Immunology of whales and dolphins.

The increasing disease susceptibility in different whale and dolphin populations has led to speculation about a possible negative influence of environmental contaminants on the immune system and therefore on the health status of marine mammals. Despite current efforts in the immunology of marine mammals several aspects of immune functions in aquatic mammals remain unknown. However, assays for evaluating cellular immune responses, such as lymphocyte proliferation, respiratory burst as well as phagocytic and cytotoxic activity of leukocytes and humoral immune responses have been established for different cetacean species. Additionally, immunological and molecular techniques enable the detection and quantification of pro- and anti-inflammatory cytokines in lymphoid cells during inflammation or immune responses, respectively. Different T and B cell subsets as well as antigen-presenting cells can be detected by flow cytometry and immunohistochemistry. Despite great homologies between marine and terrestrial mammal lymphoid organs, some unique anatomical structures, particularly the complex lymphoepithelial laryngeal glands in cetaceans represent an adaptation to the marine environment. Additionally, physiological changes, such as age-related thymic atrophy and cystic degeneration of the "anal tonsil" of whales have to be taken into account when investigating these lymphoid structures. Systemic morbillivirus infections lead to fatalities in cetaceans associated with generalized lymphoid depletion. Similarly, chronic diseases and starvation are associated with a loss of functional lymphoid cells and decreased resistance against opportunistic infections. There is growing evidence for an immunotoxic effect of different environmental contaminants in whales and dolphins, as demonstrated in field studies. Furthermore, immunomodulatory properties of different persistent xenobiotics have been confirmed in cetacean lymphoid cells in vitro as well as in animal models in vivo. However, species-specific differences of the immune system and detoxification of xenobiotics between cetaceans and laboratory rodents have to be considered when interpreting these toxicological data for risk assessment in whales and dolphins.

Molecular cloning and characterization of beluga whale (Delphinapterus leucas) interleukin-1beta and tumor necrosis factor-alpha.

Interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) are cytokines produced primarily by monocytes and macrophages with regulatory effects in inflammation and multiple aspects of the immune response. As yet, no molecular data have been reported for IL-1beta and TNF-alpha of the beluga whale. In this study, we cloned and determined the entire cDNA sequence encoding beluga whale IL-1beta and TNF-alpha. The genetic relationship of the cytokine sequences was then analyzed with those from several mammalian species, including the human and the pig. The homology of beluga whale IL-1beta nucleic acid and deduced amino acid sequences with those from these mammalian species ranged from 74.6 to 86.0% and 62.7 to 77.1%, respectively, whereas that of TNF-alpha varied from 79.3 to 90.8% and 75.3 to 87.7%, respectively. Phylogenetic analyses based on deduced amino acid sequences showed that the beluga whale IL-1beta and TNF-alpha were most closely related to those of the ruminant species (cattle, sheep, and deer). The beluga whale IL-1beta- and TNF-alpha-encoding sequences were thereafter successfully expressed in Escherichia coli as fusion proteins by using procaryotic expression vectors. The fusion proteins were used to produce beluga whale IL-1beta- and TNF-alpha-specific rabbit antisera.

Molecular cloning, phylogenetic analysis and expression of beluga whale (Delphinapterus leucas) interleukin 6.

Interleukin 6 (IL-6) is a cytokine produced primarily by the monocytes/macrophages with regulatory effects in hematopoiesis, acute phase response, and multiple aspects of the immune response. IL-6 exerts its activity through its binding to specific high affinity receptors at the surface of target cells. As yet, no molecular data have been reported for the beluga whale IL-6. In this study, we cloned and determined the entire beluga whale IL-6-encoding cDNA sequence by reverse transcription-polymerase chain reaction (RT-PCR) sequencing, and analysed its genetic relationship with those from several mammalian species including human, rodent, ruminant, carnivore and other marine species. The identity levels of beluga whale IL-6 nucleic and deduced amino acid sequences with those from these mammalian species ranged from 62.3 to 97.3%, and 42.9 to 95.6%, respectively. Phylogenetic analysis based on amino acid sequences showed that the beluga whale IL-6 was most closely related to that of the killer whale. Thereafter, beluga whale IL-6-encoding sequence was successfully expressed in Escherichia coli by using the pTHIOHisA expression vector for the production of a recombinant fusion protein. The immunogenicity of the recombinant fusion protein was then confirmed as determined by the production of a beluga whale IL-6-specific rabbit antiserum.

Molecular cloning and characterization of CD4 in an aquatic mammal, the white whale Delphinapterus leucas.

Given the importance of the cell surface recognition protein, CD4, in immune function, the cloning and characterization of CD4 at the molecular level from an odontocete cetacean, the white whale (Delphinapterus leucas), was carried out. Whale CD4 cDNA contains 2662 base pairs and translates into a protein containing 455 amino acids. Whale CD4 shares 64% and 51% identity with the human and mouse CD4 protein, respectively, and is organized in a similar manner. Unlike human and mouse, however, the cytoplasmic domain, which is highly conserved, contains amino acid substitutions unique to whale. Moreover, only one of the seven potential N-linked glycosylation sites present in whale is shared with human and mouse. Evolutionarily, the whale CD4 sequence is most similar to pig and structurally similar to dog and cat, in that all lack the cysteine pair in the V2 domain. These differences suggest that CD4 may have a different secondary structure in these species, which may affect binding of class II and subsequent T-cell activation, as well as binding of viral pathogens. Interestingly, as a group, species with these CD4 characteristics all have high constitutive expression of class II molecules on T lymphocytes, suggesting potential uniqueness in the interaction of CD4, class II molecules, and the immune response. Molecular characterization of CD4 in an aquatic mammal provides information on the CD4 molecule itself and may provide insight into adaptive evolutionary changes of the immune system.

