ABSTRACT
Reported in this study are the initial results from studies to develop rabbit models of systemic lupus erythematosus (SLE) by immunizations using two distinct peptides on branched polylysine backbones (multiple Ag peptide)-peptides. Eleven rabbits received a peptide from the Sm B/B' spliceosomal complex previously shown to be immunogenic in rabbits, and 13 rabbits received a peptide from the rabbit N-methyl-d-aspartate receptor NR2b. All 24 animals in different generations of pedigreed, noninbred rabbits produced peptide-specific responses. Anti-nuclear autoantibody responses, including anti-dsDNA, were seen in 17 of 24 rabbits. To date, two rabbits have been observed to have seizure-like events and a third nystagmus. A model for eliciting development of SLE in genetically related yet heterogeneous rabbits may more closely resemble development of human SLE than do some models in inbred mice. Through selective breeding, it may also ultimately provide additional information about the genetics and etiology of SLE and serve as a model for assessing new treatment options.
Subject(s)
Autoantigens/immunology , Autoimmunity , Disease Models, Animal , Lupus Erythematosus, Systemic/immunology , Peptides/immunology , Rabbits/immunology , Animals , Autoantibodies/blood , Autoantibodies/immunology , Enzyme-Linked Immunosorbent Assay , Female , Fluorescent Antibody Technique , Lupus Erythematosus, Systemic/genetics , Male , Pedigree , Rabbits/genetics , Receptors, N-Methyl-D-Aspartate/immunology , Spliceosomes/immunologyABSTRACT
Studies in mouse, human, and chicken suggest that activation-induced deaminase (AID) is involved in three known processes leading to antibody diversification: somatic hypermutation, gene conversion, and class-switch recombination. Developing rabbit appendix provides a particularly good site for studying all three of these B cell maturation events. We report here successful cloning of rabbit AID and isolation of AID protein from rabbit appendix-cell nuclear and cytoplasmic extracts. We succeeded in identifying and locating AID protein in cells by immunohistochemical and immunofluorescent staining techniques and examined colocalization of AID and other molecules important for Ab diversification. This report extends our knowledge about AID to a mammalian species that uses gene conversion to diversify rearranged Ig genes. Although much work remains to understand fully the mechanism of action of AID and its association with other cellular components, the rabbit system now offers a particularly useful model for future studies of these dynamics.
Subject(s)
Appendix/enzymology , Cytidine Deaminase/genetics , Cytidine Deaminase/metabolism , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Variable Region/genetics , Mutation , Amino Acid Sequence , Animals , Base Sequence , Blotting, Western , Cell Division/physiology , Chickens , Cytidine Deaminase/isolation & purification , Fluorescent Antibody Technique , Humans , Immunohistochemistry , Mice , Molecular Sequence Data , Rabbits , Sequence AlignmentABSTRACT
The Nogo-66 receptor (NgR1) is a promiscuous receptor for the myelin inhibitory proteins Nogo/Nogo-66, myelin-associated glycoprotein (MAG), and oligodendrocyte myelin glycoprotein (OMgp). NgR1, an axonal glycoprotein, is the founding member of a protein family composed of the structurally related molecules NgR1, NgR2, and NgR3. Here we show that NgR2 is a novel receptor for MAG and acts selectively to mediate MAG inhibitory responses. MAG binds NgR2 directly and with greater affinity than NgR1. In neurons NgR1 and NgR2 support MAG binding in a sialic acid-dependent Vibrio cholerae neuraminidase-sensitive manner. Forced expression of NgR2 is sufficient to impart MAG inhibition to neonatal sensory neurons. Soluble NgR2 has MAG antagonistic capacity and promotes neuronal growth on MAG and CNS myelin substrate in vitro. Structural studies have revealed that the NgR2 leucine-rich repeat cluster and the NgR2 "unique" domain are necessary for high-affinity MAG binding. Consistent with its role as a neuronal MAG receptor, NgR2 is an axonassociated glycoprotein. In postnatal brain NgR1 and NgR2 are strongly enriched in Triton X-100-insoluble lipid rafts. Neural expression studies of NgR1 and NgR2 have revealed broad and overlapping, yet distinct, distribution in the mature CNS. Taken together, our studies identify NgRs as a family of receptors (or components of receptors) for myelin inhibitors and provide insights into how interactions between MAG and members of the Nogo receptor family function to coordinate myelin inhibitory responses.
Subject(s)
Myelin-Associated Glycoprotein/metabolism , Nerve Tissue Proteins/metabolism , Receptors, Cell Surface/metabolism , Animals , Animals, Newborn , Axons/metabolism , Cells, Cultured , Cerebellum/cytology , Chlorocebus aethiops , Cricetinae , Cricetulus , GPI-Linked Proteins , Membrane Microdomains/metabolism , Myelin Proteins , Myelin-Associated Glycoprotein/physiology , N-Acetylneuraminic Acid/metabolism , Neurites/physiology , Nogo Receptor 1 , Protein Binding , Rats , Receptors, Peptide/metabolismABSTRACT
Previous studies in our laboratory suggested that there was positive selection of B cells during early development in the appendix of normal and V(H) mutant (ali/ali) rabbits. Preferential expansion and survival of B lymphocytes was affected by the Ig V(H) frameworks 1 and 3 sequences expressed on the cell surface. We demonstrated a specific interaction between rabbit CD5 and the V region of rabbit heavy chains and suggested that CD5 is a potential selecting ligand for B-cell surface immunoglobulin framework region sequences. To further investigate the role of CD5 in rabbit B-cell selection and survival we prepared recombinant constructs and obtained stable expression of the three scavenger receptor cysteine-rich (SRCR) extracellular domains of rabbit CD5. Here we describe the production and purification of this expressed recombinant CD5 protein, polyclonal antibody obtained by immunization of a goat and initial production and characterization of specific mAbs against peptides selected from each sequenced SRCR domain.
