Catfish lymphocytes expressing CC41-reactive leukocyte immune-type receptors (LITRs) proliferate in response to Edwardsiella ictaluri infection in vitro.

Monoclonal antibodies (mAbs) CC34 and CC41 recognize overlapping subsets of leukocyte immune-type receptors (LITRs). The mAb CC34 was raised against the clonal TS32.15 cytotoxic T cell line and the mAb CC41 was raised against the clonal NK cell line TS10.1. In this study, an in vitro model was developed to monitor CC34- and CC41-reactive cells in response to Edwardsiella ictaluri infection. Briefly, head kidney leukocytes and peripheral blood lymphocytes (PBL) were isolated from individual catfish and labeled with CellTrace Violet and CellTrace FarRed dye, respectively. Head kidney-derived macrophages were infected with E. ictaluri and then cocultured with autologous PBL. The combined cell cultures were then analyzed using flow cytometry. A significant increase in CC41 staining was observed in the PBL population at 2, 5 and 7 days after culture, which suggest that LITRs are involved in cell-mediated immunity to E. ictaluri.

Cloning and characterization of antiviral cytotoxic T lymphocytes in channel catfish, Ictalurus punctatus.

To determine the role of piscine anti-viral cytotoxic cells, we analyzed the response of channel catfish to Ictalurid herpesvirus 1, commonly designated channel catfish virus (CCV). Peripheral blood leukocytes (PBL) from catfish immunized with MHC-matched, CCV-infected G14D cells (G14D-CCV) showed marked lysis of G14D-CCV but little to no lysis of uninfected allogenic (3B11) or syngeneic (G14D) cells. Expansion of effectors by in vitro culture in the presence of irradiated G14D-CCV cells generated cultures with enhanced cytotoxicity and often broader target range. Cytotoxic effectors expressed rearranged TCR genes, perforin, granzyme, and IFN-γ. Four clonal cytotoxic lines were developed and unique TCR gene rearrangements including γδ were detected. Furthermore, catfish CTL clones were either CD4+/CD8- or CD4-/CD8-. Two CTL lines showed markedly enhanced killing of G14D-CCV targets, while the other two lines displayed a broader target range. Collectively, catfish virus-specific CTL display unique features that illustrate the diversity of the ectothermic vertebrate immune response.

Insights into the dynamics of memory, effector and apoptotic cytotoxic T lymphocytes in channel catfish, Ictalurus punctatus.

In this study, we used the channel catfish model clonal TS32.15 alloantigen-specific cytotoxic T cell (CTL) line to examine the dynamics of memory CTL expansion and senescence in teleosts. Although TS32.15 has been routinely cultured to study catfish CTL responses and killing mechanisms, little is known about the dynamics of the CTLs in these cultures. Here we show that this cell line consists of small non-cytotoxic T cells and larger granular effector T cells and that their ratios vary with time after stimulation. Small CTLs, when exposed to their irradiated targets, replicate and differentiate to morphologically distinct cytotoxic effectors, which do not replicate. After lysing target cells, or with prolonged absence of stimulation, the effector cells transition to a non-cytolytic senescent stage or become apoptotic. In addition, we demonstrate that natural IgM in catfish serum binds lipids, including PIP2, on early apoptotic CTLs, and that these IgM+ CTL can be cleared by catfish head kidney-derived macrophages.

A Leukocyte Immune-Type Receptor Subset Is a Marker of Antiviral Cytotoxic Cells in Channel Catfish, Ictalurus punctatus.

