Mitogen and growth factor-induced activation of a STAT-like molecule in channel catfish lymphoid cells.

This article describes the identification of a putative STAT molecule in the channel catfish (Ictalurus punctatus), the first report of such a molecule in a 'lower' vertebrate. A monoclonal antibody against human STAT6 recognizes an approximately 100 kDa molecule that becomes activated and translocates to the nucleus upon both growth factor and mitogen stimulation of catfish leukocytes. This presumed catfish STAT binds the mammalian interferon-gamma activation site, a known motif of mammalian STAT binding, as shown by electromobility shift assays. Purification of the proteins present in these DNA complexes confirms that the catfish reactive molecule binds to the interferon-gamma activation site sequence. These results suggest that STAT molecules have been highly conserved in vertebrate evolution.

T-cell receptors in channel catfish: structure and expression of TCR alpha and beta genes.

Herein are reported full length cDNA sequences for TCR alpha- and beta-chains of the channel catfish. Included are sequences belonging to four Valpha and six Vbeta families which share hallmarks in common with the Valpha and Vbeta genes of other species. Similar to the situation in other vertebrates, the catfish Calpha and Cbeta sequences exhibit distinct immunoglobulin, connecting peptide, transmembrane and cytoplasmic domains. However, the catfish TCR Calpha and Cbeta regions are shorter than those of mammals and the catfish Cbeta chain lacks a cysteine in its connecting peptide region. Two different catfish Cbeta cDNA sequences were identified, suggesting the existence of either two Cbeta loci or allotypes. Based on Southern blot analyses, each of the catfish TCR gene loci appear to be arranged in a translocon (as opposed to multicluster) organization with multiple V elements and a single or few copies of C region DNA. At the deduced amino acid level, the catfish Cbeta sequence exhibits 42% identity with the Cbeta of Atlantic salmon, 41% identity with the Cbeta of rainbow trout and 26% identity with Cbeta of the horned shark. The catfish Calpha amino acid sequence exhibits 44 and 29% identity with Calpha of the rainbow trout and southern pufferfish, respectively. TCRalpha and beta messages are selectively expressed and rearranged in a catfish clonal cell line that appears to be of the T lineage. This TCR alpha/beta expressing clonal lymphocyte line, designated 28S.1, has T-cell like function in that it constitutively produces a supernatant factor(s) with growth promoting activity. These findings should facilitate functional studies of fish TCRs and T cells in ways not previously possible with other 'lower' vertebrate models.

Expression of a mouse-channel catfish chimeric IgM molecule in a mouse myeloma cell.

Fusion genes encoding a murine VH domain and the constant region domains of the mu chain from the channel catfish, Ictalurus punctatus, were stably expressed in the lambda light chain producing mouse myeloma cell line J558L. Although the pathways of pre-mRNA processing for expression of membrane (micron and secreted (microsecond) forms of the mu chain differ between mammals and teleosts, mRNAs encoding both catfish micron and microsecond were correctly expressed in the mouse myeloma cells. The mouse-channel catfish chimeric mu chain polypeptide was able to associate covalently with the mouse lambda light chain and assemble, intracellularly, into polymers of covalent structure (microL)2-8 which resembled those seen with native catfish IgM. In contrast to native catfish IgM, the mouse-catfish chimeric IgM showed the property of binding strongly to protein A of Staphylococcus aureus. The mouse-channel catfish chimeric IgM was core-glycosylated, but did not contain terminal sialic acid. Secretion rates for the chimeric IgM were low, and the possibility could not be excluded that extracellular chimeric IgM was released from dead or dying cells. The reason(s) for the intracellular retention of the chimeric IgM (probably in the endoplasmic reticulum) are not known, but those mechanisms involving retention via cysteine residues were excluded.

Microsystem for in vitro primary and secondary immunization of channel catfish (Ictalurus punctatus) leukocytes with hapten-carrier conjugates.

Methods are described for the in vitro generation and detection of antibody-secreting cells (PFC) from channel catfish. Hapten-specific PFC can readily be enumerated by an indirect plaque assay employing rabbit antiserum to catfish Ig and guinea pig complement. A modified Mishell-Dutton-type culture system was developed for effectively generating significant in vitro anti-hapten PFC responses with catfish leukocytes at 27 degrees C. The classical hapten-carrier effect and primary responses to both TI and TD antigens were demonstrable with catfish cells. Variables found to be important with catfish cells included the serum supplement, cell densities and, to a lesser extent, antigen form. Optimistically these methods will prove useful in attempts to delineate the functional roles of different lymphocyte subpopulations in fish.

Use of particulate antigen to enhance primary in vitro antibody responses to trinitrophenyl-keyhole limpet hemocyanin.

