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Cerebrospinal fluid dendritic cells infiltrate the brain parenchyma and target the cervical lymph nodes under neuroinflammatory conditions.

Hatterer E, Touret M, Belin MF, Honnorat J, Nataf S - PLoS ONE (2008)

Bottom Line: This migratory behavior was accompanied by an accentuation of EAE clinical signs and an increased systemic antibody response against myelin oligodendrocyte glycoprotein, a major immunogenic myelin antigen.Altogether, these results indicate that CSF-circulating DCs are able to both survey the inflamed brain and to reach the cervical lymph nodes.In EAE and maybe multiple sclerosis, CSF-circulating DCs may thus support the immune responses that develop within and outside the inflamed CNS.

View Article: PubMed Central - PubMed

Affiliation: INSERM, U842, Lyon, France.

ABSTRACT

Background: In many neuroinflammatory diseases, dendritic cells (DCs) accumulate in several compartments of the central nervous system (CNS), including the cerebrospinal fluid (CSF). Myeloid DCs invading the inflamed CNS are thus thought to play a major role in the initiation and perpetuation of CNS-targeted autoimmune responses. We previously reported that, in normal rats, DCs injected intra-CSF migrated outside the CNS and reached the B-cell zone of cervical lymph nodes. However, there is yet no information on the migratory behavior of CSF-circulating DCs under neuroinflammatory conditions.

Methodology/principal findings: To address this issue, we performed in vivo transfer experiments in rats suffering from experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. EAE or control rats were injected intra-CSF with bone marrow-derived myeloid DCs labeled with the fluorescent marker carboxyfluorescein diacetate succinimidyl ester (CFSE). In parallel experiments, fluorescent microspheres were injected intra-CSF to EAE rats in order to track endogenous antigen-presenting cells (APCs). Animals were then sacrificed on day 1 or 8 post-injection and their brain and peripheral lymph nodes were assessed for the presence of microspheres(+) APCs or CFSE(+) DCs by immunohistology and/or FACS analysis. Data showed that in EAE rats, DCs injected intra-CSF substantially infiltrated several compartments of the inflamed CNS, including the periventricular demyelinating lesions. We also found that in EAE rats, as compared to controls, a larger number of intra-CSF injected DCs reached the cervical lymph nodes. This migratory behavior was accompanied by an accentuation of EAE clinical signs and an increased systemic antibody response against myelin oligodendrocyte glycoprotein, a major immunogenic myelin antigen.

Conclusions/significance: Altogether, these results indicate that CSF-circulating DCs are able to both survey the inflamed brain and to reach the cervical lymph nodes. In EAE and maybe multiple sclerosis, CSF-circulating DCs may thus support the immune responses that develop within and outside the inflamed CNS.

