Monocytes invading the brain parenchyma in response to ischemia or TBI have detrimental effect on neuronal survival and functional recovery after injury (Chen et al, 2003
; Dimitrijevic et al, 2007
; Semple et al, 2010a
). The influx of these inflammatory cells is driven by monocyte chemoattractants, such as CCL2, whose synthesis is rapidly increased in the injured cortex (Semple et al, 2010a
; Szmydynger-Chodobska et al, 2010
). In the present study, we demonstrated that neurotrauma also results in a rapid increase in production of CCL2 by the lateral ventricle CP located ipsilaterally to injury. This increase in choroidal CCL2 synthesis was not associated with posttraumatic accumulation of monocytes in the choroidal tissue, but resulted from production of CCL2 by the choroidal epithelium. Indeed, although monocytes can produce CCL2 in response to proinflammatory mediators (Colotta et al, 1992
), these inflammatory cells were rarely found in the ipsilateral CP at 6
hours post-TBI, a time point at which a maximum increase in choroidal CCL2 synthesis was observed. An increase in choroidal production of CCL2 was associated with a significant elevation of CCL2 concentration in the CSF, which was comparable to the levels of this chemokine found in the CSF of patients with severe TBI (Semple et al, 2010a
These observations raise the question about the importance of the CP as a source of CCL2 in the injured brain. Using the primary cultures of choroidal epithelial cells, we found that the rate of apical secretion of CCL2 in response to IL-1β
is relatively stable during the first 6
hours of incubation with the cytokine (see ) and amounts to 15
of surface area of epithelial monolayer. In these experiments, the epithelial monolayers were exposed to 10
pg/mL of IL-1β
, the average concentration of IL-1β
found in ventricular CSF in patients with severe TBI at 6
hours after injury (Shiozaki et al, 2005
). To extrapolate these in vitro
data to the in vivo
situation, we assumed that the apical surface of choroidal epithelium of one lateral ventricle CP is 0.7
(without taking into account the surface area of apical microvilli; unpublished data). Accordingly, the rate of CCL2 secretion by one lateral ventricle CP would amount to 10.5
ng/h. In our previous study, we have determined that with a substance continually secreted by the CP, the time needed for this substance to reach half of its maximal concentration in the CSF is ~70
minutes in rats (Batisson et al, 2006
). Given this time and assuming that the rate of CSF production in Long-Evans rats is 177μ
L/h (DePasquale et al, 1989
), the concentration of CCL2 in the CSF at 6
hours post-TBI would be 59
ng/mL. This estimated concentration of CCL2 is comparable to the average level of CCL2 (44
ng/mL) found in the CSF collected at 6
hours after injury, which clearly indicates that the CP represents a significant source of CCL2 in the injured brain. These calculations are based on the simplified compartmental model and do not take into consideration the loss of CCL2 from the CSF due to its diffusion into the brain parenchyma.
The immunohistochemical analysis of choroidal tissue demonstrated that CCL2 is produced by the epithelial cells. This chemokine did not appear to be synthesized by other types of cells normally present in the choroidal tissue, such as endothelial and epiplexus cells or stromal macrophages, in both sham-injured and traumatized rats. These results are in line with the previous studies, in which in situ
hybridization histochemistry was used to demonstrate the upregulation of CCL2 expression in the CP in response to peripheral administration of proinflammatory mediators, such as lipopolysaccharide, IL-1β
, and tumor necrosis factor-α
(Thibeault et al, 2001
). The hybridization signal shown by these authors was consistent with the expression of CCL2 by the choroidal epithelial cells. On the other hand, Mitchell et al (2009)
, who also used an in situ
hybridization technique, reported the increased expression of CCL2 in choroidal stromal cells after the carrageenan-induced peripheral inflammation, but the types of cells producing this chemokine were not identified. The reason for these discrepant results is not immediately clear, but may be associated with the different animal models used. Unlike the cerebrovascular endothelium that is an important source of CCL2 in the injured brain (Szmydynger-Chodobska et al, 2010
), the endothelial cells of choroidal microvessels did not appear to produce this chemokine. The fenestrated phenotype of choroidal endothelial cells (Strazielle and Ghersi-Egea, 2000
) may be a factor differentiating the choroidal and cerebrovascular endothelia in their ability to synthesize CCL2.
