 ~ of the total phospholipids of lymphocytes. Meade & Mertin (1978) have reviewed the role of fatty acids in immunity and the concept that it is changes in membrane phospholipids which underlie lymphocyte triggering. It is therefore of interest that CRP may react directly with a membrane component and that it has a synergistic effect with PHA (a T cell mitogen). Phosphorylcholine abolished the synergistic response to PHA and CRP. Mortensen et al. (1975) found a consistent, but statistically insignificant, enhancement of the response to PHA when human CRP was also added to the human lymphocytes. Since Hokama et al. (1973) found CRP to have an inhibitory effect upon PHA-induced mitogenicity, these conflicting results can only be explained by differences in the CRP preparations and biological systems used. Mitogenic responses, perhaps suggestive of lymphoid heterogeneity, have been reported in fishes (Etlinger et al., 1976; Chiimonczyk, 1978). Peripheral blood leucocytes of our marine teleosts were also responsive to mitogens, but only at 20°C. Although the ambient temperature of both plaice and lumpsucker is below 10°C, the lack of response at 9°C is probably indicative of the absence of stimulation in the natural environment. Our results would indicate that in the artificial system of mouse leucocytes with fish CRP there appears to be some specificity for mitogenicity directed towards T cells, with phosphorylcholine involved in the reaction. The possibility cannot be excluded however, that lumpsucker CRP may share determinants with a naturally occurring antigen to which the mice have previously been exposed. The T cell dependence of the mitogenic response to the CRP could also be explained on this basis. In the "natural" situation, the lumpsucker CRP has no mitogenic effects on its own leucocytes. This is not surprising since the individual fish from which the leucocytes were isolated, all had circulating levels of CRP over I0 times in excess of the 100#g used in cultures. We therefore conclude that in fish, CRP is not involved in lymphocyte activation and that its role, if indeed involved in the immune response, is perhaps more indirect, through complement activation or the release of pharmacological mediators (Baldo & Fletcher, 1975).
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