Expression profiles of cloned channel catfish (Ictalurus punctatus) lymphoid cell lines and mixed lymphocyte cultures.

Clonal channel catfish lymphoid cell lines and mixed lymphocyte cultures (MLCs) have proven extremely useful in examining immune responses at the cellular and molecular levels. To date clonal catfish cell lines and MLCs have been biologically and phenotypically characterized using a variety of techniques including reverse transcription polymerase chain reaction (RT-PCR), as well as Northern and Southern blotting. To expand the molecular characterization of these cultures, microarray analysis was employed. Clonal B (3B11), macrophage (42TA), and cytotoxic T cell (TS32.15 and TS32.17) lines and MLCs were examined using a cDNA array containing approximately 2500 probes derived from EST libraries prepared from the 42TA macrophage cell line, a MLC, and 5-14-day-old catfish fry. Analysis showed that each cell line displayed a unique RNA expression profile that included a variety of immune-related genes. Pearson correlation analysis indicated that one cytotoxic T cell line (TS32.15) clustered with the MLC, whereas a second cytotoxic T cell line (TS32.17) was more closely associated with a second cluster containing B cells and macrophages. This study illustrates the utility of microarray analyses in profiling RNA expression patterns in catfish lymphoid cell lines and will serve as a platform for examining catfish immune responses following virus infection or poly [I:C] treatment.

Structure of the catfish IGH locus: analysis of the region including the single functional IGHM gene.

The catfish IGH locus is large ( approximately 1 Mb) and complex, having undergone multiple internal duplications and transpositions. To define the structure of the locus that contains the single expressed IGHM gene, two overlapping bacterial-artificial-chromosome (BAC) clones spanning the most 3' end of the channel catfish immunoglobulin heavy (IGH) chain locus have been completely sequenced. The analyses created a contig of 257,153 bp containing 55 VH, 6 D, 12 JH genes and the IGH constant region genes encoding the functional secreted and membrane forms of IgM and the membrane form of IgD. This analysis revealed three major features. First, no C-region genes were found aside from the previously described IGHM1 and IGHD1, with the latter gene being the most 3' C-region gene of the catfish IGH locus. There was no evidence in the region sequenced for genes that could encode an Ig class similar to the IgZ/IgT described in zebrafish, trout and pufferfish. Second, there are a high number of VH pseudogenes, 28 out of 55 (51%). In contrast, the entire zebrafish IGH locus has 40 functional VH genes and eight pseudogenes (17%). Third, an internal duplication of a 52.4-kb block of VH genes has occurred. These observations suggest that the IGH locus of teleost fish varies significantly from species to species in the diversity of C-region genes as well as the numbers of genes encoding V regions.

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.

Genomic organization and differential expression of channel catfish MHC class I genes.

Two clones, designated Icpu-UA/3 and Icpu-UA/26, were isolated from a genomic library prepared from a single homozygous gynogenetic channel catfish. Sequence analysis showed that each clone encoded a gene product containing features conserved among MHC class I molecules. The genomic organization of both clones indicated that each domain, with the exception of the cytoplasmic, was encoded by a separate exon. Moreover, like mammals, catfish cytoplasmic regions were encoded by three exons rather than two as previously described for other teleost MHC class I genes. Analysis of nucleotide sequences upstream of catfish class I genes revealed the presence of several regulatory motifs similar to those seen in mammalian class I genes. These included a TATA box, Enhancer B, Site alpha, ISRE, and GAS elements. To determine the functional significance of these elements, EMSAs and tissue expression assays were performed. EMSAs demonstrated that an Enhancer B element within Icpu-UA/26, and an imperfect Enhancer B element and/or a GC-rich region within Icpu-UA/3 were responsible for formation of specific DNA/protein complexes. Expression studies detected Icpu-UA/26 transcripts in all tissues tested, whereas Icpu-UA/3 encoded messages were seen in a limited number of tissues. These results define the intron/exon organization of catfish MHC class I genes, suggest that Icpu-UA/3 encodes a nonclassical gene, and provide the first functional evidence that upstream sequences, similar to those seen in mammalian class I genes, play important roles in regulating teleost MHC gene expression.

Heterogeneity of channel catfish CTL with respect to target recognition and cytotoxic mechanisms employed.

