P225
PHENOTYPIC AND FUNCTIONAL PLASTICITY OF MACROPHAGES AND DENDRITIC
CELLS IN VITRO AND MONONUCLEAR PHAGOCYTE PHENOTYPE IN VIVO
Mylonas, K, Vernon, M, Clay, S, Rutherford, J, Hughes, J
MRC Centre for Inflammation Research, Edinburgh
INTRODUCTION AND AIMS: Macrophages (M) and dendritic cells (DC) are mononuclear
phagocytes and may exhibit various phenotypes in vitro and in vivo. This may depend upon the
ambient levels of the growth and differentiation factors M-CSF (for M) and GM-CSF (for DC) as
well as other cytokines. This project explored the plasticity of murine bone marrow (BM) derived M
and DC exposed to various growth factors in vitro. We also examined the level of M-CSF and GMCSF expression of kidneys following unilateral ureteric obstruction (UUO) together with the nature of
the infiltrating mononuclear phagocytes.
METHODS: Murine M or DCs were generated by culture of bone marrow (BM) with M-CSF or
GM-CSF respectively for 7 days. M and DC culture was then continued in either the original medium
or that of the opposing cell type for 5 days i.e. M culture continued with M-CSF or changed to GMCSF and vice versa for DCs. Cell phenotype and function was assessed on days 1, 3 and 5 of culture
(day 5 data presented). Cell phenotype was assessed by (i) flow cytometry for F4/80, CD11c and MHC
Class II and (ii) realtime PCR for the chemokine receptor CCR7 (highly expressed on DCs with
minimal M expression). The ability to drive T cell proliferation was determined by a mixed
lymphocyte reaction (MLR). Murine kidneys underwent UUO for 7 days. Control and UUO kidneys
were enzymatically dissociated to a single cell suspension and analysed by flow cytometry (F4/80,
CD11c, MHC Class II) and realtime PCR (M-CSF & GM-CSF expression).
RESULTS: Control M-CSF treated M were F4/80HICD11cLOWMHC class IILOW with minimal CCR7
mRNA expression whilst control GM-CSF treated DCs were F4/80LOWCD11cHIMHC class IIHI with
abundant CCR7 mRNA expression. M-CSF treated DCs expressed F4/80, CD11c, MHC class II
and CCR7. In contrast, GM-CSF treated M expressed F4/80, CD11c and CCR7 but MHC
class II expression was unchanged. Control GM-CSF treated DCs efficiently drove T cell proliferation
with M-CSF treated M being ineffective. M-CSF treated DCs induced low levels of T cell
proliferation whilst GM-CSF treated M induced comparable T cell proliferation to control DCs.
Experiments involving the labelling of cells with dyes prior to the change in growth medium excluded
generation of cells from progenitors. Culture of M and DC in a 50/50 mix of M-CSF/GM-CSF in an
attempt to mimic the complexity of the in vivo environment produced ‘hybrid cells’ with features of
both M and DC but a DC phenotype was more predominant i.e. a more marked change from baseline
was seen in M than DC. Realtime PCR analysis of d7 UUO kidneys indicated a marked  in GMCSF mRNA but no change in M-CSF mRNA expression compared to naïve non-obstructed tissue (6fold  in GM-CSF/M-CSF mRNA ratio at d7). Flow cytometric F4/80+ cells in d7 UUO kidneys
consisted 2 discrete populations, (i) F4/80+CD11c+ cells with high expression of both MHC Class II
and GM-CSF receptor suggesting that these cells may be DCs, (ii) F4/80+CD11c- cells with low MHC
Class II expression.
CONCLUSION: This study has shown that in vitro M and DC display dramatic plasticity of
phenotype and function. This suggests that mononuclear phagocytes are capable of developing various
overlapping ‘hybrid’ M/DC phenotypes dependent upon the ambient levels of M-CSF/GM-CSF.
However, mononuclear phagocytes infiltrating obstructed kidneys in vivo appear to exhibit more
discrete M and DC phenotypes despite exposure to both GM-CSF and M-CSF suggesting that
infiltrating monocytes may become polarised in vivo during renal inflammation.