Source: UNIVERSITY OF MISSISSIPPI MEDICAL CENTER submitted to
IDENTIFICATION AND MOLECULAR CHARACTERIZATION OF CYTOTOXIC CELLS IN CATFISH
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0186822
Grant No.
99-35204-7844
Project No.
MISR-1999-02019
Proposal No.
1999-02019
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Sep 15, 1999
Project End Date
Mar 31, 2004
Grant Year
1999
Project Director
Chinchar, G. V.
Recipient Organization
UNIVERSITY OF MISSISSIPPI MEDICAL CENTER
2500 NORTH STATE STREET
JACKSON,MS 39216
Performing Department
MICROBIOLOGY
Non Technical Summary
Channel catfish are the most important aquacultural species in the United States and are a significant component of the economies of several states. However, as aquacultural practices become more intensive, the likelihood of serious disease among farm-raised catfish increases. The long-range goal of this proposal is to enhance disease prevention through an expanded understanding of the catfish immune system. Specifically, this project will focus on anti-viral cell-mediated immunity (CMI) because, although little is known about CMI in fish, in other animals CMI is essential for recovery from viral infections. The goals are threefold: (1) to demonstrate that catfish possess authentic cytotoxic T cells, (2) to characterize natural killer and other cytotoxic cells, and (3) to elucidate the genetic organization and function of catfish major histocompatibility complex (MHC) class I genes. To achieve the first aim, MHC matched, 2nd generation gynogenetic fish will be used to determine if catfish utilize MHC-restricted, cytotoxic T cells to kill virus-infected targets. Successful completion of this study will facilitate the assessment and development of vaccines designed to induce both antibodies and cytotoxic effector cells in catfish. Specific aim #2 will provide fundamental information on the mechanisms of cell-mediated killing by different types of channel catfish cytotoxic cells. Finally, since currently little is known about catfish MHC and its role in immunity, the third aim will provide important information on the genetic organization and role of catfish MHC genes and
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
31137101090100%
Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3710 - Catfish;

Field Of Science
1090 - Immunology;
Goals / Objectives
Our goals are three-fold: (a) to demonstrate that catfish possess authentic cytotoxic T lymphocytes (CTL) and to determine the kinetics of their induction in vivo, (b) to characterize at the molecular and phenotypic levels natural killer (NK) and other cytotoxic cells, and (c) to elucidate the genetic organization and expression patterns of catfish major histocompatibility complex (MHC) class I genes.
Project Methods
Firstly, the ability of fish lymphocytes to kill cells infected by channel catfish herpes virus, channel catfish reovirus, and one or more indovirus species, e.g. European catfish virus will be monitored. Long-term cell lines from gynogenetic catfish will be infected with virus and used as syngeneic targets in in vitro assays to determine whether channel catfish possess authentic, virus-specific CTL. Among fish, this system is unique to channel catfish and is a reflection of the development of MHC matched families of gynogenetic fish and our ability to generate virus-permissive, long-term, cell lines from catfish lymphoid cells. Lysis of target cells will be measured by both chromium release and DNA fragmentation (JAM) assay. In a second series of experiments, CTL, along with NK cells and allospecific cytotoxic T cells, will be characterized to determine their mechanism of killing. The role of soluble and cell surface-associated lytic molecules such as perforin and Fas/Fas ligand will be assessed and the induction of cytoplasmic granules monitored. Apoptotic killing will be monitored by the release of fragmented DNA, Annexin V binding, TUNEL assay and Hoechst/DAPI staining of native and condensed chromatin. Finally, nucleotide sequence analysis will identify promoter regions upstream of MHC class I A genes and PCR and Northern blot assays will be used to determine the expression kinetics of class I genes in different fish tissues and cell lines. Furthermore, the impact of virus infection on MHC expression will be determined.

