- Email: [email protected]
Demonstration of an antibody response of the anterior kidney following intestinal administration of a soluble protein antigen in trout
~
Comp.Biochem.Physiol.Vol. 109B,No. 2/3, pp. 499-509, 1994
Copyright© 1994ElsevierScienceLtd PrintedinGreatBritain.All rightsreserved
Pergamon
0305-0491/94$7.00+ 0.00
03o5-0491(94)00063-s
Demonstration of an antibody response of the anterior kidney following intestinal administration of a soluble protein antigen in trout Dominique Dorin, Marie-France Sire and Jean-Marie Vernier Laboratoire de PhysiologieCellulaireet M6tabolique des Poissons, Bat. 447, Universit6Paris XI, 91405 Orsay Cedex, France The humoral antibody response following anal intubation of a soluble antigenic protein to trout was investigated. The transfer of human immunoglobulin G (IgGh) to the plasma was demonstrated by ELISA assays. The participation of the anterior kidney in plasma clearance of the antigen was shown by an immunofluorescence study. The anterior kidney displayed a proliferation of specific B lymphocytes and differentiation into plasma cells producing anti-IgGh IgM. The peak of plasma specific antibody concentration occurred 30 days after intubation and a second intubation led to another peak 20 days later, whose amplitude was close to that of the primary response. Key words: Anterior kidney; Antibody response; Soluble protein antigen; Trout. Comp. Biochem. Physiol. 109B, 499-509, 1994.
Introduction A specialized region in the gut of the teleost fish, the posterior gut, is responsible for the massive absorption of proteins which then undergo intracellular lysis. A portion of the protein molecules escapes intracellular degradation, however, and reaches the intercellular space as well as the interstitial space of the lamina propria (Sire and Vernier, 1992). The demonstration of a mucosal (=local) immune response after the oral administration of a soluble antigenic protein was obtained in trout (Georgopoulou and Vernier, 1986) with human immunoglobulin G (IgGh) and subsequently confirmed in the carp (Rombout and van den Berg, 1989) with ferritin.
Again using IgGh, the aim of the present work was to describe sequentially the uptake of the antigen from the posterior gut of the trout, the appearance of the IgGh in the plasma, the localization of IgGh in the anterior kidney, and the subsequent antibody response of the anterior kidney to the IgGh as a contribution to the systemic immune response. The analysis of the contribution of the anterior kidney (pronephros) was chosen since the organ has lost its secretory function and is believed to be the phylogenetic analogue of bone marrow and/ or lymph glands in the other vertebrates.
Materials and Methods
Correspondence to: J.-M. Vernier, Laboratoire de
Animals
Physiologic Cellulaire et M6tabolique des Poissons, Bat. 447, Universit6 Paris XI, 91405 Orsay Cedex, France. Tel. 1-69-41-79-33; Fax 1-69-85-34-59. Received 30 October 1993; accepted 24 April 1994.
Rainbow trout (Oncorhynchus mykiss) were reared in running water at constant temperature (12 + I°C) and fed with Aqua
499
500
Dominique Dorin et al.
14 (Aqualim, Nersac, France). Animals, weighing 250-300g, were fasted 5 days before the experiment. Anal intubation, tissue and blood sampling were operated on anesthetized animals (0.3 ml of phenoxy2-ethanol/water). The antigenic protein, IgGh (Institut Pasteur, Paris, France) dissolved in phosphate buffer saline (PBS) or the buffer alone (control animals) was introduced directly by anal intubation, using a syringe with a soft flexible tube (0.2 mm diameter) attached to its extremity. The animals were distributed in six groups: Group A: 10mglgGh/1 ml PBS (n = 18). Group B: 10 mg IgGh/1 ml PBS (n =6). Group C: no anal intubation (n = 12). Group D: 30 mg IgGh/1 ml PBS (n = 6). Three animals were killed after one week and the remaining three again received a booster of 10 mg of IgGh in 1 ml of PBS, 90 days after the first administration. Group E: 30mglgGh/1 ml PBS (n = 5). Group F: 1 ml PBS (n = 35).
Antibodies Rabbit anti-IgGh (Sigma), goat antirabbit IgG labeled with FITC (Sigma), goat anti-trout immunoglobulin M (IgM) (Kirkegaard and Perry Laboratories), rabbit anti-goat IgG labeled with fluorescein isothiocyanate (FITC) (Sigma), IgGh labeled with FITC (Institut Pasteur, Paris, France), goat anti-IgGh labeled with peroxidase (Sigma), and goat anti-trout IgM labeled with peroxidase (Kirkegaard and Perry Laboratories) were used. No crossed reaction with IgGh could be detected with goat anti-trout IgM, rabbit anti-goat IgG and goat anti-rabbit IgG.
Tissue localization of lgGh, IgM and specific IgM anti-IgGh Fragments of the posterior gut and the anterior kidney were removed and fixed in 4% paraformaldehyde in 0.1 M PBS (pH 7.4), dehydrated and embedded in paraffin. 1. Localization of lgGh and IgM. Animals of group A were used and the specimens of tissues were removed 1, 4, 6, 16, 24 hr and 3 days after administration of IgGh. Six micrometer thick sections were incubated with 1% bovine serum albumin (BSA) in PBS for 30 min at 20°C.
IgGh: after 1 hr incubation with rabbit anti-IgGh (1/100 in PBS), sections were washed with PBS/BSA for 10 min and with PBS for 2 × 10min. Finally they were incubated for 1 hr with goat anti-rabbit IgG labeled with FITC (1/20 in PBS) and washed 3 x 10 min with PBS. IgM: The immunoglobulin was visualized using goat anti-trout IgM (1/100 in PBS) and by a rabbit anti-goat IgG labeled with FITC (1/60 in PBS). 2. Localization of lgM anti-IgGh. Animals of group B were used and fragments of anterior kidney were removed 14 days after the administration of IgGh. Six micrometer thick sections were incubated with 1% BSA in PBS for 30 min at 20°C and then for 1 h with IgGh labeled with FITC (1/25 in PBS). Sections were washed with PBS for 3 × 10 min. The following controls for aspecific reactions were performed for all immunocytochemical reactions: (i) non-immunized animals (group F), (ii) the primary antibodies were omitted and sections incubated with pre-immune or non-immune serum or with PBS alone.
Culture of anterior kidney lymphocytes The anterior kidney was aseptically removed and disrupted by passing through a steel screen (80 mesh). The cell suspension was deposited on Histopaque (Sigma) and centrifuged at 1000g for 20 min, at 15°C. The viability of leucocytes was determined by Trypan Blue (0.4%) dye exclusion. Lymphocytes were adjusted at a concentration of 3 x 106 or 4 × 10 6 cells/well and cultivated with 3 ml (six-well culture plates, Corning) or 400/~1 (24-well culture plates, Corning) of culture medium (RPMI 1640, Sigma, containing 25 mM HEPES, 1% streptomycin-penicillin (120 mg/ml; 10 000 U) and 2 mM L-glutamine, Sigma) at 15°C in a humid atmosphere ( O 2 / N 2 / C O 2 , 20:75: 5). The animals of group C were utilized to optimize the culture medium conditions and to determine if the direct action of the antigenic protein (IgGh) or of the mitogenic agent for B cells, the E. coli lipopolysaccharide (LPS), can induce a polyclonal reaction of the cultivated cells. Twenty-fourwell culture plates were used with 400 #1 culture medium/well. (a) FCS 10/24: 10%
Trout immune response
501
fetal calf serum, 50/~1 of medium added (Flow Laboratories). A standard range every 24hr. (b) FCS 10/24/LPS: medium of IgGh (0, 25, 50, 75 and 100 ng/ml PBS) with 80#tool LPS/ml and 50/~mol fl- was used to calculate IgGh serum concenmercaptoethanol/ml. (c) FCS/10/72:50/~1 trations. The sensitivity threshold of the of medium added every 72 hr. (d) FCS/ method was 3 ng/ml. 10/72/IgGh: 40/~g IgGh/ml of medium. (e) TS 2/24: 2% inactivated trout serum, 50 #1 ELISA assay of total IgM Microplates were coated with IgGh medium added every 24 hr. (f) TS 2/24/LPS: medium with LPS and fl-mercaptoethanol (10 ~g/ml PBS) for 2 hr at 37°C and overnight at 4°C. After washing and saturating (see b). Cells were enumerated in four wells on with 3% BSA, dilutions of culture superdays 1, 3, 5, 7 (a, b, e, f) or on days 3, 6, natants (group C) were incubated for 2 hr 9, 12, 15 (c and d) and supematants were at 37°C. The final incubation was with a stored at - 2 0 ° C for ELISA assays of total goat anti-trout IgM labeled with peroxidase (1/100 in PBS). Subsequent steps were IgM or for specific IgM anti-IgGh. Three animals of group D were killed 1 as described above. A standard range of week after antigen administration. Anterior IgM (three at 10 ng/ml) was used to calcukidney cells (3 x 106) in 3ml of culture