Immune functions in beluga whales (Delphinapterus leucas): evaluation of natural killer cell activity.

Natural killer (NK) activity, an important non-specific defense mechanism against viral infections and tumors, was demonstrated in beluga whales using two different methods: 51Cr release and flow cytometry. Using the 51Cr release assay, NK activity in belugas was shown to be higher against K-562 than against YAC-1 cell lines. Moreover, it was enhanced by the addition of human recombinant interleukin-2 with both cell lines. NK activity evaluated by flow cytometry in the peripheral blood of eight belugas increased when the effector:target cell (E:T) ratio increased, and averaged 13.9% +/- 3.8% (range 9.9% to 17.8%) at an E:T ratio of 100:1. While NK activity could be readily detected using both methods, the lack of radio-isotopes and related laboratory room make the flow cytometric method a viable and safe alternative. The evaluation of this function in cetaceans could lead to a better understanding of the early events that lead to viral epizootics in populations of marine mammals in different parts of the world, as well as to the high prevalence of neoplasms in St. Lawrence beluga whales.

Natural antibodies to human lymphocytes and erythrocytes in the serum of Orcinus orca killer whale.

The existence of naturally occurring heterophile antibodies to antigenic determinants on human blood cell membranes has long been known. It has been shown that the serum of Orcinus orca (Killer whale) does contain similar antibody. Absorption techniques in concert with either microagglutination or complement-dependent microcytotoxicity assays revealed at least three antibody specificities erythrocyte (RBC), B-lymphocyte and T-lymphocyte. Human erythrocyte specificity has been separated from other mammalian RBC specificity, and higher microagglutination titers and/or scores were observed with human group A RBC's than with group B,O, or AB. Tests run at 4 degrees, 20 degrees and 37 degrees C). Higher microcytotoxicity and microagglutination activity was demonstrated with B versus T lymphocytes. It is hoped that the characterization of the antigenic specificity of these heterophile agglutinins will prove to be useful as a biological reagent-tool which may be applied to the identification of a new receptor on human lymphocytes and/or erythrocytes. Also, if isolated, these agglutinins could be useful in the study of the occurrence and presence of specific receptors on cell membranes and give insight as to how these receptors change in health, disease and malignancy.

Antigenic structure of sperm whale myoglobin. I. Partition of specificities between antibodies reactive with peptides and native protein.

We have resolved anti-sperm whale Mb antibodies into two distinct populations: One population is reactive only with the native molecule, and the second is reaction with both Mb and peptide fragments. CNBr cleavage of sperm whale Mb yields three peptides: peptide I (1-55), peptide II (56-131), and peptide III (132-153). Immunoadsorbent columns made with these three peptides were used to fractionate antibody to sperm whale Mb. These columns cumulatively bind 60 to 70% of the total antibody present in the four sera studied (the amount of antibody bound by each peptide was proportional to its size). The remaining 30 to 40% of antibody could only bind specifically to an immunoadsorbent column of native Mb. This result indicates that not all antigenic determinants on the native Mb molecule are represented on peptide fragments.

Antigenic structure of sperm whale myoglobin. II. Characterization of antibodies preferentially reactive with peptides arising in response to immunization with the native protein.

The antigenicity of peptide fragments derived from CNBr-cleaved sperm whale Mb has been tested in competitive inhibition assays. These peptides appeared to be very poor inhibitors in a reaction involving radiolabeled sperm whale Mb with antipeptide antibodies, requiring about 10(4)-fold excess of peptides to achieve the same level of inhibition as cold native Mb. However, cold peptides competed much better with radiolabeled peptides for antibody binding than the native protein. These antibodies, preferentially reactive with peptides, originated from sera of animals immunized with the native molecule. This indicates heterogeneity of the antibodies produced against Mb and suggests the presence of diverse antibody subpopulations reactive with different antigenic forms of the protein. Results presented in this paper illustrate that competitive radioimmunoassays preferentially measure antibodies to the radiolabeled species, and this phenomenon can be used to demonstrate distinct populations among antibodies isolated to a single peptide.

T cell clones reactive with sperm whale myoglobin. Isolation of clones with specificity for individual determinants on myoglobin.

We have been able to isolate clones of sperm whale muscle myoglobin (Mb)-reactive T cells from (C57BL/6 x A/J)F1 [(B6A)F1] mice. Four types of clones were isolated, distinguished by their patterns of recognition of Mb cyanogen bromide (CNBr) fragments and antigen presenting cell (APC) requirements. Individual T cell clones proliferated in response to one of three CNBr fragments of Mb. Dose-response curves of all clones were identical for native Mb and the appropriate fragment. T cell clones reactive to fragment 1-55 did not proliferate in response to peptide 15-22 (a peptide that binds to serum antibody directed against 1-55). These data support previous findings suggesting differences between antigen recognition by T and B cells, i.e., T cells may not recognize antigen in its native conformation and/or T and B cells may recognize distinct epitopes on the same antigen. Using T cell clones to analyze genetic control of responsiveness to Mb, we found that certain (B6A)F1 T cells recognize Mb presented by low responder strain APC. Thus, genetically determined low responsiveness in this case is probably not due to failure of APC function. We also found that responsiveness to certain Mb epitopes mapped to the I-A subregion whereas others mapped, via gene complementation, to the I-A and I-E subregions. We found no examples of responsiveness mapping to the I-C subregion and suggest an alternative explanation for previous reports mapping genetic control of responsiveness to certain Mb determinants to I-C.