Methods are described for the efficient induction of primary anti-hapten antibody responses to trinitrophenyl-keyhole limpet hemocyanin (TNP-KLH) in vitro. Mouse leukocytes stimulated in vitro with particulate (bentonite-adsorbed) TNP-KLH demonstrated 10-fold more IgM-secreting, plaque-forming cells (PFC) as measured by a hemolytic plaque assay than did leukocytes stimulated with soluble TNP-KLH. The T dependency of the enhanced antibody response was confirmed using highly enriched B and T lymphocyte populations. These methods should greatly facilitate studies regarding primary in vitro antibody responsiveness to T-dependent antigens.

A monoclonal antibody specific for neutrophils in normal and stressed channel catfish.

A murine monoclonal antibody, designated mAb 13C5, was found to react specifically with channel catfish neutrophils based upon its ability to identify a subpopulation of leukocytes that are phagocytic and stain positive with both Sudan Black B and nitro-blue tetrazolium. Cytofluorographic analysis with mAb 13C5 was used to assess trafficking of neutrophils in various tissues of catfish subjected to transport stress. Since no prestress neutrophil reservoir was apparent, it seems likely that stress-induced neutrophilia in catfish, as in endotherms, may result from demargination of a pool of capillary-bound neutrophils. MAb 13C5 was also used to successfully "pan" for neutrophil-enriched and depleted populations of peripheral blood leukocytes from transport stressed channel catfish. "Panning" indicated that the continued physical presence of elevated numbers of neutrophils is not responsible for the suppression of T and B cell in vitro proliferation responses to mitogens observed in stressed fish.

Cellular pathway(s) of antigen processing in fish APC: effect of varying in vitro temperatures on antigen catabolism.

Studies were conducted to determine the effects of varying in vitro temperatures during cellular processing of T-dependent antigens by catfish antigen-presenting cells (APC). Strategy involved pulsing of long-term monocyte lines (used as APC) with antigen at different temperatures for various periods of time and then fixing and coculturing with autologous peripheral blood leukocytes (PBL) as responders at a permissive temperature (i.e., 27 degrees C). Results showed that APC incubated with antigen at low, nonpermissive, but physiologically relevant, temperatures (11 and 17 degrees C) elicited secondary proliferative responses by autologous PBL. However, responses elicited with APC pulsed at 11 and 17 degrees C required longer exposure to the antigen prior to fixation (i.e., greater than or equal to 8 h compared to 5 h, the optimal incubation time at 27 degrees C). Further, there was sufficient cell-associated antigen during a short-term pulsing period (1-2 h) at 11 and 17 degrees C to provide efficient presentation after subsequent incubation of the APC at 27 degrees C for an additional 10 h before fixation. In contrast, APC pulsed with antigen at an extremely low, physiologically irrelevant, temperature (4 degrees C) for extended periods of time did not elicit the proliferation of autologous responders unless antigen pulsing was carried out for 1-2 h and the APC subsequently shifted to 27 degrees C for an additional 10 hr. Antigen uptake assays revealed significant and similar levels of internalized antigen at 11, 17, and 27 degrees C, but not at 4 degrees C. However, intracellular degradation and formation of trichloroacetic acid-soluble antigen catabolites at 11 and 17 degrees C appeared to occur at a slower rate compared to APC at 27 degrees C. Significantly, primary anti-hapten plaque-forming cell responses were also observed with PBL cocultured with APC pulsed with hapten-carrier conjugates at 11, 17, and 27 degrees C. Consequently, the previously observed suppression of primary T-cell responses in fish at low, physiologically relevant, temperatures is not due to impaired antigen processing or presentation.

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.

Characterization of anti-hapten antibodies generated in vitro by channel catfish peripheral blood lymphocytes.

Secondary in vitro stimulation of channel catfish peripheral blood lymphocytes with haptenated T-dependent antigen (TNP-KLH) elicited large numbers of hapten-specific Ab-producing cells and relatively high levels (10-96 micrograms/mL) of TNP-specific Ab in the culture medium. These in vitro generated Abs were compared to in vivo generated Abs from the serum of the same fish with respect to covalent structure, affinity, and isotypic composition of heavy and light chains. SDS-PAGE analysis under both reducing and nonreducing conditions revealed that the in vitro Abs were structurally similar to the serum Abs. Similarly, in vitro pulse-labeled Abs also exhibited the eight band profile characteristic of channel catfish serum Abs when run under nonreducing denaturing conditions. Scatchard analysis of equilibrium dialysis data revealed that the affinities of the culture- and serum-derived Abs were quite similar, that is, exhibited association constants of approximately 2.0 x 10(6) M-1. However, it was routinely observed that the in vitro generated Abs exhibited somewhat fewer binding sites per molecule than those derived from serum. The use of murine monoclonal Abs specifically for different isotypes of channel catfish heavy and light chains demonstrated that the isotypic composition of the culture- and serum-derived fish anti-TNP Abs were similar; exceptions occurred with cultures producing lower levels of Abs. These results strongly suggest that channel catfish in vitro Ab responses closely reflect what normally occurs in vivo.

