Monocytes represent a heterogeneous population of peripheral blood cells. Two distinct subsets of monocytes have recently been defined. One subset is characterized by high forward and side scatter profiles by flow cytometry, high expression of CD14 (LPS receptor), and is CD62L positive, CX3CR1 (fractalkine receptor) low and expresses inflammatory chemokine receptors (CCR1, CCR2, CXCR1 and CXCR2). The other subset expresses CD16(FcRIII) and is smaller and less granular, CD62L negative, CX3CR1 high and does not express the above-named inflammatory chemokine receptors, yet expresses higher levels of MHC class II, VLA-4 and ICAM-1. In healthy individuals, CD16+ monocytes comprise 5-10% of the total monocyte pool. A recently described analogous monocyte subset in mice differs from the larger subset in homing properties and gives rise to resident tissue macrophages and dendritic cells (Geissmann et al, Immunity, July 2003). The role of this monocyte subset in human disease has not been clearly defined. The percentage of CD16+ monocytes can increase up to 80% in numerous pathological conditions, including cancer, sepsis, Kawasaki disease, atopic eczema, tuberculosis and asthma. It remains unclear how the different subsets of monocytes return during the reconstitution of the immune system following myelosuppressive chemotherapy for cancer and myeloablative therapy prior to stem cell transplantation for both malignant and benign disorders. Furthermore, the turnover of tissue dendritic cells and macrophages is not established. Our hypothesis is that the relative prevalence and rate of return of monocyte subsets influence predilection for infections and possibly also for GvHD. To begin to assess this, we have characterized the phenotype of monocytes in normal volunteers and patients following allogeneic stem cell transplant by flow cytometry and measured cytokine production in response to in vitro stimulation with various infectious agents. Monocytes were isolated from PBMCs by forward-side scatter gating and exclusion of lineage markers for T cells (CD3), B cells (CD19) and NK cells (CD56). Monocyte subsets were then defined by CD14 and CD16 expression. The percentage of CD16+ monocytes within the monocyte pool varied from 1.5 22%. We assessed cytokine production in response to various microbial molecules by stimulating PBMCs in vitro for 16-20 hours with either Aspergillus fumigatus hyphae, LPS, LTA or CpG. Supernatants were assayed for IL-1, IFN-, TNF-, IL-10 and IL-6 by ELISA. Between different individuals, cytokine production differed dramatically. Most persons produced IL-1, IL-6 and TNF- in response to hyphae, LPS and LTA. Only two individuals produced IL-10 in response to hyphae. IFN- production varied widely. Our results demonstrate remarkable diversity in the percentages and absolute numbers of the monocyte subsets as well as cytokine production in response to various infectious agents among normal individuals as well as patients following stem cell transplantation. These studies suggest that monocyte subsets play a role in the immune systems response to various microbial and fungal products. Further characterization of the CD14+ and CD16+ monocyte subsets in the setting of immune reconstitution following stem cell transplantation may lead to a greater understanding of their role in the immune system, potentially leading to the development of novel therapeutic interventions.
Full conference title:
American Society of Hematology 45th Annual Meeting
- ASH 45th (2003)