Stephen L. Kaattari

Cortisol mediated suppression of salmonid lymphocyte responses invitro

The suppressive activity of cortisol on the in vitro induction of coho salmon (Oncorhynchus kisutch) B cell activation was examined. Suppression was observed with splenic and pronephric (anterior kidney) derived lymphocytes. The kinetics of cortisol-induced suppression revealed distinct differences in the sensitivity of splenic and pronephric lymphocytes. Pronephric lymphocytes were only sensitive to cortisol early in the induction of the antibody response, whereas the splenic cells were sensitive to cortisol throughout the culture period. Addition of supernatants from antigen stimulated pronephric cultures completely restored the ability of pronephric lymphocytes to produce an antibody response, suggesting that this glucocorticoid-suppression may be mediated by inhibition of lymphokine production.

Ontogeny of IgM and IgM-bearing cells in rainbow trout

We have studied the ontogenic development of immunoglobulin M (IgM) and of IgM-bearing cells in the rainbow trout, Oncorhynchus mykiss. Lymphocytes showing cytoplasmic IgM were first observed in embryos at 12 days before hatch (14 degrees C). At this stage, no cells positive for surface IgM were present. Lymphocytes bearing surface IgM were observed at 8 days before hatch (14 degrees C). Unfertilized trout eggs contained detectable amounts of IgM (11.2 +/- 2.6 micrograms/g of egg weight), indicating that transfer of IgM from mother to embryo can occur in salmonids. The levels of IgM from whole fish increase slowly after the appearance of intraembryonic cells that express surface IgM. The amount of IgM/g of tissue peaks around hatch, but this parameter shows lower values up to 2 months after hatch.

Salmonid spleen and anterior kidney harbor populations of lymphocytes with different B cell repertoires

An O-antigen extract of the fish pathogen Vibrio anguillarum was used to stimulate plaque-forming cells (PFC) in coho salmon. The kinetics of the response demonstrates peak PFC per organ on day 16 post immunization for the spleen and anterior kidney. Significant PFC were also seen in thymic tissue. PFC responses were shown to be immunoglobulin mediated and antigen specific. Inhibition profiles of responding PFC populations demonstrate that the cells of the anterior kidney are more restricted in their recognition of antigen than those of the spleen. These data lend functional support to the concept of the hematopoietic nature of the anterior kidney of teleosts.

The recombination activating gene 1 (RAG1) of rainbow trout (Oncorhynchus mykiss): cloning, expression, and phylogenetic analysis

The characterization of genes involved in the generation of the immune repertoire is an active area of research in lower vertebrate taxa. The recombination activating genes (RAG) have been shown to be essential for V (D) J recombination of T-cell antigen receptor (TCR) and immunoglobulin (Ig) genes, leading to the generation of the primary repertoire. As RAG1 is critical to the differentiation of pre-B and-T cells, its expression within an associated primary lymphoid organ can serve as a developmental marker. To examine the ontogeny of lymphocytes in Oncorhynchus mykiss, we cloned RAG1 from trout and examined its tissue-and lymphocyte-specific expression. The polymerase chain reaction, coupled with degenerate oligonucleotide primers, was used to amplify a homologous probe [(633 base pairs) (bp)] from rainbow trout genomic DNA, which in turn was used to isolate a lambda genomic clone. Sequence analysis of this genomic clone confirmed the RAG1 nature of this gene (3888 bp) and revealed an internal intron of 666 bp. When compared with other previously reported RAG1 sequences, the predicted amino acid translation (1073 aa) displayed a minimum of 78% similarity for the complete sequence and 89% similarity in the conserved region (aa 417-1042). Using northern blot analysis, we found the expression of RAG1 to be limited to surface Ig-n lymphocytes within the thymus. This data forms the basis for a proposal that the thymus of teleost species plays an essential developmental role in lymphopoiesis and thus can be regarded as a primary lymphoid organ.

5 - The Specific Immune System: Humoral Defense

This chapter discusses the mechanism of humoral defense in the fish. The mechanisms involved in specific humoral defense have been the most thoroughly explored of all the modes of piscine disease resistance. Piscine classes, such as elasmobranchii and osteichthyes, possess extremely wide and varied means of accomplishing physiological goals, including the development of prophylactic immune responses. Initial characterizations of fish antibody structure led to the general supposition that fish Ig was rather comparable to mammalian IgM. C3b as an opsonin can be recognized by a C3b receptor on phagocytic cells. The existence of a possible receptor has been demonstrated in the catfish. This was accomplished by using a yeast cell wall material, zymosan, to stabilize C3b. The first line of humoral defense, where pathogens can be most effectively blocked or neutralized, is the mucosal surface. It is found that when arrayed with an opsonin, such as C3b, phagocytic cells can recognize and digest the pathogen. It is observed that isotype switching and affinity maturation are two other common features of mammalian immunological memory that are primarily associated with the monomeric form of immunoglobulin.