late the IgM concentration. The sensitivity medium were deposited in each well of threshold of the method was 3 ng/ml. six-well culture plates and incubated for 8 days. Cells in four wells were enumerated ELISA assay of IgM anti-IgGh Blood samples from the remaining three on days 2, 4, 6 and 8 and the culture supernatants were stored at - 2 0 ° C for animals of group D were removed regularly ELISA assays of specific IgM anti-IgGh. (10, 20, 30, 45, 75, 110, 120 and 150 days) and treated as described above. The serum ELISA assay of serum IgGh was recovered and stored at - 2 0 ° C before Blood was collected from each exper- assaying IgM. Culture supernatants (group imental animal in group E and from each C, group D) were also tested for IgM control animal by cardiac puncture 0, 15, anti-IgGh. 30 min, 1, 3, 6 and 9 hr after administering Microplates were coated with IgGh the protein. Each blood sample was allowed (10 pg/ml PBS). After washing and saturatto clot for 1 hr at 20°C, then overnight ing with 3% BSA, dilutions of culture at 4°C and was individually centrifuged supernatants or sera were incubated for 2 hr at 3000g for 15min, at 4°C and serum at 37°C. The final incubation was with a was recovered and stored at -20°C. Flat goat anti-trout IgM labeled with peroxidase bottom, 96-well ELISA plates (NUNC) (1/100 in PBS). Subsequent steps were as were coated with 100#l PBS containing described above. l0 #g/ml of anti-IgGh raised in goats (2 hr Two types of controls were used, or at 37°C, overnight at 4°C). Between each sera, or culture supernatants from nonstep, the plates were washed five times with immunized animals, to determine backPBS containing 0.1% Tween 20. Non- ground, and assay without serum were the specific reactions were blocked using 3% negative controls. BSA in PBS for 1 hr at 37°C. They were then incubated with different dilutions of serum samples for 2 hr at 37°C and with Results a goat anti-IgGh labeled with peroxidase (1/5000) for 1.5 hr at 37°C. The enzymatic Transfer of intubated antigen (IgGh) to the reaction was allowed to develop for 20 rain anterior kidney using 100 #l/well of the substrate o-phenyl1. Kinetics of appearance of IgGh in the ene diamine [OPD/H2 02 (2 mg OPD in 5 ml serum (Fig. 1). Immunoenzymatic results of 0.1 M citrate buffer, pH 5.5 and 30/~l of obtained with ELISA showed that IgGh 30% H202) ] in darkness. Colour develop- was transferred to the plasma following the ment was stopped by adding 50/~l of 2N anal intubation of antigen (group E). An HE SO 4. The plates were read at 492 nm initial low amplitude peak was observed using a Titertek Multiskan Plus plate reader 15 min after administration, followed by a
DominiqueDorin et al.
502
in particular infranuclear, and was more intense in the intercellular space and the lamina propria (Fig. 2). Cellular localization could also be detected in the lamina. In controls receiving only PBS (group F), no specific fluorescence was observed (see Fig. 3 as an example, 1 hr after intestinal administration of buffer). j
O
~
O
0 ~'? ~ . . . . . . 1 2 3 4 5 B 7 B q Time (hours]
Fig. 1. Kinetics of appearance of IgOh in the plasma of trout after anal administration of IgGh (30 mg/ 1 ml) (O). Control animals: (O). Each sample corresponds to five animals. Vertical bars: standard error of the mean. second wave of transfer with a peak at 3 hr which extended beyond the 9 hr point. The antigen thus passed through the intestinal epithelium.
3. Clearance of plasma IgGh by the anterior kidney (Figs 4 and 5). Fish receiving the antigen (group A) exhibited specific cellular fluorescence observed first at 6 hr sample time, with the number of cells involved increasing up to 24 hr (Fig. 4). At all time points (1, 4, 6, 16 and 24hr), some anterior kidney cells exhibited an orange, natural non-specific fluorescence which persisted alone when the primary antibody had been omitted (Fig. 5) or which was observed in controls (group F) at all times.
Immune response to IgGh transfer 2. Transepithelial transfer of IgGh (Figs 1. Stimulation of lymphocytes and IgM 2 and 3). Immunofluorescence techniques production, lgM production by the anterior showed that starting 1 hr after anal intub- kidney. The indirect immunofluorescence ation (group A) there was very intense specific fluorescence in the vacuolar system of the apical hyaloplasm in posterior segment intestinal cells (Fig. 2). Antigen accumulation was observed at all points up to 24 h. Diffuse specific fluorescence was obtained in the rest of the hyaloplasm,
study on animals of group A showed that the anterior kidney produced IgM, localized at the periphery of certain cells (B lymphocytes) and in the hyaloplasm of plasma cells at all times chosen (1, 4, 6, 16, 24 hr and 3 days) after intubation. The reaction at three days (Fig. 6) was more intense than that
Fig. 2. Immunofluorescence demonstration of the transepithelial transfer of IgGh after intestinal administration (10 mg/l ml). After 16 hr, the antigen is still detectable in the supranuclear vacuolar system, but also in the intercellular spaces (--,) and in cells having infiltrated between the epithelial cells (*) (x 350). Fig. 3. Immunofluorescence demonstration of the transepithelial transfer of IgGh after intestinal administration (10 mg/l ml). Control animal, 1 hr after anal intubation of PBS ( x 200). Fig. 4. Immunofluorescence localization of IgGh in the anterior kidney. Twenty-four hours after intestinal administration of the antigen (10 mg/l ml), anterior kidney cells have taken up the IgG, as shown by the considerable fluorescence seen near the lumen of capillaries. This fluorescence is sometimes associated with melano-macrophages (white asterisk) ( x 200). Fig. 5. Immunofluorescence localization of IgGh in the anterior kidney. Some cells present a slight orange fluorescence, which persists alone when the primary antibody is omitted ( x 350). Fig. 6. Immunofluorescence demonstration of IgM production by the anterior kidney. IgM was localized at the periphery of certain cells (B lymphocytes?) (white arrows) and in the hyaloplasm of plasma cells (?) (black arrowheads). The reaction is more evident three days after administering IgGh (10 mg/1 mi) ( x 1000). Fig. 7. Immunofluorescence demonstration of specific IgM anti-IgGh producing cells of the anterior kidney. Fourteen days after intestinal administration of the antigen (10mg/1 ml) numerous parenchymal cells react positively (white stars) (x 350). Fig. 8. Immunofluorescence demonstration of specific IgM anti-IgGh producing cells of the anterior kidney. No cells react in control animal intubated with PBS ( x 350).
Trout immune response
observed in experimental animals killed at times 1, 4, 6, 16 and 2# h. The direct immunofluorescence study on animals of group B, 14 days after anal intubation, showed that some cells of the anterior kidney produced an IgM anti-IgGh (Fig. 7). No specific fluorescence could be observed in group F control animals (Fig. 8).
Stimulation of anterior kidney lymphocytes Seven days after the anal intubation of antigen (three animals from group D), lymphocytes from the anterior kidney were
503
isolated and cultured (3 x 10 6 cells/well). In the conditions of the study, cell viability remained high (about 95%) for one week and their number doubled after 4 days. Cells isolated from non-stimulated controls were unstable and could no longer be enumerated at 4 days (Fig. 9A). Stimulated cells secreted specific IgM anti-IgGh that accumulated in the culture medium (Fig. 9B). This secretion could have resulted from a polyclonal reaction caused by constituents of the culture medium or by the antigen itself. Figure 10 (A-F) results obtained from cultures of
Y
F i g u r e s 2 to 8 - -
Captions opposite.
504
DominiqueDorinet
6"
4-
32-
IA
0
,
i
,
i
i
,
,
,
.5 ¸
.4'
e-
•
.11,//-/
.3'
OJ O1
al,
serum (thus containing IgM), the addition of LPS did not cause a polyclonal response (Fig. 10F) and the assay of IgM anti-IgGh was negative. 2. Assay of I g M anti IgGh in the serum (Fig. 11). During the 5 month observation period of the three remaining animals of group D (antigen priming at time 0 and antigen boosting at 90 days), specific antibodies were present in the serum. The peak of the primary response was obtained after 30 days and the basal level was observed beyond 45 days. Boosting at 90 days resulted in a secondary response with a peak at 1 l0 days that was higher than the primary response, followed by a rapid return to the basal level.
