Monocyte function in patients with myelodysplastic syndrome

Abstract

Myelodysplastic syndrome (MDS) is a malignant hematopoietic stem cell disorder that frequently evolves into acute myeloid leukemia (AML). Patients with MDS are prone to infectious complications, in part due to the presence of severe neutropenia and/or neutrophil dysfunction. However, not all patients with neutropenia become infected, suggesting that other immune cells may compensate in these patients. Monocytes are also integral to immunologic defense; however, much less is known about monocyte function in patients with MDS. In the current study, we monitor the composition of peripheral blood monocytes and several aspects of monocyte function in MDS patients, including HLA-DR expression, LPS-induced inflammatory cytokine production, and phagocytosis. We find that monocytes from MDS patients exhibit relatively normal innate immune functions compared to monocytes from healthy control subjects. We also find that HLA-DR expression is moderately increased in monocytes from MDS patients. These results suggest that monocytes could compensate for other immune deficits in MDS patients to help fight infection. We also find that the range of immune functions in monocytes from MDS patients correlates with several key clinical parameters, including blast cell count, monocyte count, and revised International Prognostic Scoring System score, suggesting that disease severity impacts monocyte function in MDS patients.

Keywords: LPS; PBMC; human leukocyte antigen-antigen D related; myelodysplastic syndrome; phagocytosis.

Figure 1. Normal composition of monocyte subsets in MDS patients

Immediately after thawing frozen PBMCs from either healthy volunteers or from untreated patients with MDS, an aliquot of cells was fixed and subsequently stained for flow cytometry analysis. An identical gating strategy using FlowJo batch processing was used to analyze monocyte subsets in all samples. Small lymphocytes and dead cells were omitted from the initial cell gate (“non-lymphocytes”); then cells with high CD15 or CD20 were removed using a dump channel strategy; and finally CD14 and CD16 levels were assayed. Representative examples of this gating strategy are displayed for a healthy volunteer (A) and an untreated MDS patient (B). Panels C–E display the frequency of monocyte subsets. Panels F–H display HLA-DR levels (MFI) normalized relative to control in each of the three monocyte subsets (average of control defined as 1). Those comparisons that were significantly different have P values listed; all other comparisons were not significant.

Figure 2. Monocytes from MDS patients do not exhibit significant immune defects

Monocytes from either healthy volunteers, untreated MDS patients, or treated MDS patients were exposed to 20 ng/ml LPS for four hours and production of the indicated cytokine proteins (A,B) or mRNAs (C–E) was monitored. (F) Monocytes were incubated for two hours with fluorescently labeled E. coli particles and phagocytosis of these fluorescent particles was monitored using the Vybrant Phagocytosis assay Kit (Molecular Probes). All values normalized so that healthy volunteer data is averages to 1. None of the monitored parameters were statistically different from each other as determined by one-way Anova.

Figure 3. Association of clinical factors with monocyte function in patients with MDS

The graphs display the indicated cytokine proteins (A–D) or cytokine mRNAs (F–H) produced by monocytes (from untreated MDS patients) challenged with 20 ng/ml LPS for four hours compared to the indicated clinical factors in these patients (absolute peripheral blood monocyte count, marrow blast cell percentage, or IPSS-R score). Panel E depicts a comparison of absolute peripheral blood monocyte count and marrow blast cell percentage in these same patients. Solid lines depict the predicted variables from single variable linear regression. The p values from these analyses also are indicated. Further statistical information relevant to this figure is presented in Supplemental Table 4.