Leucocyte interleukin-1-like activity in the common seal (Phoca vitulina) and grey seal (Halichoerus grypus)

J. Comp. Path. 1995 Vol. 113, 253-261

Leucocyte Interleukin-l-like Activity in the C o m m o n Seal (Phoca vitulina) and Grey Seal

(Haliehoerus grypus) D. P. King*, A. W. M. Hay, I. Robinson~ and S. W. Evans Division of Clinical Sciences, Old Medical School, Universi~ of Leeds LS2 907T and ~RSPCA Norfolk 14~ldlife Hospital, Station Road, East Winch, King's Lynn; NooColk PE23 1NR, UK Summary

Interleukin-1 (IL-1) is an important cytokine with predominantly proinflammatory activities, which have been characterized in many mammals. This study showed the production of IL-l-like bioactivity by cultured seal leucocytes. Increasing concentrations of lipopolysaccharide (LPS) (0-1 ~tg/ ml) stimulated an increase in measurable IL-l-like activity in cell culture supernates. This activity increased for the first 24 h after LPS stimulation and the substance responsible had an apparent molecular weight of 17 kDa on gel filtration, similar to that described for other species. Specificity of the bioassay used was confirmed by blocking the bioactivity with an IL- 1 receptor antagonist (IL-1 ra). 9 t995 AcademicPress Limited

Introduction Interleukin-1 (IL-1) is a m e m b e r of the cytokine group of immunornodulatory molecules. Numerous nucleated cells produce this cytokine and cells of the m o n o c y t e / m a c r o p h a g e lineage stimulated with bacterial lipopolysaccharide (LPS) represent a major source. IL-1 is a mediator of the inflammatory response, inducing the production and release of eicosanoids, proteinases, some acute phase proteins and other enzymes that play a part in the generation of inflammatory mediators (Evans and Whicher, 1993). IL-1 also plays an important role in T-lymphocyte activation (Renauld et al., 1989) and in the co-stimulation of B lymphocytes to synthesize immunoglobulin (Hoffmann et al., 1987; Kunimoto et al., 1989). Marine animals, being at the apex of the food chain, are important indicators of the state of the aquatic environment. These mammals "bioaccumulate" numerous substances that possess immunotoxic properties in other animals, e.g. organochlorines (Reijnders, 1980; Law et al., 1988; Vos et al., 1988). Organochlorine molecules such as 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD), and its analogues, bind to a cytosolic receptor known as the arylhydrocarbon (Ah) receptor. Translocation to the nucleus and subsequent binding of this ligand-Ah receptor complex to specific regions of D N A known

* Present address: The International Program for Marine Mammal Health, Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis 95616, USA. 0021-9975/95/070253+09 $12.00/0

9 1995 Academic Press Limited

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D.P. King et a/.

as~dioxin-responsive enhancers are thought to alter the rate o f m R N A turnover in-sus~ptible cells. The IL-1 [3 gene contains a dioxin-responsive enhancer (Sutter et al., 1991) and T C D D treatment has been found to increase m R N A for IL-1 in murine cells (Steppan and Kerkliet, 1991). It has been suggested that immunotoxins contributed to the phocid distemper virus (PDV) epizootic in 1988 that was responsible for the mortality of approximately 16 000 animals (Osterhaus, 1989). Viruses of the same family, Paramyxoviridae, have also been shown to alter IL-1 production from cells of the m o n o c y t e / m a c r o p h a g e lineage in dogs (Krakowka et al., 1987) and m a n (Leopardi et al., 1992). There is a need, therefore, to study the regulation of this cytokine in TCDD-exposed or PDV-infected seals. This paper describes the first identification and partial characterization of IL-l-like activity from pinniped leucocytes. Materials and Methods

