Aquaporin-9 in the Brain Inflammatory Response: Evidence from Mice Injected with the Parkinsonogenic Toxin MPP.

More than 20 years have passed since the first demonstration of Aquaporin-9 (AQP9) in the brain. Yet its precise localization and function in brain tissue remain unresolved. In peripheral tissues, AQP9 is expressed in leukocytes where it is involved in systemic inflammation processes. In this study, we hypothesized that AQP9 plays a proinflammatory role in the brain, analogous to its role in the periphery. We also explored whether Aqp9 is expressed in microglial cells, which would be supportive of this hypothesis. Our results show that targeted deletion of Aqp9 significantly suppressed the inflammatory response to the parkinsonian toxin 1-methyl-4-phenylpyridinium (MPP+). This toxin induces a strong inflammatory response in brain. After intrastriatal injections of MPP+, the increase in transcript levels of proinflammatory genes was less pronounced in AQP9-/- mice compared with wild-type controls. Further, in isolated cell subsets, validated by flow cytometry we demonstrated that Aqp9 transcripts are expressed in microglial cells, albeit at lower concentrations than in astrocytes. The present analysis provides novel insight into the role of AQP9 in the brain and opens new avenues for research in the field of neuroinflammation and chronic neurodegenerative disease.

Human macrophage C-type lectin forms a heteromeric receptor complex with Mincle but not Dectin-2.

MCL, Mincle and Dectin-2 are C-type lectin receptors expressed by subsets of myeloid cells, and their genes cluster together in the APLEC/Dectin-2 gene complex. We have previously shown that MCL and Mincle form a heterodimer in the rat, and others have shown that MCL and Dectin-2 form a heterodimer in the mouse. In the rat, Dectin-2 is a pseudogene, but here, we examine the association of the three receptors in human. In co-transfection experiments analyzed with flow cytometry and immunoprecipitation, we here show that human MCL and Mincle form a disulphide-linked heterodimer that associates with the signalling adaptor molecule FcεRIγ, in accordance with our previous findings in the rat. In contrast to previous findings in the rat, data in this paper indicate a direct association of MCL with FcεRIγ, as previously shown for mouse MCL. We were unable to demonstrate the formation of a heterodimer between human MCL and Dectin-2. Thus, despite similarities, there may be important differences in the conformation of these receptors between rat, mouse and human, and this may have functional consequences.

Innate lymphoid cell characterization in the rat and their correlation to gut commensal microbes.

Innate lymphoid cells (ILCs) are important for tissue immune homeostasis, and are thoroughly characterized in mice and humans. Here, we have performed in-depth characterization of rat ILCs. Rat ILCs were identified based on differential expression of transcription factors and lack of lineage markers. ILC3s represented the major ILC population of the small intestine, while ILC2s were infrequent but most prominent in liver and adipose tissue. Two major subsets of group 1 ILCs were defined. Lineage- T-bet+ Eomes+ cells were identified as conventional NK cells, while lineage- T-bet+ Eomes- cells were identified as the probable rat counterpart of ILC1s based on their selective expression of the ILC marker CD200R. Rat ILC1s were particularly abundant in liver and intestinal tissues, and were functionally similar to NK cells. Single-cell transcriptomics of spleen and liver cells confirmed the main division of NK cells and ILC1-like cells, and demonstrated Granzyme A as an additional ILC1 marker. We further report differential distributions of NK cells and ILCs along the small and large intestines, and the association of certain bacterial taxa to frequencies of ILCs. In conclusion, we provide a framework for future studies of ILCs in diverse rat experimental models, and novel data on the potential interplay between commensals and intestinal ILCs.

Repeated social defeat promotes persistent inflammatory changes in splenic myeloid cells; decreased expression of ß-arrestin-2 (ARRB2) and increased expression of interleukin-6 (IL-6).