Progress 09/15/99 to 03/31/04

Outputs
We have made considerable progress in elucidating key aspects of cell-mediated immunity in the channel catfish. Building on previously supported USDA research, we (1) determined the genomic organization of channel catfish MHC class I genes, (2) examined target cell recognition and cytotoxic mechanisms of cloned catfish cytotoxic T cells (CTLs, (3) demonstrated the anti-viral activity of two antimicrobial peptides from lower vertebrates, (4) documented apoptotic events in iridovirus-infected fish cells, and (5) identified and characterized the activity of several key molecules (catfish NFAT, tumor necrosis factor [TNF], and interferon [IFN] ) mediating immune activation and cell death in catfish. A brief summary of each of these follows. (1) MHC class I genes: We determined the intron/exon arrangement of class I genes and identified possible transcription factor binding sites by Electromobility Shift Assay and sequence analysis. (2) Alloantigen-dependent CTLs: Clonal catfish CTLs were shown to fall into at least two categories: Antigen-specific CTLs that kill by a perforin-granzyme pathway, and more broadly-specific CTLs that kill via both secretory and other cytolytic pathways. Furthermore, catfish CTLs were shown to contain putative cytolytic granules that mediated killing. (3) Antimicrobial peptides (AMPs): We examined the ability of amphibian-derived AMPs to inactivate channel catfish virus and frog virus 3 (FV3). We showed that two amphibian AMPs rapidly inactivated both these viruses suggesting that AMPs may play a role, along with IFN, TNF, and NK cells, in controlling virus infections in fish and other lower vertebrates. (4) Apoptosis in FV3-infected cells: In a related study, we showed that iridovirus infection triggered apoptosis in fathead minnow cells. While this result was not surprising, it demonstrated that fish cells undergoing apoptosis utilize mechanisms similar to those found in mammalian cells. (5) Catfish cytokines: The role of cytokines and cytokine activators was examined in three studies examining catfish IFN, TNF, and NFAT. We were one of the first groups to identify IFN and TNF cDNAs in fish, and are in the process of generating recombinant IFN for the purpose of determining its biological effects. Moreover, using a cross-reactive antibody, we identified a catfish NFAT-like protein and showed that it bound to a known IL-2 promoter sequence. Collectively, these studies suggest that cytokines are involved in T cell activation events and anti-viral immunity. The studies summarized above have served as a stepping stone for current USDA supported work. We have continued to characterize catfish NK cells and additional AMPs. Recently, we identified putative immune evasion proteins in two iridovirus genomes. Finally, we identified and characterized elements of the death inducing and signaling complex (DISC) in catfish, i.e., FasL, FAS, FADD, and caspase 8. Clearly, our understanding of anti-viral immunity in catfish, and other fish species, is growing, and hopefully should begin to yield practical benefits in improved vaccines.

Impacts
Our work will help researchers better understand the basis of cell-mediated and innate immunity in channel catfish and other fish. Hopefully, these basic studies will serve as the starting point for developing effective vaccines and other therapies aimed at lessening the adverse economic impact of viral infections in fish.

