Abstract

Unlike the immune system of terrestrial mammals, particularly man and mouse, very little is known about the immune system of marine mammals, especially the cetaceans. Dolphins, whales and porpoises are of considerable interest from an evolutionary standpoint because it is thought they left land some 55 to 60 million years ago and adapted to life in a total aquatic environment. Moreover, cetaceans are thought to have evolved from primitive insectivores and to share common ancestors with either ungulates or carnivores. The question arises as to what changes or modifications have taken place in the immune system of cetaceans during their evolutionary history in adapting to life in a total aquatic environment.

The hematology of the bottle nosed dolphin has been well studied. While it is known that dolphins show a strong leukocytic response to infection and that their total white blood cell count is 10,000/mm³ (20% of which are peripheral blood lymphocytes) it is not known what the percentages of T and B cells are in dolphin peripheral blood and if different subsets of lymphocytes exist.

Two previous studies are available on the classification of T and B lymphocytes of dolphins. It was shown previously that dolphin lymphocytes can respond to the mitogens phytohemagglutinin, a T cell specific mitogen and pokeweed mitogen, a B cell specific mitogen. Another study showed that dolphin sera contained more agglutinins for human B lymphocytes than for T lymphocytes. However, no cell surface markers were used in these studies which would allow for positive identification of dolphin lymphocyte subsets. There are many studies available, however that have looked at T and B cell ratios in terrestrial mammals such as human, rodent, cow, pig, horse, sheep, dog and cat.

The purpose of this study was to identify lymphocyte subsets in the bottle nosed dolphin. To do this we used an antiserum to dolphin immunoglobulins which we raised and characterized, and a monoclonal antibody to human MHC class H molecules which we have shown to cross-react with dolphin class H homologues.

Twenty mls of blood each were sampled from male and female subjects of Tursiops truncatus from the ventral aspect of the tail stock. The blood obtained was from clinically healthy dolphins with total white blood cell counts and lymphocyte counts within normal ranges. Mononuclear cells were isolated from whole blood with Ficoll-Paque and washed with RPMI 1640. The red blood cells were lysed with ammonium chloride. The lymphocytes were counted on a hemocytometer with trypan blue exclusion as a measure of viability. The cells were washed twice in Hank's Balanced Salt Solution (HBSS) and resuspended to a final concentration of 0.5x10⁶ cells/ml.

The lymphocyte suspension was aliquoted in Eppendorf tubes and centrifuged at 10,000 rpm for 10 sec. The supernatant was aspirated and the cells were incubated with 50 µl of either dolphin anti-rabbit immunoglobulin (Dlg) or mouse anti-human class II at 10 µg/ml for 1 hour. The cells were washed 3x with HBSS and subsequently incubated with either rabbit Ig-specific goat Fab2 conjugated to fluorescein isothiocyanate, or mouse Ig-specific goat Fab2 conjugated to phycoerythrin secondary antibodies for 1 hour. Samples were then washed twice in phosphate buffered saline and fixed in 500 µl of 1% paraformaldehyde. For double labeling studies, samples were incubated with one of the primary antibodies and the appropriate secondary followed by labeling with the other primary and appropriate secondary antibody. Samples were analyzed on a Fluorescent Activated Cell Sorter (FACS). Forward/Side scatter plots were obtained for each sample. During FACS analysis, lymphocytes were gated based on their size and low degree of granularity.

FACS analysis showed that 90-98% of the lymphocytes shifted to positive fluorescence from controls in the three dolphins tested for class II molecules. Although each animal displayed a different degree of fluorescence, the degree remained consistent for each individual animal from experiment to experiment. These results indicated that the majority of dolphin peripheral blood lymphocytes are class II positive. In terrestrial mammals such as man and mouse class II molecules are expressed by a majority of B lymphocytes rather than T lymphocytes. FACS analysis showed that approximately 10-15% of dolphin peripheral blood lymphocytes are B lymphocytes, like many other terrestrial mammals, suggesting that the remaining class II positive cells are probably T lymphocytes. This was further clarified with double fluorescence with the percentage of DIg+ and class II+ or Dlg- and class II+ in close approximation with the single histogram statistics obtained for single labeling with DIg and class II-specific antibody.

In conclusion the T/B cell ration in dolphin peripheral blood lymphocytes appears to be similar to that observed in other mammals; however, unlike man and mouse, their T lymphocytes may constitutively express class II molecules. We will continue these studies by trying to positively identify dolphin T lymphocytes with T cell markers. We hope to obtain blood samples from dolphins in the wild to compare with the data we have from dolphins in captivity and to obtain blood samples from a variety of species to understand if class II expression on dolphin peripheral blood lymphocytes reflects environmental or evolutionary factors.

***A manuscript of these studies was submitted to Immunogenetics in January, 1990 for publication.