Elaine F. Lizzio

Suppression of Antibody-Producing Cells in Rainbow Trout Spleen Sections Exposed to Copper in Vitro

Abstract Immunosuppression was demonstrated in sections of rainbow trout Oncorhynchus mykiss (formerly Salmo gairdneri) spleens immunized in vitro and exposed in culture to different concentrations of copper chloride. The sections were immunized with dinitrophenyl-Ficoll and cultured in Eagles minimum essential medium with 2% fetal calf serum; half of the medium was withdrawn and replaced every other day. The passive hemolytic plaque assay was used to determine the number of antibody-producing cells 10 d after injection. In the sections cultured with the high copper concentration (100 μg/mL), all cells died; at copper concentrations of 0.1–10 μg/mL, leukocytes remained viable, but fewer antibody-producing cells were present than in organ sections cultured in medium without copper. This in vitro method reduces the number of animals needed and the length of time required to determine toxicity and immunosuppression, and it provides information on the effects of certain environmental pollutants on fish.

Immunostimulation by Levamisole in Rainbow Trout (Salmo gairdneri) in Vivo

The use of immunostimulants for boosting the defense mechanisms and protection against diseases in fish is of increasing interest to fish culturists. Several promising adjuvants, drugs and biological response modifiers have been tested in experiments with fish. Evelyn (1974) injected Freunds complete adjuvant with formalin-killed Renibacterium salmoninarum and found an elevated antibody titer compared to control fish. Kitao et al (1986) showed that the phagocytic activity was increased by the drug, FK-565, and later (Kitao et al. 1987) showed that it also caused a slight elevation of numbers of antibody-producing cells and serum antibody titers when given with specific bacterins. BCG (Bacille Calmette Guerin) extract and saponin Quil-A also have been shown to stimulate the nonspecific immune response in rainbow trout (Grayson et al. 1987).

Release of arachidonic acid metabolites by human monocytes or lymphocytes: Effect of treatment with interferon on stimulation by phorbol ester or calcium lonophore

The simultaneous production of prostaglandins, leukotrienes, and hydroxyeicosatetraenoic acids was studied in human peripheral blood monocytes obtained by counter-current centrifugal elutriation. Monocytes prelabeled for 4 hr with [3H]arachidonic acid (AA) released label into the surrounding medium in response to treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) or calcium ionophore (A23187). High-performance liquid chromatography of monocyte supernatants demonstrated that labeled compounds included those which eluted with authentic standards for thromboxane B2, 12-L-hydroxy-5,8,10-heptadecatrienoic acid (HHT), 6-keto-prostaglandin F alpha, prostaglandin E2, and 15-hydroxyeicosatetraenoic acid (HETE). 5-HETE and leukotriene B4 (LtB4) were detected only in response to ionophore treatment. Highly purified lymphocytes did not convert AA to autocoids, despite the release of free arachidonate in response to either stimulus. Pretreatment of monocytes with recombinant human interferon (IFN)-gamma or IFN-alpha for 18 hr resulted in enhanced release of labeled arachidonic acid and increased conversion to autocoids after TPA or ionophore stimulation. Absolute amounts of prostaglandin E2 produced in response to TPA or ionophore treatment were increased as well. These results demonstrate the autocoid profile released by stimulated human monocytes and illustrate the effects of IFN treatment on the production of lipoxygenase metabolites of arachidonic acid as well as cyclooxygenase products.

Immunization and culture of rainbow trout organ sections in vitro

Splenic and anterior kidney sections or whole organs were excised from large (1 kg) or small (200 g) rainbow trout (Salmo gairdneri) and placed in sterile 60 mm plastic plates containing 10 ml of Eagles minimal essential medium (EMEM) supplemented with normal or fetal calf serum for in vitro culture. The organ samples were immunized in vitro by direct injection or by mixing in the medium Yersinia ruckeri O-antigen or dinitrophenyl-Ficoll. The medium was changed once during the 10-day incubation at 15 C. The passive hemolytic plaque assay demonstrated antibody production from the plaque-forming cells (PFC); passive hemagglutination was used to measure antibody titers in the media. High numbers of PFC occurred in cultures of either kidney or spleen, demonstrating that these organs can function independently for antibody production. Splenic sections from large fish produced more PFC than comparable whole organs from small fish. EMEM supplemented with 2% normal calf serum was a satisfactory culture medium. 2-hydroxyethyl-mercaptan an ingredient used in mammalian cell culture, inhibited antibody production in trout cells. These techniques are being used in the culture of organs and cells to elucidate pathways and sequences of antigen uptake and delivery of the immunopoietic tissues in trout.

Flush exposure and injection immunization of rainbow trout to selected DNP conjugates

Rainbow trout (Salmo gairdneri) were immunized by flush exposure or intraperitoneal injection with single doses of the following dinitrophenyl (DNP) conjugates: DNP-keyhole limpet hemocyanin (KLH), DNP-Ficoll, DNP-O-antigen (from the fish pathogen, Yersinia ruckeri), DNP-sheep red blood cells, or DNP-duck red blood cells. The immune response was demonstrated by the passive hemolytic plaque assay to show splenic antibody-producing cells (APC) 14 days after antigen administration. High numbers of splenic APC specific for the hapten were found when the conjugates DNP-KLH, DNP-Ficoll, and DNP-O-antigen were given by injection and when DNP-O-antigen was given by flush exposure. The heterologous red blood cells were relatively nonimmunogenic. When the immune response to the hapten and carrier could be measured--e.g., after immunization with the DNP-O-antigen--the numbers of APC were consistently higher for the hapten.

Organ-Specific Cytokine Polarization Induced by Adoptive Transfer of Transgenic T Cells

There are two distinct phenotypes of T cell cytokine responses that lead to different effector functions and different outcomes in disease processes. Although evidence suggests a possible role of the local microenvironment in the differentiation or localization of T cells with these phenotypes, there are no examples of divergent T cell cytokine phenotypes with the same Ag specificity concurrently existing in different tissue compartments. Using a CD8+ T cell adoptive transfer model for graft-vs-host disease, we demonstrate that a potent type 2 cytokine response develops in the spleen while a potent type 1 cytokine response simultaneously develops in the testis. These experiments demonstrate for the first time that cytokine production can be oppositely polarized in different organs of the same individual. This may have important implications for organ-specific pathology in infection or autoimmunity: infections or autoimmune diseases that affect multiple organs may have heterogeneity in tissue cytokine responses that is not revealed in systemic lymphocyte cytokine responses. Therefore, attempts to modulate the immune response phenotype may ameliorate pathology in one organ while exacerbating pathology in another.