Cell Culture All cells (seal leucocytes and D 10 murine T-cell-line) were cultured in 1640 RPMI medium (Life Technologies Ltd, Renfrewshire, UK) supplemented with heatinactivated fetal calf serum (Gibco, UK) 10%, 2 mM glutamine (Gibco, UK), penicillin 100 IU/ml and streptomycin 100 gg/ml (Flow, UK). The D10 (DI0.G4.1) cell line was a gift from Dr S. Hopkins (University of Manchester, UK). The RPMI medium was further supplemented as appropriate by adding 5 x 10 -5 M 2-mercaptoethanol (Merck Ltd, Poole, UK), recombinant human IL-1 a (National Institute for Biological Standards and Control, [NIBSC], Potters Bar, UK; 1st international standard, 86/632) 20 pg/ml and recombinant human IL2 (British Biotechnology Products, Oxford, UK) 40 IU/ml. Culture of Seal Leucocytes Conditioned media from lipopolysaecharide (LPS) (E. coli serotype 0128:B 12; Sigma, Poole, UK)--stimulated common and grey seal leucocytes were prepared as previously described (King et al., 1993). Briefly, cells (106/ml) with a viability exceeding 90% (determined by dye exclusion) were cultured in 0"5 or 1-0 ml of RPMI medium for periods of 0-96 h. LPS was added at the start of culture, at the concentrations stated in the figure legends. After culture, cell-free supernates were collected by aspiration. Gel Filtration of IL-l-like Bioactivi~ Seal leucocyte-derived conditioned medium was fractionated with a Superose 6 column (Pharmacia, Uppsala, Sweden) with a protein separation range of 5-5000 kDa. Phosphate-buffered saline (pH 7"2) was used as the column buffer and the column flow rate was 1 ml/min. Eluted 1-ml fractions were assayed for IL- l-like biological activity. The column was calibrated for apparent molecular weight with chymotrypsinogen A (25 kDa), ribonuclease A (13"7 kDa), gel chromatography calibration reagents (Pharmacia, Sweden) and recombinant human IL-1~ (17"5 kDa; NIBSC, UK). IL-1 Bioassay and Inhibition of the IL-l-like Bioactivi~ Samples were prepared by centrifugation (10 000g for 10 min) and filtration (0"22 gm; Gelman Sciences, Northampton, UK). The IL-1 concentration of the seal leucocytederived supernates was determined by a method adapted from Hopkins and Humphries (1989). Murine D10 cells were washed twice in cytokine-free RPMI medium and

Leucocyte IL-l-like Acti~/ity in Seals

255

resuspended in RPMI medium (containing IL-2 at 60 IU/ml) at 105 cells/ml. This suspension (100 ttl; 10 000 cells) was then added to each well of a Falcon flat-bottomed 96-well plate (Becton Dickinson, Plymouth, UK) containing 100 ltl of diluted sample or standard. All samples and standards were titrated, in triplicate, to give a full dose response. Plates were incubated for 72 h at 37~ in a humidified atmosphere containing CO2 5%. At the end of the incubation period, cell proliferation was determined by measuring the mitochondrial metabolism of 3-(4, 5,-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) to the coloured formazan product (Mossmann, 1983). IL-1 activity is expressed as international units (IU) of IL-1. IU were calculated by comparing the seal leucocyte supernate titration curve with the curve of an IL-1 standard (lst international standard 86/632) titration included on each plate. Inhibition experiments were performed to determine the specificity of the bioassay. Neutralization of seal leucocyte D 10 stimhlatory activity was attempted with rabbit and-human IL- 1[3 anfisera (British Biotechnology, Oxford, UK). Specificity of the ILl ]3 antisera was confirmed by the inhibition of recombinant human IL-1 [~ (Gistron Biotechnology, Pine Brook, NJ, USA) proliferative activity. Blocking experiments were carried out with recombinant IL-1 receptor antagonist (IL-1 ra) (Steinkasserer et al., 1992). Diluted 10-1al volumes (dilution effect less than 5%) of antisera or ILl r a were added to wells containing either standard IL-1 (NIBSC, UK), IL-1 [3 or seal leucocyte-conditioned medium before the addition of the D 10 cells. Apart from these changes assays were performed as described above. Results