Channel catfish, Ictalurus punctatus, leukocyte immune type receptors (LITRs) represent a multigene family that encodes Ig superfamily proteins that mediate activating or inhibitory signaling. In this study, we demonstrate the use of mAb CC41 to monitor viral cytotoxic responses in catfish and determine that CC41 binds to a subset of LITRs on the surface of catfish clonal CTLs. Homozygous gynogenetic catfish were immunized with channel catfish virus (CCV)-infected MHC-matched clonal T cells (G14D-CCV), and PBL were collected at various times after immunization for flow cytometric analyses. The percentage of CC41(+) cells was significantly increased 5 d after primary immunization with G14D-CCV and at 3 d after a booster immunization as compared with control fish only injected with G14D. Moreover, CC41(+) cells magnetically isolated from the PBL specifically killed CCV-infected targets as measured by (51)Cr release assays and expressed messages for CD3γδ, perforin, and at least one of the CD4-like receptors as analyzed by RNA flow cytometry. When MLC effector cells derived from a G14D-CCV-immunized fish were preincubated with CC41 mAb, killing of G14D-CCV targets was reduced by ∼40%, suggesting that at least some LITRs have a role in target cell recognition and/or cytotoxicity. The availability of a LITR-specific mAb has allowed, to our knowledge for the first time, functional characterization of LITRs in an autologous system. In addition, the identification of an LITR subset as a cytotoxic cell marker will allow for more effective monitoring of catfish immune responses to pathogens.

The Src tyrosine kinase Lck binds to CD2, CD4-1, and CD4-2 T cell co-receptors in channel catfish, Ictalurus punctatus.

The binding of the lymphocyte specific protein tyrosine kinase (Lck) to T cell co-receptors is required for T cell development and activation. In mammals, Lck initiates signal transduction by binding to CD4 and CD8 co-receptors and phosphorylating ITAMs in the cytoplasmic tail of the CD3 molecules and the ζ chains. In addition, Lck can also bind to the adhesion molecule CD2 and trigger T cell activation. In this study, Lck and CD2 homologs were identified and characterized in channel catfish, Ictalurus punctatus. Lck and CD2 mRNAs were specifically expressed by clonal T cell lines, including both CD4(+) and CD4(-)CD8(-) CTL lines, and in mixed lymphocyte cultures (MLC). Western blot analyses using anti-trout Lck and anti-human p-Lck antibodies demonstrated that Lck protein is expressed in catfish clonal CTL and is phosphorylated at a conserved tyrosine residue. Because of the lack of CD8(+) CTL lines as well as the absence of CD8 message in MLC, we performed magnetic bead binding assays to correlate CD2, CD4, and CD8 co-receptor expression with Lck binding ability. Recombinant Lck reproducibly bound to CD2, CD4-1, and CD4-2, but not to CD8α or CD8ß. These data provide one possible explanation for the apparent low numbers of CD8(+) CTL and the presence of CD4(+) and CD4(-)CD8(-)CD2(+) CTL in catfish.

Identification of SHIP-1 and SHIP-2 homologs in channel catfish, Ictalurus punctatus.

Src homology domain 2 (SH2) domain-containing inositol 5'-phosphatases (SHIP) proteins have diverse roles in signal transduction. SHIP-1 and SHIP-2 homologs were identified in channel catfish, Ictalurus punctatus, based on sequence homology to murine and human SHIP sequences. Full-length cDNAs for catfish SHIP-1 and SHIP-2 (IpSHIP-1 and IpSHIP-2) were obtained using 5' and 3' RACE protocols. Catfish SHIP molecules share a high degree of sequence identity to their respective SHIP sequences from diverse taxa and both are encoded by single copy genes. IpSHIP-1 and IpSHIP-2 transcripts were expressed in all catfish tissues analyzed except for skin, and IpSHIP-1 message was more abundant than IpSHIP-2 message in lymphoid tissues. Catfish clonal B, cytotoxic T, and macrophage cell lines also expressed message for both molecules. IpSHIP-1 and IpSHIP-2 SH2 domains were expressed as recombinant proteins and were both found to be bound by cross-reacting rabbit anti-mouse SHIP-1 pAb. The anti-mouse SHIP-1 pAb also reacted with cell lysates from the cytotoxic T cell lines, macrophages and stimulated PBL. SHIP-1 is also phosphorylated at a conserved tyrosine residue, as shown by immunoprecipitation studies.

Channel catfish (Ictalurus punctatus) leukocytes express estrogen receptor isoforms ERα and ERß2 and are functionally modulated by estrogens.

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 ERß2. Transcripts of these isoforms were detected in tissue-associated leukocyte populations by PCR, but ERß2 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 17ß-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.

CD4+ T-helper cells stimulated in response to placental ischemia mediate hypertension during pregnancy.

