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Estrogens are recognized as modulators of immune responses in mammals and teleosts. While it is known that the effects of estrogens are mediated via leukocyte-specific estrogen receptors (ERs) in humans and mice, leucocyte-specific estrogen receptor expression and the effects of estrogens on this cell population is less explored and poorly understood in teleosts. Here in, we verify that channel catfish (Ictalurus punctaus) leukocytes express ERα and ERβ2. Transcripts of these isoforms were detected in tissue-associated leukocyte populations by PCR, but ERβ2 was rarely detected in PBLs. Expression of these receptors was temporally regulated in PBLs following polyclonal activation by concanavalin A, lipopolysaccharide or alloantigen based on evaluation by quantitative and end-point PCR. Examination of long-term leukocyte cell lines demonstrated that these receptors are differentially expressed depending on leukocyte lineage and phenotype. Expression of ERs was also temporally dynamic in some leukocyte lineages and may reflect stage of cell maturity. Estrogens affect the responsiveness of channel catfish peripheral blood leukocytes (PBLs) to mitogens in vitro. Similarly, bactericidal activity and phorbol 12-myristate 13-acetate induced respiratory burst was modulated by 17β-estradiol. These actions were blocked by the pure ER antagonist ICI 182780 indicating that response is, in part, mediated via ERα. In summary, estrogen receptors are expressed in channel catfish leukocytes and participate in the regulation of the immune response. This is the first time leukocyte lineage expression has been reported in teleost cell lines.

In mammals, expression of the three alternatively spliced exons of the tyrosine phosphatase CD45 is regulated by the developmental and activation state of the cell. In comparison, the channel catfish, Ictalurus punctatus, CD45 homolog contains 18 functional alternatively spliced exons. Since very little is known about CD45 regulation in ectothermic vertebrates, this study examines the regulation of catfish CD45 mRNA isoform expression in clonal T and B cells in response to stimulation. Results show that mitogenic stimulation using catfish serum or concanavalin A induced expression of mRNAs for small CD45 isoforms, and isoform message expression was growth curve dependent, i.e. cells in logarithmic phase express message for smaller CD45 isoforms, whereas stationary phase cells express message for longer CD45 isoforms. In addition, cells treated with the protein synthesis inhibitor cycloheximide expressed message for longer CD45 isoforms, and treatment with lactacystin, which blocks protein degradation, rescued smaller isoform message expression. Collectively these data suggested that expression of CD45 isoforms, in catfish, at least at the mRNA level, is "constitutively dynamic" and highly dependent on extracellular stimuli.

Immunoglobulin D (IgD) is an enigmatic antibody isotype that mature B cells express together with IgM through alternative RNA splicing. Here we report active T cell-dependent and T cell-independent IgM-to-IgD class switching in B cells of the human upper respiratory mucosa. This process required activation-induced cytidine deaminase (AID) and generated local and circulating IgD-producing plasmablasts reactive to respiratory bacteria. Circulating IgD bound to basophils through a calcium-mobilizing receptor that induced antimicrobial, opsonizing, inflammatory and B cell-stimulating factors, including cathelicidin, interleukin 1 (IL-1), IL-4 and B cell-activating factor (BAFF), after IgD crosslinking. By showing dysregulation of IgD class-switched B cells and 'IgD-armed' basophils in autoinflammatory syndromes with periodic fever, our data indicate that IgD orchestrates an ancestral surveillance system at the interface between immunity and inflammation.

Immunoglobulin light (IGL) chain genes encoding σ and λ from channel catfish, Ictalurus punctatus, and λ from Atlantic cod, Gadus morhua, were identified by mining of expressed sequence tag databases, 5′-RACE and RT-PCR protocols. cDNAs for each of these IGL chains encode typical variable (V), joining (J), and constant (C) regions and Southern blot analyses and genomic sequencing show that genes encoding these isotypes, like other teleost IGL genes, are found in a cluster organization of one or two V gene segments, followed by single J and C gene segments, all in the same transcriptional orientation. However, unlike the teleost κ genes, genes encoding catfish σ and λ are few in number and the two isotypes are each encoded by only two clusters. Similarly, Atlantic cod λ genes are predicted to be encoded by two or three clusters. As expected, sequence and phylogenetic analyses comparisons demonstrate that catfish Vσ and Cσ genes are most similar to Vσ and Cσ genes of other ectothermic vertebrates. Although catfish and Atlantic cod Vλ genes cluster with other vertebrate Vλ genes, their Cλ sequences cluster in a distinct group separate from other vertebrate IGL C sequences. However, support for classifying these sequences as λ, is their V and J recombination signal sequence (RSS) organization. The catfish and Atlantic cod genes have typical λ-like RSS with the Vλ RSS consisting of heptamer-23 bp spacer-nonamer and the Jλ RSS consisting of heptamer-12 bp spacer-nonamer. This is the first report demonstrating the presence of Igλ in teleosts.

This study characterizes four monoclonal antibodies (mAb) developed against the major histocompatibility complex (MHC) class II beta chain of the channel catfish, Ictalurus punctatus. Immunoprecipitations using catfish clonal B cells revealed that each of these mAbs immunoselected proteins of approximately 32 and 36 kD, which are of the appropriate sizes for MHC class II alpha and beta chains, respectively. Cell distribution studies using a fluorescence-activated cell sorter (FACS) combined with RT-PCR analyses demonstrated that MHC class II beta is expressed at a high density on catfish clonal macrophage, B and T cell lines, on alloantigen stimulated leukocytes, and on lipopolysaccharide-induced B-cell blasts. Collectively, these results demonstrate the potential importance of these antibodies as reagents in future studies dealing with the functional role of MHC class II molecules in immune recognition of self from non-self.