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.

Phylogeny of immune recognition: fine specificity of fish immune repertoires to cytochrome C.

Using the structurally defined protein antigen cytochrome C, studies were conducted in an attempt to delineate the fine specificities of channel catfish immune repertoires. We have previously reported that species variants of cytochrome C were cross-stimulatory to peripheral blood leukocytes (PBL) from catfish immunized with the pigeon variant. Molecular database analyses revealed the existence of overlapping epitopes that appear to define the specificity of the immune response to a "family" of closely related antigens. To further explore these observations, studies were conducted to determine the contribution of peptide 81-104 to the immunogenicity of cytochrome C. Current data showed that peptide 81-104 and intact cytochrome C were stimulatory to PBL from fish previously immunized with the native molecule. In contrast, PBL from fish previously primed with the peptide 81-104 responded only to the immunizing peptide as well as to some, but not all, variants of the peptide 81-104. The differences in the stimulatory capacities of the peptide variants appeared to correlate with amino acid substitutions at various positions of the peptide and changes in their predicted secondary structures.

Identification of a T lineage antigen in the catfish.

A murine monoclonal antibody produced against catfish thymocytes and immunoglobulin-negative lymphocytes in the blood identified a catfish T cell antigen designated CfT1. The CfT1 antigen was found to be expressed on thymocytes, a subpopulation of the lymphoid cells in blood and other lympho-hemopoietic tissues, and a T cell line, but was not expressed by erythrocytes, thrombocytes, myeloid cells, B cells or macrophage cell lines. Stimulation of blood mononuclear cells with the T cell mitogen, concanavalin A, resulted in an increased frequency of CfT1+ cells. Conversely, lipopolysaccharide stimulation increased the number of IgM+ B cells and decreased the frequency of CfT1+ cells. The CfT1 antigen was defined as a single chain protein of M(r) 35,000 lacking N- and O-linked sugars. The CfT1 molecule thus provides a T lineage-specific marker in this bony fish representative.

Membrane immunoglobulin-associated molecules on channel catfish B lymphocytes.

Membrane immunoglobulin (mIgM) on the surface of channel catfish B lymphocytes is non-covalently associated with 64 and 70 kDa molecules which are composed of covalent 32 kDa dimers and covalent 45/25 kDa subunits, respectively. Cross-linking of mIgM on catfish B cells leads to rapid phosphorylation of tyrosine residues in these presumed accessory as well as numerous other cytoplasmic molecules. These data indicate that fish likely use a signal transduction system containing elements similar to those of mammalian B cells.

Activation of channel catfish B cells by membrane immunoglobulin cross-linking.

This study demonstrates for the first time that teleost, specifically channel catfish, B cells proliferate in response to membrane immunoglobulin (mIgM) cross-linking. An early activation event mediated by anti-IgM ligation involved a rapid increase in intracellular calcium levels similar to the situation seen in mammalian B cells. In addition, catfish B cells, like mammalian B cells, did not exhibit such calcium changes following stimulation with lipopolysaccharide. Another consequence of catfish B cell mIgM cross linking was the rapid induction of intracellular protein phosphorylation. A number of proteins were phosphorylated on tyrosine residues within minutes after anti-Ig stimulation, indicating the activation of protein tyrosine kinases similar to the situation observed in mammalian B cells. These early intracellular activation events suggest that fish B cells, like mammalian B cells, employ a conserved signal transduction system upon mIgM ligation. This ability to transduce activation signals, coupled with the fact that catfish mIgM have a very short cytoplasmic tail, implies that catfish mIgM is probably associated with accessory molecules required for signal transduction. In this regard, several of the tyrosine phosphorylated catfish proteins exhibited relative molecular weights similar to the mammalian Ig-alpha and Ig-beta/gamma accessory molecules, and may represent candidates for the putative catfish mIgM accessory molecules.

Temperature-mediated processes in teleost immunity: differential effects of in vitro and in vivo 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.

Evidence for Jak-STAT interaction in channel catfish lymphoid cells.

Herein is described the identification of a putative Jak molecule in the channel catfish. A commercially available polyclonal antibody against human Jak1 was shown to react in Western blot analyses with molecules of approximately 140, 80, and 60 kDa from cell lysates of catfish long-term lymphoid cell lines. It is postulated that the 140 kDa protein is a catfish Jak homolog and the 80 and 60 kDa proteins may represent breakdown products of the 140 kDa protein. Both the 140 and 60 kDa proteins appear to be constitutively tyrosine phosphorylated in channel catfish long-term leukocyte lines. Furthermore, immuno-precipitation studies indicate an association between the Jak molecule and a STAT molecule in cloned T cell lines, and to a much lesser extent in cloned B cell lines, indicative of a possible autocrine stimulatory pathway in catfish long-term T cell lines.

