eBook: Breakthroughs and Innovations in Immunology Research - 13

tions that promote macrophage or dendritic cell differentiation.
We then characterized their ability to
produce cytokines and chemokines and upregulate
costimulatory molecules in response to inflammatory
stimuli. In addition, we tested and confirmed
the ability of the CX3CR1+
bone marrow precursors
to migrate in vivo upon adoptive transfer. Our studies
enable researchers to better understand these
vital cells and the roles they play in immunity.
Introduction
Chemokines and their receptors constitute a large
family of proteins whose main function is to promote
cell migration through a concentration gradient.
The chemokine molecular signature includes
four conserved cysteine residues that form two disulfide
bonds pairing the first with the third and the
second with the fourth. Based on the arrangement
of the N-terminal two cysteine residues, chemokines
can be grouped into four subfamilies: CXC, CC,
(X)C, and CX3C. CXC chemokines have one amino
acid that separates the first two cysteines. In CC
chemokines, these two cysteines are adjacent. In
the (X)C subfamily, the first and third cysteines are
missing. The last subfamily, CX3C, has one member,
CX3CL1/fractalkine, and it has three amino acids
between the two cysteines.
CX3CR1 is the receptor of CX3CL1 and is a G-protein-coupled,
seven-transmembrane domain receptor.
It is expressed by innate cells, predominantly
macrophages and microglia, but also by other
cell types such as T cell subsets. Given the nature of
the cells expressing the receptor, CX3CL1-CX3CR1
interaction plays a fundamental role in a number
of conditions, from Alzheimer's1
and mucosal immunity.3
to atherosclerosis2
Although expression of CX3CR1 in circulating and
resident populations has been well-studied using
a GFP/CX3CR1 reporter mouse model, CX3CR1+
bone marrow precursors are not as well-known.
In this report, we assess the capacity of murine
CX3CR1+
bone marrow cells to differentiate into
macrophages or dendritic cells when cultured with
GM-CSF or GM-CSF/IL-4 after positive selection.
We show the surface phenotype and cytokine and
chemokine profile of TLR-stimulated cells, as well
as their capacity to migrate after in vivo transfer.
Materials and Methods
Bone marrow single cell suspensions
There are several published methods to obtain a
single cell suspension from mouse bone marrow.4
Briefly, 8-12 weeks old C57BL/6 mice were sacrificed,
femurs were dissected, and excess tissue was
removed under aseptic conditions. Bones were
rinsed and transferred to complete media. The ends
of the bone were trimmed with sterilized scissors
to expose the marrow. Marrow was flushed using a
syringe and a 29G × ½ needle. Cells were collected
in a sterile 15 mL tube, washed, and counted using
a Cellometer (Nexcelom).
Magnetic cell separation
CX3CR1+
positive cells were isolated using BioLegend's
Magnetic Cell Separation System, MojoSort™
(Cat. No. 480055). Granulocytic cells were depleted
using magnetic nanoparticles directly conjugated
to anti-Ly-6G antibody. The negative fraction was
subsequently incubated with biotinylated anti-CX3CR1
antibody, followed by washing and incubation
with Streptavidin conjugated nanoparticles.
Cell differentiation and toll-like
receptor stimulation
After magnetic cell isolation, cells were cultured in
24-well plates for 7 days at a density of 1-2 x 106
/
mL in complete RPMI 1640 media, supplemented
with GM-CSF (10 ng/mL) or GM-CSF/IL-4 (Cat. No.
13
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