B cell receptor (BCR) accessory molecules CD79a and CD79b homologs were identified in the channel catfish, Ictalurus punctatus. Both are found as single copy genes that encode proteins containing a signal peptide, an extracellular immunoglobulin domain, a transmembrane region and a cytoplasmic tail containing an immune-receptor tyrosine-dased activation motif (ITAM). IpCD79a and IpCD79b transcripts correlate well with IgM message expression. They are highly expressed in peripheral blood leukocytes (PBL) enriched in membrane (m) IgM+ cells and catfish clonal B cell lines, but not in catfish clonal T cells, indicating that IpCD79a and IpCD79b expression is B cell restricted. Studies using catfish clonal B cells (3B11) transfected with constructs encoding epitope-tagged IpCD79a and IpCD79b revealed that IpCD79a was expressed as a 45 kDa protein and IpCD79b was expressed as a 32 kDa protein. Furthermore, co-immunoprecipitations of epitope-tagged CD79 proteins demonstrate that these molecules are non-covalently associated with mIgM. These data correlate with some of the previous immunoprecipitation data demonstrating that catfish mIgM associates with proteins of 45 and 32 kDa.

Transcriptional control of the immunoglobulin heavy chain (IGH) locus in the channel catfish, Ictalurus punctatus, is incompletely understood. It is, however, known that 2 variant octamer motifs and a microE5 motif in the core region of the enhancer (Emicro3') are important in driving transcription, and it has been suggested that interaction between transcription factors (Oct factors and E-proteins) bound to these sites contributes to enhancer function. In this study, the functional relationships between the microE5 motif, the proximal octamer motif, and the factors that bind them have been examined. The results of mutational analysis of these motifs showed that their interaction is important to driving transcription from the enhancer. Furthermore, the catfish Oct transcription factors were capable of a physical interaction with the catfish E-proteins. These results support a role for interaction between transcription factors bound to the octamer and microE5 motifs in the function of the Emicro3' enhancer.

The immunoglobulin heavy chain locus of teleost fish is driven by an enhancer (Eμ3′) that contains, in all species examined, octamer and E-box motifs (typically μE5). Thus, the transcription factors known (through experiments in fish) or predicted (from knowledge of their mammalian homologues) to be involved in the function of the enhancer are the octamer-binding (Oct) transcription factors Oct1 and Oct2, their coactivator BOB.1 and the μE5-binding E-proteins (E2A, HEB and E2-2). Comparative genomic analysis of the genes encoding these transcription factors in teleost fish permits some conclusions to be drawn concerning the fate of these genes following the genome-wide duplication event that occurred in this lineage. Although problems in the assembly of some regions of the zebrafish genome may complicate the analysis, the pattern that emerges from these studies is that Oct1, Oct2, BOB.1 and E2-2 are all single-copy genes in teleosts, whereas in the case of E2A and HEB, two copies of each gene are present. The evolutionary conservation of sequences between related genes is variable, but with the highest similarities always being observed in the domains with ligand-binding function, whether the ligand is a DNA motif or, as in the case of BOB.1, another protein domain. A major challenge that must be faced is the assignment of precise functions to these diverse transcription factors in teleosts; assumptions drawn by inference from their mammalian homologues will likely be misleading.

Two CD4-like (CD4L) molecules have been identified in channel catfish, Ictalurus punctatus. Although phylogenetically related to other vertebrate CD4 molecules, they exhibit only 19% amino acid identity to each other. IpCD4L-1 encodes a predicted protein containing four immunoglobulin domains, a transmembrane region and a cytoplasmic tail containing a p56lck binding site. In contrast, IpCD4L-2 encodes for a similar protein with three immunoglobulin domains. The gene organization of IpCD4L-1 is very similar to that of other vertebrate CD4 genes, while the genomic organization of IpCD4L-2 is different. Southern blots indicate both catfish molecules are likely single copy genes and mapping studies show that both are found on a single Bacterial Artificial Chromosome suggesting close linkage. At the message level, IpCD4L-1 and -2 are expressed in various catfish lymphoid tissues and in non-B-cell peripheral blood leukocytes (PBL). Both messages are upregulated in concanavalin A (ConA) and alloantigen stimulated PBL, but not in lipopolysaccharide (LPS)-stimulated cultures. In contrast, they are differentially expressed among the catfish clonal T cell lines. While both molecules appear to be T cell specific, their functional significance in catfish is unknown.

The recent identification of a large and diverse family of leukocyte immune-type receptors (IpLITRs) in channel catfish (Ictalurus punctatus) indicates that immunoglobulin superfamily (IgSF) members related to both mammalian Fc receptors (FcRs) and leukocyte receptor complex (LRC)-encoded proteins exist in fish. In the present study, it was found that IpLITR messages were preferentially up regulated in catfish peripheral blood leukocytes (PBL) and clonal cytotoxic T cells (CTL) after alloantigen stimulation. Detailed sequence analyses of the expressed IpLITR cDNAs from two clonal CTL lines indicated an unexpectedly large array of putative activatory and inhibitory IpLITR-types containing variable numbers of extracellular immunoglobulin (Ig)-like domains. Importantly, all expressed IpLITRs shared similar membrane distal Ig domains (i.e., D1 and D2), suggesting that they may bind a common type of ligand. Sequence alignments and comparative homology modeling revealed that IpLITR domains, D1 and D2, have similar predicted 3-D structural properties with the corresponding domains of the human LRC-encoded leukocyte Ig-like receptor (LILR) family. Furthermore, conservation of key major histocompatibility class I (MHC I)-binding residues were located at similar positions within the membrane distal tip of D1 between representative IpLITRs and group 1 LILRs. Taken together, these results suggest that fish LITRs have an orthologous relationship to LRC-encoded receptors such as the human LILRs and could potentially function as a diverse family of MHC class I-binding receptors.

