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inflammatory genes related to immunity (caspase 4, interferon-induced protein with
tetratricopeptide repeat (IFIT)3, and IL-6), signaling (interferon regulatory factor-1
(IRF1) and TNF-), and vascular cell adhesion molecule-1 (VCAM1). It has been
shown previously that IL-1 is an inducer of VCAM1 in astrocytes
[60]
, so the pattern of
genes induced by HMGB1 is consistent with activated transcription of a restricted num-
ber of inflammatory genes, different from those induced by classic pro-inflammatory
cytokines. It is noteworthy that recent observations indicate HMGB1 as a potenti-
ating factor for the pro-inflammatory functions of IL-1
[35]
. The lack of VCAM1
induction in astrocytes exposed to HMGB1 indicates that our astrocyte cultures do
not contain even low amounts of IL-1. It has also been demonstrated previously that
some of the pro-inflammatory functions of HMGB1 can derive from various con-
taminants bound to this sticky protein
[61]
. The ability of HMGB1 to interact with
inflammatory agents, such as LPS, phosphatidylserine, DNA, and some inflammatory
cytokines, indicates that if we are to dissect the direct signaling role of HMGB1 on
different cell models, we will have to utilize the cytokine in a highly purified form
and to consider possible effects played by suboptimal amounts of HMGB1-binding
molecules rather than by HMGB1 itself. In conclusion, in primary astrocytes HMGB1
induces a specific pattern of gene expression, which includes chemotactic molecules
and a proteolytic enzyme that facilitates tissue infiltration by inflammatory cells.
3.2.3 Analysis of Chemokines and Metalloproteinases Released
by HMGB1-Stimulated Astrocytes
To determine whether induction of specific chemokines was parallel to their enhanced
release, the conditioned media of primary astrocytes stimulated with HMGB1 were
subjected to ELISA assays. As reported in
Figure 3.5
, the release of CCL5 was highly
up-regulated by HMGB1, and the extracellular amounts of this chemokine depended
on the time of stimulation and on the concentration of HMGB1 added.
Owing to the role of CCL5 as an inducer of multiple chemokines and cytokines
in astrocytes
[62]
, it has been suggested that this chemokine could initiate inflam-
matory cascades in the CNS. Hence, the release of CCL5 by HMGB1-stimulated
astrocytes can contribute to the amplification of inflammatory events by autocrine/
paracrine signaling. This assumption is suggested by the presence of several types
of redundant receptors for CCL5 on the surface of astrocytes. Furthermore, HMGB1
stimulated the cell production of CCL2, CCL20, and CCL3. CCL3 and CCL5 recog-
nize the CCR5 receptor and operate as stimulators of immature dendritic cell migra-
tion. An important role for CCR5 and its ligands has been suggested by experimental
evidence indicating that desensitization of this receptor abrogates the recruitment of
dendritic cells to inflamed regions of the CNS
[63]
. Both CCL5 and CCL2 have also
been identified as chemokines that guide macrophages/microglia and T cells to sites
of injury/inflammation
[64]
. Because astrocytes are abundant in the CNS and are
localized in proximity to the blood-brain barrier (BBB), their up-regulation of CCL5
and CCL2 could directly contribute to rapid pro-inflammatory responses by attract-
ing both resident microglia and circulating monocytes. Astrocytes and microglia/
macrophages express the receptor for CCL2, named CCR2
[65]
. Hence, this chemokine