Publications

  • Antao, A.B, Wilson, M., Wang, J., Miller, N.W., Clem, L.W, and Chinchar, V.G. Genomic organization and differential expression of channel catfish MHC class I genes. Develop. Compar. Immunol. 25: 579 - 595, 2001.
  • Chinchar, V.G., Wang, J., Murti, G., Carey, C., and Rollins-Smith, L. Inactivation of frog virus 3 and channel catfish virus by escultentin-2-P and ranatuerin-2-P, two antimicrobial peptides isolated from frog skin. Virology 288: 351 - 357, 2001.
  • Hawke NA, Yoder JA, Haire RN, Mueller MG, Litman RT, Miracle AL, Stuge T, Shen L, Miller N, and Litman GW. Extraordinary variation in a diversified family of immune-type receptor genes. PNAS 98: 13832 - 13837, 2001.
  • Shen, L., Stuge, T.B., Zhou, H., Khayat, M., Barker, K.S., Quiniou, S.M.A., Wilson, M, Bengten, E., Chinchar, V.G., Clem, L.W., and Miller, N.W. Channel catfish cytotoxic cells: A mini-review. Develop. Compar. Immunol. 26: 141 - 149, 2002.
  • Chinchar, V.G. Ranaviruses (Family Iridoviridae): Emerging cold-blooded killers. Arch. Virol. 147: 447 - 470, 2002.
  • Park H, Zhou H, Bengten E, Wilson M, Chinchar VG, Clem LW, and Miller NW. Activation of channel catfish (Ictalurus punctatus) T cells involves NFAT-like transcription factors. Develop. Compar. Immunol.26: 775 - 784, 2002.
  • Zhou, H., Stuge, T.B., Miller, N.W., Bengten, E., Naftel, J.P., Bernanke, J.M., Chinchar, V.G., Clem, L.W., and Wilson, M. Heterogeneity of channel catfish CTL with respect to target recognition and cytotoxic mechanisms employed. J. Immunol. 167: 1325 - 1332, 2001.
  • Barker K, Khayat M, Miller N, Wilson M, Clem LW, and Bengten E. Immortal and mortal clonal lymphocyte lines from channel catfish: Comparison of telomere length, telomerase activity, tumor suppressor and heat shock protein expression. Develop. Compar. Immunol. 26: 45 - 51, 2002.
  • Bengten E, Quiniou SMA, Stuge TB, Katagiri T, Miller NW, Clem LW, Warr GW, and Wilson M. The IgH locus of the channel catfish, Ictalurus punctatus, contains multiple constant region gene sequences: Different genes encode heavy chains of membrane and secreted IgD. J. Immunol. 169: 2488 - 2497, 2002.
  • Chinchar VG, Bryan L, Wang J, Long S, and Chinchar GD. Induction of apoptosis in frog virus 3-infected cells. Virology 306: 303 - 312, 2003.
  • Zhou H, Bengten E, Miller NW, Clem LW, and Wilson M. Th T cell receptor beta locus of the channel catfish, Ictalurus punctatus, reveals unique features. J. Immunol. 170: 2573 - 2581, 2003.
  • Zou J, Secombes CJ, Long S, Miller NW, Clem LW, and Chinchar VG. Molecular identification and expression analysis of tumor necrosis factor in channel catfish (Ictalurus punctatus). Develop. Compar. Immunol. 27: 845 - 858, 2003.
  • Long S, Wilson M, Bengten E, Bryan L, Clem LW, Miller NW, and Chinchar VG. Identification of a gene encoding channel catfish interferon. Develop. Compar. Immunol. 28: 97 - 111, 2004.


Progress 10/01/01 to 09/30/02

Outputs
During the past year we have identified key catfish genes and gene products involved in cell-mediated death and in the intracellular pathways leading to apoptosis, a catfish interferon homolog, and cell surface molecules involved in antibody-dependent cell-mediated cytotoxicity and signal transduction. (1) We have cloned and sequenced catfish TNF, FasR, FADD, and caspases -8, -3, and -6, and identified catfish FasL based on cross-reactivity with a mouse anti-FasL monoclonal antibody. These seven gene products constitute key membrane-associated and intracellular proteins involved in cell-mediated apoptotic death. Their existence suggests that catfish, and by extension other fish species, utilize mechanisms of killing that are common to all vertebrates, including mammals. Furthermore, expression studies suggest that cell death in tissue culture may be density dependent and due to increased expression of cell surface-associated FasL. (2) We have identified a catfish interferon (CF IFN) homolog. This is only the second report of an IFN gene in any non-mammalian species. Sequence analysis showed that while putative CF IFN was at most 19% similar to mammalian IFN, it was 35% similar to a recently described IFN gene from zebrafish. Treatments that upregulate IFN in mammalian cells, i.e., exposure to dsRNA or a dsRNA-containing virus, induced both anti-viral activity and the expression of CF IFN mRNA in channel catfish ovary (CCO) cells. Moreover, COS-7 cells, transfected with an expression vector containing CF IFN, synthesized a factor that protected CCO cells from virus challenge. Based on these findings, we are confident that we have identified CF IFN and intend to determine its role in anti-viral immunity and in apoptosis. (3) The final study involves characterizing clonal NK-like cells and the identification of an immunoglobulin receptor on their surface. This receptor may mediate interaction between specific anti-viral antibody and virus-infected cells and lead to enhanced cellular lysis. Although the receptor has been identified indirectly via its interaction with antibody, we have identified an accessory molecule (the Fc epsilon R gamma chain) at the molecular level which may be associated with this, or other, membrane-associated receptors.

