In vitrodegradation ofAeromonas salmonicidaandLimulus polyphemushemocyanin by Atlantic salmon,Salmo salarL. macrophages

Fish & Shellfish Immunology (1995) 5, 427–439

In vitro degradation of Aerom onas salm onicid a and Lim ulus polyphem us hemocyanin by Atlantic salmon, Salm o salar L. macrophages KLARA STENSVÅG*, JARL BØGWALD†‡, KARI STEIRO†

AND

TROND Ø. JØRGENSEN*

*The Norwegian College of Fishery Science, University of Tromsø, Norway, and †Norwegian Institute of Fisheries and Aquaculture, Tromsø, Norway (Received 24 January 1995, accepted in revised form 3 May 1995) Cultivated Atlantic salmon head kidney macrophages endocytosed and digested iodinated formaldehyde-fixed Aeromonas salmonicida antigens e#ectively. The degradation was time-dependent as demonstrated by increased acid soluble radioactivity obtained in macrophage media with time after the addition of antigens. Gel chromatography of the degraded material revealed a peak of radioactivity corresponding to molecular weights between 1000 and 6500 Da. In vitro degradation of Limulus polyphemus hemocyanin (LPH) adsorbed to mineral particles was less e$cient compared to the degradation of whole bacterial cells, whereas degradation of soluble LPH was negligible. There were no significant di#erences in the rate of processing at 4 (6), 12 and 18) C. Inhibition studies using ammonium chloride, a substance that raises the pH of endosomes and lysosomes, showed decreased intracellular degradation of A. salmonicida in a dose-dependent manner. Monensin, a substance reported to block receptor-mediated phagocytosis, very e#ectively reduced the extent of processing whereas leupeptin and pepstatin, specific inhibitors of the major classes of cysteine and aspartic proteinases respectively, only slightly a#ected the processing. ? 1995 Academic Press Limited Key words:

Atlantic salmon macrophages, soluble and particulate antigendegradation.

I. Introduction In mammals, antigen processing and presentation by macrophages are among the central events in the stimulation of immune responses to thymus dependent antigens, and indications of similar mechanisms occurring in teleost fish (the channel catfish) were recently reported by Vallejo et al. (1990). A major role of the macrophage is to serve as part of the defence system of the organism, and in this connection its most important function is probably to ingest and kill microorganisms. The microorganism is encapsulated in a phagocytic vacuole, the so-called phagosome (Diment & Stahl, 1985; Diment et al., 1988). The internal pH of the phagosome appears to be between 5 and 6, somewhat less acidic than lysosomes (Mellman et al., 1986). Antigen degradation within endosomes is normally increased after fusion with lysosomes ‡Please address all correspondence to: Jarl Bøgwald, Norwegian Institute of Fisheries and Aquaculture, Postboks 2511, N-9002 Tromsø, Norway. 427 1050–4648/95/060427+13 $12.00/0

? 1995 Academic Press Limited

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containing a collection of hydrolytic enzymes. These enzymes degrade macromolecules to simple components which are either excreted from or utilised by the macrophage. An important question in this context is whether inhibition of lysosomal enzymes with acid pH-optima will be of any consequence for the outcome of the processing. This question may be answered by using inhibitors like monensin, a substance reported to block receptor-mediated phagocytosis and thereby antigen processing (Berg et al., 1983). Also, the various proteinase inhibitors like leupeptin and pepstatin are known to block antigen processing both in mammals (Levine & Chain, 1991) and in fish (Vallejo et al., 1990). Leupeptin and pepstatin have generally been used to inhibit cysteine proteinases and aspartic proteinases, important hydrolytic enzymes in mammalian macrophages (Jupp et al., 1988; Levine & Chain, 1991), respectively. Ammonium chloride, a lysosomotropic agent that raises the pH of endosomes and lysosomes (Ziegler & Unanue, 1982) inhibits enzymes with acid pH-optima. Monensin, which is a lipophilic compound, blocks the Na + /K + pump across membranes and also may interfere with receptor mediated phagocytosis (Tartako#, 1983). In the present study we investigated in vitro processing in Atlantic salmon head kidney macrophages of the particulate antigens Aeromonas salmonicida and Limulus polyphemus hemocyanin (the latter coated onto microsilica particles) (Rokstad, 1994). The degradation was assayed as acid soluble peptides and the in vitro temperature and the e#ect of various cellular inhibitors on this degradation was also investigated.