We have shown that hypertension in response to chronic placental ischemia is associated with elevated inflammatory cytokines and CD4(+) T cells. However, it is unknown whether these cells play an important role in mediating hypertension in response to placental ischemia. Therefore, we hypothesize that reduced uterine perfusion pressure (RUPP)-induced CD4(+) T cells increase blood pressure during pregnancy. To answer this question, CD4(+) T cells were isolated from spleens at day 19 of gestation from control normal pregnant (NP) and pregnant RUPP rats, cultured, and adjusted to 10(6) cells per 100 µL of saline for intraperitoneal injection into NP rats at day 13 of gestation. On day 18, in the experimental groups of rats, arterial catheters were inserted, and on day 19 mean arterial pressure was analyzed. Inflammatory cytokines and antiangiogenic factor soluble fms-like tyrosine kinase 1 were determined via ELISA. Mean arterial pressure increased from 104±2 mm Hg in NP rats to 124±2 mm Hg in RUPP rats (P<0.001) and to 118±1 mm Hg in rats receiving RUPP CD4(+) T cells (P<0.001). Circulating tumor necrosis factor-α and soluble fms-like tyrosine kinase 1 were elevated in recipients of RUPP CD4(+) T cells to levels similar to control RUPP rats. In contrast, virgin rats injected with NP or RUPP CD4(+) T cells exhibited no blood pressure changes compared with control virgin rats. Importantly, mean arterial pressure did not change in recipients of NP CD4(+) T cells (109±3 mm Hg). These data support the hypothesis that RUPP-induced CD4(+) T cells play an important role in the pathophysiology of hypertension in response to placental ischemia.

Identification of two IgD+ B cell populations in channel catfish, Ictalurus punctatus.

Channel catfish Ictalurus punctatus express two Ig isotypes: IgM and IgD. Although catfish IgM has been extensively studied at the functional and structural levels, much less is known about IgD. In this study, IgM(+)/IgD(+) and IgM(-)/IgD(+) catfish B cell populations were identified through the use of anti-IgM and anti-IgD mAbs. Catfish IgM(+)/IgD(+) B cells are small and agranular. In contrast, IgM(-)/IgD(+) B cells are larger and exhibit a plasmablast morphology. The use of cell sorting, flow cytometry, and RT-PCR demonstrated that IgD(+) B cell expression varies among individuals. For example, some catfish have <5% IgM(-)/IgD(+) B cells in their PBLs, whereas in others the IgM(-)/IgD(+) B cell population can represent as much as 72%. Furthermore, IgD expressed by IgM(-)/IgD(+) B cells preferentially associates with IgL σ. Comparatively, IgM(+)/IgD(+) B cells can express any of the four catfish IgL isotypes. Also, transfection studies show that IgD functions as a typical BCR, because Igδ-chains associate with CD79a and CD79b molecules, and all membrane IgD transcripts from sorted IgM(-)/IgD(+) B cells contain viable VDJ rearrangements, with no bias in family member usage. Interestingly, all secreted IgD transcripts from IgM(+)/IgD(+) and IgM(-)/IgD(+) B cells were V-less and began with a leader spliced to Cδ1. Importantly, transfection of catfish clonal B cells demonstrated that this leader mediated IgD secretion. Together, these findings imply that catfish IgM(-)/IgD(+) B cells likely expand in response to certain pathogens and that the catfish IgD Fc-region, as has been suggested for human IgD, may function as a pattern recognition molecule.

Expression of alternatively spliced CD45 isoforms by channel catfish clonal T and B cells is dependent on activation state of the cell and regulated by protein synthesis and degradation.

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.

Channel catfish soluble FcmuR binds conserved linear epitopes present on Cmu3 and Cmu4.

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.

Immunoglobulin D enhances immune surveillance by activating antimicrobial, proinflammatory and B cell-stimulating programs in basophils.

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.

Granzyme-like sequences in bony fish shed light on the emergence of hematopoietic serine proteases during vertebrate evolution.