Interestingly, both in sham-injured rats and in animals subjected to TBI, a distinct CCL2-positive staining of the apical surface of choroidal epithelium was observed. This finding was consistent with the results from in vitro
experiments, in which a constitutive, predominantly apical, secretion of CCL2 was observed in epithelial monolayers. Since CCL2 has the ability to bind to glycosaminoglycans (Kuschert et al, 1999
), it is possible that the apical localization of CCL2 was associated with the binding of this chemokine to glycosaminoglycans expressed on the apical surface of choroidal epithelium. In the ipsilateral CP, the intense Golgi complex-associated staining for CCL2 was observed. This pattern of CCL2-positive staining was found in clusters of epithelial cells scattered through the choroidal tissue, suggesting an uneven response to proinflammatory mediators and/or varying ability to produce the chemokine by individual epithelial cells. This likely resulted in uneven distribution of the chemokine gradients across the CP, which may explain our electron microscopic observations that monocytes accumulated only in certain areas of the ipsilateral CP.
The experiments involving the epithelial monolayers showed that CCL2 is secreted across both the apical and basolateral membranes of choroidal epithelium. This finding is in line with the previous studies, in which bidirectional secretion of chemokines by intestinal epithelia has been demonstrated and found to be necessary for leukocyte migration across this epithelial barrier (McCormick et al, 1995
). In our in vitro
studies, the possible leak and/or transcellular transfer of CCL2, which could affect our estimates of polarity of chemokine secretion by epithelial monolayers, was also evaluated. The results showed low paracellular permeability of control and stimulated monolayers, and suggested that neither IL-1β
nor CCL2 enhances a leak and/or transcellular transfer of the chemokine. These results validate our estimates of polarity of chemokine secretion by the choroidal epithelium.
The lack of changes in paracellular permeability of epithelial monolayers after exposure to CCL2 contrasts with increased permeability of the blood–brain barrier observed in response to this chemokine under both in vivo
and in vitro
conditions (Stamatovic et al, 2005
). These discrepant responses of the two barriers to CCL2 may be related to different levels of expression of CCR2 at the blood–brain barrier versus BCSFB, differences in the concentrations of CCL2 used, and/or differences in signal transduction associated with CCL2 binding to its cognate receptor.
The above-discussed features of choroidal epithelium, such as the ability to synthesize CCL2 in response to injury and bidirectional secretion of this chemokine across the apical and basolateral membranes of choroidal epithelial cells, strongly suggest that the BCSFB has a role in posttraumatic invasion of monocytes. This idea is further supported by our electron microscopic analysis of choroidal tissue. Similar to the movement of neutrophils (Szmydynger-Chodobska et al, 2009
), the migration of monocytes across the BCSFB appeared to involve the paracellular pathway. The movement of monocytes along the paracellular pathway was frequently associated with the widening of space between invading inflammatory cells and the adjacent epithelial cells (see ), a phenomenon not observed for migrating neutrophils (Szmydynger-Chodobska et al, 2009
). Interestingly, monocytes were sometimes found to invade the ipsilateral CP in tandem with neutrophils (see ). The synergistic interactions between monocyte and neutrophil chemoattractants (Gouwy et al, 2004
) could have a role in the movement of these two types of inflammatory cells together across the BCSFB.
Presently, it is not well defined how monocytes, or other inflammatory cells, invade the brain parenchyma after crossing the BCSFB. Also, little is known about the possible expression of cell adhesion molecules at ependymal and pial/glial linings bordering the CSF space. Monocytes have the ability to produce a variety of matrix metalloproteinases (Newby, 2008
), and the activation of these metalloproteinases have been shown to be a critical factor in the recruitment of inflammatory cells to the brain tissue (Toft-Hansen et al, 2006
). Carried by the bulk flow of CSF, monocytes may passage from the lateral cerebral ventricle to the cistern of velum interpositum located above the third cerebral ventricle, and may also enter the subarachnoid CSF space near the injury site. Our previous studies (Chodobski et al, 2003
) suggest that these parts of the brain CSF space have an important role in influx of peripheral inflammatory cells into the brain parenchyma. The investigations of experimental autoimmune encephalomyelitis in rodents, an animal model of multiple sclerosis, have also shown that peripheral inflammatory cells, such as T cells, may enter the neural tissue from the CSF by moving along the perivascular, Virchow-Robin, space (Bartholomäus et al, 2009
). Our preliminary observations (unpublished data) suggest that after injury, monocytes may use the same route to invade the brain parenchyma from the CSF.