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Related in: MedlinePlus

CSF-circulating DCs target the CLNs.In parallel experiments, DCs labeled with the cytoplasmic fluorescent marker CFSE were injected into the left lateral ventricle of control rats (n = 7) or EAE rats (n = 8) at the clinical peak of disease (day 12 post-immunization). Rats were then sacrificed on day 1 (d1) or 8 (d8) following injections. The cervical lymph nodes (CLNs) and axillary lymph nodes (ALN) were assessed by FACS analysis for the presence of CFSE+ cells. The level of autofluorescence was established on cells obtained from the CLNs or ALNs of non-injected EAE rats (n = 4) or control rats (n = 3). A–D: In injected EAE rats sacrificed on day 1 post-injection (n = 4), we found that 2.26+/−0.11% CFSE+ cells could be detected in the CLNs as compared to 1.37+/−0.03% in the axillary lymph nodes (p = 0.0202, Mann and Whitney test)(A). However, in injected EAE rats sacrificed on day 8 post-injection (n = 4), the percentage of CFSE+ cells was not statistically different between the CLNs and the ALNs (B). Pannels C and D show representative dot plots obtained from the analysis of injected EAE rats sacrificed on day 1 post-injection. E–F: In the CLNs of injected EAE rats, a great majority of CFSE+ cells express MHC class II molecules, on day 1 post-injection (94.25+/−3.3%) or 8 post-injection (87.65+/−5.2%). A representative dot plot is shown in F. G–H: When comparing injected EAE rats to injected control rats, data showed that on day 1 post-injection, a greater pourcentage of CSFE+ cells was detectable in the CLNs of injected EAE rats as compared to injected control rats (2.26+/−0.11% vs 1.49+/−0.13% in injected EAE and injected control rats respectively, p = 0.0339, Mann and Whitney test)(G). This difference did not reach significance on day 8 post-injection (1.71+/−0.03% vs 1.66+/−0.13% in injected EAE and injected control rats respectively) (H). *: p<0.05, NS: not significant.
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pone-0003321-g008: CSF-circulating DCs target the CLNs.In parallel experiments, DCs labeled with the cytoplasmic fluorescent marker CFSE were injected into the left lateral ventricle of control rats (n = 7) or EAE rats (n = 8) at the clinical peak of disease (day 12 post-immunization). Rats were then sacrificed on day 1 (d1) or 8 (d8) following injections. The cervical lymph nodes (CLNs) and axillary lymph nodes (ALN) were assessed by FACS analysis for the presence of CFSE+ cells. The level of autofluorescence was established on cells obtained from the CLNs or ALNs of non-injected EAE rats (n = 4) or control rats (n = 3). A–D: In injected EAE rats sacrificed on day 1 post-injection (n = 4), we found that 2.26+/−0.11% CFSE+ cells could be detected in the CLNs as compared to 1.37+/−0.03% in the axillary lymph nodes (p = 0.0202, Mann and Whitney test)(A). However, in injected EAE rats sacrificed on day 8 post-injection (n = 4), the percentage of CFSE+ cells was not statistically different between the CLNs and the ALNs (B). Pannels C and D show representative dot plots obtained from the analysis of injected EAE rats sacrificed on day 1 post-injection. E–F: In the CLNs of injected EAE rats, a great majority of CFSE+ cells express MHC class II molecules, on day 1 post-injection (94.25+/−3.3%) or 8 post-injection (87.65+/−5.2%). A representative dot plot is shown in F. G–H: When comparing injected EAE rats to injected control rats, data showed that on day 1 post-injection, a greater pourcentage of CSFE+ cells was detectable in the CLNs of injected EAE rats as compared to injected control rats (2.26+/−0.11% vs 1.49+/−0.13% in injected EAE and injected control rats respectively, p = 0.0339, Mann and Whitney test)(G). This difference did not reach significance on day 8 post-injection (1.71+/−0.03% vs 1.66+/−0.13% in injected EAE and injected control rats respectively) (H). *: p<0.05, NS: not significant.

Mentions: To gain quantitative insights on the migration of CSF DCs toward the CLNs, a FACS analysis was performed on the lymph nodes of EAE rats (n = 8) or control rats (n = 7) injected intra-CSF with CFSE+ DCs (Fig 8). In injected EAE rats sacrificed on day 1 post-injection (n = 4), we found that 2.26+/−0,11% CFSE+ cells could be detected in the CLNs as compared to 1.37+/−0,03% in the axillary lymph nodes (p = 0.02, Mann and Whitney test)(Fig 8A, C–D). However, in injected EAE rats sacrificed on day 8 post-injection (n = 4), no statistical difference was observed between the CLNs and the axillary lymph nodes regarding the percentage of CFSE+ cells (Fig 8B). It has to be noticed that a dilution effect may have partly biased these results as cellularity increases in the CLNs during the course of EAE (data not shown). It is also noteworthy that in the CLNs, a majority of CFSE+ cells were MHC class II+ on day 1 or 8 post-injection (Fig 8E–F). This suggests that in the CLNs, the phenotype of injected cells is stable over time, at least regarding the expression of MHC class II molecules.