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.

An FcR homolog (IpFcRI), representing the first such receptor from an ectothermic vertebrate, has been identified in the channel catfish (Ictalurus punctatus). Mining of the catfish expressed sequence tag databases using mammalian FcR sequences for CD16, CD32, and CD64 resulted in the identification of a teleost Ig-binding receptor. IpFcRI is encoded by a single-copy gene containing three Ig C2-like domains, but lacking a transmembrane segment and cytoplasmic tail. The encoded Ig domains of IpFcRI are phylogenetically and structurally related to mammalian FcR and the presence of a putative Fc-binding region appears to be conserved. IpFcRI-related genomic sequences are also present in both pufferfish and rainbow trout, indicating the likely presence of a soluble FcR in other fish species. Northern blot and qualitative PCR analyses demonstrated that IpFcRI is primarily expressed in IgM-negative leukocytes derived from the lymphoid kidney tissues and PBL. Significantly lower levels of IpFcRI expression were detected in catfish clonal leukocyte cell lines. Using the native leader, IpFcRI was secreted when transfected into insect cells and importantly the native IpFcRI glycoprotein was detected in catfish plasma using a polyclonal Ab. Recombinant IpFcRI binds catfish IgM as assessed by both coimmunoprecipation and cell transfection studies and it is presumed that it functions as a secreted FcR akin to the soluble FcR found in mammals. The identification of an FcR homolog in an ectothermic vertebrate is an important first step toward understanding the evolutionary history and functional importance of vertebrate Ig-binding receptors.

The Oct2 transcription factor is important in driving expression of the IgH locus of the channel catfish, Ictalurus punctatus. Two isoforms, catfish Oct2α and Oct2β, have been characterized at the level of expression and function, but little is known of the structure of the Oct2 gene in catfish. To gain insight into the diversity of Oct2 gene structure and expression in the teleost fish, a comparative genomic analysis of Oct2 was undertaken in the pufferfish (Fugu rubripes) and the zebrafish (Danio rerio). The orthologues of zebrafish and Fugu Oct2 were identified, and share with catfish Oct2 the expression of a limited number (two in zebrafish, three in Fugu) of isotypes produced by alternative pathways of RNA processing. The alternatively spliced variants of catfish Oct2 showed a different pattern of exon use from those of Fugu and zebrafish. The analysis also identified a novel homologue of Oct2 in both zebrafish and Fugu. This homologue, termed Oct2x, shares similarities to both Oct1 and Oct2. A phylogenetic analysis of the relationships of Oct2x gave an unexpected result, with Oct2x occupying a position basal to the Oct gene families of both vertebrates and Drosophila.

Multiple species of type I interferon (IFN) were recently identified in catfish (CF) (Ictalurus punctatus). Herein we extend these studies and report the existence of two distinct type II IFN genes in channel CF. As with zebrafish and the green spotted pufferfish, the two CF IFN-gamma genes are dissimilar in sequence but closely linked on the same chromosome. One of the genes (IFN-gamma2) encodes two distinct messages that likely arose via alternative splicing at two closely spaced splice donor sites within the first intron. Sequence analysis indicates that CF IFN-gamma genes contain the hallmarks of authentic IFN-gamma including: (1) a conserved nuclear localization site at the C terminus (CF IFN-gamma2 only), (2) an IFN-gamma signature sequence, (3) six putative helical regions within the mature protein, (4) one or more potential glycosylation sites, and (5) multiple mRNA instability motifs within the 3' untranslated region. Moreover, well-characterized CF T and NK cell clones were shown to synthesize IFN-gamma transcripts. This is the first unequivocal demonstration in any lower vertebrate species that NK and T cells synthesize IFN-gamma and is consistent with results in mammalian systems where T cells and NK cells are the major sources of type II IFN production. Collectively, these studies indicate that Siluriformes possess two evolutionarily conserved IFN-gamma genes and demonstrate that CF possess three key elements of the innate immune response: NK cells and types I and II IFN.

The channel catfish, Ictalurus punctatus, is widely recognized as an important model for studying immune responses in ectothermic vertebrates. It is one of the few fish species for which defined viable in vitro culture systems have been established and is currently the only fish species from which a variety of functionally distinct clonal leukocyte lines are available. Moreover, there is a large basis of biochemical and molecular information on the structure and function of catfish immunoglobulins (Igs). Catfish, as other teleosts, have a tetrameric homolog of IgM as their predominant serum Ig plus a homolog of IgD. They also have genetic elements basically similar to those of mammals, which encode and regulate their expression. The catfish Ig heavy (H) chain locus is a translocon-type locus with three Igdelta genes linked to an Igmu gene or pseudogene. The catfish IgH locus is estimated to contain approximately 200 variable (V) region genes representing 13 families as well as at least three diversity (D) and 11 joining (JH) genes. The catfish has two light (L) chain isotypes, F and G, both encoded by loci organized in multiple cassettes of VL-JL-CL with the VL in the opposite transcriptional orientation. Hence, all requisite components for encoding antibodies are present in the catfish, albeit with certain variations. In the future, whether or not additional unique features of Ig function and expression will be found remains to be determined.

