Bone Marrow Cellular Composition and Inflammatory Cytokine Expression in Patients with Inherited Bone Marrow Failure Syndromes

Abstract 4401

Proinflammatory cytokines, TNF-α and IFN-γ, are potent inhibitors of hematopoiesis, and may be relevant in the pathogenesis of bone marrow failure in inherited bone marrow failure syndromes (IBMFS). Increased levels of these cytokines in sera and in bone marrow CD3⁺ cells have been reported in Fanconi anemia (FA) patients. However, our study did not find increased TNF-α or IFN-γ in sera, or supernatants from phytohemagglutinin-stimulated peripheral blood mononuclear cells from IBMFS patients. To assess whether production of these cytokines is dysregulated in BM of these patients, we examined intracellular expression of TNF-α and IFN-γ in BM mononuclear cells from 16 FA, 20 dyskeratosis congenita (DC), 21 Diamond-Blackfan anemia (DBA) and 7 Shwachman-Diamond syndrome (SDS) patients by flow cytometry; 14 healthy adults were studied as controls.

To detect intracellular TNF-α and IFN-γ, BM lymphocytes and monocytes were stimulated with phorbol 12-myristate 13-acetate plus ionomycin (P+I), or lipopolysaccharide (LPS), respectively. Separately, unstimulated cells were stained with antibodies to CD45, CD3, CD19, CD14, and CD34 to determine the proportion of cellular subsets. Percentages of T cells in patients with IBMFS were comparable to the controls, while DC patients had lower proportion of B cells (p=0.02). The percentages of monocytes were lower in FA (p=0.04), DC (p=0.009), and DBA (p<0.001) patients. The proportions of CD34⁺ cells were also lower in IBMFS patients (≤0.02 for all) except for those with DBA, who had similar proportions as the controls. When we compared the effect of cytopenia (counts below normal for age), only the proportion of CD34⁺ cells in DC patients was significantly affected. DC patients with cytopenia (n=15) had lower numbers of CD34⁺ cells (p=0.007) compared with those without (n=5). We also analyzed the effect of somatic mosaicism in FA because it may correct the hematopoietic defect in these patients. FA patients without mosaicism (n=11) had lower proportions of CD19⁺, CD14⁺, and CD34⁺ cells than those with mosaicism (n=5), while the CD3⁺ cell numbers were unaffected.

We detected both intracellular TNF-α and IFN-γ in T cells, but only TNF-α in B cells in response to P+I, while LPS stimulation led to TNF-α production only in monocytes. Percentages of cytokine-producing T and B cells were significantly lower for patients with DBA when compared with healthy adult controls (p<0.006 for T cells and p=0.001 for B cells). There were no significant differences in the other syndromes. Comparison of intracellular cytokines between cytopenic and non-cytopenic patients showed that TNF-α-producing T cells were affected in FA (p=0.03), where the cytopenic patients had a higher proportion of TNF-α-positive T cells. For the LPS-stimulated monocytes, FA (p=0.01) and DBA (p=0.05) patients had significantly lower proportions of TNF-α-producing cells than the controls, and this was independent of cytopenia. There was no effect of mosaicism on cytokine production.

Contrary to previous reports, we did not find an increase in intracellular TNF-α or IFN-γ in T cells from FA patients. However, the number of TNF-α-producing monocytes in FA was lower than that in healthy adult controls. This is consistent with reported dysregulation of monocytes in FA patients. We also identified reduced cytokine expression in lymphocytes and monocytes from DBA patients, but not from DC or SDS. As expected, we found reduced proportions of CD34⁺ cells in FA, DC and SDS, syndromes associated with multilineage cytopenia, and not in DBA which is associated with pure red cell aplasia. And, we ascertained that FA patients with somatic mosaicism had significantly higher percentages of cells including CD34⁺, suggesting that the corrected stem cell pool in FA mosaics is able to maintain hematopoiesis in contrast to non-mosaic FA patients who develop progressive cytopenia over time. Overall, the effect of cytopenia on cytokine production was mild; however, this may be related to the small sample size. In conclusion, our results suggest that mechanisms other than an excess of inflammatory cytokines may be responsible for bone marrow failure in IBMFS, and this area of research deserves a further attention in larger studies.