Phylogeny of lymphocyte heterogeneity: cytotoxic activity of channel catfish peripheral blood leukocytes directed against allogeneic targets.

Channel catfish peripheral blood contains leukocytes that function as cytotoxic effectors directed against a variety of long-term cultured allogeneic, but not xenogeneic, targets. These effector cells are probably distinct from macrophages, B cells, and nonspecific cytotoxic cells. The cytotoxic activity of these effector cells was inhibited with monoclonal antibody 1H5. Although this reagent appears to react with a catfish cell surface molecule akin to the integrin LFA-1 present on the surface of nearly all leukocytes, it does not clarify the question as to whether or not these effectors are related to T cells.

Anti-viral cytotoxic cells in the channel catfish (Ictalurus punctatus).

Cytotoxic cells isolated from the head kidney and peripheral blood of the channel catfish appear to represent distinct subpopulations of effector cells. Previous studies showed that the former lyse xenogeneic natural killer (NK) cell targets, whereas the latter preferentially lyse allogeneic cells. Here we extend these studies and present data suggesting a third class of cytotoxic effectors responsible for killing virus-infected allogeneic and autologous cells. Peripheral blood leukocytes (PBLs) freshly isolated from unimmunized catfish lyse uninfected allogeneic target cells as well as virus-infected allogeneic and autologous cells. Cell depletion and unlabeled ("cold") target inhibition studies discriminated between putative effector classes and supported the view that at least two populations of cytotoxic cells are present within peripheral blood leukocytes. One population lyses allogeneic targets, whereas a second population kills channel catfish virus (CCV)-infected cells. In addition, inhibitor studies demonstrated that early virus gene products are sufficient to render infected cells susceptible to lysis. These results suggest that channel catfish possess distinct populations of NK-like, PBL-derived cytotoxic cells capable of lysing allogeneic and virus-infected target cells.

Activation of channel catfish (Ictalurus punctatus) T cells involves NFAT-like transcription factors.

Cyclosporin A (CsA) specifically inhibits mammalian T cells by preventing activation of transcription factors (termed nuclear factor of activated T cells (NFAT)) involved in cytokine gene expression. In this study, catfish peripheral blood lymphocytes (PBL) and antigen specific T cells were treated with CsA to gain insights into the intracellular processes involved in fish T cell activation. To this end, CsA was observed to inhibit the in vitro proliferation of Con A stimulated catfish PBL, and specific alloantigen stimulated T cells. However, the inhibitory effect of CsA on catfish T cells was obviated by treatment with Con A, antigen activation or culture supernatant from activated catfish T cells prior to the addition of CsA. The use of a phosphatase assay coupled with Western blot analysis employing a polyclonal antibody to mammalian NFAT indicated that CsA prevents the dephosphorylation and subsequent nuclear translocation of an NFAT-like molecule in catfish T cells. Finally, a nuclear protein selection protocol demonstrated that a catfish NFAT-like protein binds to a known murine IL-2 promoter sequence. These results suggest that cytokines are involved in the activation of teleost T cells, and argue that T cell activation processes are conserved over a wide phylogenetic distance.

Alternate pre-mRNA processing pathways in the production of membrane IgM heavy chains in holostean fish.

A single gene encodes two forms of the IgM heavy chain (mu) in vertebrates: one (microseconds) present in serum as secreted IgM and the other (microns) as the antigen receptor form of IgM present on the B-lymphocyte membrane. The mRNAs encoding microseconds and microns are derived from a single primary transcript by alternate pathways of RNA processing. In all vertebrates so far examined, with the exception of teleosts, microns mRNA is produced by splicing the transmembrane (TM) encoding exon 1 into a cryptic donor site near the 3' end of the C mu 4 exon. In contrast, teleost species splice the TM exon 1 into the regular splice donor site at the 3' boundary of the C mu 3 exon. We have examined micron mRNAs in two species of primitive bony fish, the holostean bowfin and the longnose gar. These fish utilize both the C mu 3 to TM1 (teleost) pathway and the typical cryptic C mu 4 to TM1 pathway. In addition the bowfin possesses a cryptic splice donor site near the middle of C mu 3. This is used in the production of a third species of microns-encoding mRNA, but does not participate in the production of an alternate form of the microseconds mRNA. The structure and patterns of expression of their mu genes suggest that the gar and bowfin may be more closely related than implied by the current view of fish evolution.