CD45 is a transmembrane protein tyrosine phosphatase, which in mammals plays an important role in T and B cell receptor and cytokine signaling. Recently, a catfish cDNA was shown to contain all characteristic CD45 features: an alternatively spliced amino-terminus, a cysteine-rich region, three fibronectin domains, a transmembrane region, and two phosphotyrosine phosphatase domains. However, analyses of CD45 cDNAs from various catfish lymphoid cell lines demonstrated that catfish CD45 is unique in that it contains a large number of alternatively spliced exons. Sequence analyses of cDNAs derived from the catfish clonal B cell line 3B11 indicated that this cell line expresses up to 13 alternatively spliced exons. Furthermore, sequence similarity among the alternatively spliced exons suggested duplication events. To establish the exact number and organization of alternatively spliced exons, a bacterial artificial chromosome library was screened, and the catfish functional CD45 gene plus six CD45 pseudogenes were sequenced. The catfish functional CD45 gene spans 37 kb and contains 49 exons. In comparison, the human and pufferfish CD45 genes consist of 34 and 30 exons, respectively. This difference in the otherwise structurally conserved catfish gene is due to the presence of 18 alternatively spliced exons that were likely derived through several duplication events. In addition, duplication events were also likely involved in generating the six pseudogenes, truncated at the 3' ends. A similarly 3' truncated CD45 pseudogene is also present in the pufferfish genome, suggesting that this specific CD45 gene duplication occurred before catfish and pufferfish diverged (approximately 400 million years ago).

The transcriptional enhancer (Eμ3′) of the IgH locus of the channel catfish, Ictalurus punctatus, differs from enhancers of the mammalian IgH locus in terms of its position, structure, and function. Transcription factors binding to multiple octamer motifs and a single μE5 motif (an E-box site, consensus CANNTG) interact for its function. E-box binding transcription factors of the class I basic helix-loop-helix family were cloned from a catfish B cell cDNA library in this study, and homologs of TF12/HEB were identified as the most highly represented E-proteins. Two alternatively spliced forms of catfish TF12 (termed CFEB1 and -2) were identified and contained regions homologous to the basic helix-loop-helix and activation domains of other vertebrate E-proteins. CFEB message is widely expressed, with CFEB1 message predominating over that of CFEB2. Both CFEB1 and -2 strongly activated transcription from a μE5-dependent artificial promoter. In catfish B cells, CFEB1 and -2 also activated transcription from the core region of the catfish IgH enhancer (Eμ3′) in a manner dependent on the presence of the μE5 site. Both CFEB1 and -2 bound the μE5 motif, and formed both homo- and heterodimers. CFEB1 and -2 were weakly active or inactive (in a promoter-dependent fashion) in mammalian B-lineage cells. Although E-proteins have been highly conserved in vertebrate evolution, the present results indicate that, at the phylogenetic level of a teleost fish, the TF12/HEB homolog differs from that of mammals in terms of 1) its high level of expression and 2) the presence of isoforms generated by alternative RNA processing.

CD45, also known as LCA, is a transmembrane protein tyrosine phosphatase encoded by the PTPRC gene. In mammals, it plays an important role in T and B cell receptor and cytokine signaling by maintaining receptor associated kinases in an active state. A prominent CD45 feature is alternative splicing of exons encoding the N-terminus, resulting in the generation of several isoforms. The expression of isoforms is tightly regulated and dependent on the developmental/activation state of the lymphocyte. Nevertheless, the significance of these multiple isoforms in mammals is poorly understood. In this study, the channel catfish CD45 homolog was sequenced and found to be similar to CD45 of other species. However, unlike mammalian CD45, it appears that up to 13 exons are used in producing multiple alternatively spliced CD45 variants in catfish cells. These 13 alternatively spliced exons variably encode for O-linked glycosylation sites. Several of the exons are identical or very similar, suggesting gene duplication of a block of four exons. As demonstrated by RT-PCR, many of the alternatively spliced forms of catfish CD45 are differentially expressed in lymphoid cell lines with B cells expressing larger isoforms than do T cells. Furthermore, immunoprecipitation experiments utilizing anti-catfish CD45 mAbs substantiated that different size CD45 isoforms are expressed at the protein level on catfish T and B cells.

TcR alpha, beta, and gamma chain negative cytotoxic NK-like cells were cloned from alloantigen-stimulated PBL obtained from nai;ve channel catfish. Stimulation with allogeneic cells and growth promoting factors are required for their continued in vitro proliferation and cytotoxic activity. These granular cells kill not only the stimulating allogeneic cells, but also unrelated allogeneic targets by a perforin/granzyme-mediated apoptosis pathway. In addition, they are negative for markers that define neutrophils, monocytes/macrophages, and non-specific cytotoxic cells. Although these NK-like clones kill a number of different allogeneic targets, they display interclonal variation in cytotoxicity toward a panel of allogeneic targets, i.e. some clones have no apparent target specificity, while others display a target preference. In addition, flow cytometric analyses revealed that expression of a putative FcmuR, an LFA-1-like molecule, and a putative thymocyte/T cell antigen varies among the different clones, with no clear correlation between surface antigen expression and cytotoxic activity. Although not all clones express a putative FcmuR, it was noted that they all expressed an ITAM containing FcepsilonR gamma chain homolog. This finding suggests that the catfish FcepsilonR gamma chain may potentially be used as an accessory molecule for not only FcmuRs, but also for other unknown activation receptors. These results support the hypothesis that catfish NK-like cells are heterogeneous in terms of target specificities and cell surface phenotype.