Cerebrospinal fluid dendritic cells infiltrate the brain parenchyma and target the cervical lymph nodes under neuroinflammatory conditions.

Hatterer E, Touret M, Belin MF, Honnorat J, Nataf S - PLoS ONE (2008)

CSF-circulating DCs target the CLNs.In parallel experiments, DCs labeled with the cytoplasmic fluorescent marker CFSE were injected into the left lateral ventricle of control rats (n = 7) or EAE rats (n = 8) at the clinical peak of disease (day 12 post-immunization). Rats were then sacrificed on day 1 (d1) or 8 (d8) following injections. The cervical lymph nodes (CLNs) and axillary lymph nodes (ALN) were assessed by FACS analysis for the presence of CFSE+ cells. The level of autofluorescence was established on cells obtained from the CLNs or ALNs of non-injected EAE rats (n = 4) or control rats (n = 3). A–D: In injected EAE rats sacrificed on day 1 post-injection (n = 4), we found that 2.26+/−0.11% CFSE+ cells could be detected in the CLNs as compared to 1.37+/−0.03% in the axillary lymph nodes (p = 0.0202, Mann and Whitney test)(A). However, in injected EAE rats sacrificed on day 8 post-injection (n = 4), the percentage of CFSE+ cells was not statistically different between the CLNs and the ALNs (B). Pannels C and D show representative dot plots obtained from the analysis of injected EAE rats sacrificed on day 1 post-injection. E–F: In the CLNs of injected EAE rats, a great majority of CFSE+ cells express MHC class II molecules, on day 1 post-injection (94.25+/−3.3%) or 8 post-injection (87.65+/−5.2%). A representative dot plot is shown in F. G–H: When comparing injected EAE rats to injected control rats, data showed that on day 1 post-injection, a greater pourcentage of CSFE+ cells was detectable in the CLNs of injected EAE rats as compared to injected control rats (2.26+/−0.11% vs 1.49+/−0.13% in injected EAE and injected control rats respectively, p = 0.0339, Mann and Whitney test)(G). This difference did not reach significance on day 8 post-injection (1.71+/−0.03% vs 1.66+/−0.13% in injected EAE and injected control rats respectively) (H). *: p<0.05, NS: not significant.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2552991&req=5

pone-0003321-g008: CSF-circulating DCs target the CLNs.In parallel experiments, DCs labeled with the cytoplasmic fluorescent marker CFSE were injected into the left lateral ventricle of control rats (n = 7) or EAE rats (n = 8) at the clinical peak of disease (day 12 post-immunization). Rats were then sacrificed on day 1 (d1) or 8 (d8) following injections. The cervical lymph nodes (CLNs) and axillary lymph nodes (ALN) were assessed by FACS analysis for the presence of CFSE+ cells. The level of autofluorescence was established on cells obtained from the CLNs or ALNs of non-injected EAE rats (n = 4) or control rats (n = 3). A–D: In injected EAE rats sacrificed on day 1 post-injection (n = 4), we found that 2.26+/−0.11% CFSE+ cells could be detected in the CLNs as compared to 1.37+/−0.03% in the axillary lymph nodes (p = 0.0202, Mann and Whitney test)(A). However, in injected EAE rats sacrificed on day 8 post-injection (n = 4), the percentage of CFSE+ cells was not statistically different between the CLNs and the ALNs (B). Pannels C and D show representative dot plots obtained from the analysis of injected EAE rats sacrificed on day 1 post-injection. E–F: In the CLNs of injected EAE rats, a great majority of CFSE+ cells express MHC class II molecules, on day 1 post-injection (94.25+/−3.3%) or 8 post-injection (87.65+/−5.2%). A representative dot plot is shown in F. G–H: When comparing injected EAE rats to injected control rats, data showed that on day 1 post-injection, a greater pourcentage of CSFE+ cells was detectable in the CLNs of injected EAE rats as compared to injected control rats (2.26+/−0.11% vs 1.49+/−0.13% in injected EAE and injected control rats respectively, p = 0.0339, Mann and Whitney test)(G). This difference did not reach significance on day 8 post-injection (1.71+/−0.03% vs 1.66+/−0.13% in injected EAE and injected control rats respectively) (H). *: p<0.05, NS: not significant.
Mentions: To gain quantitative insights on the migration of CSF DCs toward the CLNs, a FACS analysis was performed on the lymph nodes of EAE rats (n = 8) or control rats (n = 7) injected intra-CSF with CFSE+ DCs (Fig 8). In injected EAE rats sacrificed on day 1 post-injection (n = 4), we found that 2.26+/−0,11% CFSE+ cells could be detected in the CLNs as compared to 1.37+/−0,03% in the axillary lymph nodes (p = 0.02, Mann and Whitney test)(Fig 8A, C–D). However, in injected EAE rats sacrificed on day 8 post-injection (n = 4), no statistical difference was observed between the CLNs and the axillary lymph nodes regarding the percentage of CFSE+ cells (Fig 8B). It has to be noticed that a dilution effect may have partly biased these results as cellularity increases in the CLNs during the course of EAE (data not shown). It is also noteworthy that in the CLNs, a majority of CFSE+ cells were MHC class II+ on day 1 or 8 post-injection (Fig 8E–F). This suggests that in the CLNs, the phenotype of injected cells is stable over time, at least regarding the expression of MHC class II molecules.