c~ca .2' / Discussion
The antigen IgGh was introduced in the lumen of the posterior gut of trout, and 6 0 0 12345678 intestinal mucosa was studied by immunoDays fluorescence. At all observation times, there was a spectacular localization of fluorescent Fig. 9. Kinetics of the antibody response in vitro. antibody in all the components of the apical Lymphoid cells isolated from the pronephros of trout, kept at 12°C, 7 days after intestinal adminis- vacuolar system of intestinal cells. The protration of 30 rag/1 ml of IgGh were placed in culture tein was first present in small apical vacuoles medium (O). Control animals were intubated with and was later found in the larger vacuoles PBS (O). (A) Changes of lymphoid cells during one closer to the nucleus. It then accumulated in week of culture. (B) Levelsof specificIgM anti-IgGh the large deep vacuoles, where it was hydroin the culture medium. lyzed as shown previously (Georgopoulou et al., 1986). anterior kidney lymphocytes isolated from Diffuse fluorescence observed throughout untreated animals (group C), show that this the hyaloplasm, at the level of the interis not the case. The medium supplemented cellular space and the lamina propria, with 2% inactivated trout serum, with daily reflected the transepithelial transfer of IgGh addition of fresh medium, caused no poly- molecules towards this structure. The pathclonal response in contrast to observations ways followed for this type of protein transin the same conditions with 10% fetal calf fer have been described in ultrastructural serum (FCS) (Fig. 10A). In culture condi- studies, using the enzymatic properties of tions with FCS that did not cause a poly- H R P (Georgopoulou et al., 1988). In the clonal response, the addition of antigen did case of the transfer of bovine somatotropin not cause one either (Fig. 10B), total IgM (Le Bail et al., 1989) and recombinant trout production was unchanged (Fig. 10C), and somatotropin, immunocytochemical techthe assay of IgM anti-IgGh was negative. niques were used (ST/protein A-colloidal After the addition of 10% FCS, which gold antibodies). The present study has causes a polyclonal response, the addition shown the cellular localization of immunoof LPS caused a highly significant increase fluorescence within the lamina propria, (P < 0.001) in the number of lymphocytes corroborating the results of Georgopoulou after 24 hr of culture (Fig. 10D) but the and Vernier (1986), who showed that IgGh production of total IgM was unchanged could cause an immune response in the (Fig. 10E) and the assay of IgM anti-IgGh intestinal mucosa. was negative. Finally, when the medium was ELISA assays of plasma IgGh clearly supplemented with 2% inactivated trout show that the protein was present in the ,
i
i
i
i
,
,
.
Trout immune response
505
,
76. U*I
¢**,I O
5'
u)
4(J tO.
U
m
.
._J
4-
O
3-
~J
2-
i0
A
0
D
Oays .
4~
%
.
,
x
2"
CI1 u O tO. ~J
2 1-
0 0 Days
B °
. 5. 3=
4,
.
U)
01
3-
01 11.
2-
(J O O.
E
I-
£.
0
0
0
0 Days
Oays
Fig. 10. Effects of the addition of different factors to the culture medium. (A) Effects of adding serum and renewing medium on the proliferation of lymphocytes of the anterior kidney in trout. ( 0 ) 10% Fetal calf serum (FCS) added daily to the medium. (It) 10% FCS added to the medium every 3 days. (A) 2% inactivated trout serum added daily to the medium. (B, C) Effect of the addition of IgGh to the culture medium (10% FCS, medium added every 3 days). ( 0 ) 40/~g IgGh/ml of medium, (O) without IgGh. (B) Changes of lymphoid cells during two weeks of culture. (C) Levels of IgM in the culture medium. (D, E) Effect of adding E. coli lipopolysaccharide (LPS) to the culture medium (10% FCS, medium added every day). ( 0 ) 80/~mol of LPS/ml of medium, (O) without LPS. (D) Changes of lymphoid cells during one week of culture. (E) Levels of IgM in the culture medium, (F) Effect of adding E. coli lipopolysaccharide (LPS) to the culture medium (2% inactivated trout serum, medium added every day). ( 0 ) 80/~mol of LPS/ml of medium, (O) without LPS. Changes of lymphoid cells during one week of culture.
506
Dominique Dorin et 1.00,
0.75 E c-
'~ 0.50
0 25 0.00
30
75 I '110
1~0
Days Fig. 11. Humoral antibody production in animals after anal priming (day 0, 30mg/1 ml IgGh) and boosting (day 90, 10rag/1 ml). Each sample corresponds to three animals and each test was performed in triplicate. (--) mean of the control fish sampled during the experiment.
plasma. IgGh could be detected in the plasma as early as 15 min. This initial and rapid wave of transfer is undoubtedly a result of the anal route of administration. Using small quantities of protein, this procedure had been used to show the rapid and unique wave of transfer of bovine somatotropin (Le Bail et al., 1989) and of somatotropin (Moriyama et al., 1990), as well as recombinant trout somatotropin (Dorin et al., 1993). The last two studies used very low doses and showed the clearcut dosedependency of the transfer wave. In the present study, a second wave was observed with a peak at 3 hr, which did not end by 9 hr. The second wave was apparently related to the intubation of massive quantities of IgGh (30 mg vs. several tens to several hundreds of #g in the prior work cited). Plasma clearance of transferred IgGh molecules during the initial minutes was very rapid, suggesting the participation of specialized cells in various organs (anterior kidney, spleen, liver, etc.). In general, transferred proteins retain their functional characteristics, catalysis in the case of enzymes such as HRP, and biological activity of various hormones tested (Sire and Vernier, 1992). This was recently confirmed in carp (Hertz et al., 1991) using orally administered insulin which conserved a large proportion of its biological activity after being transferred to the plasma. Concerning immunoglobulins, Nakamura et al. (1990) showed that orally administered
al.
rabbit IgG is transported to the circulatory system in goldfish, preserving its specific antigen-binding activity. The anterior kidney in teleost fish is a key component of the immune system. It is a major site for the production of erythroid, lymphoid and myeloid cells and also takes up antigens (Bayne, 1986). The cell types required for this uptake and subsequent processing of the antigen are present in the organ, particularly macrophages, whether or not they are included in melanomacrophage centers. Following the intravenous, intramuscular or intraperitoneal injection of particulate or soluble antigen in a number of species, the antigen was localized in the anterior kidney (Ferguson et al., 1982; Lamers and De Haas, 1985; Tsujii, 1988; Tsujii and Seno, 1990). The immunofluorescence study of the anterior kidney after the anal intubation of IgGh showed that specific fluorescence involved an increasing number of cells dispersed in the parenchyma throughout the 24 hr observation period. Some fluorescent cells were melano-macrophages, but it was difficult to identify the other cell types involved. These results, comparable to those observed in two species of cyprinid fish after the intraperitoneal injection of carbon particles (Lamers and Parmentier, 1985) would seem to reflect the pre-eminent role of the anterior kidney in the plasma clearance of IgGh prior to its immune response. An alternative hypothesis has been formulated by Rombout et al. (1989a), who reported that the anal intubation of ferritin in carp resulted in a substantial increase in the number of macrophages in the posterior gut. This is due to invasion by small macrophages, starting 8 hr after the intubation, their number peaking at 24 hr. Beyond this point, small ferritin-loaded macrophages departed from the epithelium. The hypothesis is thus that large macrophages in the epithelium are resident cells responsible for the induction of a local or mucosal response, whereas the small macrophages carried by blood flow are responsible for the systemic immune response following their return to the spleen or the anterior kidney. Although this hypothesis cannot be excluded in the context of our study, the time-course of the appearance of IgGh in the plasma and in the anterior kidney favor the direct participation
Trout immune response
of the organ in the plasma clearance of the antigen. It has been known for some time that the anterior kidney of the rainbow trout can form immunocompetent cells. Thus, using the rosette test (immunocytoadherence), Chiller et al. (1969) intraperitoneally injected sheep red blood cells in trout weighing more than 200g and demonstrated the presence of antibody producing cells, lymphocytes and plasma cells, in the anterior kidney. Under the same experimental conditions, the kinetic study of lysis plaques indicated that for the primary response, the plaques appeared after 2 days post-injection, with peaks of plaque forming cells (PFC) situated around day 14. Among the sera tested that could supply the required complement factors, only those from salmonids were effective. In Coho salmon the time-course of response to the intraperitoneal injection of an O-antigen extract of the fish pathogen Vibrio anguillarum demonstrated peak PFC per anterior kidney on day 16 post-immunization (Kaattari and Irwin, 1985). PFC responses were shown to be immunoglobulin-mediated and by indirect evidence, antigen-specific. In an ultrastructural study in carp, Rombout et al. (1989b) showed that 25% of anterior kidney cells, morphologically identified as lymphocytes, were Ig-reactive at their surface after the use of a mouse monoclonal antibody against carp serum Ig and of a goat anti-mouse/gold. Our aim was to determine if the introduction of a soluble antigen, IgGh, in the intestinal lumen would result in the stimulation of immunocompetent cells putatively present in the anterior kidney and if this stimulation would result in the production of specific antibodies. The indirect immunofluorescence study after antigen intubation showed that some cells presented a fluorescent border, while in others fluorescence was localized throughout the hyaloplasm, probably representing B-cells and plasma cells, respectively. The lumen of some capillaries also reacted positively. Qualitative and nonspecific, this technique simply shows that kidney is indeed a lymphoid organ and that it is even a site of B lymphocyte maturation. Three days after intubation of the antigen, however, observation (Fig. 6) showed a high number of positive cells, supporting
507
the hypothesis that at this time, the anterior kidney is engaged in the primary phase of the immune response against IgGh. Lymphocytes from the anterior kidney, cultured 7 days after intubation, multiplied and secreted IgM specific for the antigen (anti-IgGh) into the culture medium. The different controls done enable us to conclude that these IgM secreted are not the result of a polyclonal reaction, due for example to growth factors present in the medium or to the antigen itself. In addition, obtaining a polyclonal reaction does not enable the presence of IgM anti-IgGh to be revealed in the culture medium. It may thus be concluded that the anterior kidney participates in the humoral immune response by the proliferation of specific B lymphocytes which differentiate into antiIgGh IgM producing plasma cells. The signals required for lymphocyte multiplication and their differentiation into plasma cells remain to be elucidated. Anal intubation with IgGh (priming) caused a classical primary antibody response with a peak after 30 days. A second intubation after 90 days (boosting) caused a new response, more rapid than the first but with comparable amplitude. These results can be compared to those of Tatner et aL (1987), obtained after the intraperitoneal injection of the same antigen in the same species. These authors considered IgGh as a thymusdependent antigen, as it is in mammals. Thymectomy five months before injection had no effect on the response, while the same operation nine months before injection resulted in an attenuation of the primary response. It was suggested that the life span of helper T cells, required for the response to a thymus-dependent antigen and present peripherally, is very long. These authors observed no memory response, but the analysis in this case is more delicate, since temperature could have interfered as a result of the duration of the experimentation. Our results, obtained in conditions of controlled temperature, do not argue for a strong immunological memory either; only the rapidity of the response to the antigen was greater. Overall, the antibody response to IgGh, reputed to be a thymus-dependent antigen, is similar to that obtained with a thymus independent antigen in mammals. In this case, at least in vitro, the primary
508
Dominique Dorin et al.