II. Materials and Methods SALMON MACROPHAGES

Atlantic salmon, Salmo salar L., weighing 0·5–1 kg, were maintained in circular tanks in a mixture of fresh and sea water (salinity 1·5%) at 10–12) C and fed commercial salmon feed pellets (Ewos, Skårer, Norway). Fish were killed by a blow to the head and the head kidneys were removed and transferred to petri dishes containing a stainless steel mesh and cold medium (Leibovitz L-15, Flow Laboratories), adjusted to 360–380 mOsm kg "1 (isotonic) and supplemented with 15 mM ca#eine as anticoagulant. The tissue was minced and gently squeezed through the mesh and dispersed by a pipette to obtain a homogenous cell suspension. The cell suspension (1·5–2 ml) was placed on top of a 54% Percoll solution (3 ml) (Pharmacia, Uppsala, Sweden) and centrifuged at 500#g for 15 min to isolate the leucocyte fraction. The cells were washed once in 30 ml of Leibovitz L-15 supplemented with glucose (0·33 g "1) and HEPES (pH 7·3, 3·57 g "1) (denoted L-15 medium) and seeded in 24 well tissue culture plates (Nunc, Denmark, 2#106 cells well "1), 1 ml medium in each well. Non-adherent cells were carefully removed by washing the cultures three times with L-15 medium, 1 h after seeding and incubation at 18) C. The cells (3–5#105 per well) were cultured at 18) C in L-15 medium, supplemented with SSR 2 (‘synthetic serum replacement’, Medi-Cult AS, Copenhagen, Denmark), or in L-15 medium supplemented with 5% foetal calf serum (FCS) in air.

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ANTIGENS

Aeromonas salmonicida subspecies salmonicida (FT 1940) were grown in brain heart infusion medium (BHI, 3·7% Difco) at 12) C on an orbital shaker. Forty-eight hours after inoculation, the bacteria were killed in 0·5% formalin and kept for at least 24 h before washing twice in water to remove medium components. Limulus polyphemus hemocyanin (LPH) was purchased from Sigma. RADIOLABELLING OF ANTIGENS

Bacteria were radioiodinated with Na125I in the presence of 1,3,4,6tetrachloro-3,6-diphenyl-glycoluril (IODO-GEN, Pierce Chemical Co, Oud Beijerland, The Netherlands) by the method of Markwell and Fox (1978). In short, 0·3 ml of a bacterial suspension (A600 =4·0) was incubated for 5–10 min on ice with 40 MBq carrier free Na125I (Energiteknikk, Oslo, Norway) in a glass tube precoated with iodo-gen. The radioiodinated bacteria were washed twice by centrifugation in water containing 30 mM ‘cold’ NaI (11 000 g, 10 min) and thereafter 4 times in L-15 medium until the supernatant contained less than 1% of the radioactivity associated with the bacteria (Gamma counter, LKB Wallac, Turku, Finland). The specific activity obtained was in the order of 1–5#106 cpm per 106 bacteria. LPH (1·0 mg in 0·3 ml 0·1 M Tris-HCl pH 8·0) was mixed with 40 MBq carrier-free Na125I in an iodogen-coated glass tube as above and incubated for 10 min on ice. The reaction mixture was transferred to a dialysis tubing and dialyzed overnight against 30 mM ‘cold’ NaI in 20 mM Tris bu#er, pH 8·0. The radioiodinated LPH was finally dialyzed overnight against 20 mM Tris bu#er, pH 8·0. The specific activity obtained was 1·8#109 cpm mg "1 protein. LIMULUS POLYPHEMUS HEMOCYANIN ADSORBED TO MINERAL PARTICLES