Production of IL-l-like Bioactivity by Seal Leucocytes IL-l-like biological activity was measured by the proliferation of the D10 murine T-cell-line (in the presence of saturating concentrations of IL-2). Addition of cell-free leucocyte-derived seal supernates induced significant proliferation of the D10 cell line. Further experiments were performed to characterize this bioactivity. The production o f IL-l-like activity was time-dependent. Fig. 1 shows the production of IL- l-like activity by leucocytes from three different c o m m o n seals cultured in the presence of LPS at 1 gg/ml. In each experiment IL-l-like activity continued to accumulate in the culture supernate over a 48-h period. Increasing concentrations of LPS were added to seal leucocyte cultures. Comparison with untreated cells showed that LPS did not stimulate any significant proliferation of the isolated seal leucocytes as measured by M T T metabolism (data not shown). However, LPS concentration-dependent increases in IL-l-like activity of the leucocyte supernates were produced with leucocytes from four out of five seals tested (Table 1). These experiments also showed variation in background IL-l-like activity and degree of LPS stimulation between the different animals tested.

Size Fractionation of IL-l-like Bioactivity Superose 6 gel chromatography o f seal leucocyte supernates led to the estimation of molecular weight of the substance responsible for IL-1-activity. Biological activity of c o m m o n seal IL-1 (Fig. 2 ) w a s confined to five fractions, peak activity being present at approximately 17 kDa. O n one occasion the molecular weight profile of IL-l-like activity in supernate derived from grey

256

D. P. King et al. 1000 8OO

~

600 400

2OO

0

300

200

100

300 I

r 100

0

3

6

12

24

48

Time (h) Fig. 1. The production of IL-l-like bioactivity from common seal leucocytes. In this experiment LPS at 1 lag/ml was present in the culture medium. The results are shown for leucocytes isolated from three different common seals.

seal leucocytes was examined. A profile similar to that of common seal supernate was obtained (data not shown).

257

Leucocyte IL-l-like Activity in Seals Table LPS-induced

o f IL-l-like activity f r o m

synthesis

for

Seal species

48

1 common

and

grey seal leucocytes cultured

h

IL-l-like activity (IU/m 0 in the presence of the stated concentration of LPS None (contro0

0"1 I~g/ ml

1 Itg/ ml

76-4 6"2 1"0 58-8 60"6

92'8 15"6 29"0 32"6 125-0

154 21'5 90-0 47"0 222.2

31 "5 +_32"2

50-6 + 50'2

95"2 + 89

Common Grey Grey Grey Grey Mean value for grey seals (n = 4) Mean data shown are + SD.

50t @

25 kDa 17-5 kDa 13-7 kDa

-1

40-

I 30

O (3O O,1

0.1

20

9 10

i

i

i

f

I

10 Fig. 2.

i

t

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,

15 20 Elution volume (ml)

'

25

'

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0.01 30

Common seal IL- l-like activity present in Superose 6 gel chromatography fractions. Seal leucocytes were stimulated with LPS at 1 gg/ml. Elution volumes of molecular weight markers: chymotrypsinogen A (25 kDa), recombinant human IL-1 (17-5 kDa) and ribonuclease A (13-7 kDa) are indicated.

Assay Specificity H u m a n IL-1 [3 antiserum was used to confirm assay specificity. Addition of IL-1 [3 antiserum did not affect the seal IL-l-like proliferation of D10 cells, although the concentration of antiserum added was sufficient to remove the proliferative response of recombinant IL-1 [3 (data not shown). It was possible, however, to inhibit IL-l-like proliferation with recombinant

258

D . P . K i n g e t al. 1.2

(a)

1.0

0.8 CD

9~ 0.6

~ o.4 0 0.2

0.0

i

10

1 0.1 Recombinant I L l a (IU/ml)

0.01

1.2 1.0 0.8-

0.6, O

0.40.20

Fig. 3.

10 100 Redprocaldilufion ofleucocytesupernate

"~'

i

1000

Inhibition of (a) recombinant human IL-1 ~ (NIBSC international standard), and (b) seal leucocytederived IL-l-like activity by recombinant IL-1 ra. Concentrations of IL-1 ra used were; ( 0 ) 0, (O) 2 ng/ml, (Ill) 20 ng/ml and ([-3)200 ng/ml. Points shown are mean + SD (n = 3 determinations).

