ABSTRACT

The described procedure is a simple and rapid method for isolating pure lymphocytes from carp gut epithelium. The advantage of the described technique of i-IELs extraction is that it allows for the extraction of the cells from each carp, making it possible to study their characteristics on an individual basis in fish. In this procedure, gut intraepithelial lymphocytes are separated from the epithelium layer and other lymphoid cells by Percoll gradient centrifugation. Since this procedure results in high recovery and purity in a preparation of i-IELs , there is no need for multiple nylon wool filtrations. The obtained i-IELs population could be utilised for molecular, biochemical or phenotypic characterisation, as well as for functional evaluation, such as proliferative and cytotoxic activities.

Key words: carp, intraepithelial lymphocytes, gut, fish

Introduction

Mammal gut-associated lymphoid tissue (GALT) comprises three major lymphoid areas: Peyer's patches (PP), lamina propria (LP) and an intraepithelial compartment, which contains the intraepithelial lymphocytes (IEL). It is well known that PP and LP cells contain more than 40% of B cells and a large percentage of CD4⁺ T cells. Additionally, lymphocyte populations of the PP and LP continuously re-circulate in the blood and lymphatic systems.

On the other hand, intestinal intraepithelial lymphocytes (i-IELs) undergo minimal re-circulation and do not require the thymus for their maturation (POUSSIER and JULIUS, 1994), which suggests that their role is specific for their tissue site. The majority of i-IELs in all vertebrates (in rodents, through chickens to humans) express CD8⁺ (suppressor/cytotoxic) phenotype (ERNST et al., 1986). Also, i-IELs contain high percentages (approximately 30-50%) of T cells expressing gdTCR (LEFRANCOIS, 1991), whereas gdTCR is present on approximately 5% of thymocytes and circulating T cells, except in calves, where peripheral blood lymphocytes express more than 20% gdTCR (WATERS et al., 1995).

While the i-IELs precise biologic functions remain enigmatic, numerous findings made during the past few years demonstrate that mammal i-IELs may participate in immunity (FINDLY et al., 1993; MOMBAERTS et al., 1993; BOZIC et al., 1998) as well as in non-immune functions (BOISMENU and HAVRAN, 1994; 1997; BOISMENU et al., 1996).

Apart from numerous differences in the immune system between mammals and lower vertebrates, notably fish (MANNING, 1994; RAST et al., 1997; MAGOR and VASTA, 1998), i-IELs obviously exist in the gut of teleosts (HART et al., 1986, 1988; ROMBOUT et al., 1993). Indeed, i-IELs have recently been isolated from carp gut (ROMBOUT et al., 1998) and it seems likely that this gut lymphocytes population represent a very large number of putative T cell types. However, the procedure of isolation has not been described comprehensively. Therefore, we describe a simple and rapid technique for isolation of carp i-IELs in detail here.

Materials and methods

Fish

Twelve-month-old carp, Cyprinus carpio L., with initial masses of approximately 200 g, were kept in an aquarium (100 l) under laboratory conditions in running, filtered tap water with constant flow rate and temperature (19±1 °C). The fish were fed daily with commercially prepared pellet food (Ribnjak "Berlek", Ptuj, Slovenia), amounting to 2% of their body mass. Ten fish were used in the experiment.

Necropsy procedures

After euthanasia by spinal rupture, the gut was excised for isolation of intestinal intraepithelial lymphocytes. In order to isolate i-IELs, immediately after excision, collected intestines were placed in a tissue culture medium (TCM), consisting of RPMI 1640 without Ca²⁺/Mg²⁺, plus 10% foetal calf serum (FCS).

Percoll solutions

For separation of i-IELs from enterocytes and other cell types, the material was centrifugated in Percoll gradients. Before the preparation of Percoll gradients, it must be prepared in a stock solution. A 1× stock solution was used by mixing 90 ml Percoll (Sigma) with 10 ml of 10 × phosphate-buffered saline (PBS). These solutions must be freshly prepared.

For 40% Percoll a solution was mixed consisting of 40 ml of 1× stock solution and 60 ml RPMI 1640 without Ca²⁺/Mg²⁺. Also, for a 70% Percoll solution, 70 ml of 1× stock solution and 30 ml RPMI 1640 was mixed without Ca²⁺/Mg²⁺.