Impacts
These studies will further our understanding of the molecular mechanisms of anti-viral immunity in catfish. Moreover, identification of catfish interferon is an exciting advance and, given the utility of the catfish system, should provide insight into the ways that interferon mediates anti-viral immunity in this and other commercially important fish species.

Publications

  • Park J, Zhou H, Bengten E, Wilson M, Chinchar VG, Clem LW, and Miller NW. 2002. Activation of channel catfish (Ictalurus punctatus) T cells involves NFAT-like transcription factors. Develop. Compar. Immunol. 26: 775 - 784.
  • Bengten, E, Quiniou S, Khayat M, Katagiri T, Miller NW, Clem LW, Warr GW, and Wilson M. 2002. The IgH locus of the channel catfish contains multiple constant region gene sequences: Different genes encode heavy chains of membrane and secreted IgD. J. Immunol. 169:2488-2497.
  • Cioffi C C, Pollenz RS, Middleton DL, Wilson MR, Miller NW, Clem LW, Warr GW, and Ross DA. 2002. Oct2 transcription factor of a teleost fish: Activation domains and function from an enhancer. Arch. Biochem. Biophys. 404:55-61
  • Shen L, Stuge TB, Evenhuis JP, Bengten E, Wilson M, Chinchar VG, Clem LW, and Miller NW. 2003. Channel catfish NK-like cells are armed with IgM via a putative FcmR. Develop. Compar. Immunol. (In press).
  • Zhou, H., E. Bengten, N. W. Miller, L. W. Clem, and M. Wilson. 2003. The T cell receptor beta locus of the channel catfish, Ictalurus punctatus, reveals unique features. J. Immunol. (In press)
  • Zou J, Secombes C, Long S, Miller NW, and Chinchar VG. 2003. Molecular identification and expression analysis of tumor necrosis factor in channel catfish. (submitted)
  • Shen L, Stuge TB, Bengten E, Wilson M, Chinchar VG, Naftel JP, Bernanke JM, Clem LW, and Miller NW. 2003. Characterization of clonal NK-like cells from channel catfish. (submitted)
  • Long S, Wilson M, Bengten E, Clem LW, Miller NW, and Chinchar VG. 2003. Identification of a gene encoding channel catfish interferon. (manuscript in preparation)


