International Hepatology

International Hepatology Communications communications 3 (1995) 260-266

Gadolinium chloride-induced Kupffer cell phagocytosis blockade promotes increased

antibody (IgGl) response to orally administered ovalbumin in the rat

Carmen Vidal* ‘, Arturo Gonzhlez-Quintela2, Valentin Cuervas-Mons

Department of Medicine, Clinica Puerta de Hierro, Universidad Autdnoma, Madrid, Spain

Received 22 August 1994; accepted 5 December 1994

Abstract

The effect of Kupffer cell phagocytosis blockade (KCPB) on the production of specific IgE and IgGl antibodies against oral ovalbumin (OVA) was evaluated in an experimental animal model. Two cycles of 15 mg of OVA (each cycle consisting of 3 doses of 5 mg given on 3 con- secutive days, beginning on days 1 and 20) were administered to 5 female Wistar rats by the intragastric route (Group I). In another 5 female Wistar rats, under the same sensitization pro- tocol, KCPB was induced with gadolinium chloride (25 mg/kg body weight, i.v.) 24 h before each antigen cycle (Group II). Antibodies were determined by passive cutaneous anaphylaxis in serum obtained on days 13, 16, 32, 35 and 50. No specific IgE antibodies were detected at any time in any group. Only one animal showed detectable IgGl specific antibodies in Group I, but the response was weak and disappeared on day 32. On the contrary, antibody response appeared more frequently and was more sustained in animals of Group II. Thus, 3 and 4 of them showed IgGl antibodies on days 32 and 35, respectively, persisting up to day 50 (P < 0.05 with respect to the other group). The results suggest that Kupffer cells may represent a barrier to oral sensitization since gadolinium chloride-induced KCPB increases specific antibody responses to intragastrically administered OVA.

Keywords: Hepatic phagocytosis; Kupffer cells; Specific antibodies

* Corresponding author, Praza do Xuncal 1, 2’ A, Perillo. Oleiros, 15172 La Coruiia, Spain. Tel: 34 81 63 63 95; Fax: 34 81 53 12 55.

’ Present address: Allergy Unit, Complejo Hospitalario Santiago de Compostela, Spain. 2 Present address: Department of Medicine, Complejo Hospitalario Santiago de Compostela, Spain.

0928-4346/95/$09.50 0 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0928-4346(94)00188-B

C. Vi&l et al. /International Hepatology Communications 3 (1995) 260-266 261

1. Introduction

The state of specific immunologic unresponsiveness that can be achieved after feeding certain antigens has long been recognized [l]. The classic studies of Chase [ 1] and Cantor and Dumont [2] were the first to demonstrate a reduced cell-mediated and humoral response, respectively, after oral administration of haptens.

The precise basis of this oral tolerance remains to be clarified. The generation of T suppressor cells, deficient T helper cells, anti-idiotypic antibody formation, presence of immune complexes and induction of secretory antibodies by the acces- sory cells of Peyer’s patches have been proposed as possible mechanisms [3]. Studies in the early 1960s suggested that the liver might play a critical role, since the injection of picryl chloride into the mesenteric vein produced solid tolerance, while jugular vein or intraperitoneal injection of the same agent was not as tolerogenic [4]. The position of the liver between the gastrointestinal tract and the general circulation prevents sensitization to potential antigens absorbed into the portal system. In this setting, Kupffer cells, the fixed macrophages of the liver, would appear as pivotal, both removing immunogenic aggregates due to their phagocytic capacity and allow- ing tolerogenic monomers or fragments to reach peripheral lymphoid tissue [3].

Along this line, previous studies of our group demonstrated that the Kupffer cell phagocytosis blockade induced by gadolinium chloride increases the production of IgE and IgGl specific antibodies to a protein antigen (ovalbumin) when repeatedly injected through the portal vein in a rat model [5]. The present study was designed to evaluate the influence of the same model of Kupffer cell phagocytosis blockade on the production of specific IgE and IgGl immune response against ovalbumin when this antigen is inoculated intragastrically in an experimental animal model.

2. Materials and methods

2.1. Animals Female Wistar rats, weighing 150-170 g were used in the sensitization experi-

ments. Unimmunized male Wistar rats, weighing 200-240 g, served as recipients in passive cutaneous anaphylaxis. All animals were purchased from the Animal Breed- ing Unit at Hospital Puerta de Hierro, Autonomous University, Madrid, Spain, and were fed on 2837~CAT Biosure (Laboratory Animal Foodstuff, Barcelona, Spain) which contains no ovalbumin. The International Council for Laboratory Animal Science guidelines for the care and use of the animals were followed.

2.2. Antigen Crystalized ovalbumin (Abel16 Laboratories, Madrid, Spain) was dissolved in

physiologic saline solution at concentrations of 5 and 10 mg/ml and stored at 4°C until used.