response is weak and the secondary response is comparable to the first, limited to the production of IgM. Arkoosh and Kaattari (1991), however, demonstrated an immunological memory in rainbow trout: more than a single injection of the priming antigen T N P - K L H (trinitrophenylated-keyhole limpet hemocyanin) was required to produce an enhanced in vitro response to this Tdependent antigen. A second priming injection however, was not required to produce an enhanced secondary response to the T-independent form of antigen TNP-LPS (trinitrophenylated-lipopolysaccharide). In contrast to the situation in mammals, there was no evidence for affinity maturation during the primary or secondary response. M e m o r y in trout may be due strictly to a simple expansion o f the antigen-specific precursor pool without the selective development of cell clones producing antibodies with increasing affinity. In mammals, this process is classically linked to the transformation of IgM to IgG. The antibody response of fish, restricted to IgM, thus presented no increase in affinity. Concerning this antibody response, Mughal and M a n n i n g (1986) showed that IgGh was strongly antigenic in the mullet and that priming by oral administration was equally as effective as priming by injection. An antibody response in carp was obtained after anal intubation with ferritin ( R o m b o u t et al., 1986). Following a primary injection with SRBC in this species, the spleen contained only 5% of the total number o f PFC, while more than 90% were observed in the anterior kidney (van Muiswinkel et al., 1991). In addition, Bayne (1986) reported that in teleost fish it was impossible to depress the humoral antibody response by splenectomy, since the anterior kidney could complete all the phases of the immune response. The present work has enabled us to demonstrate the reality of a humoral immune response following the intestinal transfer of an antigenic protein. The anterior kidney participates in this response. Acknowledgements--This work was supported by a
grant from CEC (ECLAIR-AGRE 0020). We thank C. Ballagnyfor technical assistance and C. Le Mentec for typing the manuscript. Thanks are also extended to N. Narradon for photographic assistance.
References Arkoosh M. R. and Kaattari S. L. (1991) Development of immunological memory in rainbow trout (Oncorhynchus mykiss). I. An immunochemicaland cellular analysis of the B cell response. Dev. comp. Immunol. 15, 279-293. Bayne C. J. (1986) Pronephric leucocytesof Cyprinus carpio: isolation, separation and characterization. Vet. Immunol. Immunopath. 12, 141-151. Chiller J. M., Hodgins H. O., Chambers V. C. and Weiser R. S. (1969) Antibody response in rainbow trout (Salmo gairdneri). I. Immunocompetent cells in the spleen and anterior kidney. J. Immunol. 102, 1193-1207. Dorin D., Martin P., Sire M. F., Smal J. and Vernier J. M. (1993) Protein uptake by intestinal macrophages and eosinophilic granulocytes in trout: an in vivo study. Biol. Cell 79, 37-44. Ferguson H. W., Claxton M. J., Moccia R. D. and Wilkie E. J. (1982) The quantitative clearance of bacteria from the bloodstream of rainbow trout (Salmo gairdneri ). Vet. Pathol. 19, 687-699. Georgopoulou U. and Vernier J. M. (1986) Local immunological response in the posterior intestinal segment of the rainbow trout after oral administration of macromolecules. Dev. comp. Immunol. 10, 529-537. Georgopoulo U., Sire M. F. and Vernier J. M. (1986) Immunologicaldemonstration of intestinal absorption and digestion of protein macromolecules in the trout (Salmo gairdneri ). Cell Tissue Res. 247, 387-395. Georgopoulou U., Dabrowski K., Sire M. F. and Vernier J. M. (1988) Absorption of intact proteins by the intestinal epithelium of trout, Salrno gairdneri. A luminescence enzyme immunoassay and cytochemical study. Cell Tissue Res. 251, 145-152. Hertz Y., Tchelet A., Madar Z. and Gertler A. (1991) Absorption of bioactive human growth hormone after oral administration in the common carp (Cyprinus earpio) and its enhancement by deoxycholate. J. comp. Physiol. 161B, 159-163. Kaattari S. L. and Irwin M. J. (1985) Salmonid spleen and anterior kidney harbor populations of lymphocytes with different B cell repertoires. Dev. comp. lmmunol. 9, 433-44A,. Lamers C. H. J. and De Haas M. J. H. (1985) Antigen localization in the lymphoid organs of carp (Cyprinus carpio). Cell Tissue Res. 242, 491498. Lamers C. H. J. and Parmentier, H. K. (1985) The fate of intraperitoneally injected carbon particles in cyprinid fish. Cell Tissue Res. 242, 499-503. Le Bail P. Y., Sire M. F. and Vernier J. M. (1989) Intestinal transfer of growth hormone into the circulatory system of the rainbow trout, Salmo gairdneri: interference by granule cells. J. exp. Zool. 251, 101-107. Moriyama S., Takahashi A., Hirano T. and Kawauchi H. (1990) Salmon growth hormone is transported to the circulation of rainbow trout, Oncorhynchus mykiss, after intestinal administration. J. comp. Physiol. 160B, 251-257. Mughal M. S. and Manning M. J. (1986) The immune system of juvenile thick-lipped grey mullet, Chelon
Trout immune response labrosus Risso: antibody responses to soluble protein antigens. J. Fish Biol. 29, 177-186. Muiswinkel W. B. van, Lamers C. H. J. and Rombout J. H. W. M. (1991) Structural and functional aspects of the spleen in bony fish. Res. lmrnunol. 142, 362-366. Nakamura O., Kobayashi M., Suzuki Y., Aida K. and Hanyu I. (1990) Transport of orally administered rabbit IgG into blood circulation of goldfish. Nippon Suisan Gakkaishi 59, 1749-1753. Rombout J. H. and Berg A. A. van den (1989) Immunological importance of the second gut segment of carp. I. Uptake and processing of antigens by epithelial cells and macrophages. J. Fish Biol. 35, 13-22. Rombout J. H. W. M., Berg A. A. van den, Berg C. T. G. A. van den, Witte P. and Egberts E. (1989a) Immunological importance of the second gut segment of carp. III. Systemic and/or mucosal immune responses after immunization with soluble or particulate antigen. J. Fish Biol. 35, 179-186.
CBPB IC9/2-3--V
509
Rombout J. H. W. M., Bot H. E. and Taverne-Thiele J. J. (1989b) Immunological importance of the second gut segment of carp. II. Characterization of mucosal leucocytes. J. Fish Biol. 35, 167-178. Sire M. F. and Vernier J. M. (1992) Intestinal absorption of protein in teleost fish. Comp. Biochem. Physiol. 130A, 771-781. Tatner M. F., Adams A. and Leschen W. (1987) An analysis of the primary and secondary antibody response in intact and thymectomized rainbow trout, Salmo gairdneri Richardson, to human gamma globulin and Aeromonas salmonicida. J. Fish Biol. 31, 17%195. Tsujii T. (1988) The role of renal macrophages in the aglomerular kidney of the sea-horse (teleost) in the removal of exogenous macromolecules from circulating blood. Arch. Histol. Cytol. 51, 327-339. Tsujii T. and Seno S. (1990) Melano-macrophages centers in the aglomerular kidney of the sea horse (Teleosts): morphologic studies on its formation and possible function. Anat. Rec. 226, 460-470.