A tumor necrosis factor (TNF) alpha-like gene, encoding a propeptide of 230 amino acids and a mature (soluble) peptide of 162 amino acids, was identified in channel catfish (Ictalurus punctatus). While the catfish protein shared features in common with both mammalian TNFalpha and TNFbeta homologs, overall sequence identity/similarity was slightly higher vs. TNFalpha genes when mature TNF sequences were compared. Phylogenetic analysis placed catfish and other fish TNF sequences within their own cluster apart from mammalian TNFalpha and beta genes, and supported the suggestion that TNFalpha and beta genes separated after the divergence of mammals and teleosts. In contrast to trout and carp, but similar to flounder, catfish TNF was present as a single copy gene. Expression studies demonstrated that catfish TNFalpha mRNA was present in all tested tissues (i.e. liver, spleen, head kidney, mesonephros, gill, thymus, and PBLs) from an unstimulated fish. Moreover, catfish TNF was constitutively expressed in actively proliferating, but otherwise unstimulated, macrophage (42TA) and T cell (G14D; TS32.17) lines, but not in B cell (1G8 or 3B11) or fibroblast lines. TNF expression was upregulated in PBLs, and in G14D and 42TA cells, but not in 3B11 cells, by PMA/calcium ionophore treatment. These results demonstrate that a catfish homolog of TNFalpha has been identified, and indicate that catfish TNFalpha is expressed in catfish in a manner similar to that seen in mammals.

A bacterial artificial chromosome (BAC) library was constructed by cloning HindIII-digested high molecular weight DNA from a gynogenetic channel catfish, Ictalurus punctatus, into the vector pBeloBAC11. Approximately 53 500 clones were arrayed in 384-well plates and stored at -80°C (CCBL1), while clones from a smaller insert size fraction were stored at -80°C without arraying (CCBL2). Pulsed-field gel electrophoresis of 100 clones after NotI digestion revealed an average insert size of 165 kb for CCBL1 and 113 kb for CCBL2. Further characterization of CCBL1 demonstrated that 10% of the clones did not contain an insert. CCBL1 provides a 7.2-fold coverage of the channel catfish haploid genome. PCR-based screening demonstrated that 68 out of 74 unique loci were present in the library. This represents a 92% chance to find a unique sequence. These libraries will be useful for physical mapping of the channel catfish genome, and identification of genes controlling major traits in this economically important species.

Two-color flow cytometry demonstrated that 4-8% of channel catfish PBL are positive for both F and G IgL chain isotypes, suggesting that they passively acquire serum IgM via a putative FcmicroR. These cells show spontaneous killing toward allogeneic targets, and in vitro stimulation of PBL with allogeneic cells results in an increase of double IgL chain positive cells with a concomitant increase in nonspecific cytotoxicity. Long-term cultures of alloantigen-stimulated PBL contain both sIgM(+) and sIgM(-) cytotoxic cells that transcribe message for the catfish homolog of the FcepsilonR gamma chain, but not for Igmicro and TCR-alpha,-beta, or -gamma chains. Immunoprecipitation of lysates from sIgM(+) NK-like cells with anti-IgM co-immunoprecipitated a putative FcmicroR of approximately 64 kDa. Finally, removal of IgM from sIgM(+) NK-like cells and replacement with anti-hapten antibody enabled antibody-armed effectors to kill haptenated targets that were refractory to killing by effectors armed with normal IgM. This is the first report suggesting that teleost NK-like cells express a putative FcmicroR which participates in antibody-dependent cell-mediated cytotoxicity.

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.

The delta-chain of catfish IgD was initially characterized as a unique chimeric molecule containing a rearranged VDJ spliced to C micro 1, seven C domain-encoding exons (delta1-delta7), and a transmembrane tail. The presence of cDNA forms showing splicing of delta7 to an exon encoding a secretory tail was interpreted to indicate that membrane (deltam) and secreted (deltas) forms were likely expressed from a single gene by alternative RNA processing. Subsequent cloning and sequence analyses have unexpectedly revealed the presence of three delta C region genes, each linked to a micro gene or pseudogene. The first (IGHD1) is located 1.6 kb 3' of the functional C micro (IGHM1). The second (IGHD3) is positioned immediately downstream of a pseudo C micro (IGHM3P), approximately 725 kb 5' of IGHM1. These two delta genes are highly similar in sequence and each contains a tandem duplication of delta2-delta3-delta4. However, IGHD1 has a terminal exon encoding the transmembrane region, whereas IGHD3 has a single terminal exon encoding a secreted tail. The occurrence of IGHD3 immediately downstream of a micro pseudogene indicates that the putative deltas product may not be expressed as a chimeric micro delta molecule. Western blots and protein sequencing data indicate that an IGHD3-encoded protein is expressed in catfish serum. Thus, catfish deltam transcripts appear to originate from IGHD1, whereas deltas transcripts originate from IGHD3 rather than, as previously inferred, from a single expressed delta gene. The third delta (IGHD2) is associated with a pseudo C micro (IGHM2P); its presence is inferred by Southern blot analyses.

