Samuel F. Schluter

Evolution of the acute phase response: iron release by echinoderm (Asterias forbesi) coelomocytes, and cloning of an echinoderm ferritin molecule.

That the plasma concentration of certain divalent cations change during an inflammatory insult provides a major host defense response in vertebrate animals. This study was designed to investigate the involvement of iron sequestration in invertebrate immune responses. A ferritin molecule was cloned from an echinoderm coelomocyte cDNA library. The amino acid sequence showed sequence homology with vertebrate ferritin. The cDNA contained a conserved iron responsive element sequence. Studies showed that stimulated coelomocytes released iron into in vitro culture supernatants. The amount of iron in the supernatants decreased over time when the amebocytes were stimulated with LPS or PMA. Coelomocytes increased expression of ferritin mRNA after stimulation. In vertebrates, cytokines can cause changes in iron levels in macrophages. Similarly, echinoderm macrokines produced decreases in iron levels in coelomocyte supernatant fluids. These results suggest that echinoderm ferritin is an acute phase protein and suggest that sequestration of iron is an ancient host defense response in animals.

Phylogenetic emergence and molecular evolution of the immunoglobulin family.

Publisher Summary The combinatorial immune system defined by the presence of antigen specific recognition units from the immunoglobulin family, the genetic machinery necessary for recombination, and cells of the lymphoid series expressing these receptors is fully functional from the earliest extant gnathostomes to mammals; however, the definitive evidence for genes specifying immunoglobulins, T-cell receptors (TCR), or recombination activating genes has not been documented for more primitive agnathan vertebrates, lower deuterostomes, or protostome, or acoelomate invertebrates. This chapter focuses on the appearance and molecular evolution of antibodies and T-cell receptors (TCR), which are the antigen-specific recognition elements of the combinatorial immune system. The combinatorial immune system of jawed vertebrates arose as an evolutionary big bang that involves the generation and duplication of V and C domains from an unknown precursor, and also involves the incorporation of joining segment genes, recombination signal sequences, and transmnembrane/cytoplasmic segments. The most recent major step in the evolution of immune system was the emergence in mammals of germinal centers within the lymph nodes, correlating with the IgM to IgG switch and affinity maturation following from somatic mutation and antigenic selection. The distinct heavy chain isotypes, such as IgY of chickens, reptiles, and amphibians and IgW of sharks have also arisen through gene duplication in evolution, and these may show distant relationships to mammalian immunoglobulins, such as IgD and IgE.

Molecular origins and evolution of immunoglobulin heavy-chain genes of jawed vertebrate

Cartilaginous fish are the most ancient extant jawed vertebrates possessing bona fide immunoglobulin (Ig) and T-cell receptor molecules. The study of these animals is critical for understanding the origins of the vertebrate immune system. Here, Samuel Schluter, Ralph Bernstein and John Marchalonis review the latest data concerning heavy-chain variable genes and associated isotypes in these animals, and propose a model for the early origins of Igs.

Antibody production in sharks and humans: a role for natural antibodies.

Although gene segments specifying Igs of all vertebrates show clear homology, their arrangements differ markedly, thereby suggesting that the mechanisms for the generation of diversity and for the regulation of gene expression may be quite distinct. In the sandbar shark, light chain gene segments are distributed as apparently independent clusters consisting of V, J, and C elements that require rearrangement for expression. The usual distance between V and C in the clusters is 3 kb but larger clusters occur. The V, J, and C elements are clearly homologous to those of human lambda chains. Shark Igs resemble mammalian IgM in structure and gene similarity. IgM may comprise as much as 50% of serum proteins in the shark. By contrast, IgM in humans comprises less than 5%. Human autoantibodies usually are IgM. These show little dependence on thymic function for expression and tend to increase with age. We have carried out a study of the capacity of Igs of unimmunized sharks and people (normals and patients suffering from autoimmune diseases) to react against a panel of antigens, including those usually considered autoantibodies, such as thyroglobulin and single-stranded DNA. Sharks and humans possess IgM antibodies that react with thyroglobulin and ssDNA. Affinity-purified natural shark antibodies to thyroglobulin or ssDNA constitute small fractions of total IgM. They illustrate extensive cross-reactivity comparable to that shown by polyspecific IgM autoantibodies produced by human B cells (CD5+) that appear early in ontogeny.

Evolutionary factors in the emergence of the combinatorial germline antibody repertoire.

