Do ectothermic vertebrates have a home in which to affinity mature their antibody responses?

There has been a longstanding question of whether affinity maturation occurs in ectotherms, and if it does, where in tissues this happens. Although cold-blooded vertebrates (ectotherms) lack histologically discernible germinal centers, they have a fully functional Ig gene mutator enzyme (activation-induced cytidine deaminase: AID or Aicda). Protein and Ig cDNA transcript analyses provide evidence that ectotherms can, under certain conditions, demonstrate antibody affinity maturation, and somatic hypermutation of their Ig genes during secondary immune responses. Here, we review the evidence for antibody affinity maturation and somatic hypermutation of Ig V(D)J exons. We argue that past evidence of long-term intact antigen retention, and recent studies of in situ expression of AID transcripts, point to fish melanomacrophage clusters as sites functionally analogous to a germinal center. Recent work in zebrafish provides a way forward to test these predictions through V(D)J repertoire analyses on isolated, intact melanomacrophage clusters. This work has implications not only for vaccine use in aquaculture, but also for antibody affinity maturation processes in all ectothermic vertebrates.

The zebrafish IgH locus contains multiple transcriptional regulatory regions.

Many fish have, in addition to IgM and IgD, a third isotype called IgZ or IgT. The ζ-chain locus is embedded among the Ig heavy chain V-, D- and J-elements in a manner reminiscent of the TcR δ/α locus. Isotype selection thus occurs during VDJ recombination, a process that is facilitated by intralocus transcription. Using in silico analyses and enhancer reporter vectors we identified 3 new regions within the zebrafish IgH locus through which transcription can be activated in catfish B-cell lines. Two of these, termed Eζi (Jζ to Cζ1 intronic) and Eζ3' regions flank the ζ-chain constant domain exons. A third region, Eδ3', resides downstream of the δ-chain exons. All regions contain predicted binding sites for transcription factors that contribute to B-cell specific transcription in fish and mammals. Each region also has proximal matrix attachment regions, which may further contribute to transcriptional activation and chromatin remodeling. We discuss possible roles for these regions during VDJ recombination.

Evolution of effectors and receptors of innate immunity.

The bony fishes are derived from one of the earliest divergent vertebrate lineages to have both innate and acquired immune systems. They are considered by some to be an ideal model to study the underpinnings of immune systems precisely because of their phylogenetic position and the fact that their adaptive immune systems have not been elaborated to the extent seen in mammals. By the same token, examination of innate immune systems in invertebrates and early chordates can provide insight into how homologous systems operate in fish and higher vertebrates. Herein, we provide an overview of the molecular evidence that we hope helps clarify the evolutionary relationships of innate immune molecules identified in bony fishes. The innate immune systems being considered include select chemokines (CC and CXC chemokines and their receptors), cytokines (IL-1, IL-8, interferons, TGF-beta, TNF-alpha), acute phase proteins (SAA, SAP, CRP, alpha2M, and the complement components--C3-C9, MASP, MBL, Bf), NK cell receptors, and molecules upstream and downstream of the Toll signaling pathways.

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

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

Catfish Oct2 binding affinity and functional preference for octamer motifs, and interaction with OBF-1.

The DNA-binding (POU) domain of the catfish Oct2 transcription factor was shown, by electromobility shift assays and surface plasmon resonance techniques, to have an affinity for the consensus octamer motif (ATGCAAAT) that was slightly higher than its affinity for a variant motif (ATGtAAAT). This observation is consistent with the transcriptional activation potentials of catfish Oct2 alpha and Oct2 beta, which were shown to activate transcription in catfish B and T cell lines to an equivalent extent from both the consensus and variant octamer motifs. When tested in a mouse plasmacytoma cell line, catfish Oct2 alpha and Oct2 beta, as well as mouse Oct2, showed higher transcriptional activation with the variant, as compared to the consensus, octamer motif. Catfish Oct2 was shown to function synergistically with the mammalian co-activator, OBF-1, activating octamer-dependent transcription in catfish T cells. The strong transcriptional activity of OBF-1 in catfish cells was dependent on the presence of octamer motif(s) at the proximal (promoter) rather than the distal (enhancer) position.