The use of allogeneic and autologous lymphoid cell lines has facilitated studies of cytotoxic T lymphocytes (CTL) and natural killer (NK)-like cells in channel catfish. Naïve catfish leukocytes were shown to spontaneously kill allogeneic cells and virally-infected autologous cells without the need for prior sensitization, and allogeneic cytotoxic responses were greatly enhanced by in vitro alloantigen stimulation. Both catfish CTL and NK-like cells have been successfully cloned from these alloantigen-stimulated cultures, and represent the first cytotoxic cell lines derived from any ectothermic vertebrate. These cloned cytotoxic cells contain granules and likely induce apoptosis in sensitive targets via a putative perforin/granzyme mechanism. In addition, some catfish CTL clones may also kill targets by an additional mechanism, possibly by Fas/FasL-like interactions. Importantly, these cytotoxic cells do not express the marker for catfish nonspecific cytotoxic cells (NCCs), and thus represent cell types distinct from NCCs. The use of monoclonal antibodies against the catfish F and G immunoglobulin light chain isotypes revealed the presence of a putative Fc receptor for IgM (Fc mu R) on some catfish NK-like cells that appears to 'arm' these cells with surface IgM. In addition, a potentially important monoclonal antibody (CC41) developed against catfish NK-like cells was found to recognize an approximately 150kDa molecule on the surface of catfish cytotoxic cells. These studies clearly demonstrate that catfish possess an array of different cytotoxic cells. The availability of various cloned cytotoxic cell lines should enable unambiguous functional studies to be performed in ways not currently possible with any other fish species.

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.

The transcriptional enhancer (Eµ3′) of the IgH locus of the channel catfish, Ictalurus punctatus, shows strong B cell-specific activity and differs from the mammalian Eµ enhancer in both location and structure. It occurs between the µ and δ genes and contains numerous transcription factor binding sites, predominantly octamer and µE5 motifs of consensus and variant sequences. It lacks the classical µA-µE3(CBF)-µB core array of binding motifs seen within mammalian IgH Eµ enhancers. To determine the functionally important motifs, a series of mutant enhancers was created using sequence-targeted polymerase chain reaction. Whereas the mutation of consensus and variant octamer motifs (individually or in multiples) decreased enhancer function, mutation of a single consensus µE5 motif destroyed the function of this enhancer in mammalian plasmacytomas. Mutation of this consensus µE5 site, combined with mutations of certain octamer sites, destroyed function in catfish B cells. Experiments using artificial enhancers containing multimers of motifs or short regions of the native enhancer suggested that the minimal Eµ3′ enhancer (a) contains a consensus µE5 site and two octamer sites, (b) is B cell-specific, and (c) is active across species. The dependence of an Ig enhancer on sites that bind basic helix-loop-helix and Oct transcription factors has not previously been observed and confirms large differences in structure and function between fish and mammalian IgH enhancers.

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-2Rα-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.

In order to characterize the Major histocompatibility complex (MHC) class II A genes of the channel catfish (Ictalurus punctatus) a cDNA library was screened and PCR was performed. Four different full-length cDNA sequences for MHC class II A genes were obtained from a clonal B cell line derived from an outbred fish. Two different genomic sequences and corresponding cDNAs were obtained from a presumably homozygous gynogenetic catfish. The A genes have five exons and four phase one introns. The first exon encodes the 5' untranslated region (UTR) and leader peptide; the second and third exons encode the alpha1 and alpha2 domains, respectively. The connecting peptide, transmembrane and cytoplasmic domains, as well as part of the 3' UTR, are encoded by the fourth exon and the rest of the 3' UTR is encoded by the fifth exon. Southern blot analyses using an exon three probe revealed two to four hybridizing fragments with considerable restriction fragment length polymorphisms evident among randomly selected outbred channel catfish. These findings are consistent with the presence of at least two functional polymorphic MHC class II A gene loci. An unusual aspect of the channel catfish MHC class II alpha chain is its lack of N-linked glycosylation sites.

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 α and β 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 αβ+ allospecific cytotoxic cells, 2) TCR αβ+ nonspecific cytotoxic cells, 3) allospecific TCR αβ+ noncytotoxic cells, 4) TCR αβ− nonspecific cytotoxic cells, and 5) TCR αβ− allospecific cytotoxic cells. The demonstration of cloned, TCR αβ+, allospecific cytotoxic effectors provides the strongest evidence to date for the existence of cytotoxic T cells in fish.

Four cDNAs encoding the major histocompatibility complex (MHC) class I alpha chain were isolated from a channel catfish clonal B-cell cDNA library. Sequence analysis suggests these cDNAs represent three different MHC class I loci. All cDNAs encoded conserved residues characteristic of the MHC class I alpha chain: namely, those involved in peptide binding, salt bridges, disulfide bond formation, and glycosylation. Southern blot analyses of individual outbred and second-generation gynogenetic fish indicated the existence of both polygenic and polymorphic loci. Northern blot studies demonstrated that catfish B, T, and macrophage cell lines transcribed markedly higher levels of class I alpha and beta2-microglobulin (beta2m) mRNA than fibroblast cell lines. In addition, immunoprecipitation data showed that a 41 000 Mr glycoprotein (presumably class I alpha) was associated with beta2m on the surface of catfish B cells. This latter finding is the first direct evidence for the cell surface association of beta2m with the MHC class I alpha chain on teleost cells and supports the notion that functional MHC class I proteins exist in teleosts.

Immunoglobulins (Ig) can occur in two physical forms, soluble (secreted) and membrane bound. The soluble form is secreted from B cells, and is present in the blood and other fluids where it plays a role as an immune effector molecule. The membrane-bound form of the Ig molecule is inserted into the B-cell membrane, where it serves as a receptor for antigen. The function of the membrane-bound Ig as a receptor for antigen requires additional accessory molecules, the membrane Ig plus accessory molecules are referred to, collectively, as the B-cell receptor (BCR) complex. The secreted and membrane-bound forms of an Ig result from alternative patterns of RNA processing of the primary transcript from the heavy chain gene. IgM is the only class of Ig known to be conserved in all vertebrate species (perhaps exclusive of the agnathan fish). While the structure of the IgM heavy (mu) chain gene has been highly conserved in vertebrate evolution, the patterns of alternative RNA processing of the mu transcript show surprising diversity. In particular, the bony fish (teleosts) produce membrane mu-chain message by a splicing pathway that is quite different from that seen in other vertebrates; it results in the production of membrane IgM that lacks the C mu 4 domain. How this unusual RNA splicing pattern could have evolved and its implications for the function of the BCR in the bony fishes are considered here.

