Rearrangement of immunoglobulin genes in shark germ cells.

The variable (V), (diversity [D]), and joining (J) region recombinases (recombination activating genes [RAGs]) can perform like transposases and are thought to have initiated development of the adaptive immune system in early vertebrates by splitting archaic V genes with transposable elements. In cartilaginous fishes, the immunoglobulin (Ig) light chain genes are organized as multiple VJ-constant (C) clusters; some loci are capable of rearrangement while others contain fused VJ. The latter may be key to understanding the evolutionary role of RAG. Are they relics of the archaic genes, or are they results of rearrangement in germ cells? Our data suggest that some fused VJ genes are not only recently rearranged, but also resulted from RAG-like activity involving hairpin intermediates. Expression studies show that these, like some other germline-joined Ig sequences, are expressed at significant levels only early in ontogeny. We suggest that a rejoined Ig gene may not merely be a sequence restricting antibody diversity, but is potentially a novel receptor no longer tied to somatic RAG expression and rearrangement. From the combined data, we arrived at the unexpected conclusion that, in some vertebrates, RAG is still an active force in changing the genome.

Antibody repertoire development in cartilaginous fish.

There are 3 H chain and 3 L chain isotypes in the cartilaginous fish, all encoded by genes in the so-called cluster (VDDJ, VJ) organization. The H chain isotypes IgM and IgNAR, are readily detected at the protein level in most species. The third is readily identified at the protein level in skates (IgR) but only via immunoprecipitation or at the transcript level in sharks (IgW). High levels of diversity in CDR3 and up to 200 germline genes have been detected for IgM depending upon the species examined. IgNAR displays very high levels of CDR3 diversity but almost none in the germline. At least IgNAR and L chain genes have been shown to hypermutate to very high levels, apparently in response to antigen. The mutation footprints are similar to those in mammals except that the shark genes uniquely mutate nucleotide residues in tandem. A conspicuous feature of cartilaginous fish Ig genes is the presence of germline-joined genes, which are a result of RAG activity in germ cells. Such genes are expressed early in ontogeny and then extinguished or expressed at lower levels. 19S IgM and IgW expression precede that of 7S IgM and IgNAR during ontogeny. The 'switch' from 19S to 7S IgM, the regulation of expression of the Ig clusters, and the microenvironments for mutation/selection of cartilaginous fish B cells are all areas of ongoing research.

Histology and immunology of the placenta in the Atlantic sharpnose shark, Rhizoprionodon terraenovae.

The Atlantic sharpnose shark, Rhizoprionodon terraenovae, is viviparous species that forms a yolk sac placenta to facilitate exchange between mother and embryo. However, very little is known about the immunological aspects of this organ in sharks. To begin to understand this, we used histology, histochemistry and immunohistochemistry to investigate the sharpnose shark placenta throughout gestation. We report the presence of lymphoid aggregates in the maternal portion of the placenta during all stages of gestation, and their increasing size and vascularity near term. Immunoglobulin is found in the maternal tissues of the placenta, but its presence in embryonic tissue and potential transfer from maternal circulation remains unclear. Placental cells resembling mammalian uterine NK cells and melanomacrophages of lower vertebrates are described for the first time. Similarities with mammalian placentae point to shared aspects in the co-evolution of reproductive and immune systems, even between two phylogenetically diverse groups in which placentation arose by convergent evolution.

Evolution and developmental regulation of the major histocompatibility complex.

The ontogeny and evolution of the major histocompatibility complex (MHC) are blossoming fields that grant insight into the origins of the adaptive immune system and into the strategies adopted by particular groups of vertebrates for expression of MHC during development. This review surveys general topics concerning MHC evolution, with special emphasis on the significance of linkage of gene families within the MHC; a model is proposed in which the MHC class III region is the "primordial immune complex" with its members giving rise to classical MHC molecules. The developmental expression of MHC, both of the classical and non-classical genes, is described in detail with a concentration on differential expression by extraembryonic tissues in mammals and by tissues in "transition" during metamorphosis in amphibians.

IgM-mediated opsonization and cytotoxicity in the shark.

Two types of cytotoxic reactions have been observed using cells from the nurse shark: spontaneous cytotoxicity mediated by cells of the macrophage lineage and antibody-dependent killing carried out by a different effector cell population. Previous data showed that removal of phagocytic cells using iron particles abolished macrophage-mediated killing, but not antibody-dependent reactions. The current study used single cell assays and showed that the effector of antibody-driven reactions was the neutrophil. Surprisingly, the mechanism of killing was shown to be phagocytosis mediated by both 7S and 19S immunoglobulin M (IgM). Reactions proceeded with as little as 0.01 microg of purified 19S or 7S IgM and were complete within 4-6 h. In contrast, purified immunoglobulin did not adsorb to macrophages and had no effect on target cell binding or cytotoxicity. Pretreatment of cells with cytochalasin D abolished the phagocytic reaction, but not spontaneous cytotoxicity. These data show that antibody-mediated killing results from opsonization and phagocytosis; the mechanism of macrophage killing is currently unknown. In addition, these data show that the shark neutrophil, not the macrophage lineage, carries a receptor for Fc mu.