Radioiodinated LPH in 20 mM Tris bu#er, pH 8·0, was mixed with mineral particles (Microsilica, Elkem, Fiskaa Verk, Kristiansand, Norway) and incubated overnight at 4) C with gentle agitation. Unadsorbed material was removed by centrifugation and particles were resuspended in 20 mM Tris bu#er, pH 8·0 (Bøgwald et al., 1992). The washing procedure was repeated five times until the amount of radioactivity in the supernatant was less than 1% of the radioactivity associated with the particles. Adsorbed protein was determined by measuring the radioactivity associated with the particles and by protein measurement by the method of Smith et al. (1985). MACROPHAGE-DEGRADATION OF RADIOIODINATED BACTERIAL ANTIGENS

Bacteria (5#106 per well) were added to macrophage cultures 20 h after seeding. Before adding the antigens, the macrophages were washed once and 1·0 ml fresh medium (L-15 medium supplemented with SSR-2 or 5% FCS) was added. The culture plates were centrifuged at 160 g for 10 min at 18) C to obtain a close and immediate contact between the macrophages and the bacteria. Immediately thereafter, the ‘zero’ timepoint of cultures were collected. Further harvesting of macrophage cultures was performed as indicated

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in the Results section. Fixed macrophages (70% ethanol for 10 min) were used as controls. On harvesting, the medium was carefully removed, transferred to Eppendorf vials and frozen ("20) C). The cells were washed once in L-15 medium and solubilised in 0·1% Triton X-100 (Sigma) in phosphate bu#ered saline, pH 7·3 (PBS) for 10 min. Remnants of cells sticking to the plastic surface were scraped o# by a rubber policeman, and the whole transferred to Eppendorf vials and frozen. Macrophage media and lysates were analysed for radioactivity in the gamma counter. High molecular weight or undegraded protein antigens were precipitated by trichloroacetic acid (TCA, 10% final concentration), centrifuged at 11 000 g for 10 min and the supernatants were measured for radioactivity. The di#erence in radioactivity before and after precipitation is a measure of the proportion of degradation.

MACROPHAGE-DEGRADATION OF RADIOIODINATED LIMULUS POLYPHEMUS HEMOCYANIN (LPH)

The soluble and microsilica adsorbed LPH (0·1 ìg per well) were added to macrophage cultures 20 h after seeding. The cultures were maintained in L-15 medium supplemented with 5% FCS in air. At various timepoints after the addition of particles, culture supernatants and cell-lysates were collected and precipitated in 10% TCA (final concentration). After centrifugation, the supernatants were analysed for radioactivity.

INHIBITORS

Inhibitors were added to the macrophage cultures immediately before the addition of antigens and antigen degradation was measured after 24 h. Monensin (Sigma) was dissolved in methanol (100 mg ml "1) and diluted in culture medium to 1 and 5 ìM immediately prior to use. Ammonium chloride was dissolved in H2O to 1 M and diluted in L-15 medium to final concentrations of 5, 25 and 50 mM. The cysteine proteinase inhibitor, leupeptin (Acetyl-Leu-Leu-Arg-al, Sigma) was dissolved in L-15 medium to 10 mM and added to macrophage cultures in final concentrations of 10, 50, 100, 250 and 500 ìM. The aspartic proteinase inhibitor pepstatin (Sigma) was dissolved in dimethyl sulfoxide (DMSO) to 10 mM and diluted to final concentrations of 1, 10, 50, 100, 250 and 500 ìM in L-15 medium immediately prior to use.