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.

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.

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.

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.

The Ig heavy chain enhancer of the channel catfish (Ictalurus punctatus) has an unusual position and structure, being found in the 3′ region of the μ gene and containing eight functional octamer motifs of consensus (ATGCAAAT) and variant sequences. The presence of multiple octamer motifs suggests that an Oct2 homologue may play an important role in driving expression of the Ig heavy chain locus in a teleost fish. To test this hypothesis, two catfish Oct2 cDNAs (α and β) were cloned by screening a catfish B cell cDNA library. Catfish Oct2 α and β isoforms are derived by alternative RNA splicing; as determined by Southern analysis, Oct2 is a single copy gene. In comparisons with mammalian Oct2, the catfish Oct2 isoforms show high sequence conservation in their N-terminal regions and POU domains, but extensive divergence in their C-terminal regions. Catfish Oct2 α and β are tissue restricted, bind both consensus and variant octamer motifs, and activate transcription in both catfish and murine cells. In contrast, mouse Oct2 activated transcription in mouse but not catfish cells. Catfish Oct2 β is a more potent transcriptional activator than Oct2 α. In transient expression assays, catfish Oct2 β showed a marked preference for the octamer variant, ATGtAAAT, which occurs twice in the catfish enhancer. Mouse Oct2 also showed increased activity with the variant octamer when tested in mouse B cells. Gel-shift analysis competition assays indicated that catfish Oct2 binds the consensus octamer motif with an apparently higher affinity than it does the variant motif.

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 ∼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-γ activation site; IRF, interferon response factor; EMSA, electromobility shift assay. 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-γ activation site sequence. These results suggest that STAT molecules have been highly conserved in vertebrate evolution.

In previous work, lysis of allotargets was routinely observed with PBL from nonimmune channel catfish. In the work reported here, greatly increased (approximately 100-fold) cytotoxic responses were generated by stimulation of channel catfish PBL with irradiated cells of allogeneic cloned B cell lines in mixed leukocyte cultures (MLC). This increased cytotoxicity did not appear to be simply a consequence of cell proliferation since stimulation of catfish PBL proliferative responses with polyclonal mitogens did not result in increased lysis. Somewhat surprisingly, the MLC-generated cytotoxicity did not exhibit allospecificity; i.e., allogeneic targets from other fish were as susceptible to lysis as were the cells used as stimulators. This apparent lack of allospecificity in MLC-generated cytotoxicity was confirmed by "cold" target inhibition assays. However, autologous targets were not killed, clearly demonstrating that MLC-generated effectors could distinguish "self" from "nonself" at the level of lysis/recognition. Although their origin is unresolved, the MLC-generated effectors may be a source of highly enriched fish cytotoxic cells and thus facilitate directly addressing questions pertaining to the evolution of such cells.

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 (δ) 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 μ, and seven constant domains encoded by a δ gene homolog. Identification of the catfish gene as δ is based on the following properties: sequence relatedness to mammalian δ; a location within the IgH locus that is immediately downstream of the μ gene; separate terminal exons for the secretory and membrane forms; coexpression with the complete μ 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.

Long term cell lines can be readily established at high frequency with PBLs from normal channel catfish. Depending upon the mode of stimulation, morphologically and functionally distinct catfish lymphoid cell lines resembling B cells, T cells and monocytes have been developed. These fish cell lines appear unique from their putative mammalian counterparts in that they are immortalized without the need for exogenous factors or overt attempts at transformation.

Immunoglobulin M heavy chain (mu) sequences of two holostean fish, the bowfin, Amia calva, and the longnose gar, Lepisosteus osseus, were amplified from spleen mRNA by RACE-PCR, cloned, and sequenced. Each mu chain showed the conserved four constant domain structure typical of a secreted mu chain. Southern blot analyses with specific heavy chain variable (VH) and constant (CH) region probes suggest that both fish possess an IgH locus that resembles that of the teleosts, amphibians, and mammals in its organization. The overall sequence similarity of gar and bowfin mu chains was 60% and 48% at the nucleotide and amino acid levels, respectively, while similarity to the mu chains of teleosts and elasmobranchs was lower. The bowfin mu chain possesses a distinctive proline-rich sequence at the C mu 1/C mu 2 boundary; a shorter proline-rich sequence is present at this position in the gar mu chain. Both gar and bowfin show, in their C mu 4 sequences, motifs that could serve as cryptic splice donor sites for the production of mRNA encoding the membrane-bound form of the mu chains, and the bowfin also shows a potential cryptic splice donor site in the C mu 3 exon.

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.

Techniques are described for the generation of channel catfish long term leukocyte cell lines. These techniques include the isolation of peripheral blood leukocytes, purification of B cells by anti-immunoglobulin panning, mitogen stimulation, and in vitro maintenance and cloning of leukocyte cultures. Once stimulated in vitro, channel catfish leukocytes proliferate continuously without the need for exogenous growth factors or feeder cells. The various leukocyte cultures generated are heterogeneous and contain mixtures of monocyte/macrophage-like, T-like, or B cells. Clonal cell lines can be obtained from these mixed cultures by limiting dilution cloning in the presence of conditioned medium. A critical component of the culture medium is the use of channel catfish serum which is required for supporting and maintaining in vitro leukocyte proliferation.