BACKGROUND: Previous studies suggest that persistent exposure to social stress in mammals may be associated with multiple physiological effects. Here, we examine the effects of social stress in rats, i.e. repeated social defeat, on behavior, hypothalamic-pituitary-adrenal (HPA)-axis and immune system. METHODS: A resident-intruder paradigm, where an intruder rat was exposed to social stress by a dominant resident rat for 1 hour each day for 7 consecutive days was used. The day after the last stress exposure in the paradigm the data were analyzed. Variation in social interaction was observed manually, whereas locomotion was analyzed off-line by a purpose-made software. Gene expression in the pituitary gland, adrenal gland and myeloid cells isolated from the spleen was measured by qPCR. RESULTS: The exposure to social stress induced decreased weight gain and increased locomotion. An increased nuclear receptor subfamily group C number 1 (NR3C1) expression in the pituitary gland was also shown. In myeloid cells harvested from the spleen, we observed decreased expression of the ß2-adrenergic receptor (ADRB2) and ß-arrestin-2 (ARRB2), but increased expression of interleukin-6 (IL-6). Subsequent analyses in the same cells showed that ARRB2 was negatively correlated with IL-6 following the stress exposure. CONCLUSION: Our results show that that the experience of social stress in the form of repeated social defeat in rats is a potent stressor that in myeloid cells in the spleen promotes persistent inflammatory changes. Future research is needed to examine whether similar inflammatory changes also can explain the impact of social stress, such as bullying and harassment, among humans.

Interstitial cells in calcified aortic valves have reduced differentiation potential and stem cell-like properties.

Valve interstitial cells (VICs) are crucial in the development of calcific aortic valve disease. The purpose of the present investigation was to compare the phenotype, differentiation potential and stem cell-like properties of cells from calcified and healthy aortic valves. VICs were isolated from human healthy and calcified aortic valves. Calcification was induced with osteogenic medium. Unlike VICs from healthy valves, VICs from calcified valves cultured without osteogenic medium stained positively for calcium deposits with Alizarin Red confirming their calcific phenotype. Stimulation of VICs from calcified valves with osteogenic medium increased calcification (p = 0.02), but not significantly different from healthy VICs. When stimulated with myofibroblastic medium, VICs from calcified valves had lower expression of myofibroblastic markers, measured by flow cytometry and RT-qPCR, compared to healthy VICs. Contraction of collagen gel (a measure of myofibroblastic activity) was attenuated in cells from calcified valves (p = 0.04). Moreover, VICs from calcified valves, unlike cells from healthy valves had lower potential to differentiate into adipogenic pathway and lower expression of stem cell-associated markers CD106 (p = 0.04) and aldehyde dehydrogenase (p = 0.04). In conclusion, VICs from calcified aortic have reduced multipotency compared to cells from healthy valves, which should be considered when investigating possible medical treatments of aortic valve calcification.

Dendritic Cell Activating Receptor 1 (DCAR1) Associates With FcεRIγ and Is Expressed by Myeloid Cell Subsets in the Rat.

Dendritic cell activating receptor-1 (DCAR1) is a cell-surface receptor encoded by the Antigen Presenting Lectin-like gene Complex (APLEC). We generated a mouse monoclonal antibody against rat DCAR1, and used this to characterize receptor expression and function. Rat DCAR1 was expressed on minor subsets of myeloid cells in lymphoid tissue, but was uniformly expressed at a high level by eosinophils, and at a low level by neutrophils. It was expressed by eosinophils in the peritoneal cavity and the lamina propria of the gut, and by subsets of macrophages or dendritic cells at these sites. Polarization of peritoneal macrophages showed that DCAR1 expression was absent on M1 macrophages, and increased on M2 macrophages. DCAR1 could be expressed as a homodimer and its associated with the activating adaptor protein FcεRIγ. This association allowed efficient phagocytosis of antibody-coated beads. Additionally, cross-linking of DCAR1 on the surface of rat eosinophils lead to production of reactive oxygen species. These data show that DCAR1 is an activating receptor. Its expression on M2 macrophages and eosinophils suggests that it may play a role in the immune response to parasites.

Evidence of functional Cd94 polymorphism in a free-living house mouse population.

The CD94 receptor, expressed on natural killer (NK) and CD8+ T cells, is known as a relatively non-polymorphic receptor with orthologues in humans, other primates, cattle, and rodents. In the house mouse (Mus musculus), a single allele is highly conserved among laboratory strains, and reports of allelic variation in lab- or wild-living mice are lacking, except for deficiency in one lab strain (DBA/2J). The non-classical MHC-I molecule Qa-1b is the ligand for mouse CD94/NKG2A, presenting alternative non-americ fragment of leader peptides (Qa-1 determinant modifier (Qdm)) from classical MHC-I molecules. Here, we report a novel allele identified in free-living house mice captured in Norway, living among individuals carrying the canonical Cd94 allele. The novel Cd94LocA allele encodes 12 amino acid substitutions in the extracellular lectin-like domain. Flow cytometric analysis of primary NK cells and transfected cells indicates that the substitutions prevent binding of CD94 mAb and Qa-1b/Qdm tetramers. Our data further indicate correlation of Cd94 polymorphism with the two major subspecies of house mice in Europe. Together, these findings suggest that the Cd94LocA/NKG2A heterodimeric receptor is widely expressed among M. musculus subspecies musculus, with ligand-binding properties different from mice of subspecies domesticus, such as the C57BL/6 strain.

