VETERINARSKI ARHIV 69 (2),
97-103, 1999

ISSN 1331-8055 Published in
Croatia

Isolation of carp (Cyprinus
carpio L.) intestinal intraepithelial lymphocytes

Frane Bozic*

Department of Pharmacology and
Toxicology, Faculty of Veterinary Medicine,
University of Zagreb, Zagreb,
Croatia

* Contact address:
Frane Bozic, DVM,

Department of Pharmacology and Toxicology, Veterinary Faculty, University of
Zagreb, Heinzelova 55, 10000 Zagreb, Croatia,
Phone: 385 1 23 90 163; Fax:
385-1-214-697; E-mail: [email-masked]

BOZIC, F.: Isolation of carp
(Cyprinus carpio L.) intestinal intraepithelial lymphocytes. Vet. arhiv
69, 97-103, 1999.

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.

References

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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.

SAZETAK

Opisani postupak jednostavna je i brza
metoda za izolaciju cistih limfocita iz crijevnoga epitela šarana. Prednost
opisane metode izdvajanja limfocita je u tome što se njome moze izdvojiti
dostatan broj stanica iz samo jedne ribe. Crijevni intraepitelni limfociti
razdvojeni su od epitelijalnih elemenata i ostalih limfnih stanica
centrifugiranjem u gradijentu gustoce Perkola. Radi velikoga broja i iznimne
cistoce izoliranih stanica, nepotrebno je procišcavanje filtracijom kroz
višeslojnu najlonsku vunu. Dobivena populacija crijevnih intraepitelnih
limfocita moze se rabiti u svrhu molekulske, biokemijske ili fenotipske analize,
jednako kao i za istrazivanje njihove funkcijske uloge, primjerice proliferacije
i citotoksicnosti.

Kljucne rijeci: šaran,
intraepitelni limfociti, crijevo, ribe

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