Although life began on earth approximately 3.5 billion years ago, the combinatorial immune response apparently arose in a “big bang” approximately 450 million years ago, [1–4] coincident with the emergence of jawed vertebrates. Preceding this event was the so-called Cambrian explosion occurring approximately 545 million years ago that resulted in the seemingly rapid appearance of virtually all living forms as represented by the fossil record [5, 6]. However, molecular investigations seeking to calibrate evolutionary clocks and analyze phylogenetic relationships indicate that the explosive phases of evolution implied by the fossil record may have been preceded by extended periods of inconspicuous innovation [5, 6] in possible living organisms thatdid not become part of the currently available fossil record. The necessary elements of the combinatorial immune system, immunoglobulins (Igs), T-cell receptors (TCR), MHC products and recombinase activator genes (RAG) are clearly present in even the most primitive jawed vertebrates, the chondrichthian fishes [7–10] which appeared in evolution approximately 450 million years ago. Definitive evidence for these elements is thus far lacking in agnathan vertebrates and in lower deuterostomes. Nevertheless, many primordial elements upon which the combinatorial system is built may well have preceded the split in evolution between protostomes and deuterostomes and their origins may even extrapolate back to ancient times corresponding to the origin and evolution of bacteria.

Characterization of arrangement and expression of the T cell receptor γ locus in the sandbar shark

Ig and T cell receptor (TCR) genes consist of separate genomic elements, which must undergo rearrangement and joining before a functional protein can be expressed. Considerable plasticity in the genomic arrangement of these elements has occurred during the evolution of the immune system. In tetrapods, all Ig and TCR chain elements are arranged as translocons. In teleosts, the Ig heavy and TCR chains are translocons, but light chain genes may occur as clusters. However, in chondrichthyes, all of the Ig light and heavy chain genes are arranged as clusters. These clusters vary in number from <10 to several hundred, depending on isotype and species. Here, we report that the germ-line gene for the TCR γ chain in a chondrichthyan, the sandbar shark (Carcharhinus plumbeus), is present as a single locus arranged in a classic translocon pattern. Thus, the shark utilizes 2 types of genomic arrangements, the unique cluster organization for Ig genes and the “conventional” translocon organization for TCR genes. The TCR γ translocon contains at least 5 V region genes, 3 J segment genes, and 1 C segment. As expected, the third hypervariable segment (CDR3), formed by the rearrangement of the Vγ and Jγ segments, contributed the major variability in the intact V region structure. Our data also suggest that diversity may be generated by mutation in the V regions.

Evolution of variable and constant domains and joining segments of rearranging immunoglobulins.

The rearranging immunoglobulins (Igs) are a family of recognition and defense proteins found in all vertebrate classes. These proteins consist of two types of polypeptide chains; each of these contains a variable (V) domain, a joining (J) segment, and a constant (C) region, which can itself consist of one to four domains. The distinction betweeen light and heavy chains is an ancient one phylogenetically that is reflected in the structures of V, J, and C regions. Despite the early emergence of these genetic elements, conservatism is apparent in the peptide structures encoded by V, J, and C exons. C regions of heavy chains did not evolve as single units; rather the individual domains show their own clustering patterns, which apparently are independent of heavy‐chain designation or species. C‐region domains of light chains and the T cell receptor β chain are similar to one another and to the most carboxyl‐terminal domain of heavy chains. Comparison of the light chains of sharks, bullfrogs, chickens, and mammals indicated that a phylogenetic distinction can be made between x and λ light chains. V and J segments of the rearranging T cell receptors α, γ, and δ are homologous to the corresponding segments of Igs, but their C regions form a group that is markedly distinct from those of conventional Igs and Tcr β.—Marchalonis, J. J.; Schluter, S. F. Evolution of variable and constant domains and joining segments of rearranging immunoglobulins. FASEB J. 3: 2469‐2479; 1989.

Synthetic Autoantigens of Immunoglobulins and T-Cell Receptors: Their Recognition in Aging, Infection, and Autoimmunity