IL-1 ra (Fig. 3). These experiments showed that over a dose range, IL-1 ra was equally efficient at inhibiting seal leucocyte and recombinant h u m a n ILl a-stimulated proliferation. The concentrations of LPS used in these experiments did not result in any significant proliferation of the D 10 cell line.

Leucocyte IL-l-like Activity in Seals

259

Discussion This study shows that isolated seal blood leucocytes produced an IL-l-like moiety with properties similar to those of the sequenced and characterized molecules found in man and rodents. A characterized murine T-cell-line was used to detect this IL-l-like activity. IL-l-like activity in cell-free leucocyte supernates was shown to increase over 48 h, suggesting that the activity was cell-derived and could not be attributed to extraneous stimuli. The D 10 cell-line used has been reported to respond to IL-1 derived from several species. Successful neutralization of IL-l-like activity with IL-1 ra confirmed that the proliferation measured was dependent upon the IL-1 receptor. As this inhibition occurs at the level of the cell line receptors, it is not surprising that the IL-1 ra used was found to be equipotent against seal and human IL-l-like molecules. The failure to neutralize this activity with anti-human IL- 1 [3 may be explained as follows. First, as IL- 1 exists in both and [3 forms (Cameron et al., 1986), it is unlikely that an anti-IL-l[3 would remove all biological activity. Second, the anti-human IL-113 did not crossreact with seal IL-1 [3. This suggests that immunogenic epitopes expressed by the protein are probably not phylogenetically well conserved. Gel chromatography showed that the molecular weight of this IL-l-like activity was approximately 17 kDa, which is similar to that described for both isoforms of the human protein (Cameron et al., 1986). Cats (Goitsuka et al., 1987), cattle (Canning and Neill, 1989) and horses (May et al., 1990) possess ILl molecules with molecular weights of 15-20, 17"8 and 17-18 kDa, respectively. It is likely that the IL-l-like activity determined in this study (with respect to quantity of protein present) was an underestimate. This is suggested by several factors, such as reduced mass activity of seal IL-1 and the presence of IL-1 binding molecules in the supernates (Borth and Luger, 1989). Measurement of IL-l-like activity from culture supernates will assist in the study of pinniped disease and may also shed some light on the immunotoxic effects of environmental pollutants. This is especially relevant in North Sea common seals, in which a distemper virus epizootic in 1988 was responsible for the death of approximately 16 000 animals (Osterhaus, 1989). Viruses of the Paramyxoviridae have been shown to alter IL-1 production from cells of the monocyte/macrophage lineage in dogs (Krakowka et al., 1987) and man (Leopardi et al., 1992). It is not known whether exposure of marine mammals to pollutants may impair their immune response to the phocid distemper virus. In conclusion, by means of the DG 10.G4 bioassay, it was possible to identify an IL-l-like molecule produced by isolated leucocytes of two species of seal. Some properties of this IL-l-like activity were consistent with those described for IL-1 in other mammalian species.

Acknowledgments The authors thank all the staff of the RSPCA Wildlife Unit, Norfolk, UK for providing blood samples. IL-1 ra used in this study was kindly provided by Dr A. Steinkasserer, MRC ImmunochemistryUnit, Oxford, UK. This work was supported by a Greenpeace Environmental Trust grant.

260

D.P. King et al. References

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Steppan, L. B. and Kerkliet, N. I. (1991). Influence of 2, 3, 7, 8-tetrachlorodibenzop-dioxin (TCDD) on the production of inflammatory cytokine mRNA by C57B 1/ 6 macrophages. Toxicologist, 11, 35-39. Sutter, T. R.,Guzman, K., Dold, K. M. and Greenlee, W. F. (1991). Targets for dioxin: genes for plasminogen activator inhibitor-2 and interleukin 1[3. Science, 254, 415-418. Vos, J. G., van Loveren, H., Wester, P. W. and Vethaak, A. D. (1988). The effects of environmental pollutants on the immune system. European Environment Review, 2, 1-7.

Received, December 23rd, 1994] Accepted, June 3rd, 1995 J