B7H6 is a functional ligand for NKp30 in rat and cattle and determines NKp30 reactivity toward human cancer cell lines.

NK cells kill cancer cells and infected cells upon activation by cell surface receptors. Human NKp30 is an activating receptor expressed by all mature NK cells. The B7 family member B7H6 has been identified as one ligand for NKp30. Several alternative ligands have also been reported, and the field remains unsettled. To this end, we have identified full-length functional B7H6 orthologs in rat and cattle, demonstrated by phylogenetic analysis and transfection experiments. In cell-cell contact-dependent assays, chimeric NKp30 reporter cells responded strongly to B7H6 in rat and cattle. Likewise, rat NKp30 expressing target cells induced strong activation of B7H6 reporter cells. Together, these observations demonstrate that B7H6 is conserved as a functional ligand for NKp30 in mammalian species separated by more than 100 million years of evolution. B7H6 and NKp30 are pseudogenes in laboratory mice. The rat thus represents an attractive experimental animal model to study the NKp30-B7H6 interaction in vivo. B7H6 was widely expressed among human cancer cell lines, and the expression level correlated strongly with the activation of human NKp30 reporter cells. Furthermore, siRNA knockdown of B7H6 abolished NKp30 reporter responses, suggesting that B7H6 is the major functionally relevant expressed ligand for NKp30 on these cancer cell lines.

Rat embryonic fibroblasts immortalized by MRPS18-2 protein are target for NK-cells.

Overexpression of the human mitochondrial ribosomal protein MRPS18-2 (S18-2) led to immortalization of primary rat embryonic fibroblasts (REFs). The derived cells (18IM) expressed embryonic stem cell markers. Noteworthy, genes encoding the COX family proteins were up-regulated significantly. It is known that the COX family proteins are involved in the regulation of immune response. In the present work we demonstrate that 18IM cells behave like stem cells when subjected to directed differentiation in vitro. However, unlike stem cells, 18IM cells do not develop tumors in vivo, in SCID mice. This phenomenon is observed due to the strong natural killer (NK) cell immunogenicity. 18IM cells were better recognized by NK cells, compared with primary REFs, as was shown by a standard NK killing assay. Our data explain asymmetry in behavior of stem-like cells in vivo and in vitro, and this support the notion that stem and/or cancer-initiating cells are preferred targets for NK-cells. Concluding, the S18-2 protein is a putative target for cancer vaccines.

The Activating Human NK Cell Receptor KIR2DS2 Recognizes a ß2-Microglobulin-Independent Ligand on Cancer Cells.

The functions of activating members of the killer cell Ig-like receptor (KIR) family are not fully understood, as the ligands for these receptors are largely unidentified. In this study, we report that KIR2DS2 reporter cells recognize a ligand expressed by cancer cell lines. All cancer targets recognized by KIR2DS2 were also recognized by KIR2DL2 and KIR2DL3 reporters. Trogocytosis of membrane proteins from the cancer targets was observed with responding reporter cells, indicating the formation of KIR2DS2 ligand-specific immunological synapses. HLA-C typing of target cells showed that KIR2DS2 recognition was independent of the HLA C1 or C2 group, whereas targets cells that were only recognized by KIR2DL3 expressed C1 group alleles. Anti-HLA class I Abs blocked KIR2DL3 responses toward C1-expressing targets, but they did not block KIR2DS2 recognition of cancer cells. Small interfering RNA knockdown of ß2-microglobulin reduced the expression of class I H chain on the cancer targets by >97%, but it did not reduce the KIR2DS2 reporter responses, indicating a ß2-microglobulin-independent ligand for KIR2DS2. Importantly, KIR2DL3 responses toward some KIR2DS2 ligand-expressing cells were also undiminished after ß2-microglobulin knockdown, and they were not blocked by anti-HLA class I Abs, suggesting that KIR2DL3, in addition to the traditional HLA-C ligands, can bind to the same ß2-microglobulin-independent ligand as KIR2DS2. These observations indicate the existence of a novel, presently uncharacterized ligand for the activating NK cell receptor KIR2DS2. Molecular identification of this ligand may lead to improved KIR-HLA mismatching in hematopoietic stem cell transplantation therapy for leukemia and new, more specific NK cell-based cancer therapies.

