Molecular cloning and functional characterization of Cynomolgus monkey (Macaca fascicularis) CC chemokine receptor, CCR3.

We have cloned and performed the first functional characterization of the chemokine receptor, CCR3, of Cynomolgus monkey (Macaca fascicularis). The deduced amino acid sequence of the cloned Cynomolgus CCR3 was found to be more similar to that of a previously-reported Rhesus (Macaca mulatta) CCR3 (99.4%) than that of a reported Cynomolgus CCR3 (98.0%). Stably-transfected Cynomolgus CCR3 bound human eotaxin (CCL11) with similar kinetics (Kd 240 pM) and was responsive to human CCR3 ligands (eotaxin [CCL11], eotaxin-2 [CCL24], and MCP4 [CCL13]) in Ca(2+) mobilization and chemotaxis assays, thus provides a useful alternative species model system for the analysis of modulators of eotaxin--CCR3 induced signaling and activation.

Identification of genetic loci associated with paralysis, inflammation and weight loss in mouse experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE), a model for human multiple sclerosis, is an inducible inflammatory and demyelinating disease of the central nervous system (CNS). Susceptibility to this disease is heritable and is demonstrated by the development of an ascending paralysis accompanied by a loss in body wt 2-3 weeks following immunization with proteins derived from CNS myelin. In a previous genetic analysis of susceptibility to EAE in a cross between susceptible SJL/J mice and resistant B10.S mice, we found suggestive evidence of linkage with disease susceptibility at the telomeric end of chromosome 2 and in the central region of chromosome 3. To define these associations more precisely and to investigate the genetic factors controlling measurable phenotypes of EAE, we performed a new analysis with a larger number of mice. The results now indicate that the chromosome 2 locus significantly influences EAE-related weight loss (P = 6.7 x 10(-5)) and that the chromosome 3 locus is linked with the development of paralysis. In addition, an intriguing inheritance pattern was revealed in which female backcross mice generated from B10.S female x (B10.S x SJL/J)F(1) male parents experienced significantly more EAE-related weight loss (P = 1.2 x 10(-4)) than females generated from F1 female x B10.S male parents. After controlling for this inheritance, a new locus at the centromeric end of chromosome 8 was identified that significantly influences both the development of paralysis (P = 8.2 x 10(-6)) and the incidence of CNS inflammation (P = 7.0 x 10(-5)) in EAE.

Mapping and identification of autoimmunity genes.

Over the past several years, intense effort has been made to map the chromosomal locations of genes involved in the susceptibility to autoimmune diseases. The first phase of this mapping effort-performed in most cases by using microsatellite markers to scan the genome for loci that are linked with disease-has generated first-draft maps for numerous autoimmune diseases in humans, mice and rats, pointing to as many as 20 different loci in some diseases. The second phase is now beginning, with efforts focused on narrowing the loci sufficiently to allow the positional cloning of disease-associated alleles. From these mapping data it is clear that some of these loci overlap between various autoimmune diseases and preliminary results suggest that indeed there is a sharing of 'autoimmunity genes' between various autoimmune diseases.

Cloning, expression, and genetic mapping of Sema W, a member of the semaphorin family.

The semaphorins comprise a large family of membrane-bound and secreted proteins, some of which have been shown to function in axon guidance. We have cloned a transmembrane semaphorin, Sema W, that belongs to the class IV subgroup of the semaphorin family. The mouse and rat forms of Sema W show 97% amino acid sequence identity with each other, and each shows about 91% identity with the human form. The gene for Sema W is divided into 15 exons, up to 4 of which are absent in the human cDNAs that we sequenced. Unlike many other semaphorins, Sema W is expressed at low levels in the developing embryo but was found to be expressed at high levels in the adult central nervous system and lung. Functional studies with purified membrane fractions from COS7 cells transfected with a Sema W expression plasmid showed that Sema W has growth-cone collapse activity against retinal ganglion-cell axons, indicating that vertebrate transmembrane semaphorins, like secreted semaphorins, can collapse growth cones. Genetic mapping of human SEMAW with human/hamster radiation hybrids localized the gene to chromosome 2p13. Genetic mapping of mouse Semaw with mouse/hamster radiation hybrids localized the gene to chromosome 6, and physical mapping placed the gene on bacteria artificial chromosomes carrying microsatellite markers D6Mit70 and D6Mit189. This localization places Semaw within the locus for motor neuron degeneration 2, making it an attractive candidate gene for this disease.

Mapping autoimmunity genes.

Rat and mouse models for the major human autoimmune/inflammatory diseases are under intense genetic scrutiny. Genome-wide linkage studies reveal that each model is regulated by multiple genetic loci. Many of these loci colocalize to homologous genomic regions associated with several different autoimmune diseases of mice, rats and humans. Candidate genes are being identified. Polymorphic alleles associated with these chromosomal segments may represent predisposing genetic elements common to a number of human diseases with very different clinical presentations.

Inheritance of susceptibility to experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis is a disease of the central nervous system that can be readily induced in a variety of species by immunization with myelin proteins. It is one of the most commonly studied models of cell-mediated autoimmune disease and consequently has been important in the elucidation of many immunological functions in vivo. Nevertheless, very little is understood about the genetic and environmental factors that control the disease. Several groups have begun undertaking systematic genetic analyses in mice to identify loci that associate with increased susceptibility to disease. In this review, we will summarize the work that has been done to understand the genetic predisposition that makes certain inbred animals susceptible to disease and others resistant, and we will discuss some of the difficulties in the genetic analysis that have arisen due to the complexities of this disease model.

Genetic analysis of susceptibility to experimental autoimmune encephalomyelitis in a cross between SJL/J and B10.S mice.

Experimental autoimmune encephalomyelitis (EAE), a model for human multiple sclerosis, is a T cell-mediated autoimmune disease that can be induced in experimental animals by immunization with myelin Ags. Inbred strains of mice show varying degrees of susceptibility to EAE, indicating that susceptibility is an inherited trait. To define the genetic factors that control susceptibility to EAE, we performed linkage analysis on the first backcross (BC1) between highly susceptible SJL/J mice and resistant B10.S mice, both of which are of the H-2s haplotype. Mice were immunized for disease with encephalitogenic myelin proteolipid protein peptide 139 to 151, and analysis was performed on 68 backcross mice showing the severe disease phenotype (disease score > or = 3)and 68 backcross mice of the resistant phenotype (no clinical or histologic signs of disease) using microsatellite markers covering >98% of the genome. We found the strongest linkage (p = 0.001) with clinical disease at two loci: one at the telomeric end of chromosome 2, and another near the center of chromosome 3. In addition, several other regions showing some evidence of linkage (p < or = 0.05) with clinical disease were found.