Previously we sequenced a partial cDNA clone encoding the 3' region of the message for the membrane receptor form of the heavy (mu) chain of the channel catfish which indicated that the first transmembrane (TM1) exon is spliced directly to the C mu 3 exon and not into a cryptic site within the CH4 exon, as occurs in other vertebrates. Studies utilizing polymerase chain reaction analysis of mRNA and further analysis of cDNA clones now confirm that the only detectable splicing pattern used in micron production by the channel catfish utilizes this C mu 3----TM1 pathway of pre-mRNA splicing.

Studies were conducted to address further the role(s) of antigen processing and presentation in the induction of immune responses in a phylogenetically lower vertebrate, specifically a teleost, the channel catfish. In particular, studies were aimed at determining the subcellular compartments involved in antigen degradation by channel catfish antigen-presenting cells (APC) as well as ascertaining the reexpression of immunogenic peptides on the surfaces of APC. The results showed that exogenous protein antigens were actively endocytosed by APC as detected by flow cytometry. Use of radiolabeled antigen and subcellular fractionation protocols also showed that antigen localized in endosomes/lysosomes. Furthermore, there was an apparent redistribution of antigen between these organelles and the plasma membrane during the course of antigen pulsing. Functional assays for the induction ofin vitroantigen-specific proliferation of immune catfish peripheral blood leukocytes (PBL) showed that membrane preparations from antigen-pulsed autologous APC were highly stimulatory. The magnitude of responses elicited with such membrane preparations was very similar to that of PBL cultures stimulated with native antigen-pulsed and fixed intact APC or prefixed intact APC incubated with a peptide fragment of the nominal antigen. Current data further corroborate our previous findings that steps akin to antigen processing and presentation are clearly important in the induction of immune responses in lower vertebrates like fish, in a manner similar to that seen in mammalian systems. Consequently, it would appear that many immune functions among the diverse taxa of vertebrates are remarkably conserved.

Recent in vitro studies clearly demonstrate the importance of antigen processing and presentation in the generation of immune responses to T-dependent antigens (i.e. proteins and hapten-carrier conjugates) in phylogenetically lower vertebrates such as teleosts. Similar to the situation in mammals, antigens are processed and presented by accessory or antigen-presenting cells (APC), such as monocytes or macrophages, to specific lymphocytes in a seemingly alloantigen- (presumably major histocompatibility complex [MHC] or MHC-like) restricted fashion. Results show that processing involves proteolysis, which presumably occurs within acidic subcellular compartments. The requirement for processing can be circumvented by the presentation of peptide fragments of the native antigen on paraformaldehyde-fixed APC. Moreover, usage of structurally defined proteins, such as cytochrome C, as model antigens reveals that their species variants are cross-stimulatory to immune fish lymphocytes. Molecular analyses of such antigens reveal the existence of overlapping epitopes that seem to define the specificity of the immune response to such “families” of antigens but not to other unrelated (yet structurally defined) antigens. Consequently, these studies corroborate the hypothesis that immune functions in the divergent classes of vertebrates are highly conserved. Further, results from such studies also show that these immunologic processes appear to occur under low temperature regimes previously reported to suppress primary immune responses. Hence, these studies provide direct evidence that low temperature-induced immunosuppression in fish does not involve impaired APC functions. In light of the above observations indicating similarities between fish and mammalian systems, implications for fish vaccine design are also discussed.

A linear epitope on catfish IgM has been identified as the docking site for the catfish soluble FcmuR (IpFcRI). Western blot analyses and latex bead binding assays identified the consensus octapeptide motif FxCxVxHE located at the second cysteine that forms the intrachain disulfide bond of the catfish Cmu3 and Cmu4 immunolglobulin (Ig) domains as the IpFcRI binding sites. Furthermore, molecular modeling of catfish Cmu3 and Cmu4 confirmed that the octapeptide in both of these domains is accessible for IpFcRI interactions. In addition, since this octapeptide motif is also found in other vertebrate Ig domains, IpFcRI binding to Ig heavy (H) and light (L) chains from rainbow trout, chicken, mouse, rabbit, and goat were examined by Western blot analyses and latex bead binding assays. IpFcRI readily bound reduced rainbow trout (Igmu), chicken (Ignu), mouse (Igmu, Iggamma1, Iggamma2a, Iggamma2b, and Igalpha), rabbit (Igmu and Iggamma) and goat (Iggamma) IgH chains, and mouse Igkappa and Iglambda, and chicken Iglambda IgL chains. IpFcRI also bound mouse IgM, IgA and IgG subclasses when examined under native conditions.

This study characterizes three monoclonal antibodies (mAbs) developed against the constant (C) region of the immunoglobulin light (IgL) sigma chain isotype of the channel catfish, Ictalurus punctatus. Microsphere bead assays and Western blot analyses utilizing different recombinant (r) proteins show that these anti-catfish IgL sigma chain mAbs each specifically recognize the denatured form of IgL sigma. Importantly, Western blotting of catfish sera using the anti-IgL sigma mAbs also identified an IgL chain-sized immunoreactive band(s) of approximately 27kDa. It is anticipated that these mAbs in combination with the already existing anti-catfish Ig heavy (H) and IgL chain mAbs will be useful in future studies examining the functional roles of the different catfish IgL isotypes.