Natural Killer Cell Reduction and Uteroplacental Vasculopathy.

Uterine natural killer cells are important for uteroplacental development and pregnancy maintenance. Their role in pregnancy disorders, such as preeclampsia, is unknown. We reduced the number of natural killer cells by administering rabbit anti-asialo GM1 antiserum in an established rat preeclamptic model (female human angiotensinogen×male human renin) and evaluated the effects at the end of pregnancy (day 21), compared with preeclamptic control rats receiving normal rabbit serum. In 100% of the antiserum-treated, preeclamptic rats (7/7), we observed highly degenerated vessel cross sections in the mesometrial triangle at the end of pregnancy. This maternal uterine vasculopathy was characterized by a total absence of nucleated/living cells in the vessel wall and perivascularly and prominent presence of fibrosis. Furthermore, there were no endovascular trophoblast cells within the vessel lumen. In the control, normal rabbit serum-treated, preeclamptic rats, only 20% (1/5) of the animals displayed such vasculopathy. We confirmed the results in healthy pregnant wild-type rats: after anti-asialo GM1 treatment, 67% of maternal rats displayed vasculopathy at the end of pregnancy compared with 0% in rabbit serum-treated control rats. This vasculopathy was associated with a significantly lower fetal weight in wild-type rats and deterioration of fetal brain/liver weight ratio in preeclamptic rats. Anti-asialo GM1 application had no influence on maternal hypertension and albuminuria during pregnancy. Our results show a new role of natural killer cells during hypertensive pregnancy in maintaining vascular integrity. In normotensive pregnancy, this integrity seems important for fetal growth.

Mincle, the receptor for mycobacterial cord factor, forms a functional receptor complex with MCL and FcεRI-γ.

Upon receptor activation, the myeloid C-type lectin receptor Mincle signals via the Syk-CARD9-Bcl10-MALT1 pathway. It does so by recruiting the ITAM-bearing FcεRI-γ. The related receptor macrophage C-type Lectin (MCL) has also been shown to be associated with Syk and to be dependent upon this signaling axis. We have previously shown that MCL co-precipitates with FcεRI-γ, but were unable to show a direct association, suggesting that MCL associates with FcεRI-γ via another molecule. Here, we have used rat primary cells and cell lines to investigate this missing link. A combination of flow cytometric and biochemical analysis showed that Mincle and MCL form heteromers on the cell surface. Furthermore, association with MCL and FcεRI-γ increased Mincle expression and enhanced phagocytosis of Ab-coated beads. The results presented in this paper suggest that the Mincle/MCL/FcεRI-γ complex is the functionally optimal form for these C-type lectin receptors on the surface of myeloid cells.

Rat macrophage C-type lectin is an activating receptor expressed by phagocytic cells.

Macrophage C-type lectin (MCL) is a membrane surface receptor encoded by the Antigen Presenting Lectin-like gene Complex (APLEC). We generated a mouse monoclonal antibody for the study of this receptor in the rat. We demonstrate that rat MCL is expressed on blood monocytes and neutrophils, as well as on several tissue macrophage populations, including alveolar and peritoneal cavity macrophages. We also demonstrate MCL expression on a subset of resident spleen macrophages. Immunohistochemistry analysis of the spleen showed staining specifically in the marginal zone and red pulp. Exposure to pro-inflammatory mediators or to yeast cell wall extract (zymosan) increased surface MCL expression on peritoneal macrophages. We characterized a rat myeloid cell line, RMW, which expresses high levels of MCL. We found that MCL co-immunoprecipitated with the activating adaptor protein FcεRIγ in these cells. Moreover, beads coated with anti-MCL antibody increased phagocytosis in the RMW cells. Together, these observations indicate that rat MCL is a receptor that activates phagocytosis in myeloid cells under inflammatory conditions.

