eBook: Breakthroughs and Innovations in Immunology Research - 17

(TLR9 agonist), they responded by upregulating
MHC II, CD80, and CD86. This is expected in antigen
presenting cells (APCs) as they express several
TLRs and undergo further maturation when
stimulated with TLR agonists. A classical landmark
of APC maturation is the upregulation of MHC II
and co-stimulatory molecules (CD80, CD86) upon
TLR stimulation. We also observed a difference in
the expression pattern when comparing the two
cultures, with unstimulated GM-CSF derived cells
(grey histograms) expressing lower levels of MHC
II and CD80, but higher levels of CD115. However,
LPS (red histograms) and CpG (blue histograms)
stimulated cells are capable of upregulating MHC
II and costimulatory molecules to the same extent.
Interestingly, GM-CSF-derived cells downregulate
CD115 to the same low levels observed in GM-CSF/
IL-4-derived cells, which further supports the evidence
that the cells derived from the different culture
conditions develop into different cell types.
Cytokine and chemokine profiles
following TLR stimulation
Following the culturing methods described above,
we examined the cytokine profile for these cells.
Using our LEGENDplex™ multiplexing bead-based
system, we analyzed both inflammatory cytokine
and chemokine production from the macrophage
and dendritic cell populations following
stimulation with LPS or CpG. As shown in Figure
5, macrophages express a distinct cytokine profile
compared to dendritic cells. While macrophages
upregulated expression of IL-1α, IL-1β, IL-12p40,
IL-12p70, IL-27, IFN-β, IP-10, KC, MIP-1α, and MIP1β,
dendritic cells produced increased amounts
of IL-23, BLC, and MIP-3α. In addition, LPS elicited
a stronger cytokine and chemokine profile compared
to CpG stimulation.
Figure 5. LEGENDplex™ cytokine and chemokine profile of mouse
bone marrow-derived macrophages and dendritic cells. Numbers
reflect fold-increase based on pg/mL values of stimulated cells over
non-stimulated cells.
MojoSort isolated cells retain functional
ability to migrate
We also assessed the function of these bone marrow
CX3CR1+
precusors in vivo by labelling them
with CFSE and injecting them into recipient mice.
Control mice were treated with PBS. After 3 days,
spleen, intestine (data not shown) and bone marrow
were harvested and stained. As seen in Figure
6, CX3CR1+
, CFSE-labeled cells from the donor mice
successfully migrated to the bone marrow, demonstrating
that their migratory capacity was unaffected
by the MojoSort™ protocol.
Discussion
The function of macrophages and dendritic cells is
critical for a number of disease outcomes, as well as
normal, physiological conditions making it vital to
properly characterize them. Helft et al.8
have previously
looked at cell culturing methods and the heterogeneity
of bone marrow-derived macrophages
17
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