Harvesting of carp i-IELs

Intestinal intraepithelial lymphocytes were isolated from carp gut by a modified procedure previously described for rodents i-IEL isolation (LEVENTON et al., 1983; MOSLEY and KLEIN, 1992; LEFRANCOIS, 1993). This procedure was adapted to the fish system. After fish euthanasia, proximal and distal segments of gut were removed from one individual carp and were placed in a Petri dish containing RPMI 1640 plus 10% FCS. Intestines were held in forceps and the tip of a 20-G-needle with syringe filled with cold TCM, was forced into the lumen between forceps, flushing the entire gut thoroughly in TCM to remove faecal material, with 30 ml (total) cold TCM. Intestines were replaced in a Petri dish containing fresh TCM and connective tissue was removed from intestinal samples. Gut samples were cut longitudinally, and then into small segments, 1-1.5 cm in length. These segments were then transferred to 50-ml conical tubes with 40 ml cold PBS without Ca²⁺/Mg²⁺ and were washed by inverting the tubes several times. This step was repeated three times. The intestinal pieces were then transferred into a 100-ml Erlenmeyer flask containing 80 ml PBS without Ca²⁺/Mg²⁺, plus 1 mM ethylenediamine tetraacetic acid disodium salt (EDTA) (Sigma). The samples were stirred in this solution for 30 min at 37 ⁰C. The solution, together with gut pieces, was then transferred to a 50-ml conical centrifuge tube and vortexed for 10 sec at maximum setting. When the pieces had settled, the supernatant was transferred to another 50-ml conical tube and pelleted by centrifugation (400 × g for 8 min). Remaining intestinal tissues were then removed. The collected pellet was re-suspended in 40% Percoll (Sigma). The cells in the 40% Percoll (10 ml) were overlaid onto 2 ml of 70% Percoll solution, placed in a 15-ml conical tube and i-IELs were separated from enterocytes by centrifugation (600 × g for 20 min) at room temperature. The top half of the gradient was aspirated to within approximately 2 cm of the interface and i-IELs were then harvested from 40:70 interface with a Pasteur pipette. Cells were diluted 1:3 with cold RPMI 1640 without Ca²⁺/Mg²⁺, and centrifugated for 8 min at 800 × g to pellet. The purified i-IELs (> 96% pure) were more than 98% viable as determined by 0.2% trypan blue exclusion staining. Freshly isolated i-IELs were enumerated under a light microscope.

Results

It is evident that there was an abundant number of i-IELs in the gut of carp. The quantity mean (± SD) of i-IEL collected from one individual carp used in the basic protocol was 5.0 ± 0.4 × 10⁷ for proximal and second gut segment. The mean (± SD) number of viable cells was 4.9 ± 0.5 × 10⁷. Although the count was variable depending on the initial amount of intestinal mucous, probably due to individual differences, the yield of i-IEL was generally consistent between different extractions (Fig. 1). Purity exceeded 96% (contamination due to epithelial and other cells was negligible) and viability was over 98%, as determined with trypan blue exclusion staining.

Fig. 1. Recovery of intraepithelial lymphocytes from the proximal and distal gut of carp. Absolute number obtained from individual fish (N=10) revealed consistency between different i-IEL extractions.

Discussion

The described procedure is a simple and rapid method for isolating extremely pure lymphocytes from carp gut epithelium. The advantage of the described technique of i-IELs extraction is that it allows for extraction of cells from carp on a per fish basis. Thus, a large amount of i-IELs can be obtained from individual fish, making it possible to study their characteristics on an individual basis. It is important to note that the high reproducibility was observed after centrifugation of cells in Percoll gradients at 600 × g for 20 min or at 1500 × g for 30 min, as has been previously proposed (ROMBOUT et al., 1998). The obtained absolute number of i-IELs in this current study was, however, significantly smaller than i-IEL numbers recently isolated from carp gut epithelium by others (ROMBOUT et al., 1998). These differences in the numbers of lymphocytes are probably due to techniques of i-IELs isolation. Namely, the present i-IELs extraction procedure differs from the i-IEL isolation method performed by ROMBOUT et al. (1998), in that the lamina propria remained intact after digestion of the epithelium (MOSLEY and KLEIN, 1992), but scraping off gut mucosa from the underlying tissue could cause damage to the lamina propria and subsequently result in contamination from lymphocytes in that compartment and resulting in a greater number of cells in i-IEL preparation (our unpublished results). Additionally, it is important that procedures of i-IEL isolation do not penetrate into the lamina propria also, due to changes in the phenotype of i-IELs (ERNST et al., 1986).

This study provides evidence that, in contrast to higher vertebrates, the gut of carp contains more intestinal intraepithelial lymphocytes, although the higher vertebrates, such as calves, possess a larger population of lymphocytes within their intestinal epithelium (WATERS et al., 1995) than do carp. However, this is not the case in rodents, such as mice and rats. The i-IEL yield was found to be variable for these rodent species, not exceeding 15 x 10⁶ in rats (TEITELBAUM et al., 1995) and 10 x 10⁶ in mice (LEVENTON et al., 1983; MOSLEY and KLEIN, 1992; LEFRANCOIS, 1993). We consider the abundant i-IEL numbers recovered by the procedure described here and elsewhere (ROMBOUT et al., 1998) to be an important immunological hallmark in teleost fish. Together with excessive mucous production, compared to higher vertebrates, a very large population of i-IEL could benefit fish survival in a non-hostile aquatic environment. Interestingly, preliminary observations in this laboratory indicated that a certain drop in i-IEL numbers could be accompanied by enteropathy found in stressed carp.

Despite the numerical importance of carp i-IELs, and the fact that they arise in the immediate vicinity of the largest permanent antigenic stimulus in the whole organism, data on their origin, site of development and phenotypic characteristics, are generally lacking. Indeed, the gdTCR⁺ CD8⁺ T cells in i-IEL of carp (ROMBOUT et al., 1998) have recently been suggested.

In conclusion, the abundant number of carp i-IELs, as a possible front line of defence at epithelial barriers, could constitute an excellent model system for novel, functional studies of fish immunity, with the aim of benefitting aquaculture. Also, fish i-IELs represent a potential model system for comparative studies of immunology, which could assist in unravelling the complexities of the mammalian immune system.

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Received: 2 November 1998
Accepted: 10 April 1999

BOZIC, F.: Izdvajanje intraepitelnih limfocita iz crijeva sarana (Cyprinus carpio L.). Vet. arhiv 69, 97-103, 1999.