Rat and mouse CD94 associate directly with the activating transmembrane adaptor proteins DAP12 and DAP10 and activate NK cell cytotoxicity.

Signaling by the CD94/NKG2 heterodimeric NK cell receptor family has been well characterized in the human but has remained unclear in the mouse and rat. In the human, the activating receptor CD94/NKG2C associates with DAP12 by an ionic bond between oppositely charged residues within the transmembrane regions of NKG2C and DAP12. The lysine residue responsible for DAP12 association is absent in rat and mouse NKG2C and -E, raising questions about signaling mechanisms in these species. As a possible substitute, rat and mouse NKG2C and -E contain an arginine residue in the transition between the transmembrane and stalk regions. In this article, we demonstrate that, similar to their human orthologs, NKG2A inhibits, whereas NKG2C activates, rat NK cells. Redirected lysis assays using NK cells transfected with a mutated NKG2C construct indicated that the activating function of CD94/NKG2C did not depend on the transmembrane/stalk region arginine residue. Flow cytometry and biochemical analysis demonstrated that both DAP12 and DAP10 can associate with rat CD94/NKG2C. Surprisingly, DAP12 and DAP10 did not associate with NKG2C but instead with CD94. These associations depended on a transmembrane lysine residue in CD94 that is unique to rodents. Thus, in the mouse and rat, the ability to bind activating adaptor proteins has been transferred from NKG2C/E to the CD94 chain as a result of mutation events in both chains. Remarkable from a phylogenetic perspective, this sheds new light on the evolution and function of the CD94/NKG2 receptor family.

Paired opposing leukocyte receptors recognizing rapidly evolving ligands are subject to homogenization of their ligand binding domains.

Some leukocyte receptors come in groups of two or more where the partners share ligand(s) but transmit opposite signals. Some of the ligands, such as MHC class I, are fast evolving, raising the problem of how paired opposing receptors manage to change in step with respect to ligand binding properties and at the same time conserve opposite signaling functions. An example is the KLRC (NKG2) family, where opposing variants have been conserved in both rodents and primates. Phylogenetic analyses of the KLRC receptors within and between the two orders show that the opposing partners have been subject to post-speciation gene homogenization restricted mainly to the parts of the genes that encode the ligand binding domains. Concerted evolution similarly restricted is demonstrated also for the KLRI, KLRB (NKR-P1), KLRA (Ly49), and PIR receptor families. We propose the term merohomogenization for this phenomenon and discuss its significance for the evolution of immune receptors.

Development and function of CD94-deficient natural killer cells.

The CD94 transmembrane-anchored glycoprotein forms disulfide-bonded heterodimers with the NKG2A subunit to form an inhibitory receptor or with the NKG2C or NKG2E subunits to assemble a receptor complex with activating DAP12 signaling proteins. CD94 receptors expressed on human and mouse NK cells and T cells have been proposed to be important in NK cell tolerance to self, play an important role in NK cell development, and contribute to NK cell-mediated immunity to certain infections including human cytomegalovirus. We generated a gene-targeted CD94-deficient mouse to understand the role of CD94 receptors in NK cell biology. CD94-deficient NK cells develop normally and efficiently kill NK cell-susceptible targets. Lack of these CD94 receptors does not alter control of mouse cytomegalovirus, lymphocytic choriomeningitis virus, vaccinia virus, or Listeria monocytogenes. Thus, the expression of CD94 and its associated NKG2A, NKG2C, and NKG2E subunits is dispensable for NK cell development, education, and many NK cell functions.

The complete inventory of receptors encoded by the rat natural killer cell gene complex.

The natural killer cell gene complex (NKC) encodes receptors belonging to the C-type lectin superfamily expressed primarily by NK cells and other leukocytes. In the rat, the chromosomal region that starts with the Nkrp1a locus and ends with the Ly49i8 locus is predicted to contain 67 group V C-type lectin superfamily genes, making it one of the largest congregation of paralogous genes in vertebrates. Based on physical proximity and phylogenetic relationships between these genes, the rat NKC can be divided into four major parts. We have previously reported the cDNA cloning of the majority of the genes belonging to the centromeric Nkrp1/Clr cluster and the two telomeric groups, the Klre1-Klri2 and the Ly49 clusters. Here, we close the gap between the Nkrp1/Clr and the Klre1-Klri2 clusters by presenting the cDNA cloning and transcription patterns of eight genes spanning from Cd69 to Dectin1, including the novel Clec2m gene. The definition, organization, and evolution of the rat NKC are discussed.

