Splenic Ossification in Sickle Cell Anemia Mice

Introduction: The chronic anemia in sickle cell disease (SCD) leads to highly expanded medullary erythropoiesis, which results in extramedullary hematopoiesis (EMH). EMH is almost entirely extramedullary erythropoiesis (EME) in SCD, with spleens of SCD mice enlarged and the architecture nearly effaced by EME. We observed foci of metaplastic ossification foci develop in SCD spleens of aged SCD mice with associated trilineage hematopoiesis

Methods: The Townes SCD mice (termed SS mice) and their normal counterparts (termed AA mice) were kindly provided by Dr. Timothy M Townes (University of Alabama, Birmingham). Spleens from 28 AA and 28 SS mice, aged 54±1.3 and 52±1 weeks of age, respectively, were harvested, individually weighed, photographed and fixed for histopathology. Interleukin-5 (IL-5) levels were analyzed Mouse Luminex Screening Assay in washings of BM and spleen from 4 mice of each group. Two-tailed unpaired student's t-tests were used to compare groups.

Results: The spleens of SCD mice were 6 times the size of normal spleens. We observed 1.4±0.2 (mean±SEM; range 1-3) foci of metaplastic ossification per spleen in SS mice, which were 1-4 mm in diameter. These foci were present in areas of EME and only found in 16 of 28 (60%) spleens of 1 year-old SS mice but absent in spleens of 1 year-old AA mice. Ossification foci contained mature osteoid tissue encasing typical bone marrow (BM) elements (myeloid and lymphoid cells, megakaryocytes, erythroid cells, hemosiderin laden macrophages, histiocytes, stromal/mesenchymal cells and micro-vessels), unlike the rest of the spleen which had EME and occasional splenic lymphoid follicles, and represented cancellous bone and BM in spleen. Apart from the ossification foci, the rest of the SCD splenic tissue showed near replacement by erythroid precursors and sinusoids congested with sickled RBC, with sparsely scattered lymphoid follicles. IL-5 levels were not elevated either in plasma or bone marrow supernatant. No such foci were seen in spleens of strain and age-matched control AA mice as well as 2-4 month-old SS or AA mice

Conclusion: We show that aged SCD mice develop foci of ossification, akin to intramedullary hematopoiesis developing within the extensive EME. Ossification is the process of new bone formation, which was unique to spleens of SCD mice, but not seen in normal spleens of 1 year old control mice, aged concurrently. EMH induced splenic ossification has been reported in malignancies and in an IL-5 transgenic mouse model (Macias MP et. al., J Clin Invest, 2001). A comprehensive review of the literature in SCD mice, which have been studied for over two decades, showed that most studies on SCD mice were performed on young mice; and studies where aged mice were studied, histopathology of spleens was not examined. We did not find reports of splenic ossification in any other hemoglobinopathy or benign hematological disorder. Therefore, this phenomenon may be a late consequence of chronic EME in the context of inflammation, and bone marrow scarring. We propose that the life-long anemia and chronic stress-erythropoiesis causes this phenomenon. With disease modifying therapies, patients with SCD have intact/enlarged spleens, radiological evidence of calcification has been reported, and it is conceivable that these may represent splenic ossification, which can have clinical consequences.

No relevant conflicts of interest to declare.