CD300f modulates monocyte and dendritic cell functions

Azlan, Maryam (2013). CD300f modulates monocyte and dendritic cell functions PhD Thesis, School of Medicine, The University of Queensland.

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Author Azlan, Maryam
Thesis Title CD300f modulates monocyte and dendritic cell functions
School, Centre or Institute School of Medicine
Institution The University of Queensland
Publication date 2013
Thesis type PhD Thesis
Supervisor David Munster
Georgina Clark
Total pages 155
Language eng
Subjects 1199 Other Medical and Health Sciences
1107 Immunology
Formatted abstract
CD300f is a member of the CD300 family of immunoregulatory molecules that associate with the inhibitory tyrosine kinase Src homology phosphatase-1 (SHP-1) and the activatory p85α subunit of phosphoinositide 3-kinase (PI3K). CD300f is expressed on the surface of myeloid cells. The exact role of human CD300f in mediating myeloid cell functions is unknown. The main aim of this PhD project is to understand the role of human CD300f in modulating monocyte and dendritic cell (DC) functions. Monocytes and DC are leucocyte subsets that participate in host immune responses. Monocytes are of myeloid origin and in humans are subdivided into ‘classical’ CD14++CD16- monocytes, ‘intermediate’ CD14+CD16+ monocytes and ‘non-classical’ CD14+CD16++ monocytes. Human peripheral blood DC consists of plasmacytoid DC (pDC) and myeloid DC (mDC). The mDC are subdivided into three subsets; CD16+ DC, CD1c+ DC and CD141+ DC.

In the absence of a specific ligand at the commencement of this PhD project, we generated a monoclonal antibody (mAb) against the extracellular domain of human CD300f, namely MMRI-23. By conducting immunoprecipitation, Western blotting and blocking experiments, MMRI-23 mAb was confirmed to bind to an epitope on the extracellular CD300f Ig-like domain. MMRI-23 mAb was used to identify the presence of CD300f on the cell surface of leucocytes. The CMRF-81 mAb was used as an isotype control mAb alongside MMRI-23 mAb in all experiments. In this study, flow cytometric and mRNA analyses have been used to show that MMRI-23 is expressed on the so-called ‘inflammatory’ CD14+CD16+ monocytes (CD16+ monocytes), M1 in vitro-derived macrophages and CD16+ DC. Furthermore, CD300f is expressed more on the CD16+ monocytes compared to CD14+CD16- monocytes (CD16- monocytes). CD300f is also expressed at higher levels on CD16+ DC but not on CD1c+ DC and CD141+ DC.

To investigate the function of CD300f, four leucocyte subsets have been successfully purified from human peripheral blood mononuclear cells (PBMC) simultaneously which are; whole CD14+ monocytes, CD16+ monocytes, CD16- monocytes and CD16+ DC. Combining positive immunomagnetic selection with subsequent flow cytometric cell sorting yielded useful quantities of purified monocyte and DC subsets of high viability and purity.

The MMRI-23 mAb was then used as a surrogate ligand to crosslink cell surface CD300f on the purified monocyte and DC subsets. Crosslinking cell surface CD300f using MMRI-23 mAb on the CD14+ monocytes resulted in downregulation of myeloid markers CD14 and CD33, but enhanced endocytosis. Crosslinking with MMRI-23 mAb increased endocytosis by CD16+ monocytes but decreased endocytosis by CD16+ DC. The CD300f signalling induced endocytosis was inhibited by the PI3K inhibitor Wortmannin.

The role of CD300f was investigated in chemotaxis by monocyte and DC. CD300f signalling on CD14+ monocytes promoted chemotaxis towards the chemokines CCL19, CCL21 and CXCL12. CD300f signalling mainly induced the CD16+ monocytes to migrate towards CXCL12. The U937 cell line was then used as a model to study the mechanism of MMRI-23 mAb induced chemotaxis towards CXCL12. MMRI-23 mAb crosslinking results in increased chemotaxis of U937 cells towards CXCL12 and was inhibited by Wortmannin indicating that CD300f induces chemotactic signalling through PI3K.

Upon recognition of pathogen, monocyte and DC subsets secrete specific cytokines. The role of CD300f in secretion of cytokines was tested. MMRI-23 mAb induced CD300f signalling increased secretion of proinflammatory cytokines TNF-α, IL-1β and IL-8 by CD14+ monocytes. However, other monocyte subsets did not produce significant amounts of cytokines when crosslinked with MMRI-23 mAb indicating that CD300f may require additional stimuli to induce cytokine secretion.

Interestingly, crosslinking cell surface CD300f resulted in decreased viability of CD14+ monocytes and CD16- monocytes but not CD16+ monocytes and CD16+ DC. The decreased viability of monocyte subsets in the presence of MMRI-23 mAb was unaltered by Wortmannin which suggests that CD300f signalling induced inhibition of monocyte survival is not PI3K dependent.

This study demonstrated that CD300f signalling induced endocytosis by CD14+ monocytes and CD16+ monocytes but decreased endocytosis by CD16- monocytes and CD16+ DC. In addition, CD300f signalling decreased the viability of CD14+ monocytes and CD16- monocytes but not other cell types. These results suggest the possible dual functionality of CD300f in humans. However, the possible mechanism whereby CD300f delivers activatory or inhibitory signals requires further investigation.

Although the ligand for human CD300f is unknown, studies in mouse CD300f have shown that the phosphatidylserine and extracellular ceramides are the ligands for mouse CD300f. Mouse CD300f inhibited phosphatidylserine binding to Annexin V while the binding of ceramide to CD300f inhibited FCεRI-mediated activation of bone marrow-derived mast cells. These results indicate that human CD300f may also bind more than one ligand due to the existence of two possible ligand binding sites as demonstrated by the crystal structure of CD300f. The inhibition of mast cell function by CD300f, if demonstrated in humans may be a potential therapeutic application in limiting allergic responses.

Overall my study has demonstrated that CD300f signalling results in changes of phenotype, endocytosis, chemotaxis and cell survival of monocyte and DC subsets. This is the first study demonstrating the role of human CD300f in mediating monocyte and DC functions. It is a significant addition to the currently limited knowledge of human CD300f which will lead to better understanding of inflammatory monocyte and DC biology and possibly clinical utility.
Keyword CD300f
Dendritic cells (DC)

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Created: Wed, 05 Feb 2014, 11:48:48 EST by Mrs Maryam Azlan on behalf of Scholarly Communication and Digitisation Service