Catherine F. Ellsaesser

Cortisol-induced hematologic and immunologic changes in channel catfish (Ictalurus punctatus)

1. Intravenous injections of physiologic doses of cortisol resulted in both hematologic and immunologic changes in channel catfish peripheral blood leucocytes. These changes mimicked those seen when catfish were acutely stressed by handling and transport. 2. Eighteen hours after the administration of cortisol, decreases in the number of circulating lymphocytes and concomitant increases in the number of circulating neutrophils were observed, i.e. to the same levels seen previously in stressed fish. 3. Functional analysis of peripheral blood leucocytes from cortisol-injected fish indicated that the remaining lymphocytes were no longer capable of responding to mitogenic stimuli. 4. This suppression of mitogenic stimuli was not seen when peripheral blood leucocytes were cultured in vitro with physiologic doses of cortisol. 5. This latter observation suggests that the cortisol alone was probably not directly responsible for the loss of responsiveness but possibly acted in vivo as an initiator of other events that eventually resulted in the observed immunosuppression.

Temperature-mediated processes in teleost immunity: Differential effects of invitro and invivo temperatures on mitogenic responses of channel catfish lymphocytes☆

The in vitro mitogenic responses of channel catfish peripheral blood leucocytes to ConA and LPS were differentially affected by both in vitro and in vivo temperatures. The magnitude of the response to LPS was relatively independent of both in vitro culture temperature and in vivo acclimation temperature. The magnitude of the response to ConA was suppressed at lower in vitro temperatures although this suppression could be reduced by lower in vivo acclimation temperatures. In vitro temperature-shift experiments indicated that channel catfish PBL could respond to ConA at a lower in vitro temperature if first stimulated with ConA at a higher in vitro temperature. The converse, however was not true in that channel catfish PBL did not respond at a higher in vitro temperature after an initial stimulation with ConA at a lower in vitro temperature. This latter failure to respond could not be attributed to the induction of a suppressor cell (or factor) by exposure to ConA at a lower temperature. These studies, when coupled with other available data on channel catfish PBL subpopulations, are interpreted as supporting the hypothesis that low temperature immunosuppression in fish may result from preferential inhibitory effects on T cells rather than B cells.

Phorbol ester/calcium ionophore activate fish leukocytes and induce long-term cultures

This study documents that phorbol ester (TPA) and calcium ionophore (A23187) in combination are potent mitogens for channel catfish peripheral blood leukocytes (PBL), stimulating both catfish T and B cells. Unlike T-cell responses to Concanavalin A (ConA), these responses to TPA/A23187 did not appear to require monocytes and were not strongly inhibited by low culture temperature. These results support the notion that catfish lymphocytes utilize the bifurcating phosphatidylinositol bisphosphate second-messenger system for transmembrane signaling during the activation process, as do mammalian lymphocytes. Furthermore, it was unexpectedly found that TPA/A23187 stimulation of normal catfish PBL reproducibly (greater than or equal to 95%) resulted in the generation of long-term leukocyte cultures that did not require restimulation or the addition of exogenous factors for continued proliferation. These TPA/A23187-induced leukocyte cultures were refractory to cloning and appeared to contain 10-40% monocytes and 50-80% putative T cells with no detectable B cells or neutrophils.

Analysis of Channel Catfish Peripheral Blood Leucocytes by Bright-Field Microscopy and Flow Cytometry

Abstract Peripheral blood leucocytes of channel catfish Ictalurus punctatus were characterized by Wrights stain, differential cytochemical stains, and immunofluorescence; lymphocytes, thrombocytes, neutrophils, and monocytes were identified. The morphologically similar neutrophils and monocytes were distinguished with a Sudan black B stain for neutrophils and an assay of nonspecific esterase activity for monocytes. Monocytes also phagocytized polystyrene beads. Leucocytes were physically separated by cytofluorography based upon their forward and 90° light-scattering properties. One population of sorted cells contained almost exclusively lymphocytes, a second contained predominantly thrombocytes, and the third contained both neutrophils and monocytes. Multiparameter cytofluorography with mouse monoclonal antibodies to channel catfish immunoglobulin revealed that a vast majority of leucocytes exhibiting surface immunofluorescence were lymphocytes, though only about 40% of the lymphocytes contained demonstr...