CELL-FREE PROCESSING OF A. SALMONICIDA ANTIGENS

Head kidney macrophages were incubated with uncoated microsilica and cultured in air in L-15 medium supplemented with SSR 2. At various timepoints, medium was collected (conditioned medium) and frozen until used. The conditioned media (cell-free) were incubated with radioiodinated A. salmonicida bacteria, precipitated with TCA and the amount of acid soluble radioactivity was measured.

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Fig. 1. Time-dependent degradation of Aeromonas salmonicida antigens by cultured head kidney macrophages from Atlantic salmon at 18) C. The results are presented as the mean percentage of TCA soluble radioactivity relative to added radioiodinated antigen to each culture. The data represent duplicate cell cultures from each fish and two separate fish. (a) Cell-associated TCA soluble radioactivity (macrophage lysates). (b) TCA soluble radioactivity from macrophage media. /, Live cells; ., fixed cells. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)

HPLC of processed A. salmonicida antigens was performed by gel filtration on a 7·8#300 mm Protein Pak 60 (Waters) column. The column was equilibrated with 20 mM Tris-HCl bu#er, pH 7·4. Macrophage medium supernatants (before TCA precipitation) were diluted in equilibration bu#er (1:2) before application on the column. The 280 nm spectra were recorded and the amount of radioactive antigens in each fraction was determined. Myoglobin (Biorad, MW 17000), aprotinin (Sigma, MW 6500) and vitamin B12 (Biorad, MW 1350) were used for calibration of the column. III. Results TIME DEPENDENT DEGRADATION OF AEROMONAS SALMONICIDA ANTIGENS AT 18) C

The time course of macrophage-mediated degradation of A. salmonicida antigens is shown in Fig. 1. The amount of acid soluble radioactive label as a % of total radioactivity in macrophage lysates is shown in Fig. 1(a). Maximal acid soluble radioactivity was found 4–6 h after the addition of antigens. Fig. 1(b) shows the amount of radioactivity (% of total radioactivity added) in supernatants of TCA precipitated macrophage media. The acid soluble radioactivity increased with increasing incubation time and reached 80% 24 h after the addition of bacteria. The background levels represented by radioactivity in TCA soluble material taken from cell cultures prefixed with ethanol,

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Fig. 2. Time-dependent degradation of soluble and particulate Limulus polyphemus hemocyanin in Atlantic salmon head kidney macrophages at 18) C. Results are the mean of radioactivity (cpm) in TCA soluble fractions of macrophage media from four culture wells of each fish and three separate fish. /, Particularized LPH, live macrophages; ., particularized LPH, fixed macrophages; 4, soluble LPH, live macrophages; 0, soluble LPH, fixed macrophages.

indicated that less than 10% of the radioactive antigen had degraded spontaneously over the time course of the experiment.

DEGRADATION OF SOLUBLE VERSUS PARTICULATE ANTIGENS

As shown in Fig. 2 there was an e#ective degradation of particulate LPH in macrophage cultures as demonstrated by high amounts of TCA soluble radioactivity in the culture medium. Soluble LPH was not degraded or taken in by salmon macrophages in detectable amounts during the 192 h (8 days) incubation period (Fig. 2).

INHIBITION OF ANTIGEN DEGRADATION

Inhibitors were added to macrophage cultures immediately before the addition of A. salmonicida. After harvesting, the macrophage media and cell lysate were mixed with TCA to a final concentration of 10% and centrifuged. The supernatants were analysed for radioactivity as a measure of the extent of degradation of the antigens. Incubations of macrophages with leupeptin or pepstatin caused only slight inhibition of the degradation of bacterial antigens in head kidney macrophages [only leupeptin is shown, Fig. 3(a)]. On the other hand ammonium chloride [Fig. 3(b)] and monensin [Fig. 3(c)] showed a strict dose dependent inhibition of antigen degradation in parallel macrophage cultures. The capacity of macrophages to degrade particulate LPH (adsorbed to microsilica) after addition of inhibitors followed the same pattern as with A. salmonicida antigens (data not shown).