2.3. Antibody assays Specific IgE and IgGl were assayed by passive cutaneous anaphylaxis according

to the method previously described [5]. Briefly, 0.1 ml dilutions of sera obtained

262 C. Vidal et al. /International Hepatology Communications 3 (1995) 260-266

from ovalbumin fed-rats were injected intradermally into unimmunized animals. Three and 48 h later for IgGl and IgE determinations, respectively, 2.5 mg of oval- bumin added to 0.5 ml of 0.5% Evan’s blue dye (Fluka Lab., Buchs, Switzerland) was administered through the jugular vein. Local inflammatory reactions in places where specific IgGl and IgE were present appeared as blue spots, the diameters of which were measured. Titres were expressed as the reciprocal of the final dilution yielding a positive reaction (at least 5 mm in diameter).

2.4. Kupffer cell phagocytosis blockade (KCPB) In the corresponding group, KCPB was induced with gadolinium chloride

(Aldrich Chemical Co., Milwaukee, USA) at a dose of 25 mg/kg of body weight, dissolved in 0.5 ml of distilled water and administered through the jugular vein, under light ether anesthesia. The impairment of blood clearance of a tin radiocolloid (Amerscan Hepatate II@, Amersham Int., UK) was used to verify the blocking effect of gadolinium chloride. Phagocytic index (calculated according to the method of Biozzi [6] modified as previously described [S]) was evaluated in six untreated con- trol animals and on days 1,3,8, 14 and 23 after a 25 mg/kg dose of gadolinium chlo- ride in different groups of five rats each.

Gd OVA Gd OVA

8111 I I 1111 I I I I 11.1 I I I Ill1 II

p

0123 13 16 19 202122 32 35 50

DAY

Blood samples

DAY GROUP I (n= 5)

(intragastric OVA) GROUP II (n= 5)

(intragastric OVA pluo Gd)

13th 015 215 (4.4)

16th o/5 o/5

32th l/5 f4’ 315 (19A4)

35th o/5 415 (19,9,4,4)*

50th o/5 415 (19,4,4.3)*

Fig. 1. Scheme of the experimental protocol (top) and results of the study (bottom). For the latter, the proportion of animals with detectable specific IgGl antibodies at each moment is represented. The titres obtained in each of the animals are represented as super-indices between brackets. (OVA, ovalbumin; Gd, gadolinium chloride; l P < 0.05 with respect to the other group).

C. Vi&l et al. /International Heparology CommunicaGons 3 (1995) 260-266 263

2.5. Zntragastric administration of the antigen After a fasting period of 12 h, the antigen was given by gastric intubation using

a 14 gauge needle with its end blunted to prevent abrasion and minimize trauma (Abbocath-T@ , Abbot Ireland Ltd., Ireland). All of the rats were lightly anesthetized to facilitate the administration of ovalbumin solution.

2.6. Experimental protocol As can be seen in Fig. 1, two cycles of 15 mg (10 mg/ml) of ovalbumin (divided

in 3 doses of 5 mg, administered on 3 consecutive days, beginning on days 1 and 20 of the experimental protocol) were given to 10 Wistar rats. Animals were divided into 2 groups. Group I received ovalbumin alone, while in rats of Group II, gadolinium chloride was administered 24 h before initiating each 15 mg cycle of oral ovalbumin. Adjuvants were not used in order to avoid additional interference in macrophage activity.

Blood samples (1.5 ml) for titration of serum antibodies were obtained on days 13, 16, 32, 35 and 50. Sera were stored at -20°C until tested for IgE and IgGl anti- bodies.

2.7. Statistics Fisher’s exact test and the Mann-Whitney U-test were used to compare results

between groups.

3. Results

3.1. KCPB Phagocytic index in control rats and at different times after the injection of

gadolinium chloride is represented in Fig. 2. It was found to be significantly depress- ed 24 h, 3 and 14 days after gadolinium chloride administration with respect to base- line values. The phagocytic index was gradually recovered and almost normalized on day 23.

3.2. Antibody response No specific IgE antibodies were detected at any time in any group. IgGl anti-

bodies were detected in 2 out of 5 rats of Group II 12 days after the first cycle of ovalbumin, but the response was weak and disappeared 3 days later. As can be seen in Fig. 1, 12 days after the second ovalbumin administration, IgGl antibodies were detected in 4 out of 5 rats of Group II with a maximum titre of l/16. The same rats presented detectable IgGl specific antibodies up to 37 days after the administration of the antigen., In contrast, one animal of Group I showed IgGl specific antibodies at day 12 after the second ovalbumin cycle but they were not detected at days 15 and 37. Differences between Group I and II at these times were statistically signifi- cant (P < 0.05).

4. Discussion

Blockade of phagocytosis has been widely used to gain insight into in vivo Kupffer

264 C. Vi&d et al. /International Hepatology Communications 3 (1995) 260-266

Phagocytic index (xi 00) T 10 I-

8

6

Baseline 1 3 8 14 23

Time (days)

Fig. 2. Phagocytic index in normal Wistar rats (baseline) and at different intervals after the administration of intravenous gadolinium chloride (25 mglkg body weight). Mean values and standard deviations are represented. Dotted and open points represent P < 0.01 and P < 0.05 with respect to baseline values, respectively.

cell functions [6]. Gadolinium chloride is a lanthanide which seems to be particularly useful for this purpose, as it induces the inhibition of the initial phases of phagocyto- sis by Kupffer cells [7]. Moreover, recent studies showed that gadolinum chloride not only blocks but also transiently eliminates large Kupffer cells from the periportal zone of the rat liver acinus after a single intravenous injection [8]. Machrophage populations other than Kupffer cells are less vulnerable to the action of the intrave- nous injection of gadolinium chloride since phagocytic activity does not decrease (and even increases) in the spleen, lungs and bone marrow [7]. The present study, for which a radiocolloid that is taken up mainly by the liver was used, further con- firms the significant, prolonged and reversible inhibition of reticuloendothelial phagocytosis after a single gadolinium chloride intravenous injection. Previous stud- ies have demonstrated that the same effect can be achieved after a second injection when liver Kupffer cell repopulation has taken place and liver phagocytosis restored WI.