Progress 10/01/00 to 09/30/01

Outputs
During the past year we have made significant progress in three areas relating to the aims of this award. (1) The genomic organization of two channel catfish MHC class I genes has been determined and the promoter regions responsible for gene regulation have been identified. Expression studies (Northern blots) and electromobility shift assays have been performed and show that promoter elements upstream of the two identified genes are likely functional and support the suggestion that one is a classical MHC gene, and the other is a non-classical MHC gene. This is the first comprehensive report describing the genomic organization of class I MHC genes in fish and the first to functionally characterize teleost MHC promoter elements. We intend to build on these results and fine-structure map the regulatory elements responsible for controlling MHC class I expression in catfish. (2) We have generated, for the first time in fish, clonal lines of NK-like cells and antigen-specific cytotoxic T cells (CTL). Furthermore, we have shown, through a series of inhibitor and electron microscopic studies, that CTL kill by one of two mechanisms, a ligand-based system, perhaps similar to Fas/FasL, and a secretory system, perhaps analogous to perforin/granzyme. Based on these findings, we feel confident that we will be able to identify virus-specific catfish CTL. In addition, we have shown that some catfish NK-like effector cells display a putative immunoglobulin-binding Fc-like receptor. This receptor appears responsible for binding exogenously-synthesized catfish immunoglobulin and may play a role in target cell killing, perhaps by a process analogous to antibody-dependent cell-mediated cytotoxicity. (3) In an extension of the above studies, we have characterized at the molecular level several genes involved in CTL-mediated cytotoxicity. We have cloned and sequenced catfish Fas, caspase 8, FADD, and TNF. Moreover, we have identified, by BLAST analysis of cDNA libraries prepared from activated lymphoid cell lines, catfish genes similar to granzymes, perforin, TNF receptor, and immunoglobulin-like receptors. The latter are perhaps analogous to the killer cell immunoglobulin-like receptors (KIR) of mammals. Furthermore, we have identified, by serological cross-reactivity, FasL in catfish cells. Taken together, these results indicate that catfish possess many of the membrane-bound and membrane proximal proteins responsible for cell-mediated apoptotic death and strengthen the argument that catfish CTL effectors utilize both secretory (perforin/granzyme) and ligand-based (Fas/FasL) pathways in their lysis of target cells.

Impacts
Since both NK cells and CTL are thought to play major roles in resolving ongoing viral infections, the above studies will provide the basis for a better understanding of target cell killing by catfish cytotoxic effectors. The long-term goal of this research is to devise vaccine strategies that enhance CTL responses and thus better protect fish from viral disease.

Publications

  • Hawke NA, Yoder JA, Haire RN, Mueller MG, Litman RT, Miracle AL, Stuge T, Shen L, Miller NW, and Litman GW. Extraordinary variation in the structure and organization of a diversified family of immune-type receptor genes. Proc. Nat. Acad. Aci. USA 98: 13832 - 13837 (2001).
  • Shen L, Stuge TB, Zhou H, Khayat M, Barker KS, Quiniou SMA, Wilson M, Bengten E, Chinchar VG, Clem LW, and Miller NW. Channel catfish cytotoxic cells: A mini-review. Develop. Compar. Immunol. 26: 141 - 149 (2002).
  • Long S, Wilson M, Bengten E, Khayat M, Hawke N, Clem LW, Miller NW, and Chinchar VG. Identification of apoptosis genes in the channel catfish and their role in cell-mediated immunity. FASEB, Annual Meeting, April 2002 (submitted).
  • Antao AB, Wilson MR, Wang J, Bengten E, Miller NW, Clem LW, and Chinchar VG. Genomic organization and differential expression of channel catfish MHC class I genes. Develop. Compar. Immunol. 25: 579 - 595 (2001).
  • Park H, Zhou H, Katagiri T, Wilson M, Bengten E, Clem LW, and Miller NW. Activation of channel catfish T cells involves NFAT-like transcription factors. FASEB J. 15 (4): A702 (2001).
  • Stuge TB, Wilson MR, Zhou H, Barker KS, Bengten E, Chinchar G, Miller NW, and Clem LW. Development and analysis of various clonal alloantigen-dependent cytotoxic cell lines from channel catfish. J. Immunology 164: 2971 - 2977 (2000).
  • Zhou H, Stuge TB, Miller NW, Bengten E, Naftel JP, Bernanke JM, Chinchar VG, Clem LW, and Wilson M. Heterogeneity of channel catfish CTL with respect to target recognition and cytotoxic mechanisms employed. J. Immunology 167: 1325 - 1332 (2001).