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Fig. 3. Inhibition of antigen processing of Aeromonas salmonicida antigens in cultured head kidney macrophages from Atlantic salmon at 18) C. The results are presented as the mean percentage of TCA soluble radioactivity in inhibited relative to non-inhibited (control) duplicate culture wells from each of two fish. Inhibitors were added immediately before the addition of antigens. (a) Leupeptin; (b) Ammonium chloride; (c) Monensin. EFFECT OF TEMPERATURE ON DEGRADATION OF A. SALMONICIDA AND LPH ANTIGENS

Fig. 4(a) shows the time-dependent degradation of A. salmonicida antigens (whole cell bacteria) in salmon macrophages incubated at 6, 12 and 18) C. The release of TCA soluble material in the supernatant appeared to occur at a slightly faster rate at 18) C, though not significantly di#erent from 6 and 12) C. Fig. 4(b) shows a similar experiment using Limulus polyphemus hemocyanin adsorbed to microsilica. There were no significant di#erences in the processing capability of macrophages cultured at 4, 12 and 18) C. Three days after the addition of antigens the degradation (amount of TCA soluble material) had reached a plateau of about 60%. CELL-FREE PROCESSING OF A. SALMONICIDA ANTIGENS

Macrophages are able to secrete a variety of lysosomal enzymes (Nathan, 1987). This means that macrophages might be able to degrade antigens extracellularly. To investigate the involvement of extracellular degradation by macrophages, cell-free supernatants (conditioned media) were collected and subsequently incubated for 24 h with radioiodinated bacteria. Our results show that the increased TCA-soluble radioactivity was negligible (data not shown), and confirm the view of a strict intracellular degradation in fish macrophages (Vallejo et al., 1990; 1991a). HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)

Macrophage medium, obtained from cultures after 24 h incubation with radioiodinated formalin-fixed A. salmonicida whole cell bacteria (before TCA precipitation), was applied to a gel filtration column. Three peaks containing radioactive material emerged (Fig. 5). The first peak represents material eluted with the void volume as determined with Blue dextran. The second peak represents a high-molecular weight peptide fraction, free radioactive

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Fig. 4. Time-dependent degradation of radioiodinated antigens by cultured head kidney macrophages from Atlantic salmon at various temperatures (4/6, 12, 18) C) expressed as radioactivity in TCA soluble fraction as % of total radioactivity added. (a) Aeromonas salmonicida bacteria. Results are the mean of six culture wells. -, Live cells 6) C; ,, fixed cells 6) C; /, live cells 12) C; ., fixed cells 12) C; 4, live cells 18) C; 0, fixed cells 18) C. (b) Limulus polyphemus hemocyanin adsorbed to microsilica. Results are the mean of duplicate culture wells (3 individual fish). -, Live cells 4) C; ,, fixed cells 4) C; other symbols as for (a).

iodide and/or free radiolabelled tyrosine. The third peak is exclusively obtained from media sampled from live macrophages (and not with glutaraldehyde/ethanol fixed macrophages or zero time cultures). The third peak corresponds to peptides with a molecular weight between 1000 Da (total volume of the column) and 6500 Da as determined with aprotinine and vitamin B12 as standards. Little or no correlation of radioactive peaks and absorbance at 280 nm was observed (data not shown). IV. Discussion Correlations between antigen uptake and degradation by fish macrophages and subsequent induction of antigen-specific immune responses have only

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Fig. 5. High Performance Liquid Chromatography (HPLC) on Protein Pac 60 silica column of macrophage media after degradation of radioiodinated Aeromonas salmonicida bacteria (24 h). Media were sampled from live (live phagocytes, ——) or ethanol fixed macrophage cultures (fixed phagocytes, · · ·) obtained from two parallel culture wells from each of two individual fish, and applied to the column (before TCA precipitation) and after dilution in equilibration bu#er (1:2). Results are expressed as radioactivity (cpm) in each fraction sampled from the column.