THE RECOMBINATION ACTIVATING GENE 2 (RAG2) OF THE RAINBOW TROUT ONCORHYNCHUS MYKISS

We have previously described the isolation and expression ofRAG1 in trout to provide an initial understanding regarding the tissues involved inV(D)J recombination of antigen receptors in this teleost. Here we report that the recombination activating gene 2 (RAG2) of rainbow trout has now been cloned and characterized. The rainbow trout genomicRAG2 gene (1602 base pairs) displays an average of 60% and 75% similarity at the nucleotide and amino acid level when compared with clones from other species and was found to contain an acidic region in the carboxyl terminal end, which is typical ofRAG2 sequences. The proximity ofRAG1 and −2 within this teleost is similar to that found in other vertebrates. The genes are convergently transcribed and share a 3′ untranslated (UT) region [2.8 kilobases (kb)] which is much shorter than that found in higher vertebrates (6–8 kb). The entire 3′ UT region was also sequenced and used in conjunction with cDNA clones to identify the polyadenylation sites for bothRAG genes. Northern blot analysis of one-year-old trout demonstrated strong expression ofRAG2 in the thymus, with a much weaker signal being detected in the pronephros. Using reverse transcriptase-polymerase chain reaction, we detected the highest expression of bothRAG1 and −2 in the thymas followed by the pronephros, with much fainter signals being observed in the spleen, mesonephros, and liver. Finally, both genes are expressed in embryos begining at approximately day 10 post-fertilization. Taken together, these findings indicate that the thymus and pronephros most likely serve as the primary lymphoid tissues in trout, based uponRAG expression. In addition, the trout sequences may provide further insight into the evolution and origins of theRAG genes as well as that of the immune system itself.

Development of immunological memory in rainbow trout (Oncorhynchus mykiss). I. An immunochemical and cellular analysis of the B cell response

In vivo and in vitro analyses of the antibody responses of rainbow trout (Oncorhynchus mykiss) confirmed the existence of immunological memory in this species. An enhanced in vitro secondary antibody response was found to be due strictly to an expansion of the antigen-sensitive precursor pool without a concomitant increase in clone size. In contrast to the development of immunological memory in mammalian species, there was no evidence for affinity maturation during the primary or secondary response. A distinct shift in the fine specificity profiles of the antibodies, however, did occur during the generation of the secondary response. Additionally, more than a single injection of the priming antigen, TNP-KLH was required to produce an enhanced in vitro response to this T-dependent antigen. However, a second priming injection was not required to produce an enhanced secondary response to the T-independent form of antigen, TNP-LPS. These results indicate that memory in trout may be due to a simple expansion of the antigen-specific precursor pool without many of the qualitative changes in antibody or B cell function associated with the expression of memory in mammals.

Effectiveness of an Oral Enteric Coated Vibrio Vaccine for use in Salmonid Fish

An oral enteric-coated Vibrio anguillarum vaccine was developed by initially coating lyophilized bacteria onto 0.9 mm diameter dextrose sugar beads followed by an Eudragit L-30D coat to serve as enteric protection. Vaccine efficacy was determined by both an in vivo challenge with live pathogen and measurements of serum and mucus antibody levels by ELISA. Survival rates after challenge were 80.3%, 83.3% and 70.3% among the vaccine group, positive and negative controls respectively. Serum and mucus antibody levels were found to be significantly greater in the vaccine group (p less than 0.01). In cases where equivalent survival among tested groups is observed, determination of antibody titer by ELISA may be a preferable indicator of vaccine efficacy.

Fish B lymphocytes: Defining their form and function

Abstract Fish B lymphocytes have been defined, as in mammalian systems, as those lymphocytes that express immunoglobulin on their surface and secrete specific antibody in response to antigenic stimuli. Such cells have been identified in a number of fish species. However, the physical characterization of these cells, their distribution, the form of their response, and the antibody product itself have not precisely followed the mammalian paradigm. The implications of these unique features of fish B cell form and function are the focus of this review. The antigenic phenotype of the B cell and the possible function of distinct subpopulations are discussed. The origin of the fish B lymphocyte itself is still a great enigma. The developmental and ontological pathway(s) taken by fish B cell progenitors are still virtually unknown, but considerable data have accrued that allow investigators to postulate possible sources of such lymphocytic stem cells (i.e. bone marrow equivalents), such as the anterior kidney. As opposed to the ontological development of B cells, the nature of the intercellular cooperation and lymphokine requirements for an antibody response seem to parallel mammalian counterparts quite closely. Finally, the coordination of all these cells, factors, and products into a complex function, such as the generation of immunological memory, present an excellent venue for comparative immunologists. In the expression of this complex and highly regulated process, fish have demonstrated some basic similarities to mammals, but differ in other important features. Understanding how fish have circumvented the need for these aspects of memory allows us not only to understand fish function, but enables us to place specific mammalian functions in a broader context. Such analyses will provide new theoretical approaches to the understanding of mammalian as well as fish immune responses.

Polyclonal activation of salmonid B lymphocytes

The process of in vitro polyclonal activation of coho salmon (Oncorhynchus kisutch) lymphocytes was examined with respect to the induction of mitogenesis, total immunoglobulin production, and the production of specific antibodies or plaque forming cells. These studies demonstrate that antigen specific stimulation of antibody production is not linked to mitogenic activity, or total immunoglobulin production, while the polyclonal activation of specific antibody production is closely linked to these functions. Stimulation of immunoglobulin production by phytohemagglutinin suggests that this mitogen may not be limited to T cell activation in salmonids or, alternatively, it may induce the production of lymphokines capable of polyclonally activating B cells. Further, fetal calf serum was found to cause production of large amounts of immunoglobulin in vitro without antigenic stimulation.