Two types of catfish alloantigen-dependent cytotoxic T cells were cloned from PBL from a fish immunized in vivo and stimulated in vitro with the allogeneic B cell line 3B11. Because these are the first clonal cytotoxic T cell lines derived from an ectothermic vertebrate, studies were undertaken to characterize their recognition and cytotoxic mechanisms. The first type of CTL (group I) shows strict alloantigen specificity, i.e., they specifically kill and proliferate only in response to 3B11 cells. The second type (group II) shows broad allogeneic specificity, i.e., they kill and proliferate in response to several different allogeneic cells in addition to 3B11. "Cold" target-inhibition studies suggest that group II CTL recognize their targets via a single receptor, because the killing of one allotarget can be inhibited by a different allotarget. Both types of catfish CTL form conjugates with and kill targets by apoptosis. Killing by Ag-specific cytotoxic T cells (group I) was completely inhibited by treatment with EGTA or concanamycin A, and this killing is sensitive to PMSF inhibition, suggesting that killing was mediated exclusively by the secretory perforin/granzyme mechanism. In contrast, killing by the broadly specific T cytotoxic cells (group II) was only partially inhibited by either EGTA or concanamycin A, suggesting that these cells use a cytotoxic mechanism in addition to that involving perforin/granzyme. Consistent with the presumed use of a secretory pathway, both groups of CTL possess putative lytic granules. These results suggest that catfish CTL show heterogeneity with respect to target recognition and cytotoxic mechanisms.

Thioredoxin acts as a B cell growth factor in channel catfish.

To identify differentially expressed genes from channel catfish macrophages, a cDNA library from LPS-stimulated catfish macrophages was screened by subtractive hybridization. This screening yielded a 552-bp cDNA coding for catfish thioredoxin (CF-TRX). The deduced amino acid sequence revealed that CF-TRX contains 107 amino acids and is 59% homologous to human adult T cell leukemia-derived factor/TRX, originally described as an IL-2R alpha-inducing factor. Northern blot analyses showed that CF-TRX is expressed in catfish T and macrophage cell lines, but weakly in B cell lines. Similar results were also observed in Western blot analyses using a mAb specific for recombinant CF-TRX (rTRX). The use of rTRX in functional studies demonstrated that rTRX induces in vitro proliferative responses of catfish PBL that were synergistically enhanced by the addition of culture supernatants from catfish T cell lines. In addition, cell separation studies and flow cytometric analyses revealed that the cells proliferating in rTRX-stimulated cultures were mostly B cells. These results suggest that CF-TRX may have an important role in the activation and proliferation of channel catfish B cells.

Altered expression of P(2) receptor mRNAs in the basilar artery in a rat double hemorrhage model.

BACKGROUND AND PURPOSE: Extracellular ATP might induce cerebral vasospasm after subarachnoid hemorrhage through P(2) receptor. To investigate the roles of P(2) receptor subtypes in vasospasm, we examined the changes in mRNA expression of P(2) receptor subtypes in basilar arteries from double cisternal blood injection rat models. METHODS: One hundred male Sprague-Dawley rats, each weighing 350 to 400 g, were divided into 2 groups of 50. In the first group (n=50), the autologous arterial blood (0.2 to 0.3 mL) was injected into the cisterna magna on days 0 and 2. The rats were killed on day 3, 5, or 7 (n=10 in each group). In the sham group (n=10), the rats were injected with saline (0.3 mL) instead of blood. Ten rats were killed without blood or saline injection and served as control. The basilar arteries from rats in each group were used for reverse transcription and polymerase chain reaction. In another group of 50 rats, the same experiment was conducted, and the basilar arteries were collected for transmission electron microscopic study. RESULTS: In the subarachnoid hemorrhage groups, transmission electron microscopy showed the reduction in vessel perimeter on days 5 and 7 to be approximately 30% to 40%. The P(2X1) mRNA level was significantly decreased on day 3 and recovered on days 5 and 7. The P(2Y1) mRNA level was transiently increased on day 5, and the P(2Y2) mRNA level was elevated from day 5 to day 7 (P:<0.05). CONCLUSIONS: The differential expression of the P(2) receptors indicates that P(2X1) subtype might not play an important role in vasospasm. The upregulation of P(2Y1) and P(2Y2) receptors might enable ATP to produce contraction at low levels of concentration.

Transcriptional enhancers of immunoglobulin light chain genes in Atlantic cod (Gadus morhua).

The organization of immunoglobulin heavy (H) chain genes in teleosts resembles that of mammals and amphibians, whereas light (L) chain genes are arranged in multiple clusters of variable (VL), joining (JL), and constant (CL) region segments. Sequence analysis of two Atlantic cod genomic clones (14,966 and 13,116 bp in length) revealed a very compact IgL chain locus with the VL genes in opposite transcriptional orientation to the JL and the CL genes. This suggests the possibility of rearrangements between clusters by inversion. Each cluster spans approximately 2.1 kb and distances between clusters vary between 2.1 and 4.8 kb. To gain insight into the transcriptional regulation of this complex, multiclustered locus, chloramphenicol acetyl transferase reporter constructs containing 14 different DNA segments from the two genomic clones were transfected into channel catfish B and non-B-cell lines, as well as into mouse B-cell lines. These studies showed strong enhancer activity downstream of the CL region in three out of six L chain gene clusters when assayed in fish, but not in mouse B cells. Interestingly, both mouse and human lambda enhancers exhibited strong activity in the fish B cells, while the mouse 3' kappa enhancer did not. This suggests that transcription factors similar to those involved in mammalian lambda expression are present in B cells from teleosts.