been demonstrated in channel catfish (Vallejo et al., 1990; Vallejo et al., 1991a, 1991b). In these experiments exogenous protein antigens were taken up by peripheral blood monocytes and subjected to intracellular proteolysis. The peptide fragments formed were detected as TCA soluble material or by electrophoresis. In the present work we used head kidney macrophages from Atlantic salmon and studied the degradation of Limulus polyphemus hemocyanin (LPH) and the fish pathogen Aeromonas salmonicida. We observed a time dependent degradation of the antigens as determined by increased amounts of TCA soluble radioactive material. The absolute amounts of acid soluble antigens in the culture medium were strikingly much higher than in the cell lysate. This fact indicates that the antigens were phagocytosed, degraded and e#ectively released from the cells most probably by exocytosis (Andrew et al., 1985). The appearance of acid soluble antigens in both cell lysates and cell media supports this view as the relative amount of radioactivity was at its highest at an earlier timepoint in cell lysate (peak at 4–6 h) than in the medium. The macrophage-degraded bacterial antigens obtained in media were analysed by HPLC gel filtration chromatography. The results obtained indicate that the processed antigens have a molecular weight between 1000 and 6500 Da. To further study the mechanism of degradation, the inhibitors monensin, ammonium chloride and specific proteinase inhibitors were used. Monensin is a lipophilic substance (ionophore) that may influence intracellular transport and inhibition of receptor-mediated endocytosis (Tartako#, 1983; Vallejo et al.,

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1990; 1992b). Our results show inhibition of antigen degradation in a dosedependent manner. Again, this indicates that antigen uptake and/or intracellular transport is a prerequisite for antigen processing. Also, the lysosomotropic agent, ammonium chloride, inhibited the process of antigen degradation. Ammonium chloride does not inhibit phagocytosis per se (Vallejo et al., 1992b; Espelid & Jørgensen, 1992), but raises the pH of endosomes and lysosomes and thus inhibits enzymes with acid pH-optima. Although nonlysosomal forms of cathepsin D and some of the cysteine proteinases have recently been described (Diment et al., 1988; Guagliardi et al., 1990), the strong inhibitory e#ect provided by ammonium chloride makes it unlikely that extra-lysosomal degradation takes place in Atlantic salmon macrophages. Certain classes of endopeptidase inhibitors have been shown to block the processing of protein antigens, i.e. those specific for cysteine and aspartic proteinases (Levine & Chain, 1991). These inhibitors are frequently used in high concentrations in cell culture, and are assumed to enter the processing compartments by endocytosis. In our hands, leupeptin and pepstatin, inhibitors of cysteine and aspartic proteinases respectively, only showed a slight inhibitory e#ect on the protein degradation in salmon macrophages. The non-inhibitory e#ect of leupeptin is contradictory to what is seen using channel catfish peripheral blood monocytes (Vallejo et al., 1990). Although the latter authors did not assay degradation per se, but used an antigen processing dependent proliferation assay in their study, this discrepancy is not solved. Since inhibitors, like pepstatin, are often only slightly soluble in aqueous media and must be added in small aliquots in organic solvents it is di$cult to estimate the actual concentration of this inhibitor in cell cultures. In addition, it is even more di$cult to estimate the intracellular concentrations of the inhibitors in the compartments involved in antigen processing. The Atlantic salmon used in these experiments were adapted to a temperature of 10–12) C whereas the temperature used for culturing head kidney macrophages from this species is normally 17–18) C. Thus, a more adapted temperature for culturing the cells would be 10–12) C. Antigen processing was therefore compared at 18 and 12) C. In addition, antigen processing at 4) C was used to investigate the e$ciency of antigen degradation at a low temperature normally experienced by Atlantic salmon in the winter season. The rate of antigen degradation in salmon macrophages was very little influenced by temperatures in the range 4–18) C. The cells were able to process antigen at 4–6) C, even though the fish were adapted to 10–12) C. Similar studies done with channel catfish monocytes have also shown that the antigen uptake and degradation was e#ective even at low temperatures (below 18) C) previously shown to be nonpermissive for primary catfish T cell reponses (Miller & Clem, 1984; Bly & Clem, 1991; Vallejo et al., 1992a). As also expected for channel catfish cells, at extreme low temperatures for this species (4) C), monocytes were not able to endocytose antigen (Vallejo et al., 1992a). Dannevig & Berg (1985) studied in vivo uptake of formaldehyde treated human serum albumin in the char (Salmo alpinus L.) and in vitro degradation by measuring increase in acid soluble radioactivity in a cell suspension isolated from the head kidney. The authors found a very rapid in vitro degradation of in vivo endocytosed ligand. When cells were harvested 75 min after intravenous administration