Comp.Biochem.Physiol.Vol. 109B,No. 2/3, pp. 499-509, 1994
Copyright© 1994ElsevierScienceLtd PrintedinGreatBritain.All rightsreserved
Pergamon
0305-0491/94$7.00+ 0.00
03o5-0491(94)00063-s
Demonstration of an antibody response of the anterior kidney following intestinal administration of a soluble protein antigen in trout Dominique Dorin, Marie-France Sire and Jean-Marie Vernier Laboratoire de PhysiologieCellulaireet M6tabolique des Poissons, Bat. 447, Universit6Paris XI, 91405 Orsay Cedex, France The humoral antibody response following anal intubation of a soluble antigenic protein to trout was investigated. The transfer of human immunoglobulin G (IgGh) to the plasma was demonstrated by ELISA assays. The participation of the anterior kidney in plasma clearance of the antigen was shown by an immunofluorescence study. The anterior kidney displayed a proliferation of specific B lymphocytes and differentiation into plasma cells producing anti-IgGh IgM. The peak of plasma specific antibody concentration occurred 30 days after intubation and a second intubation led to another peak 20 days later, whose amplitude was close to that of the primary response. Key words: Anterior kidney; Antibody response; Soluble protein antigen; Trout. Comp. Biochem. Physiol. 109B, 499-509, 1994.
Introduction A specialized region in the gut of the teleost fish, the posterior gut, is responsible for the massive absorption of proteins which then undergo intracellular lysis. A portion of the protein molecules escapes intracellular degradation, however, and reaches the intercellular space as well as the interstitial space of the lamina propria (Sire and Vernier, 1992). The demonstration of a mucosal (=local) immune response after the oral administration of a soluble antigenic protein was obtained in trout (Georgopoulou and Vernier, 1986) with human immunoglobulin G (IgGh) and subsequently confirmed in the carp (Rombout and van den Berg, 1989) with ferritin.
Again using IgGh, the aim of the present work was to describe sequentially the uptake of the antigen from the posterior gut of the trout, the appearance of the IgGh in the plasma, the localization of IgGh in the anterior kidney, and the subsequent antibody response of the anterior kidney to the IgGh as a contribution to the systemic immune response. The analysis of the contribution of the anterior kidney (pronephros) was chosen since the organ has lost its secretory function and is believed to be the phylogenetic analogue of bone marrow and/ or lymph glands in the other vertebrates.
Materials and Methods
Correspondence to: J.-M. Vernier, Laboratoire de
Animals
Physiologic Cellulaire et M6tabolique des Poissons, Bat. 447, Universit6 Paris XI, 91405 Orsay Cedex, France. Tel. 1-69-41-79-33; Fax 1-69-85-34-59. Received 30 October 1993; accepted 24 April 1994.
Rainbow trout (Oncorhynchus mykiss) were reared in running water at constant temperature (12 + I°C) and fed with Aqua
499
500
Dominique Dorin et al.
14 (Aqualim, Nersac, France). Animals, weighing 250-300g, were fasted 5 days before the experiment. Anal intubation, tissue and blood sampling were operated on anesthetized animals (0.3 ml of phenoxy2-ethanol/water). The antigenic protein, IgGh (Institut Pasteur, Paris, France) dissolved in phosphate buffer saline (PBS) or the buffer alone (control animals) was introduced directly by anal intubation, using a syringe with a soft flexible tube (0.2 mm diameter) attached to its extremity. The animals were distributed in six groups: Group A: 10mglgGh/1 ml PBS (n = 18). Group B: 10 mg IgGh/1 ml PBS (n =6). Group C: no anal intubation (n = 12). Group D: 30 mg IgGh/1 ml PBS (n = 6). Three animals were killed after one week and the remaining three again received a booster of 10 mg of IgGh in 1 ml of PBS, 90 days after the first administration. Group E: 30mglgGh/1 ml PBS (n = 5). Group F: 1 ml PBS (n = 35).
Antibodies Rabbit anti-IgGh (Sigma), goat antirabbit IgG labeled with FITC (Sigma), goat anti-trout immunoglobulin M (IgM) (Kirkegaard and Perry Laboratories), rabbit anti-goat IgG labeled with fluorescein isothiocyanate (FITC) (Sigma), IgGh labeled with FITC (Institut Pasteur, Paris, France), goat anti-IgGh labeled with peroxidase (Sigma), and goat anti-trout IgM labeled with peroxidase (Kirkegaard and Perry Laboratories) were used. No crossed reaction with IgGh could be detected with goat anti-trout IgM, rabbit anti-goat IgG and goat anti-rabbit IgG.
Tissue localization of lgGh, IgM and specific IgM anti-IgGh Fragments of the posterior gut and the anterior kidney were removed and fixed in 4% paraformaldehyde in 0.1 M PBS (pH 7.4), dehydrated and embedded in paraffin. 1. Localization of lgGh and IgM. Animals of group A were used and the specimens of tissues were removed 1, 4, 6, 16, 24 hr and 3 days after administration of IgGh. Six micrometer thick sections were incubated with 1% bovine serum albumin (BSA) in PBS for 30 min at 20°C.
IgGh: after 1 hr incubation with rabbit anti-IgGh (1/100 in PBS), sections were washed with PBS/BSA for 10 min and with PBS for 2 × 10min. Finally they were incubated for 1 hr with goat anti-rabbit IgG labeled with FITC (1/20 in PBS) and washed 3 x 10 min with PBS. IgM: The immunoglobulin was visualized using goat anti-trout IgM (1/100 in PBS) and by a rabbit anti-goat IgG labeled with FITC (1/60 in PBS). 2. Localization of lgM anti-IgGh. Animals of group B were used and fragments of anterior kidney were removed 14 days after the administration of IgGh. Six micrometer thick sections were incubated with 1% BSA in PBS for 30 min at 20°C and then for 1 h with IgGh labeled with FITC (1/25 in PBS). Sections were washed with PBS for 3 × 10 min. The following controls for aspecific reactions were performed for all immunocytochemical reactions: (i) non-immunized animals (group F), (ii) the primary antibodies were omitted and sections incubated with pre-immune or non-immune serum or with PBS alone.
Culture of anterior kidney lymphocytes The anterior kidney was aseptically removed and disrupted by passing through a steel screen (80 mesh). The cell suspension was deposited on Histopaque (Sigma) and centrifuged at 1000g for 20 min, at 15°C. The viability of leucocytes was determined by Trypan Blue (0.4%) dye exclusion. Lymphocytes were adjusted at a concentration of 3 x 106 or 4 × 10 6 cells/well and cultivated with 3 ml (six-well culture plates, Corning) or 400/~1 (24-well culture plates, Corning) of culture medium (RPMI 1640, Sigma, containing 25 mM HEPES, 1% streptomycin-penicillin (120 mg/ml; 10 000 U) and 2 mM L-glutamine, Sigma) at 15°C in a humid atmosphere ( O 2 / N 2 / C O 2 , 20:75: 5). The animals of group C were utilized to optimize the culture medium conditions and to determine if the direct action of the antigenic protein (IgGh) or of the mitogenic agent for B cells, the E. coli lipopolysaccharide (LPS), can induce a polyclonal reaction of the cultivated cells. Twenty-fourwell culture plates were used with 400 #1 culture medium/well. (a) FCS 10/24: 10%
Trout immune response
501
fetal calf serum, 50/~1 of medium added (Flow Laboratories). A standard range every 24hr. (b) FCS 10/24/LPS: medium of IgGh (0, 25, 50, 75 and 100 ng/ml PBS) with 80#tool LPS/ml and 50/~mol fl- was used to calculate IgGh serum concenmercaptoethanol/ml. (c) FCS/10/72:50/~1 trations. The sensitivity threshold of the of medium added every 72 hr. (d) FCS/ method was 3 ng/ml. 10/72/IgGh: 40/~g IgGh/ml of medium. (e) TS 2/24: 2% inactivated trout serum, 50 #1 ELISA assay of total IgM Microplates were coated with IgGh medium added every 24 hr. (f) TS 2/24/LPS: medium with LPS and fl-mercaptoethanol (10 ~g/ml PBS) for 2 hr at 37°C and overnight at 4°C. After washing and saturating (see b). Cells were enumerated in four wells on with 3% BSA, dilutions of culture superdays 1, 3, 5, 7 (a, b, e, f) or on days 3, 6, natants (group C) were incubated for 2 hr 9, 12, 15 (c and d) and supematants were at 37°C. The final incubation was with a stored at - 2 0 ° C for ELISA assays of total goat anti-trout IgM labeled with peroxidase (1/100 in PBS). Subsequent steps were IgM or for specific IgM anti-IgGh. Three animals of group D were killed 1 as described above. A standard range of week after antigen administration. Anterior IgM (three at 10 ng/ml) was used to calcukidney cells (3 x 106) in 3ml of culture late the IgM concentration. The sensitivity medium were deposited in each well