Abstract Immunoglobulins and their close relatives, the antigen-specific T-cell receptors, are recognition proteins that express structures which readily serve as self-immunogens. Healthy humans can produce antibodies against variable region-defined recognition structures termed idiotypes, as well as against constant region structures, and the levels of these can increase markedly in autoimmune disease; e.g., rheumatoid factors are autoantibodies directed against a conformational determinant of the γ heavy chain. More recent analyses employing synthetic peptide technologies and construction of recombinant T-cell receptors document that autoantibodies directed against both variable and constant region markers of the α/β T-cell receptor occur in healthy individuals. Alterations in levels of antibody, usage of IgM or IgG isotypes, and specificity for particular peptide-defined regions vary with natural physiological processes (aging, pregnancy), with artificial allografting, with retroviral infection, and with the inception and progression of autoimmune disease (e.g., rheumatoid arthritis, systemic lupus erythematosus). Two of the major autoimmunogeneic regions of the Tcr α/β are “constitutive” markers inasmuch as all Individuals tested produce antibodies against these regions. The most frequently observed autoantibodies are against Tcr Vβ CDR1 and Fr3 markers. It is hypothesized that these are normally involved in immunoregulation. Autoantibodies usually are not detected against CDR2 region determinants, or the “private idiotypes” defined by the CDR3 region, or the highly conserved FR4 segment specified by the joining gene segment. However, autoantibodies against the CDR2 of the Tcrα chain occur in some SLE patients, and healthy pregnant women produce antibodies against the common peptide determinant expressed by the joining gene and the beginning of the Cα or Cβ domain. Although the precise role of the naturally occurring autoantibodies in immunoregulation remains to be determined, modification of the course of autoimmune diseases in experimental rodent models (experimental allergic encephalomyelitis) has been successfully carried out by immunization with synthetic peptides corresponding to the CDR2 and Fr3/CDR3 segments, and immunization of humans with synthetic Vβ CDR2 segments may prove helpful in multiple sclerosis.

Natural human antibodies to synthetic peptide autoantigens: correlations with age and autoimmune disease.

Clinically healthy humans as well as patients suffering from various autoimmune diseases produce natural antibodies against a variety of self-components. Such antibodies have been proposed to carry out a physiologic role in maintaining the integrity of self, as well as potentially destructive roles in the generation of autoimmune diseases. Because human autoantigens, particularly membrane proteins, are usually present in extremely small amounts, it is generally impossible to obtain enough to carry out a detailed characterization of the antibodies or the antigenic determinants recognized. To circumvent this difficulty, we developed synthetic autoantigens predicted from the gene sequence of two functionally critical membrane proteins; the band 3 anion transport protein which is found on all cells, and the T-cell receptor (beta chain) which is the antigen-specific receptor on thymus-derived lymphocytes. We have investigated the natural human IgM and IgG antibody responses to peptides selected on the basis of predicted molecular surface exposure and previously known antigenicity, and correlate levels of binding with changes in age and by comparison with autoimmune diseases. We report that the IgM response to synthetic autoantigens tends to be higher than that of IgG molecules, but significant IgG binding occurs to some peptides. This situation is particularly noticeable in comparison of rheumatoid arthritis patients with normal individuals. Distinct peptide portions of individual molecules are recognized differently by the autochthonous immune system as manifested by age dependence of the response and differential levels of IgM and IgG activity. The synthetic autoantigens that tend to generate the highest amounts of natural antibody are those that are either exposed on the surface of the cell (band 3 peptides) or are exposed in the predicted 3-dimensional folding of the molecule (T-cell receptor beta peptides). Rheumatoid arthritis patients tend to give higher IgM reactivities to both band 3 and Tcr beta peptides than do normals, with this effect being less pronounced in the distinct autoimmune disease systemic lupus erythematosus. Studies of normal humans ranging in age from 20 to 90 years suggest two major patterns for the IgM natural antibody response to synthetic peptides giving high response. The first is that the level of IgM reactivity is high early in life and remains high throughout. The second pattern is one in which the reaction is high in younger individuals, but diminishes substantially in the latter decades of life.(ABSTRACT TRUNCATED AT 400 WORDS)

Cloning of shark RAG2 and characterization of the RAG1/RAG2 gene locus

The recombination‐activating genes (RAG) encode a site‐specific recombinase that is centrally responsible for the rearrangement of genomic V(D)J exons necessary to form functional immunoglobulin and T‐cell receptor genes. To help elucidate the origins of the RAG genes, we have cloned the RAG2 gene from the sandbar shark (Carcharhinus plumbeus) and characterized the entire RAG1/RAG2 gene locus. The shark RAG2 protein consists of 520 amino acids, is ~50% identical with RAG2 proteins from other vertebrates, and contains the same three domains identified in mammalian RAG2. Residues critical for RAG2 function are conserved in the shark sequence. In common with other vertebrate species, the shark RAG2 coding region lacks introns and is closely linked in opposite orientation to the RAG1 gene. The intergenic region is 9.4 kb, which is considerably larger than of teleosts (2–3 kb) and is comparable to that of tetrapods. This length is partially explained by the presence of several SINE and LINE fragments. The ancestors of the sharks were apparently the first vertebrates in phylogeny to have RAG genes, and our results confirm that the RAG genes have been highly conserved during evolution both in terms of sequence and gene organization.