Evolution of the MHC: antigenicity and unusual tissue distribution of Xenopus (frog) class II molecules.

Antibodies that recognize Xenopus class II molecules have been developed. Mouse monoclonal antibodies were prepared by immunizing BALB/c mice with frog MHC antigens that had been partially purified with alloantisera, and by immunizing mouse spleen cells in vitro with activated Xenopus T lymphocytes. In addition, five mouse monoclonal antibodies specific for human class II antigens were found to cross-react with Xenopus class II antigens. A.TH mice, which do not express E class II molecules, always produce immunoprecipitating antibodies reactive with frog class II molecules after immunization with frog lymphocytes; other mouse strains rarely produce such antibodies. Two of the monoclonal antibodies raised against frog class II molecules recognize the denatured class II beta chain on Western blots, and the other three appear to recognize only the class II heterodimeric complex. The antibodies display differential reactivity with the allelic class II products of Xenopus. The monoclonal antibodies react with all adult lymphocytes in the spleen and peripheral blood, T cells and B cells having equivalent levels of class II antigens per cell. Class II molecules are "differentiation antigens" on adult thymocytes as the expression is greatest on the mature medullary population. The number of class II molecules/lymphocyte increases after culturing in medium containing fetal bovine serum. Sequential immunoprecipitation and isoelectric focusing experiments have shown that cell surface class II molecules immunoprecipitated with the monoclonal antibodies are the same as those immunoprecipitated with the cross-reactive antiserum specific for DR antigens which was previously used to identify frog class II molecules.

MHC restriction of T-cell proliferative responses in Xenopus.

The MHC restriction of Xenopus allogeneic MHC- and antigen-specific T-cell proliferative responses was assessed. Xenopus MHC-specific monoclonal antibodies that recognize class I and class II molecules were tested for inhibitory effects on the generation of secondary T-cell proliferative responses. Antigen-specific T-cell lines were inhibited by anti-class II but not anti-class I monoclonal antibodies. Secondary alloantigen-specific proliferative responses also demonstrated MHC class II restriction. Allogeneic MHC- and antigen-specific T-cell lines demonstrated differential sensitivity to anti-class II monoclonal antibodies directed at discrete class II epitopes. These results indicate that Xenopus T cells interact with antigen-presenting cells similarly to mammals, and directly confirm previous data indicating that MHC class II restriction of proliferative responses is present in amphibians.

The immune system of ectothermic vertebrates.

The adaptive immune system, as defined by T cell receptors, immunoglobulins, and the major histocompatibility complex (MHC), has been described definitively at the level of teleost fish. Cartilaginous fish, which display many of the hallmarks of such an adaptive system, nevertheless have several features of their responses that seem primitive. Data are presented suggesting that some adaptive mechanisms in cartilaginous fish, including MHC restriction and somatic diversification, are present to the same "degree' as compared to mammals, and that these animals may possess other molecules and functions previously overlooked. MHC linkage studies in amphibians suggest that the entire genetic complex, including class I, class II, and class III genes, arose early in the vertebrate line (at least 350 x 10(6) years ago) and has been maintained intact, at least for those genes involved in immunity. Studies of MHC in polyploid Xenopus have demonstrated that there is a maximal number of expressed MHC genes 'permitted' to be expressed in any individual, regardless of the number of potential MHC-bearing chromosomes present in the species. A speculative hypothesis is presented on the origins of adaptive immunity based on ectothermic models.

Isolation and characterization of a cDNA encoding a Xenopus 70-kDa heat shock cognate protein, Hsc70.I.

We isolated a full-length cDNA clone encoding a Xenopus laevis 70-kDa heat shock cognate protein, hsc70.I. The protein coding region exhibits a high degree of identity with a number of mammalian hsc70 proteins, such as rat hsc71 (92%), whereas the identity to Xenopus hsp70 is only 80%. These data suggest that the inducible and constitutive forms of hsp70 diverged long before the emergence of amphibians. The Xenopus hsc70.I contains a number of conserved elements, including the ATP-binding domain, a nuclear localization signal and the carboxy-terminal EEVD motif, which has been implicated in several activities associated with chaperonin function. Northern blot analyses revealed that maternal hsc70.I mRNA is present in cleavage and early blastula stages of Xenopus development. After the onset of zygotic transcription at the midblastula stage, the levels of hsc70.I message increase through to the tadpole stages. Furthermore, in contrast to hsp70 mRNA, the relative levels of hsc70.I mRNA are not enhanced after heat shock in embryos and in the kidney epithelial cell line, A6. The levels of hsc70.I mRNA are high in adult spleen and testis, with moderate levels in eye, heart, liver and brain and comparatively low levels in hindlimb muscle.