of threshold of the method was 3 ng/ml. six-well culture plates and incubated for 8 days. Cells in four wells were enumerated ELISA assay of IgM anti-IgGh Blood samples from the remaining three on days 2, 4, 6 and 8 and the culture supernatants were stored at - 2 0 ° C for animals of group D were removed regularly ELISA assays of specific IgM anti-IgGh. (10, 20, 30, 45, 75, 110, 120 and 150 days) and treated as described above. The serum ELISA assay of serum IgGh was recovered and stored at - 2 0 ° C before Blood was collected from each exper- assaying IgM. Culture supernatants (group imental animal in group E and from each C, group D) were also tested for IgM control animal by cardiac puncture 0, 15, anti-IgGh. 30 min, 1, 3, 6 and 9 hr after administering Microplates were coated with IgGh the protein. Each blood sample was allowed (10 pg/ml PBS). After washing and saturatto clot for 1 hr at 20°C, then overnight ing with 3% BSA, dilutions of culture at 4°C and was individually centrifuged supernatants or sera were incubated for 2 hr at 3000g for 15min, at 4°C and serum at 37°C. The final incubation was with a was recovered and stored at -20°C. Flat goat anti-trout IgM labeled with peroxidase bottom, 96-well ELISA plates (NUNC) (1/100 in PBS). Subsequent steps were as were coated with 100#l PBS containing described above. l0 #g/ml of anti-IgGh raised in goats (2 hr Two types of controls were used, or at 37°C, overnight at 4°C). Between each sera, or culture supernatants from nonstep, the plates were washed five times with immunized animals, to determine backPBS containing 0.1% Tween 20. Non- ground, and assay without serum were the specific reactions were blocked using 3% negative controls. BSA in PBS for 1 hr at 37°C. They were then incubated with different dilutions of serum samples for 2 hr at 37°C and with Results a goat anti-IgGh labeled with peroxidase (1/5000) for 1.5 hr at 37°C. The enzymatic Transfer of intubated antigen (IgGh) to the reaction was allowed to develop for 20 rain anterior kidney using 100 #l/well of the substrate o-phenyl1. Kinetics of appearance of IgGh in the ene diamine [OPD/H2 02 (2 mg OPD in 5 ml serum (Fig. 1). Immunoenzymatic results of 0.1 M citrate buffer, pH 5.5 and 30/~l of obtained with ELISA showed that IgGh 30% H202) ] in darkness. Colour develop- was transferred to the plasma following the ment was stopped by adding 50/~l of 2N anal intubation of antigen (group E). An HE SO 4. The plates were read at 492 nm initial low amplitude peak was observed using a Titertek Multiskan Plus plate reader 15 min after administration, followed by a
DominiqueDorin et al.
502
in particular infranuclear, and was more intense in the intercellular space and the lamina propria (Fig. 2). Cellular localization could also be detected in the lamina. In controls receiving only PBS (group F), no specific fluorescence was observed (see Fig. 3 as an example, 1 hr after intestinal administration of buffer). j
O
~
O
0 ~'? ~ . . . . . . 1 2 3 4 5 B 7 B q Time (hours]
Fig. 1. Kinetics of appearance of IgOh in the plasma of trout after anal administration of IgGh (30 mg/ 1 ml) (O). Control animals: (O). Each sample corresponds to five animals. Vertical bars: standard error of the mean. second wave of transfer with a peak at 3 hr which extended beyond the 9 hr point. The antigen thus passed through the intestinal epithelium.
3. Clearance of plasma IgGh by the anterior kidney (Figs 4 and 5). Fish receiving the antigen (group A) exhibited specific cellular fluorescence observed first at 6 hr sample time, with the number of cells involved increasing up to 24 hr (Fig. 4). At all time points (1, 4, 6, 16 and 24hr), some anterior kidney cells exhibited an orange, natural non-specific fluorescence which persisted alone when the primary antibody had been omitted (Fig. 5) or which was observed in controls (group F) at all times.
Immune response to IgGh transfer 2. Transepithelial transfer of IgGh (Figs 1. Stimulation of lymphocytes and IgM 2 and 3). Immunofluorescence techniques production, lgM production by the anterior showed that starting 1 hr after anal intub- kidney. The indirect immunofluorescence ation (group A) there was very intense specific fluorescence in the vacuolar system of the apical hyaloplasm in posterior segment intestinal cells (Fig. 2). Antigen accumulation was observed at all points up to 24 h. Diffuse specific fluorescence was obtained in the rest of the hyaloplasm,
study on animals of group A showed that the anterior kidney produced IgM, localized at the periphery of certain cells (B lymphocytes) and in the hyaloplasm of plasma cells at all times chosen (1, 4, 6, 16, 24 hr and 3 days) after intubation. The reaction at three days (Fig. 6) was more intense than that
Fig. 2. Immunofluorescence demonstration of the transepithelial transfer of IgGh after intestinal administration (10 mg/l ml). After 16 hr, the antigen is still detectable in the supranuclear vacuolar system, but also in the intercellular spaces (--,) and in cells having infiltrated between the epithelial cells (*) (x 350). Fig. 3. Immunofluorescence demonstration of the transepithelial transfer of IgGh after intestinal administration (10 mg/l ml). Control animal, 1 hr after anal intubation of PBS ( x 200). Fig. 4. Immunofluorescence localization of IgGh in the anterior kidney. Twenty-four hours after intestinal administration of the antigen (10 mg/l ml), anterior kidney cells have taken up the IgG, as shown by the considerable fluorescence seen near the lumen of capillaries. This fluorescence is sometimes associated with melano-macrophages (white asterisk) ( x 200). Fig. 5. Immunofluorescence localization of IgGh in the anterior kidney. Some cells present a slight orange fluorescence, which persists alone when the primary antibody is omitted ( x 350). Fig. 6. Immunofluorescence demonstration of IgM production by the anterior kidney. IgM was localized at the periphery of certain cells (B lymphocytes?) (white arrows) and in the hyaloplasm of plasma cells (?) (black arrowheads). The reaction is more evident three days after administering IgGh (10 mg/1 mi) ( x 1000). Fig. 7. Immunofluorescence demonstration of specific IgM anti-IgGh producing cells of the anterior kidney. Fourteen days after intestinal administration of the antigen (10mg/1 ml) numerous parenchymal cells react positively (white stars) (x 350). Fig. 8. Immunofluorescence demonstration of specific IgM anti-IgGh producing cells of the anterior kidney. No cells react in control animal intubated with PBS ( x 350).
Trout immune response
observed in experimental animals killed at times 1, 4, 6, 16 and 2# h. The direct immunofluorescence study on animals of group B, 14 days after anal intubation, showed that some cells of the anterior kidney produced an IgM anti-IgGh (Fig. 7). No specific fluorescence could be observed in group F control animals (Fig. 8).
Stimulation of anterior kidney lymphocytes Seven days after the anal intubation of antigen (three animals from group D), lymphocytes from the anterior kidney were
503
isolated and cultured (3 x 10 6 cells/well). In the conditions of the study, cell viability remained high (about 95%) for one week and their number doubled after 4 days. Cells isolated from non-stimulated controls were unstable and could no longer be enumerated at 4 days (Fig. 9A). Stimulated cells secreted specific IgM anti-IgGh that accumulated in the culture medium (Fig. 9B). This secretion could have resulted from a polyclonal reaction caused by constituents of the culture medium or by the antigen itself. Figure 10 (A-F) results obtained from cultures of
Y
F i g u r e s 2 to 8 - -
Captions opposite.
504
DominiqueDorinet
6"
4-
32-
IA
0
,
i
,
i
i
,
,
,
.5 ¸
.4'
e-
•
.11,//-/
.3'
OJ O1
al,
serum (thus containing IgM), the addition of LPS did not cause a polyclonal response (Fig. 10F) and the assay of IgM anti-IgGh was negative. 2. Assay of I g M anti IgGh in the serum (Fig. 11). During the 5 month observation period of the three remaining animals of group D (antigen priming at time 0 and antigen boosting at 90 days), specific antibodies were present in the serum. The peak of the primary response was obtained after 30 days and the basal level was observed beyond 45 days. Boosting at 90 days resulted in a secondary response with a peak at 1 l0 days that was higher than the primary response, followed by a rapid return to the basal level.