NK cell receptors in rodents and cattle.

Natural killer (NK) cells discriminate between normal syngeneic cells and infected, neoplastic or MHC-disparate allogeneic cells. The reactivity of NK cells appears to be regulated by a balance between activating receptors that recognize non-self or altered self, and inhibitory receptors recognizing normal, self-encoded MHC class I molecules. Subfamilies of NK receptors undergo rapid evolution, and appear to co-evolve with the MHC. We here review present views on the evolution and function of NK cell receptors, with an emphasis on knowledge gained in cattle and rodents.

KLRE/I1 and KLRE/I2: a novel pair of heterodimeric receptors that inversely regulate NK cell cytotoxicity.

NK cells identify infected, neoplastic, or MHC-disparate target cells via several different receptors. The NK cell receptor KLRE1 lacks known signaling motifs but has nevertheless been shown to regulate NK cell-mediated cytotoxicity. Here we demonstrate that KLRE1 forms functional heterodimers with either KLRI1 or KLRI2. Cotransfection with KLRE1 was necessary for surface expression of the NK cell receptor chains KLRI1 and KLRI2 in 293T cells. Moreover, KLRE1 can be coimmunoprecipitated with KLRI1 or KLRI2 from transfected NK cell lines. By flow cytometry, KLRE1 and KLRI1 showed colinear expression on NK cells, suggesting surface expression as heterodimers. Unlike other killer cell lectin-like receptors, KLRE1/KLRI1 and KLRE1/KLRI2 heterodimers predominantly migrated as single chains in SDS-PAGE, indicating noncovalent association. KLRI1 was coimmunoprecipitated with the tyrosine phosphatase Src homology region 2 domain-containing phosphatase 1. In accordance with an inhibitory function, anti-HA Ab induced reduced killing of FcR-bearing targets by KLRI1-HA-transfected NK cell lines in a redirected cytotoxicity assay. Reciprocally, KLRI2-HA transfectants displayed increased killing in this assay. Finally, Ab to KLRE1 induced inhibition in KLRI1-transfected cells but increased cytotoxicity in KLRI2 transfectants, demonstrating that KLRE/I1 is a functional inhibitory heterodimer in NK cells, whereas KLRE/I2 is an activating heterodimeric receptor.

Association of arthritis with a gene complex encoding C-type lectin-like receptors.

OBJECTIVE: To identify susceptibility genes in a rat model of rheumatoid arthritis (RA) and to determine whether the corresponding human genes are associated with RA. METHODS: Genes influencing oil-induced arthritis (OIA) were position mapped by comparing the susceptibility of inbred DA rats with that of DA rats carrying alleles derived from the arthritis-resistant PVG strain in chromosomal fragments overlapping the quantitative trait locus Oia2. Sequencing of gene complementary DNA (cDNA) and analysis of gene messenger RNA (mRNA) expression were performed to attempt to clone a causal gene. Associations with human RA were evaluated by genotyping single-nucleotide polymorphisms (SNPs) in the corresponding human genes and by analyzing frequencies of alleles and haplotypes in RA patients and age-, sex-, and area-matched healthy control subjects. RESULTS: Congenic DA rats were resistant to OIA when they carried PVG alleles for the antigen-presenting lectin-like receptor gene complex (APLEC), which encodes immunoregulatory C-type lectin-like receptors. Multiple differences in cDNA sequence and mRNA expression precluded cloning of a single causal gene. Five corresponding human APLEC genes were identified and targeted. The SNP rs1133104 in the dendritic cell immunoreceptor gene (DCIR), and a haplotype including that marker and 4 other SNPs in DCIR and its vicinity showed an indication of allelic association with susceptibility to RA in patients who were negative for antibodies to cyclic citrullinated peptide (anti-CCP), with respective odds ratios of 1.27 (95% confidence interval [95% CI] 1.06-1.52; uncorrected P = 0.0073) and 1.37 (95% CI 1.12-1.67; uncorrected P = 0.0019). Results of permutation testing supported this association of the haplotype with RA. CONCLUSION: Rat APLEC is associated with susceptibility to polyarthritis, and human APLEC and DCIR may be associated with susceptibility to anti-CCP-negative RA.