Hematopoietic serine proteases (SPs) are stored in the granules of different leukocytes and these enzymes are important effector molecules in the immune system of mammals. However, very little is known about the presence of these proteins in lower vertebrates. Herein, the primary structures of five novel fish SPs, from the Atlantic cod (Gadus morhua) and the channel catfish (Ictalurus punctatus), are presented. One of the cod SPs is a homologue to human GzmA and K. The other fish SPs identified are termed 'Gzm-like' and are distantly related to a large heterogeneous group of hematopoietic SPs, including most of the T-cell Gzms (B-H), the mast cell chymases, the mast cell/basophil proteases of the mouse mast cell protease-8 subfamily (M8-family) and the neutrophil cathepsin G. Extensive BLAST-searches in genome and expressed sequence tag (EST) databases identified 40 additional teleost SPs related to the mammalian hematopoietic SP family. Subsequent phylogenetical analyses clearly demonstrate that the diversification into different subgroups within the GzmB/chymase/cathepsin G-related family has occurred independently in bony fishes and in mammals. In contrast, our findings suggest that the three subgroups, including (1) GzmK and the potent apoptosis-inducing GzmA, (2) the neutrophil proteases (proteinase 3, N-elastase and azurocidin), and (3) adipsin, have all evolved as distinct groups before the separation of tetrapods from the ray-finned fish approximately 420 million years ago.

Genomic organization of the channel catfish CD45 functional gene and CD45 pseudogenes.

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).

Identification and characterization of a FasL-like protein and cDNAs encoding the channel catfish death-inducing signaling complex.

To elucidate cytolytic mechanisms in the channel catfish, lysates from catfish lymphoid and fibroblast cell lines were screened by Western blot analysis using a panel of antibodies reactive with components of the mammalian apoptotic pathway. Strong reactivity with three proteins (approximate Mr 70,000, 37,000, and 15,000) was seen using an antibody targeted to mammalian Fas ligand (FasL). The sizes of the two smaller proteins are consistent with their tentative designation as membrane-bound (37,000 Mr) and soluble (15,000 Mr) FasL. Treatments known to induce FasL in mammalian systems (e.g., PMA/calcium ionophore, UV-irradiation) induced expression of the 37,000- Mr protein in catfish T-cell lines. Moreover, expression of the 37,000- Mr protein in clonal T cells was up-regulated by increasing cell density. At the nucleotide level, homologues of Fas receptor (FasR), FADD, and caspase 8 were identified and characterized. These gene products likely constitute the teleost equivalent of the death-inducing signaling complex (DISC). FADD was constitutively expressed in all (T, B, macrophage, and fibroblast) cell lines examined as well as in peripheral blood lymphocytes (PBL), whereas FasR and caspase 8 were expressed in all cell lines except CCO, a FasL-positive fibroblast line. In contrast to FasL, expression of FasR and caspase 8 was inversely proportional to cell density. Collectively these studies identified four membrane-proximal proteins involved in the initiation of apoptosis in channel catfish and suggest that mechanisms of cell-mediated cytotoxicity in teleosts are similar to those used by mammals.

Immortal and mortal clonal lymphocyte lines from channel catfish: comparison of telomere length, telomerase activity, tumor suppressor and heat shock protein expression.

Channel catfish autonomous (immortal) and nonautonomous (mortal) leukocyte lines were phenotyped with respect to telomere length and the expression of telomerase, Hsp70 and p53, potentially important factors in cellular immortalization. The autonomous cells constitutively expressed telomerase whereas the nonautonomous cells expressed this activity only transiently. This observation, coupled with the low telomerase activity level seen in freshly isolated leukocytes, suggests that telomerase expression in catfish leukocytes is activation induced. In contrast both types of cell lines exhibited quite similar patterns of significantly shortened telomeres, suggesting that telomerase does not stabilize catfish telomeres until a critical short length is reached. Northern analyses indicated that, like telomerase, Hsp70 gene expression was constitutive in autonomous cells and transient in nonautonomous cells. In contrast, p53 mRNA levels appeared similarly low and noncycling in both long-term cultured types of catfish cells, regardless of the culture situation. Furthermore it was noted, by Western analyses, that both types of cells display multiple sized forms of p53 proteins. This latter observation implies that truncation of p53 protein is probably not directly involved in the in vitro immortalization process of channel catfish leukocytes.

Channel catfish cytotoxic cells: a mini-review.

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.