Role Of Tumor Necrosis Factor α In Bone Marrow Niche Dysfunction Of Patients With Systemic Lupus Erythematosus (SLE)

Abstract

Objectives and background SLE is a chronic systemic autoimmune disorder associated with autoantibodies and cytopenias. There are few studies of the pathogenesis of anemia of chronic inflammation in lupus. This study addresses the pathogenesis of the hematological manifestations of lupus. We have shown that SLE bone marrow (BM) exhibits striking death of niche and hematopoietic cells associated with tumor necrosis factor-α (TNFα) over-production. Here, we further examined the pathogenesis of hematological BM niche dysfunction. Methods Pathology records over the past 10 years from the University of Florida were reviewed and BM aspirates/core biopsies from 6 SLE patients were identified for further study. Wright-Giemsa stained BM aspirate smears and cytospin preparations, hematoxylin and eosin (H&E)-stained and reticulin stained BM core biopsies were reviewed. Immunohistochemistry (IHC) for TNFα, cleaved caspase-3, and CD71 was performed on core biopsies and expression levels were quantified morphometrically. BM specimens from individuals undergoing staging for lymphoma were selected as controls. Results Five of 6 SLE patients had nephritis and 3 were direct Coombs+ (one with hemolytic anemia). Mean hemoglobin was 9.1g/dL in SLE patients and 11.9g/dL in controls. Mean WBC was 3800/mm3 in patients and 8200/mm3 in controls, and mean platelet counts were 133,000/mm3 and 258,000/mm3, respectively. BM aspirates exhibited numerous apoptotic cells, erythroid dyspoiesis, plasmacytosis, hemophagocytosis, and phagocytosis of nuclear material by mature neutrophils (LE cells). Numerous LE cells were seen in 5/6 SLE BM aspirates. Compared to normal BM biopsies, SLE BM biopsies exhibited hypocellularity, erythroid dyspoiesis, polyclonal plasmacytosis, mild reticulin fibrosis, BM stromal damage/disorganization and 3 out of 6 with interstitial lymphoid aggregates. IHC with anti-cleaved caspase-3 antibodies, a specific marker of cell death, revealed numerous caspase-3+ cells in a range of 20-40% of total BM cells in contrast to control BMs of <5%. Interestingly, a large majority of osteoblasts or lining cells in BM osteal niches were caspase-3+ in SLE BM, whereas caspase-3+ cells were rare in control BM biopsies. Using double IHC (TUNEL assay plus anti-CD71 or myeloperoxidase antibodies), prominent apoptotic erythroid precursors with less extensive cell death of the myeloid and megakaryocytic lineages was found in SLE BM core biopsies. In SLE, caspase-3+ cells occupied 9-12% of the total BM area vs. 1-2% in control BM (p<0.001). As TNFα promotes Fas-mediated apoptosis and may damage hematopoietic precursors and/or stromal cells, we examined its production in SLE BM. IHC with anti-TNFα antibodies revealed intense intra- and extracellular staining surrounding neutrophils and monocytes, and the extent of TNFα staining was dramatically higher in SLE patients’ BM biopsies than in controls. Staining was particularly intense adjacent to presumptive osteoblasts lining the surface of bone trabeculae, though there also was staining in interstitial regions. There was little TNFα staining of control BM. Morphometric analysis revealed 10-18% of the area in SLE BM intensely stained with anti-TNFα vs. 1-3.5% in controls (P < 0.001, Student t-test), suggesting that TNFα overproduction may cause BM niche dysfunction. Conclusion BM TNFα-mediated niche cell apoptosis is likely to be involved in the pathogenesis of SLE-associated hematological abnormalities. Disclosures: No relevant conflicts of interest to declare.