Bottom Line: This migratory behavior was accompanied by an accentuation of EAE clinical signs and an increased systemic antibody response against myelin oligodendrocyte glycoprotein, a major immunogenic myelin antigen.Altogether, these results indicate that CSF-circulating DCs are able to both survey the inflamed brain and to reach the cervical lymph nodes.In EAE and maybe multiple sclerosis, CSF-circulating DCs may thus support the immune responses that develop within and outside the inflamed CNS.

View Article: PubMed Central - PubMed

Affiliation: INSERM, U842, Lyon, France.

ABSTRACT

Background: In many neuroinflammatory diseases, dendritic cells (DCs) accumulate in several compartments of the central nervous system (CNS), including the cerebrospinal fluid (CSF). Myeloid DCs invading the inflamed CNS are thus thought to play a major role in the initiation and perpetuation of CNS-targeted autoimmune responses. We previously reported that, in normal rats, DCs injected intra-CSF migrated outside the CNS and reached the B-cell zone of cervical lymph nodes. However, there is yet no information on the migratory behavior of CSF-circulating DCs under neuroinflammatory conditions.

Methodology/principal findings: To address this issue, we performed in vivo transfer experiments in rats suffering from experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. EAE or control rats were injected intra-CSF with bone marrow-derived myeloid DCs labeled with the fluorescent marker carboxyfluorescein diacetate succinimidyl ester (CFSE). In parallel experiments, fluorescent microspheres were injected intra-CSF to EAE rats in order to track endogenous antigen-presenting cells (APCs). Animals were then sacrificed on day 1 or 8 post-injection and their brain and peripheral lymph nodes were assessed for the presence of microspheres(+) APCs or CFSE(+) DCs by immunohistology and/or FACS analysis. Data showed that in EAE rats, DCs injected intra-CSF substantially infiltrated several compartments of the inflamed CNS, including the periventricular demyelinating lesions. We also found that in EAE rats, as compared to controls, a larger number of intra-CSF injected DCs reached the cervical lymph nodes. This migratory behavior was accompanied by an accentuation of EAE clinical signs and an increased systemic antibody response against myelin oligodendrocyte glycoprotein, a major immunogenic myelin antigen.

Conclusions/significance: Altogether, these results indicate that CSF-circulating DCs are able to both survey the inflamed brain and to reach the cervical lymph nodes. In EAE and maybe multiple sclerosis, CSF-circulating DCs may thus support the immune responses that develop within and outside the inflamed CNS.

Show MeSH
Related in: MedlinePlus