Telomerase expression and telomere length in immortal leukocyte lines from channel catfish.

Normal channel catfish leukocytes readily undergo spontaneous in vitro immortalization yielding functionally active diploid cell lines. Since telomerase activation appears to be a critical step in the establishment of immortal mammalian cells, studies were undertaken to determine if and when telomerase expression occurs during the in vitro immortalization process of channel catfish leukocytes. To this end, freshly isolated peripheral blood leukocytes (PBL) from normal fish were shown to exhibit low to undetectable levels of telomerase activity and within four days after culture initiation showed dramatic increases in telomerase activity which typically remained high for at least four weeks. This activity then declined, concomitant with decreases in cellular proliferation and increases in cell death. Cells which escaped this culture "crisis" re-expressed high levels of telomerase activity indefinitely. Although telomerase activity was expressed early in the immortalization process, clonal cell lines derived from these cultures had relatively short telomeres. These results suggest that telomerase expression in catfish leukocytes is activation-induced, and its expression does not necessarily stabilize telomere length until a critically, albeit ill-defined, short length is reached.

Development and analysis of various clonal alloantigen-dependent cytotoxic cell lines from channel catfish.

To determine the phenotypes of cytotoxic cells in channel catfish, clonal alloantigen-dependent leukocyte lines were established from mixed leukocyte cultures. Each clone was analyzed for expression of TCR alpha and beta genes by RT-PCR and for target cell specificity by 51Cr-release assay. Based on the above criteria, the following five different cell types were identified among the 19 clones analyzed: 1) TCR alphabeta+ allospecific cytotoxic cells, 2) TCR alphabeta+ nonspecific cytotoxic cells, 3) allospecific TCR alphabeta+ noncytotoxic cells, 4) TCR alphabeta- nonspecific cytotoxic cells, and 5) TCR alphabeta- allospecific cytotoxic cells. The demonstration of cloned, TCR alphabeta+, allospecific cytotoxic effectors provides the strongest evidence to date for the existence of cytotoxic T cells in fish.

Hemolysate induces tyrosine phosphorylation and collagen-lattice compaction in cultured fibroblasts.

Hemolysate, a proposed causative agent for cerebral vasospasm after subarachnoid hemorrhage, produces contraction of cerebral arteries by activation of tyrosine kinases. In addition, hemolysate increases fibroblast-collagen compaction that could play a role in cerebral vasospasm. We studied the effect of hemolysate on tyrosine phosphorylation and fibroblast-collagen compaction in cultured canine basilar and human dermal fibroblasts using tyrosine kinase inhibitors and tyrosine antibodies. Hemolysate enhanced tyrosine phosphorylation of two proteins, 64 and 120 kDa, in cultured canine basilar artery and human dermal fibroblast cells. The effect of hemolysate was time-dependent and concentration-dependent. Oxyhemoglobin and ATP, the two major components of hemolysate, produced similar tyrosine phosphorylation, however, with a different time course. Tyrosine kinase inhibitors genistein and tyrphostin A51 abolished the effect of hemolysate in both cerebral and dermal fibroblasts. Hemolysate increased fibroblast-populated collagen-lattice compaction and tyrosine kinase inhibitors genistein and tyrphostin A51 attenuated the effect of hemolysate. We conclude that hemolysate activates tyrosine kinase that leads to the increase of fibroblast compaction. This effect of hemolysate may contribute to cerebral vasospasm.

Identification and characterization of the tumor suppressor p53 in channel catfish (Ictalurus punctatus).

Herein is presented the sequence of a catfish full-length p53 cDNA obtained from a cloned B cell line cDNA library. Southern blot analyses determined that a restriction fragment linked polymorphism (RFLP) existed with PstI among outbred catfish. Western blot analyses demonstrated that, when compared to PBLs, the catfish leukocyte lines express higher levels of p53 protein. Additionally, the results of Western blot analyses and in vitro translation experiments suggest that the catfish leukocyte lines may produce truncated forms of p53 due to internal initiation.

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.

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.

Functional and molecular characterization of teleost leukocytes.