Our results show an increased IgGl specific response against oral ovalbumin when liver phagocytosis is inhibited with gadolinium chloride prior to the antigen adminis- tration. Previous studies, in experimental animal models, showed that antibody responses to oral antigens are enhanced in circumstances in which liver function is altered. Thus, after the induction of hepatic cirrhosis with oral phenobarbitone and inhaled carbon tetrachloride, the susceptibility of rats to oral administration of a protein antigen is enhanced [9]. To explain that situation, the authors resorted to

C. Vidal et al. /International Hepatology Communications 3 (1995) 260-266 265

the disrupted antigen sequestration by Kupffer cells which would favour normal peripheral antigen processing. Indirect evidence for this hepatic effect is provided by the presence of increased antibody titres to gut-associated bacteria, specific anti- bodies to food antigens (normally located in the gastrointestinal tract) and polyclonal hypergammaglobulinemia which are commonly seen in patients with ad- vanced liver diseases closely related to the impairment of the phagocytic activity of Kupffer cells [lo].

The surgical portacaval shunt which anatomically bypasses the liver by diverting portal blood flow from the gastrointestinal tract away from the liver directly into the systemic circulation has been used as another model to investigate the role of the liver in oral tolerance. For instance, Cantor and Dumont showed, as early as 1967, that the state of specific humoral unresponsiveness normally achieved after feeding a potent hapten (pycril chloride) disappeared when portal blood was diverted from the liver after undergoing portacaval transposition [2]. Years later, Callery et al. obtained similar results using a model of delayed cellular hypersensitivity and a pro- tein antigen (ovalbumin), thus indicating a critical role of the liver in oral tolerance [ll]. According to our results, Kupffer cells may be at least a component of the cellular bases of this phenomenon.

It has been reported that reticuloendothelial phagocytosis blockade could pro- mote the production of antibodies to parenterally administered antigens in experi- mental animal models [12]. To our knowledge, the present study is the first to demonstrate increased sensitization to a protein antigen (ovalbumin) repeatedly ad- ministered by the oral route after KCPB. In a previous work, we observed that the liver represented a barrier to sensitization to ovalbumin administered directly into the portal vein of heterozygous Gunn rats [5]. If it is assumed that Kupffer cells rep- resent mere scavengers, it could be supposed that KCPB could permit antigens to bypass the liver and reach other organs (i.e. the spleen) thus increasing serum anti- bodies. However, we have previously shown that gadolinium-induced KCPB not only abolished the inhibitory effect of the liver in inducing an IgE or IgGl immune response to ovalbumin introduced into the portal system, but also markedly increas- ed the antibody production to repeated doses of the antigen injected by the same route. In the same study, the rise in antibody production after gadolinium chloride- induced KCPB did not seem to be the result of a systemic modulation, since a similar rise in antibody response was not observed when ovalbumin was injected through the jugular vein after KCPB.

Along this line, the results of the present study also suggest that the liver may act as a barrier to antigens located in the gastrointestinal tract, preventing them for in- ducing systemic humoral responses. Thus, oral administration of ovalbumin failed to elicit specific IgE and IgGl antibodies, but when KCPB was induced with gadolinium chloride, serum specific IgGl antibodies were detected in 4 out of 5 rats. The titres obtained were low, but it should be taken into account that adjuvants were not used in the sensitization protocol. The reason why specific IgE antibodies were not detected in the present study remains unclear. In this sense, the different results obtained with respect to our previous studies [5] may be due to the strain of rat employed (Wistar rat vs. Gunn rat) and the route of administration of the antigen.

266 C. Vi&l et al. /International Hepatology Communications 3 (199.5) 260-266

It is important to note that when ovalbumin is inoculated intragastrically, inter- actions with lymphoid organs in the intestinal tract cannot be avoided. Finally, the dosage of antigen and the schedule of feeding are important parameters in the induc- tion of either sensitization or tolerance and, perhaps, the experimental protocol was good enough for producing IgGl but not IgE antibodies.

In conclusion, although systemic effects of gadolinium chloride cannot be ruled out from this study, results support the idea that the liver (and particularly Kupffer cells) may be an important part of the enteric mucosal immune barrier. Further stud- ies are needed to verify this fact, as well as the intrahepatic or extrahepatic mechanisms responsible for increased antibody production after gadolinium chloride-induced KCPB.

Acknowledgements

The authors thank Mrs. Martha Messman for the English translation of the manu- script.

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