Experiment
Autoimmune hemolytic anemia (AIHA) occurs in 7-22% of patients with systemic lupus erythematosus (SLE) and often preceeds the diagnosis of SLE. Lupus-prone NZW mice develope a genetically imposed severe spontaneous autoimmune hemolytic anemia that is very similar to the corresponding human disease.
In this study NZB.NZW congenic mice containing an introgressed fragment of NZW on Chromosome 4 encompassing the Lbw2 locus was used to investigate the role in AIHA. QTL Lbw2 has previoulsy been linked to survival, glomerulonephritis, and splenomegaly.
Female NZB/BlScr(NZB) and NZB.NZW-Lbw2 congenic mice were assesed for Coomb's positivity monthly for a total of one year. Results revealed a striking reduction in anti-erythrocyte Ab (AEA) in the Lbw2 congenic mice campared with NZB mice. At 12 months all NZB mice were affected and <40% of the congenics were positive. The amount of AEAs on red blood cells (RBCs) were also lower in the Lbw2 congenics. In contrast, the NZB and Lbw2 congenic mice showed no differrence in total IgM and IgG serum AB levels which at 12 months were comparable to nonautoimmune C56BL/6 mice. Findings indicated that the NZB Lbw2 congenic interval primarily promoted the production of AB levels in red blood cells.
Histologic and cellular characteristics of spleens were analyzed. One year old Lbw2 congenic NZB mice displayed subtantially lower spleen weights that were strikingly only slightly greater than those of non-autoimmune C57BL/6 mice. Spleen sections from NZB mice revealed a marked accumulation of RBCs within the red pulp and severe distortion of the normal architecture. In the NZB bacground, Lbw2 was associated with splenomegaly resulting from AEA related accumulation of RBCs.
To more precisely map the location of the Lbw2 gene responsible for the suppression of AEAs, a panel of Lbw2 interval specific subcongenic mice were created (NZB.NZW-Lbw2NZW/LacScr/Scr) and screened for the presence of AEAs. [Fig 6A.]
All subcongenic strains demonstrated suppression of AEA production compared to the NZB mice. However, the subcongenic containing the largest interval, designated SA (62.0-134.0 Mb, Supp Table 1) showed the same degree of suppression as the full Lbw2 congenic, indicating that it probably contained all relevant Lbw2 genes.
The remaining three subcongenics (designated SB, SC, and SD) demonstrated only partial suppression of AEAs indicating that Lbw2 contains at least three subloci:
The centromeric most QTL, Lbw2a (lupus-NZB x NZW 2a), was defined by the region of SA not shared with the other subcongenics, 62.0-89.9 Mb. The approximate 28 Mb interval contains 85 known genes that include several immunologically relevant genes such as the type I interferon gene family, Tlr4 and Tnfsf8 which encodes CD30L.
QTL Lbw2b (lupus-NZB x NZW 2b) was defined by the SC interval, (86.0-115.7 Mb), which spans approximately 30 Mb and contains approximately 191 protein coding genes including Jak1, Jun and Cdkn2c.
QTL Lbw2c (lupus-NZB x NZW 2c) was defined by the centromeric end of SD (118.9 Mb) and the telomeric end of SA (134.2 Mb). [Fig 7; Supp Table 1.] This interval overlapped SA/SB and SD and was approximately 13.5 Mb in size and contained approximately 243 unique protein coding genes including Tlr12, Lck and Ermap and Epb4.1.
The additive contributions of at least 3 genetic varaints on Chromosome 4 were required for full susceptibility to AEA production in NZB mice.
The same panel of subcongenic mice were analyzed to localize splenomegaly; all subcongenics exhibited lower spleen weights than NZB; demonstrating that the NZB Lbw2 locus confers enhanced susceptibility to splenomegaly. Two AEA-promoting subloci, Lbw2b and Lbw2c, were linked with splenomegaly, a finding consistent with the association of AEA and splenomegaly. [Fig 6C.]
The subcongenic mice were also used to more precisely map the location of the gene affecting total and B-1a cell numbers in the peritoneum. The smallest of the three subcongenics, SC, demonstrated a significant decrease in both total peritoneal and B-1a cells at most stages, localizing genetic control of the phenotype to the Lbw2b locus. [Fig 7.] An unexpected result was the lack of significant effect of Lbw2 on B cell proliferation.
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