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.

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.

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

During 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 (greater than 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.

Channel catfish as an unconventional model for immunological studies.

The channel catfish, Ictalurus punctatus, is an economically important species which is readily available, acclimates well to the laboratory setting, and is amenable to considerable experimental manipulation. Although the channel catfish is still a relatively circumscribed species in terms of comprehensive physiologic and/or endocrinologic studies, our current understanding of the basic immunobiology and immunochemistry of the channel catfish is significantly further advanced than for any other teleost species. In this respect the channel catfish is not only proving useful in the general areas of comparative immunology but it is also showing considerable promise as a model system for definitive studies on problems which bridge the fields of immunology and endocrinology, i.e., understanding the effects of environmental temperature and stress on the immune system.

Hydatidiform mole macromolecules inhibit interleukin-2-mediated murine lymphocyte proliferation in vitro.

Macromolecules extracted from hydatidiform mole trophoblast inhibit mitogen-induced lymphocyte proliferation. To characterize the mechanism of this immunomodulation, we determined the effects of hydatidiform mole vesicle fluid (HMF) and tissue extracts (HME) on lymphokine function in vitro. Utilization of interleukin-1 (IL-1) and interleukin-2 (IL-2) were determined by using a lymphoma cell line (LBRM-33-1A5) and a murine T cell line (CTLL2), respectively. HMF suppressed (P less than .05) IL-2-dependent CTLL2 cell proliferation at 500 (36.4% of controls) and 50 (74.9% of controls) micrograms/ml. HME also suppressed CTLL2 proliferation (P less than .05) at 500 (46.0% of controls), 100 (67.2% of controls), 50 (71.5% of controls), and 10 (85.4% of controls) micrograms/culture ml. In contrast, HMF exhibited no effect on IL-1-stimulated LBRM-33-1A5 production of IL-2. However, 500 micrograms/ml of HME inhibited (P less than .05) IL-2 production (63.0% of controls) in the IL-1 utilization assay. This suppressive effect was probably due to a carry over of HME from the LBRM-33-1A5 culture to the target cells (CTLL2) used to measure IL-2 production. Molecular weight chromatography of an HME sample eluted an IL-2 inhibitor in a low molecular weight (35-50 kd) and high molecular weight (greater than 250 kd) fraction. These data suggest that one way in which macromolecules derived from hydatidiform mole could interfere with in vitro immunologic responses is by modulating interleukin-2 function.

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.

Blood serum chemistry measurements of normal and acutely stressed channel catfish.

1. An automated blood serum chemistry analytical system designed for human usage was employed to establish the levels of 26 different components present in sera obtained from various experimental groups of channel catfish. 2. Comparisons of samples from feral and commercial production pond fish during warm months indicated statistically significant differences in the serum levels of sodium, CO2, urea nitrogen, direct bilirubin, cholesterol, creatinine and protein. 3. Laboratory acclimated and production pond fish exhibited differences in serum electrolytes (sodium, potassium, chloride, phosphorus), serum metabolites (urea nitrogen, creatinine, triglycerides), serum enzymes [gamma-glutamyl transferase, glutamate-oxaloacetate transaminase (GOT), lactate dehydrogenase (LDH), alkaline phosphatase, and amylase], and serum iron. 4. Seasonal (temperature?) differences in production pond fish were noted for 12 serum components including potassium, magnesium, CO2, glucose, creatinine, albumin, iron, alkaline phosphatase, and glutamate-pyruvate transaminase (GPT). 5. Comparisons of samples obtained from laboratory-acclimated fish before and 18 hours after acute handling and transport stress revealed significant differences in only three serum parameters: glucose, LDH, and creatine phosphokinase (CPK). 6. These studies suggest that "normal" values established by any method of sera analysis may be different in the same species depending on the diet, season, and presence of environmental stressors.

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.

Phylogeny of lymphocyte heterogeneity: identification and separation of functionally distinct subpopulations of channel catfish lymphocytes with monoclonal antibodies.

The purpose of this study was to identify monoclonal antibodies reactive with channel catfish T cells. Since a variety of commercially available anti-human and mouse T cell reagents failed to react with channel catfish leucocytes, the development of anti-fish T cell monoclonal antibodies was undertaken. One of these reagents, designated mAb 13C10, reacted with channel catfish surface immunoglobulin negative, but not positive, lymphocytes. This reagent, when used in "panning" protocols, was able to isolate those channel catfish lymphocytes which provide helper activity for antibody synthesis to a thymus dependent antigen. In addition, this antibody was observed to react with most thymocytes, neutrophils, and thrombocytes, few hepatocytes, and with some brain cells (possibly neurons). It was concluded that this antibody reacts with relatively high molecular weight antigens on channel catfish T cells and that it may be a pan anti-T cell reagent (possibly akin to Thy-2) for this species.

Analysis of developmentally defective chemical signaling mutants of Polysphondylium violaceum.

Six aggregation-defective mutants of Polysphondylium violaceum dependent on external addition of the pheromone D factor for aggregation were isolated after nitrosoguanidine mutagenesis. With a screening technique based on synergistic development, D-factor-dependent mutants can be separated from other kinds of aggregateless mutants. Genetic complementation analyses of the newly isolated mutants showed them to be mutant at the aggA locus. Individual mutants exhibited different sensitivities to D factor(s), responding maximally over a 300-fold range of concentrations.