Allorecognition in colonial tunicates: protection against predatory cell lineages?

The MHC molecules have been historically perceived as transplantation antigens, though it is now recognized that their primary, if not sole, role is in eliminating parasites and in surveillance and clearance of aberrant self. Indeed, pregnancy in mammals would represent the closest to a natural transplantation process that occurs in vertebrates. However, among the immediate ancestors to the vertebrates, natural intraspecific allorecognition processes are common. Among members of the colonial tunicate Botryllus schlosseri, two individuals that share a single allele of the highly polymorphic fusibility/histocompatibility (Fu/HC) locus are able to fuse with one another. Could this Fu/HC be related to the MHC such that the MHC really did have its origins as a transplantation antigen? Presently we review the genetics and biology of natural transplantation processes in colonial tunicates, comparing it with allorecognition as mediated through the vertebrate T-cell receptor, killer cell inhibitory receptor/Ly49, and MHC. Experimental approaches to determining if the molecules regulating allorecognition in tunicates have any ancestral relationship to the vertebrate MHC are discussed, as is a genomic approach to isolating novel mediators of allorecognition. We also explore the biological basis for allorecognition in colonial tunicates and recent work that highlights the costs of not maintaining a system for allorecognition.

Characterization of Oct2 from the channel catfish: functional preference for a variant octamer motif.

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 mu 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 (alpha and beta) were cloned by screening a catfish B cell cDNA library. Catfish Oct2 alpha and beta 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 a and beta 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 beta is a more potent transcriptional activator than Oct2 alpha. In transient expression assays, catfish Oct2 beta 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.

Functional motifs in the IgH enhancer of the channel catfish.

The transcriptional enhancer (Emu3') within the Ig heavy chain (IgH) locus of the channel catfish differs from those found in mammalian IgH loci in both its location and structure. However, upon transfection into fish or mouse lymphocytes, it activates transcription to an extent equivalent to that of the mouse IgH intronic enhancer (Emu). Potential transcription factor binding motifs in Emu3' are more numerous than in mammalian IgH enhancers, and are dispersed over 1.6 kilobases. We transfected catfish and mouse lymphoid cells with reporters under the control of artificial promoters containing motifs from the catfish enhancer. We demonstrate that 9 of 11 octamer motifs identified in the catfish enhancer, representing five variations of the consensus octamer (ATGCAAAT), are functional in both a catfish B-cell line (1B10) and the mouse plasmacytoma J558L. Only those octamer variants in which one of the first four bases is altered are active. Clear species differences in the strengths of the variant octamer motifs were evident, and in catfish B cells the ATGtAAAT motif was over threefold more active than the consensus octamer. The one muA and two muB motifs in Emu3' do not contribute to transcriptional activation. These results suggest that the relative functional contributions of IgH enhancer motifs has changed significantly during vertebrate evolution.

An Ig heavy chain enhancer of the channel catfish Ictalurus punctatus: evolutionary conservation of function but not structure.

The teleost fishes are among the earliest evolutionary lineages to have an Ig heavy chain (IgH) locus whose organization approximates that of mammals. To understand transcriptional control of the IgH locus in a teleost fish and to gain insight into the evolution of the control elements, the enhancer activity in the IgH locus of the channel catfish, Ictalurus punctatus, was investigated. Segments of the locus extending from upstream of the proximal JH gene to 2.5 kb downstream of the second transmembrane (TM2) exon of the mu gene were tested in transient transfection expression assays in murine myeloma and T cell lines, and in catfish B lymphoblastoid, monocyte-like, and putative T cell lines. In marked contrast to mammals, no enhancer activity was observed in the catfish JH to C mu intron, but strong enhancer activity (approaching that of the murine IgH intronic enhancer) was identified in a 1.8-kb segment that included the TM2 exon. This catfish enhancer was active in a B lineage-specific manner in both catfish and murine cells. It was not localized in a small core region, but appeared to contain multiple, dispersed cooperative elements rich in octamer- and mu E5-related motifs. Although the catfish IgH enhancer shares functional characteristics with the mammalian IgH intronic and 3' enhancers, its unusual organization does not permit any obvious inferences concerning evolutionary relationships between the catfish enhancer and any one of the murine IgH enhancers.

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

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