The coupling of immunologically relevant in vitro assay systems, cell separation techniques, and the development of distinct clonal leukocyte lines has established the existence of T, B, natural killer, and accessory cell equivalents in teleosts. B cells are directly defined by monoclonal antibodies to teleost immunoglobulin (Ig) and identification of Ig H and L chain genes. As in mammals, fish B cells show Ig H-chain gene rearrangements, allelic exclusion, produce both membrane-bound and secreted forms of Ig, and transduce intracellular proliferative signals upon anti-Ig cross-linking. It has also been found that some fish B cells express a unique chimeric Ig chain with sequence homology to mammalian Ig delta. Teleost T cells are still indirectly defined as sIg- lymphocytes due to a lack of definitive surface markers. These mIg- lymphocytes are the responding cells in mixed leukocyte cultures, proliferate specifically to autologously processed and presented antigen, provide helper function for in vitro antibody responses, and produce interleukin-like factors upon activation. Recent identification of teleost T-cell receptor alpha and beta genes has now permitted the unequivocal genetic demonstration that some of these mIg- cells are bona fide T cells. It is anticipated that such long-term clonal cell lines will be indispensable tools for dissecting the physiology, biochemistry and molecular biology of teleost immune responses.

A novel chimeric Ig heavy chain from a teleost fish shares similarities to IgD.

IgD is considered to be a recently evolved Ig, being previously found only in primates and rodents. Here we describe, from a teleost fish (the channel catfish, Ictalurus punctatus), a novel complex chimeric Ig heavy chain, homologous, in part, to the heavy chain (delta) of IgD. In addition to alternative secretory or membrane-associated C termini, this chimeric molecule contains a rearranged variable domain, the first constant domain of mu, and seven constant domains encoded by a delta gene homolog. Identification of the catfish gene as delta is based on the following properties: sequence relatedness to mammalian delta; a location within the IgH locus that is immediately downstream of the mu gene; separate terminal exons for the secretory and membrane forms; coexpression with the complete mu chain in some but not all B cells. These results (i) suggest that IgD is an ancient immunoglobulin that was present in vertebrates ancestral to both the mammals and the ray-finned fishes, and (ii) raise the possibility that this Ig isotype may have served an as yet unidentified important function early in the evolution of the immune system.

Ig light chain gene in the Siberian sturgeon (Acipenser baeri).

Elasmobranch and teleost fish have their Ig light (L) chain loci organized in multiple clusters (VL-JL-CL). The VL segments of teleosts are in opposite transcriptional orientation to the CL genes, suggesting that in teleosts and elasmobranchs there may have been separate evolutionary events leading to this organization. To address this problem, the IgL locus from the Siberian sturgeon (Acipenser baeri) (representative of a branch between elasmobranchs and teleosts) was investigated. Sequence analysis of cDNA clones shows that sturgeon VL genes are most similar to those of teleosts, but that sturgeon CL genes are more similar to those of the sharks. Southern blot analyses of sturgeon erythrocyte DNA with VL- and CL-specific probes showed that there are more than 20 VL segments in both the tetraploid Siberian sturgeon and the diploid sterlet (Acipenser ruthenus), but only a few CL segments in the genome of the Siberian sturgeon and up to four CL segments in that of the sterlet. Screening of an unamplified genomic library gave more than 300 VL-positive and four CL-positive clones. None of these contained inserts positive for both probes. PCR analysis of a genomic CL clone using IC and CL-specific primers suggested that upstream of the CL segment there are at least seven JL segments. it is concluded that sturgeons have a kappa-like organization of their IgL locus and that the clustered organization of IgL loci in bony fish and sharks arose from two distinct evolutionary events.

Immunoglobulin VH regions in Atlantic cod (Gadus morhua L.): their diversity and relationship to VH families from other species.

Variable regions (VH) of Atlantic cod cDNA clones have been isolated by the polymerase chain reaction (PCR) technique, using primers for the first constant domain of heavy chain (CH1) and lambda gt11. Based upon sequence analysis and comparisons these clones have been divided into three different VH families. The approximate number of members in each family was estimated by genomic Southern blot. Comparisons of complementarity determining regions (CDRs) and frameworks (FRs) show that FR2 and the last part of FR3 are the most conserved regions. The CDR3 is very heterogeneous and gives a major contribution to VH diversity. Possible relationships between VH sequences from 17 species are shown graphically. Different VH families are often more conserved between species than within any one species. Two genomic VH clones have been isolated and partially sequenced. The VH genes have an octamer and TATA motif in the 5' region, followed by an 18-amino-acid-long hydrophobic leader, and the mature VH coding region. The characteristics heptamer-nonamer recombination signals for VH to D joining are present 3' of the VH segment.