Primitive vertebrate immunity: what is the evolutionary derivative of molecules that define the adaptive immune system?

The adaptive immune system is capable of responding to an infinite number of antigens with the antigen-specific receptors immunoglobulin (Ig) and the T cell receptor (TCR). Ig binds soluble antigens while TCR recognizes antigen bound in clefts of polymorphic self-encoded major histocompatibility complex (MHC) class I and class II molecules. All of these molecules are wholly or partially composed of Ig superfamily domains. TCR and Ig use V-set Ig superfamily domains, always in heterodimeric forms, in antigen recognition. Although the ways in which TCR and Ig bind antigen are fundamentally different, the structure of the heterodimeric V domains is probably identical. The antigen-binding cleft of MHC proteins has a structure unlike Ig superfamily domains, although several investigators have proposed that this cleft is evolutionarily derived from Ig domains. We believe the MHC cleft is a primitive structure, perhaps related to the peptide-binding domains of intracellular chaperone proteins. A model is proposed whereby chaperone proteins were the primordial MHC molecules, presenting peptides derived from invariant proteins residing inside cells for recognition by lymphocytes with minimally diverse receptors. Such a system may be reflected today by the epithelial immune system, apparently governed by monomorphic MHC molecules and lymphocytes with unconventional antigen-specific receptors.

Advances in immunology.

Much was accomplished in the last decade in understanding how the adaptive immune system evolved to combat pathogens. Essential features of antigen presentation and T lymphocyte recognition were deciphered, setting the stage for further studies that elucidated basic elements of lymphocyte differentiation (including positive and negative selection during lymphocyte ontogeny) and the major interactions that occur among cells in secondary lymphoid organs in an ongoing immune response. The major challenges of today are found in the burgeoning fields of programmed cells death, enzymology of recombination and somatic mutation, development of memory, and the recognition of pathogens by unconventional lymphocytes.

Expression of MHC class II antigens during Xenopus development.

Larval and adult forms of the amphibian Xenopus differ in their MHC class II expression. In tadpoles, class II epitopes can be detected by monoclonal antibodies only on B cells, macrophages (whatever their location), spleen reticulum, thymus epithelium, and the pharyngobuccal cavity. In contrast, all adult T cells express class II on their surface. The transitions in class II expression occur at metamorphosis and are accompanied by other changes. The skin is invaded by class II positive dendritic cells, and the skin glands differentiate and also express class II. The gut, which expressed class II in discrete areas of the embryonic tissue, becomes invaded with B cells, and its epithelium also becomes class II positive.

MHC class I antigens as surface markers of adult erythrocytes during the metamorphosis of Xenopus.

An alloantiserum produced against Xenopus MHC class I antigens has been used to distinguish different erythrocyte populations at metamorphosis. By analysis using a fluorescence-activated cell sorter (FACS) analyzer, tadpole (stage 55) and adult erythrocytes have distinct volume differences and tadpole cells have no MHC antigens on the cell surface. Both tadpole and adult erythrocytes express a "mature erythrocyte" antigen marker, recognized by its monoclonal antibody (F1F6). During metamorphosis, immature erythrocytes, at various stages of differentiation, which express adult levels of cell-surface MHC antigens by 12 days after tail resorption, are found in the bloodstream. These immature cells are biosynthetically active, produce adult hemoglobin, and mature by 60 days after the completion of metamorphosis. Percoll gradient-density fractionation has shown that all of the cells in the new erythrocyte series express adult levels of MHC antigens but there is only a gradual increase in the amount of "mature erythrocyte" antigen. Tadpole erythrocytes, which are biosynthetically active during larval stages, produce small amounts of surface MHC antigens before the metamorphic climax and then become metabolically inactive. They are completely cleared from the circulation by 60 days after metamorphosis. Erythrocytes from tadpoles arrested in their development for long periods of time express intermediate levels of MHC antigens, suggesting a "leaky" expression of these molecules in the tadpole cells. The most abundant erythrocyte cell-surface proteins from tadpoles and adults, as judged by two-dimensional gel electrophoresis, are very different.

Comparative genomics of the MHC: glimpses into the evolution of the adaptive immune system.

MHC gene organization (size, complexity, gene order) differs markedly among different species, and yet all nonmammalian vertebrates examined to date have a true "class I region" with tight linkage of genes encoding the class I presenting and processing molecules. Three paralogous regions of the human genome contain sets of linked genes homologous to various loci in the MHC class I, class II, and/or class III regions, providing insight into the organization of the "proto MHC" before the emergence of the adaptive immune system in the jawed vertebrates.