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and further incubated in vitro at 15) C for 90 min, 60–70% of the endocytosed ligand appeared as acid soluble products. In their experimental design the in vitro degradation was clearly temperature dependent. At 0) C about 20% of the ligand was acid soluble compared to 50–60% and 80% at 7) C and 20) C respectively. In this context it would be interesting to investigate whether macrophages, isolated from salmon adapted to very low temperatures (0) C), would have gained the capacity to e#ectively process antigens even at this low in vitro temperature. In mammals, it has been shown that particulate antigens (carrier adsorbed) are more e#ective than soluble antigens in eliciting an immune response after intraperitoneal injection (Gallily & Garvey, 1968; Altman & Dixon, 1989). Several kinds of particles have been used as carriers including mineral particles (Hennessen, 1965; Gallily & Garvey, 1968), latex particles (Litwin & Singer, 1965), polymethyl methacrylate particles (Kreuter et al., 1988) and liposomes (Allison & Gregoriades, 1974). Both LPH adsorbed to mineral particles (miocrosilica) and whole A. salmonicida cells demonstrated a high susceptibility to processing. In contrast to LPH adsorbed to microsilica, soluble LPH was only slightly degraded for the period of time tested. These results are in agreement with observations by Dannevig et al. (1990; 1994). These authors studied both in vivo and in vitro uptake of soluble and colloidal substances. They demonstrated in vitro uptake of colloidal human serum albumin by head kidney macrophages whereas uptake of soluble ovalbumin and dinitrophenylated human serum albumin could not be detected. Interestingly, they found in vivo uptake of soluble ligands (dinitrophenyl-human serum albumin) in both sinusoidal endothelial cells and macrophages in the head kidney. Also, Vallejo et al. (1990) using channel catfish purified blood monocytes and autoradiography, demonstrated uptake and degradation of soluble 125I-hemocyanin and monocyte dependent processing/presentation and stimulation of T cells by the same antigen. Although their experimental situation di#ered from the one we have used (Atlantic salmon, head kidney macrophages etc.) this might reflect species variations of cells of the mononuclear phagocyte system. This work was supported by the Norwegian Research Council, grant no. 1401– 2800.098.

References Allison, A. C. & Gregoriadis, G. (1974). Liposomes as immunological adjuvants. Nature 252, 252. Altman, A. & Dixon, F. J. (1989). Immunomodifiers in vaccines. In Vaccine Biotechnology. Advances in Veterinary Science and Comparative Medicine (J. L. Bittle & F. L. Murphy, eds) pp. 301–343. San Diego, CA: Academic Press. Andrew, P. W., Jackett, P. S. & Lowrie, D. B. (1985). Killing and degradation of microorganisms by macrophages. In Mononuclear Phagocytes: Physiology and Pathology (R. T. Dean & W. Jessup, eds) pp. 311–335. Amsterdam: Elsevier Science Publishers B.V. Berg, T., Blomho#, R., Næss, L., Tolleshaug, H. & Drevon, C. A. (1983). Monensin inhibits receptor-mediated endocytosis of asialoglycoproteins in isolated rat hepatocytes. Experimental Cell Research 148, 319–330.

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