c~ca .2' / Discussion
The antigen IgGh was introduced in the lumen of the posterior gut of trout, and 6 0 0 12345678 intestinal mucosa was studied by immunoDays fluorescence. At all observation times, there was a spectacular localization of fluorescent Fig. 9. Kinetics of the antibody response in vitro. antibody in all the components of the apical Lymphoid cells isolated from the pronephros of trout, kept at 12°C, 7 days after intestinal adminis- vacuolar system of intestinal cells. The protration of 30 rag/1 ml of IgGh were placed in culture tein was first present in small apical vacuoles medium (O). Control animals were intubated with and was later found in the larger vacuoles PBS (O). (A) Changes of lymphoid cells during one closer to the nucleus. It then accumulated in week of culture. (B) Levelsof specificIgM anti-IgGh the large deep vacuoles, where it was hydroin the culture medium. lyzed as shown previously (Georgopoulou et al., 1986). anterior kidney lymphocytes isolated from Diffuse fluorescence observed throughout untreated animals (group C), show that this the hyaloplasm, at the level of the interis not the case. The medium supplemented cellular space and the lamina propria, with 2% inactivated trout serum, with daily reflected the transepithelial transfer of IgGh addition of fresh medium, caused no poly- molecules towards this structure. The pathclonal response in contrast to observations ways followed for this type of protein transin the same conditions with 10% fetal calf fer have been described in ultrastructural serum (FCS) (Fig. 10A). In culture condi- studies, using the enzymatic properties of tions with FCS that did not cause a poly- H R P (Georgopoulou et al., 1988). In the clonal response, the addition of antigen did case of the transfer of bovine somatotropin not cause one either (Fig. 10B), total IgM (Le Bail et al., 1989) and recombinant trout production was unchanged (Fig. 10C), and somatotropin, immunocytochemical techthe assay of IgM anti-IgGh was negative. niques were used (ST/protein A-colloidal After the addition of 10% FCS, which gold antibodies). The present study has causes a polyclonal response, the addition shown the cellular localization of immunoof LPS caused a highly significant increase fluorescence within the lamina propria, (P < 0.001) in the number of lymphocytes corroborating the results of Georgopoulou after 24 hr of culture (Fig. 10D) but the and Vernier (1986), who showed that IgGh production of total IgM was unchanged could cause an immune response in the (Fig. 10E) and the assay of IgM anti-IgGh intestinal mucosa. was negative. Finally, when the medium was ELISA assays of plasma IgGh clearly supplemented with 2% inactivated trout show that the protein was present in the ,
i
i
i
i
,
,
.
Trout immune response
505
,
76. U*I
¢**,I O
5'
u)
4(J tO.
U
m
.
._J
4-
O
3-
~J
2-
i0
A
0
D
Oays .
4~
%
.
,
x
2"
CI1 u O tO. ~J
2 1-
0 0 Days
B °
. 5. 3=
4,
.
U)
01
3-
01 11.
2-
(J O O.
E
I-
£.
0
0
0
0 Days
Oays
Fig. 10. Effects of the addition of different factors to the culture medium. (A) Effects of adding serum and renewing medium on the proliferation of lymphocytes of the anterior kidney in trout. ( 0 ) 10% Fetal calf serum (FCS) added daily to the medium. (It) 10% FCS added to the medium every 3 days. (A) 2% inactivated trout serum added daily to the medium. (B, C) Effect of the addition of IgGh to the culture medium (10% FCS, medium added every 3 days). ( 0 ) 40/~g IgGh/ml of medium, (O) without IgGh. (B) Changes of lymphoid cells during two weeks of culture. (C) Levels of IgM in the culture medium. (D, E) Effect of adding E. coli lipopolysaccharide (LPS) to the culture medium (10% FCS, medium added every day). ( 0 ) 80/~mol of LPS/ml of medium, (O) without LPS. (D) Changes of lymphoid cells during one week of culture. (E) Levels of IgM in the culture medium, (F) Effect of adding E. coli lipopolysaccharide (LPS) to the culture medium (2% inactivated trout serum, medium added every day). ( 0 ) 80/~mol of LPS/ml of medium, (O) without LPS. Changes of lymphoid cells during one week of culture.
506
Dominique Dorin et 1.00,
0.75 E c-
'~ 0.50
0 25 0.00
30
75 I '110
1~0
Days Fig. 11. Humoral antibody production in animals after anal priming (day 0, 30mg/1 ml IgGh) and boosting (day 90, 10rag/1 ml). Each sample corresponds to three animals and each test was performed in triplicate. (--) mean of the control fish sampled during the experiment.
plasma. IgGh could be detected in the plasma as early as 15 min. This initial and rapid wave of transfer is undoubtedly a result of the anal route of administration. Using small quantities of protein, this procedure had been used to show the rapid and unique wave of transfer of bovine somatotropin (Le Bail et al., 1989) and of somatotropin (Moriyama et al., 1990), as well as recombinant trout somatotropin (Dorin et al., 1993). The last two studies used very low doses and showed the clearcut dosedependency of the transfer wave. In the present study, a second wave was observed with a peak at 3 hr, which did not end by 9 hr. The second wave was apparently related to the intubation of massive quantities of IgGh (30 mg vs. several tens to several hundreds of #g in the prior work cited). Plasma clearance of transferred IgGh molecules during the initial minutes was very rapid, suggesting the participation of specialized cells in various organs (anterior kidney, spleen, liver, etc.). In general, transferred proteins retain their functional characteristics, catalysis in the case of enzymes such as HRP, and biological activity of various hormones tested (Sire and Vernier, 1992). This was recently confirmed in carp (Hertz et al., 1991) using orally administered insulin which conserved a large proportion of its biological activity after being transferred to the plasma. Concerning immunoglobulins, Nakamura et al. (1990) showed that orally administered
al.
rabbit IgG is transported to the circulatory system in goldfish, preserving its specific antigen-binding activity. The anterior kidney in teleost fish is a key component of the immune system. It is a major site for the production of erythroid, lymphoid and myeloid cells and also takes up antigens (Bayne, 1986). The cell types required for this uptake and subsequent processing of the antigen are present in the organ, particularly macrophages, whether or not they are included in melanomacrophage centers. Following the intravenous, intramuscular or intraperitoneal injection of particulate or soluble antigen in a number of species, the antigen was localized in the anterior kidney (Ferguson et al., 1982; Lamers and De Haas, 1985; Tsujii, 1988; Tsujii and Seno, 1990). The immunofluorescence study of the anterior kidney after the anal intubation of IgGh showed that specific fluorescence involved an increasing number of cells dispersed in the parenchyma throughout the 24 hr observation period. Some fluorescent cells were melano-macrophages, but it was difficult to identify the other cell types involved. These results, comparable to those observed in two species of cyprinid fish after the intraperitoneal injection of carbon particles (Lamers and Parmentier, 1985) would seem to reflect the pre-eminent role of the anterior kidney in the plasma clearance of IgGh prior to its immune response. An alternative hypothesis has been formulated by Rombout et al. (1989a), who reported that the anal intubation of ferritin in carp resulted in a substantial increase in the number of macrophages in the posterior gut. This is due to invasion by small macrophages, starting 8 hr after the intubation, their number peaking at 24 hr. Beyond this point, small ferritin-loaded macrophages departed from the epithelium. The hypothesis is thus that large macrophages in the epithelium are resident cells responsible for the induction of a local or mucosal response, whereas the small macrophages carried by blood flow are responsible for the systemic immune response following their return to the spleen or the anterior kidney. Although this hypothesis cannot be excluded in the context of our study, the time-course of the appearance of IgGh in the plasma and in the anterior kidney favor the direct participation
Trout immune response
of the organ in the plasma clearance of the antigen. It has been known for some time that the anterior kidney of the rainbow trout can form immunocompetent cells. Thus, using the rosette test (immunocytoadherence), Chiller et al. (1969) intraperitoneally injected sheep red blood cells in trout weighing more than 200g and demonstrated the presence of antibody producing cells, lymphocytes and plasma cells, in the anterior kidney. Under the same experimental conditions, the kinetic study of lysis plaques indicated that for the primary response, the plaques appeared after 2 days post-injection, with peaks of plaque forming cells (PFC) situated around day 14. Among the sera tested that could supply the required complement factors, only those from salmonids were effective. In Coho salmon the time-course of response to the intraperitoneal injection of an O-antigen extract of the fish pathogen Vibrio anguillarum demonstrated peak PFC per anterior kidney on day 16 post-immunization (Kaattari and Irwin, 1985). PFC responses were shown to be immunoglobulin-mediated and by indirect evidence, antigen-specific. In an ultrastructural study in carp, Rombout et al. (1989b) showed that 25% of anterior kidney cells, morphologically identified as lymphocytes, were Ig-reactive at their surface after the use of a mouse monoclonal antibody against carp serum Ig and of a goat anti-mouse/gold. Our aim was to determine if the introduction of a soluble antigen, IgGh, in the intestinal lumen would result in the stimulation of immunocompetent cells putatively present in the anterior kidney and if this stimulation would result in the production of specific antibodies. The indirect immunofluorescence study after antigen intubation showed that some cells presented a fluorescent border, while in others fluorescence was localized throughout the hyaloplasm, probably representing B-cells and plasma cells, respectively. The lumen of some capillaries also reacted positively. Qualitative and nonspecific, this technique simply shows that kidney is indeed a lymphoid organ and that it is even a site of B lymphocyte maturation. Three days after intubation of the antigen, however, observation (Fig. 6) showed a high number of positive cells, supporting
507
the hypothesis that at this time, the anterior kidney is engaged in the primary phase of the immune response against IgGh. Lymphocytes from the anterior kidney, cultured 7 days after intubation, multiplied and secreted IgM specific for the antigen (anti-IgGh) into the culture medium. The different controls done enable us to conclude that these IgM secreted are not the result of a polyclonal reaction, due for example to growth factors present in the medium or to the antigen itself. In addition, obtaining a polyclonal reaction does not enable the presence of IgM anti-IgGh to be revealed in the culture medium. It may thus be concluded that the anterior kidney participates in the humoral immune response by the proliferation of specific B lymphocytes which differentiate into antiIgGh IgM producing plasma cells. The signals required for lymphocyte multiplication and their differentiation into plasma cells remain to be elucidated. Anal intubation with IgGh (priming) caused a classical primary antibody response with a peak after 30 days. A second intubation after 90 days (boosting) caused a new response, more rapid than the first but with comparable amplitude. These results can be compared to those of Tatner et aL (1987), obtained after the intraperitoneal injection of the same antigen in the same species. These authors considered IgGh as a thymusdependent antigen, as it is in mammals. Thymectomy five months before injection had no effect on the response, while the same operation nine months before injection resulted in an attenuation of the primary response. It was suggested that the life span of helper T cells, required for the response to a thymus-dependent antigen and present peripherally, is very long. These authors observed no memory response, but the analysis in this case is more delicate, since temperature could have interfered as a result of the duration of the experimentation. Our results, obtained in conditions of controlled temperature, do not argue for a strong immunological memory either; only the rapidity of the response to the antigen was greater. Overall, the antibody response to IgGh, reputed to be a thymus-dependent antigen, is similar to that obtained with a thymus independent antigen in mammals. In this case, at least in vitro, the primary
508
Dominique Dorin et al.