Progress 10/01/99 to 09/30/00

Outputs
During the past year we have made progress in identifying mechanisms involved in cytotoxicity mediated by channel catfish immune cells. Work has centered on three areas. (1) The genetic organization of catfish class I genes has been determined and putative upstream promoter elements have been identified. In general, catfish MHC class I genes are organized in a manner similar to mammalian class I genes, and appear to contain regulatory motifs analogous to those of mammalian genes. Electromobility shift assays demonstrated that catfish contain elements similar to mammalian Enhancer B and site alpha motifs, and sequence comparisons suggest the existence of interferon stimulated response elements, interferon gamma activation sites, and perhaps an Enhancer A element. Future studies will build on this work and determine the role that specific promoter elements play in controlling MHC class I gene expression. Successfully completed, these studies will further understanding of MHC class I genes and their products, and should provide us with a better understanding of the role of class I gene products in cell-mediated cytotoxicity. (2) We have identified a family of gynogenetic catfish that should prove suitable for studying anti-viral cytotoxic responses, and have ascertained that cell mediated killing occurs by both apoptotic and necrotic mechanisms. Moreover, we have provided unequivocal proof that catfish possess alloantigen-specific cytotoxic cells and have developed long-term cultures of NK-like effectors and cytotoxic T cells. Cloned cytotoxic effector cells will give us the opportunity to dissect cytotoxic responses at the molecular level. For example, we may be able to determine if catfish possess NK inhibitory receptors, and to determine the identity of molecules implicated in cytotoxic responses such as Fas, FasL, perforin, immunoglobulin receptors, and granzymes. (3) In keeping with our interest in identifying molecules responsible for cytotoxicity, a putative FasL-like molecule has been identified in catfish based on cross-reactivity with an anti-human FasL monoclonal antibody. Preliminary data indicate that the catfish homolog of FasL is regulated in ways similar to its mammalian counterpart, i.e. it is upregulated following mitogen stimulation or UV-irradiation. Interestingly, the putative FasL-like protein appears to be upregulated in response to an increase in cell density. Since overgrown cultures appear to undergo apoptosis, it is possible that the trigger for apoptotic death is the increased presence of FasL on the cell surface. The identity of the putative FasL is currently being sought by screening a catfish cDNA expression library. Successfully completed these studies will elucidate key molecules regulating the death and survival of immune cells.

Impacts
Channel catfish are the most commercially important aquaculture species raised in the United States. However, because of intensive farming practices, the potential exists for devastating epidemics due to infectious disease. Because understanding how fish respond to viruses and other pathogens is critical to protecting this valuable industry, the goal of this award is to elucidate the mechanisms of cellular immunity.

Publications

  • Hogan RJ, Taylor WR, Cuchens MA, Naftel JP, Clem LW, Miller NW, and Chinchar VG. (1999) Induction of target cell apoptosis by channel catfish cytotoxic cells. Cell. Immunol. 195: 110 - 118.
  • Antao AB, Chinchar VG, McConnell TJ, Miller NW, Clem LW, and Wilson MR. (1999) MHC class I genes of the channel catfish: Sequence analysis and expression. Immunogenetics 49: 303 - 311.
  • Hogan RJ, Waldbieser GC, Goudie CA, Antao A, Godwin UB, Wilson MR, Miller NW, Clem LW, McConnell TJ, Wolters WR, and Chinchar VG. (1999) Molecular and immunologic characterization of gynogenetic channel catfish (Ictalurus punctatus). Marine Biotechnology 1: 317 - 327.
  • Stuge TB, Wilson MR, Zhou H, Barker KS, Bengten E, Chinchar G, Miller NW, and Clem LW. (2000) Development and analysis of various clonal alloantigen-dependent cytotoxic cell lines from channel catfish. J. Immunol. 164: 2971 - 2977.
  • Barker KS, Quiniou S, Wilson M, Bengten E, Warr G, Clem LW, and Miller NW. (2000). Telomerase expression and telomere length in immortal leukocytes lines from channel catfish. Develop. Comp. Immunol. 24: 583 - 595.