Phylogeny of lymphocyte heterogeneity: the thymus of the channel catfish

The number of thymocytes (approximately 3 x 10(7)) that were recoverable from fingerling channel catfish remained constant from about 3 to 10 months of age, i.e. from September to April following hatching the previous June. Between 11 and 12 months, i.e. May and June, the thymus dramatically increased in size with 3 x 10(9) thymocytes being recoverable from the tissue of individual fish. The thymus remained enlarged for several months (throughout the summer) but at about 15 months (in September) began to involute such that by 17 months (November) no thymus tissue could be seen macroscopically. This natural involution could be accelerated by subjecting the fish to handling and transport stress. Thymocytes of channel catfish aged 4 to 16 months exhibited reactivity with monoclonal antibodies against peripheral T cells but not B cells. Thymocytes responded to the mitogen Concanavalin A only in the presence of added accessory cells (peripheral blood monocytes) or a monocyte-derived supernatant (presumably containing IL-1) at permissive temperatures (27 degrees C). Thymocytes could also be induced to divide at nonpermissive temperatures (17 degrees C) when incubated in the presence of the following combinations of stimulants, a) the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and the calcium ionophore A23187, b) TPA and ConA, or c) A23187 and ConA. In those cases where TPA or A23187 were used, accessory cells or their products were not needed. Collectively, these results support the notion that channel catfish thymocytes functionally mimic those lymphocytes in the peripheral blood previously designated as T cells.

Spontaneous development of functionally active long-term monocytelike cell lines from channel catfish.

SummaryDuring the course of studies involving the in vitro manipulation of channel catfish peripheral blood leukocytes, spontaneous proliferation was observed with unexpectedly high frequency. Propagation of these spontaneously proliferating cells has resulted in the development of long-term (>11 mo.) cell lines which stain positively for nonspecific esterase and peroxidase, are phagocytic for latex beads, and morphologically resemble mammalian monocytes or macrophages. These long-term cell lines also exhibit two important additional functional features. First, induction with lipopolysaccharide results in the secretion of relatively high levels of catfish high and low molecular weight species of interleukin-1 active on channel catfish and mouse T cells, respectively. Second, these cell lines are efficient antigen-presenting cells to autologous peripheral blood leukocytes for antigen specific in vitro proliferative and antibody responses. This antigen-presenting function is blocked by inhibitors known to prevent antigen processing and presentation by mammalian monocytes. Allogeneic mixtures of cell line (used as antigen-presenting cells) and responding peripheral blood leukocytes, however, resulted in strong mixed leukocyte reaction but not in specific antibody responses. The availability of such cell lines should facilitate further studies on accessory cell functions in fish immune responses.

Functionally distinct high and low molecular weight species of channel catfish and mouse IL-1

Culture supernatants from channel catfish monocytes exhibit IL-1-like activity for mouse and catfish T cells. Gel filtration analyses of these supernatants indicated that there were at least two forms of IL-1-like activity, i.e. a high molecular weight form (70 kD) active on channel catfish, but not mouse, T cells and a low molecular weight form (approximately 15 kD) with activity for mouse, but not catfish, T cells. Both sizes of catfish IL-1 exhibited alpha and beta determinants as shown by Western blot analyses using antisera to human IL-1 alpha and IL-1 beta. Further evidence for the IL-1 nature of these molecules was obtained by antibody inhibition assays wherein antisera to human IL-1 alpha and IL-1 beta each neutralized approximately 50% of the catfish activities, were additive to some extent, and could be reversed by the addition of the proper human recombinant protein. In culture supernatants of murine P388D1 cells functional activities for catfish and mouse T cells were found only in high and low molecular weight fractions, respectively. Western blots with antiserum to mouse IL-1 alpha revealed IL-1 determinants in both high and low molecular fractions of the mouse cell culture supernatants. These data suggest that catfish and mammalian IL-1 molecules may be quite similar with the caveat being that functional activity for catfish T cells requires a large protein, presented as an aggregate, a polymer, or simply a single chain 70 kD protein. However, only the low molecular weight forms (30 and 15 kD) are active on mouse T cells.

A new method for the cytochemical staining of cells immobilized in agarose

SummaryA quick, simple and inexpensive technique utilizing an isotonic matrix of semi-solid agarose for immobilizing channel catfish (Ictalurus punctatus) peripheral blood leucocytes onto microscope slides before cytochemical staining is described. The technique was demonstrated to be superior to the more conventional means of immobilizing cells for procedures requiring either fixed (Sudan Black B and nonspecific esterase stains) or viable (Nitro-Blue Tetrazolium stain) cells. Although the agarose immobilization technique was developed with channel catfish leucocytes, its utility with other fragile cell populations seems likely.