response is weak and the secondary response is comparable to the first, limited to the production of IgM. Arkoosh and Kaattari (1991), however, demonstrated an immunological memory in rainbow trout: more than a single injection of the priming antigen T N P - K L H (trinitrophenylated-keyhole limpet hemocyanin) was required to produce an enhanced in vitro response to this Tdependent antigen. A second priming injection however, was not required to produce an enhanced secondary response to the T-independent form of antigen TNP-LPS (trinitrophenylated-lipopolysaccharide). In contrast to the situation in mammals, there was no evidence for affinity maturation during the primary or secondary response. M e m o r y in trout may be due strictly to a simple expansion o f the antigen-specific precursor pool without the selective development of cell clones producing antibodies with increasing affinity. In mammals, this process is classically linked to the transformation of IgM to IgG. The antibody response of fish, restricted to IgM, thus presented no increase in affinity. Concerning this antibody response, Mughal and M a n n i n g (1986) showed that IgGh was strongly antigenic in the mullet and that priming by oral administration was equally as effective as priming by injection. An antibody response in carp was obtained after anal intubation with ferritin ( R o m b o u t et al., 1986). Following a primary injection with SRBC in this species, the spleen contained only 5% of the total number o f PFC, while more than 90% were observed in the anterior kidney (van Muiswinkel et al., 1991). In addition, Bayne (1986) reported that in teleost fish it was impossible to depress the humoral antibody response by splenectomy, since the anterior kidney could complete all the phases of the immune response. The present work has enabled us to demonstrate the reality of a humoral immune response following the intestinal transfer of an antigenic protein. The anterior kidney participates in this response. Acknowledgements--This work was supported by a
grant from CEC (ECLAIR-AGRE 0020). We thank C. Ballagnyfor technical assistance and C. Le Mentec for typing the manuscript. Thanks are also extended to N. Narradon for photographic assistance.
References Arkoosh M. R. and Kaattari S. L. (1991) Development of immunological memory in rainbow trout (Oncorhynchus mykiss). I. An immunochemicaland cellular analysis of the B cell response. Dev. comp. Immunol. 15, 279-293. Bayne C. J. (1986) Pronephric leucocytesof Cyprinus carpio: isolation, separation and characterization. Vet. Immunol. Immunopath. 12, 141-151. Chiller J. M., Hodgins H. O., Chambers V. C. and Weiser R. S. (1969) Antibody response in rainbow trout (Salmo gairdneri). I. Immunocompetent cells in the spleen and anterior kidney. J. Immunol. 102, 1193-1207. Dorin D., Martin P., Sire M. F., Smal J. and Vernier J. M. (1993) Protein uptake by intestinal macrophages and eosinophilic granulocytes in trout: an in vivo study. Biol. Cell 79, 37-44. Ferguson H. W., Claxton M. J., Moccia R. D. and Wilkie E. J. (1982) The quantitative clearance of bacteria from the bloodstream of rainbow trout (Salmo gairdneri ). Vet. Pathol. 19, 687-699. Georgopoulou U. and Vernier J. M. (1986) Local immunological response in the posterior intestinal segment of the rainbow trout after oral administration of macromolecules. Dev. comp. Immunol. 10, 529-537. Georgopoulo U., Sire M. F. and Vernier J. M. (1986) Immunologicaldemonstration of intestinal absorption and digestion of protein macromolecules in the trout (Salmo gairdneri ). Cell Tissue Res. 247, 387-395. Georgopoulou U., Dabrowski K., Sire M. F. and Vernier J. M. (1988) Absorption of intact proteins by the intestinal epithelium of trout, Salrno gairdneri. A luminescence enzyme immunoassay and cytochemical study. Cell Tissue Res. 251, 145-152. Hertz Y., Tchelet A., Madar Z. and Gertler A. (1991) Absorption of bioactive human growth hormone after oral administration in the common carp (Cyprinus earpio) and its enhancement by deoxycholate. J. comp. Physiol. 161B, 159-163. Kaattari S. L. and Irwin M. J. (1985) Salmonid spleen and anterior kidney harbor populations of lymphocytes with different B cell repertoires. Dev. comp. lmmunol. 9, 433-44A,. Lamers C. H. J. and De Haas M. J. H. (1985) Antigen localization in the lymphoid organs of carp (Cyprinus carpio). Cell Tissue Res. 242, 491498. Lamers C. H. J. and Parmentier, H. K. (1985) The fate of intraperitoneally injected carbon particles in cyprinid fish. Cell Tissue Res. 242, 499-503. Le Bail P. Y., Sire M. F. and Vernier J. M. (1989) Intestinal transfer of growth hormone into the circulatory system of the rainbow trout, Salmo gairdneri: interference by granule cells. J. exp. Zool. 251, 101-107. Moriyama S., Takahashi A., Hirano T. and Kawauchi H. (1990) Salmon growth hormone is transported to the circulation of rainbow trout, Oncorhynchus mykiss, after intestinal administration. J. comp. Physiol. 160B, 251-257. Mughal M. S. and Manning M. J. (1986) The immune system of juvenile thick-lipped grey mullet, Chelon
Trout immune response labrosus Risso: antibody responses to soluble protein antigens. J. Fish Biol. 29, 177-186. Muiswinkel W. B. van, Lamers C. H. J. and Rombout J. H. W. M. (1991) Structural and functional aspects of the spleen in bony fish. Res. lmrnunol. 142, 362-366. Nakamura O., Kobayashi M., Suzuki Y., Aida K. and Hanyu I. (1990) Transport of orally administered rabbit IgG into blood circulation of goldfish. Nippon Suisan Gakkaishi 59, 1749-1753. Rombout J. H. and Berg A. A. van den (1989) Immunological importance of the second gut segment of carp. I. Uptake and processing of antigens by epithelial cells and macrophages. J. Fish Biol. 35, 13-22. Rombout J. H. W. M., Berg A. A. van den, Berg C. T. G. A. van den, Witte P. and Egberts E. (1989a) Immunological importance of the second gut segment of carp. III. Systemic and/or mucosal immune responses after immunization with soluble or particulate antigen. J. Fish Biol. 35, 179-186.
CBPB IC9/2-3--V
509
Rombout J. H. W. M., Bot H. E. and Taverne-Thiele J. J. (1989b) Immunological importance of the second gut segment of carp. II. Characterization of mucosal leucocytes. J. Fish Biol. 35, 167-178. Sire M. F. and Vernier J. M. (1992) Intestinal absorption of protein in teleost fish. Comp. Biochem. Physiol. 130A, 771-781. Tatner M. F., Adams A. and Leschen W. (1987) An analysis of the primary and secondary antibody response in intact and thymectomized rainbow trout, Salmo gairdneri Richardson, to human gamma globulin and Aeromonas salmonicida. J. Fish Biol. 31, 17%195. Tsujii T. (1988) The role of renal macrophages in the aglomerular kidney of the sea-horse (teleost) in the removal of exogenous macromolecules from circulating blood. Arch. Histol. Cytol. 51, 327-339. Tsujii T. and Seno S. (1990) Melano-macrophages centers in the aglomerular kidney of the sea horse (Teleosts): morphologic studies on its formation and possible function. Anat. Rec. 226, 460-470.