Immunohistochemical (IHC) Detection of Natural Cytotoxic
Cells in Fish
Kathryn G. Smith; Matthew A. Sand; Niraj
K. Tripathi, BVScAH; Louri J. Caldwell, HTL (ASCP); Donald
L. Evans, PhD; and Kenneth S. Latimer, DVM, PhD
Undergraduate Honors Biology Program (Smith) and Undergraduate, Honors
Microbiology Program (Sand), The University of Georgia, Athens, GA 30602; Department
of Pathology (Tripahti, Caldwell, Latimer) and Department of Medical Microbiology and Parasitology (Evans),
College of Veterinary Medicine, The University of Georgia, Athens, GA 30602
With the evolution of multicellular
organisms, differentiated cells developed to protect these organisms against
pathogens. These differentiated
cells provided the basis of immunity. More complex organisms, such as human
beings, have a more intricate immune system. Therefore, comparative studies
of less complex organisms
provide additional information on immune system development and function. Basic
knowledge and understanding of the of the human immune system have come from
this kind of
comparative research.1
One of the most basic understandings
of immunity is that it provides a two-part defense system; humoral immunity
and cell-mediated
immunity. Humoral immunity involves the production of antibodies
against nonself antigens. Cell-mediated immunity requires viable effector cells
that present
antigens and destroy transformed or infected cells. Natural cytotoxic cells,
like cytotoxic T-lymphocytes, seek and destroy transformed or infected cells.
However, natural
cytotoxic cells recognize and destroy their targets without the need for specific
antigen presentation (this is in contrast to cytotoxic T-lymphocytes that require
specific antigen
presentation to recognize target cells).3 Thus, natural cytotoxic
cells are essential for functional cell-mediated immunity and provide one of
the first lines of
defense to eliminate transformed or infected cells.
The present study was done to determine whether
immunohistochemistry (IHC) could identify natural cytotoxic cells in formalin fixed,
paraffin embedded tissue sections of freshwater and marine fish. The ultimate goals of
this study are to identify and quantitate natural cytotoxic cells in fish tissues and to
determine if this cell population has a seasonal fluctuation that may affect overall
immunity.
Fresh tissue specimens of an ornamental Koi (Cyprinus carpio)
and black sea bass (Centropristis striata) were obtained and fixed in
10% neutral-buffered formalin solution. Formalin fixation did not exceed 24 hours
(Figs. 1 & 2). The tissues were processed routinely, embedded in paraffin wax, and sectioned at
4 µm. Hematoxylin and eosin-stained slides were reviewed and sections of anterior
(head)
kidney and spleen were selected for IHC staining.2 Replicate
tissue sections were cut for IHC, dewaxed, rehydrated through graded alcohols
to buffer, and
subjected to steam treatment to promote antigen retrieval. The primary antibody,
designated 5C6, is a monoclonal IgM antibody that has been shown to specifically
recognize natural cytotoxic cells in the blood of fish via flow cytometry (Fig.
3).3 This
primary antibody was applied at a 1:10 dilution. Appropriate incubation was allowed
for
primary antibody attachment to the targeted site. Following a brief wash in buffer
solution, a secondary biotinylated antibody was added. After appropriate incubation and
washing the avidin-biotin peroxidase complex was applied. A chromagen solution of
3,3’-diaminobenzidine tetrahydrochloride was used (Fig. 4). Following chromagen
development, the sections were stained briefly in Gill’s hematoxylin, dehydrated
through graded alcohols to xylene, coverslipped, and examined microscopically.
Sites of primary antibody attachment were identified by the presence of an insoluble
brown reaction
product.
 |
 |
| Fig. 1. Ornamental Koi (Cyprinus
carpio), a freshwater member of the carp family. |
Fig. 2. Black sea bass (Centropristis
striata), a marine fish that can be raised commercially by mariculture techniques. |
 |
 |
| Fig.
3. Representation of an IgM antibody composed of a pentameric ring structure
linked by disulfide bridges and a "J" chain. Courtesy of DAKO Corporation, Carpenteria, CA. |
Fig. 4. Schematic of
immunohistochemical staining using the avidin-biotin complex (ABC) technique. Courtesy
of DAKO Corporation, Carpenteria, CA. |
Piscine natural cytotoxic
cells were readily indentified in sections of anterior kidney and spleen
following IHC (Fig. 5 & 6). While the human
implications of this research are mostly evolutionary, the ability to study populations
of natural cytotoxic cells will facilitate research on the health and immunity
of fish, a
necessity for sustaining and advancing both aquaculture and mariculture.
 |
 |
| Fig.
5. Histologic sections of anterior kidney demonstrating identification
of natural cytotoxic
cells following immunohistochemical staining. A. Hematoxylin and eosin-stained
tissue section. B. Natural cytotoxic cells stain brown; ABC immunoperoxidase
technique with DAB
chromagen and Gill’s hematoxylin counterstain. |
 |
 |
| Fig.
6. Histologic sections of spleen demonstrating identification of natural
cytotoxic cells
following immunohistochemical staining. A. Hematoxylin and eosin-stained
tissue section. B. Natural cytotoxic cells stain brown; ABC immunoperoxidase
technique with DAB chromagen
and Gill’s hematoxylin counterstain. |
References
1. Pastoret PP, Griebel P, Bazin H, Govaerts A (eds): Handbook of
Vertebrate Immunology, Academic Press, San Diego, CA, 1998.
2. Naish S, Boenisch T, Farmilo AJ, Stead RH: Handbook of
Immunochemical Staining Methods, DAKO Corporation, Carpinteria, CA, 1989.
3. Jaso-Friedmann L, Evans DL, Grant CC, St John A, Harris DT,
Koren HS: Characterization by monoclonal antibodies of a target cell antigen complex
recognized by nonspecific cytotoxic cells. J Immunol 141:2861-2868, 1988. |