Limits...
Rescue from acute neuroinflammation by pharmacological chemokine-mediated deviation of leukocytes.

Berghmans N, Heremans H, Li S, Martens E, Matthys P, Sorokin L, Van Damme J, Opdenakker G - J Neuroinflammation (2012)

Bottom Line: However, in view of unsatisfactory results and severe side effects, complementary therapies are needed.We have examined the effect of chlorite-oxidized oxyamylose (COAM), a potent antiviral polycarboxylic acid on EAE.These results demonstrate novel actions of COAM as an anti-inflammatory agent with beneficial effects on EAE through cell deviation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Rega Institute for Medical Research, Department of Microbiology and Immunology, University of Leuven, Leuven, Belgium.

ABSTRACT

Background: Neutrophil influx is an important sign of hyperacute neuroinflammation, whereas the entry of activated lymphocytes into the brain parenchyma is a hallmark of chronic inflammatory processes, as observed in multiple sclerosis (MS) and its animal models of experimental autoimmune encephalomyelitis (EAE). Clinically approved or experimental therapies for neuroinflammation act by blocking leukocyte penetration of the blood brain barrier. However, in view of unsatisfactory results and severe side effects, complementary therapies are needed. We have examined the effect of chlorite-oxidized oxyamylose (COAM), a potent antiviral polycarboxylic acid on EAE.

Methods: EAE was induced in SJL/J mice by immunization with spinal cord homogenate (SCH) or in IFN-γ-deficient BALB/c (KO) mice with myelin oligodendrocyte glycoprotein peptide (MOG₃₅₋₅₅). Mice were treated intraperitoneally (i.p.) with COAM or saline at different time points after immunization. Clinical disease and histopathology were compared between both groups. IFN expression was analyzed in COAM-treated MEF cell cultures and in sera and peritoneal fluids of COAM-treated animals by quantitative PCR, ELISA and a bioassay on L929 cells. Populations of immune cell subsets in the periphery and the central nervous system (CNS) were quantified at different stages of disease development by flow cytometry and differential cell count analysis. Expression levels of selected chemokine genes in the CNS were determined by quantitative PCR.

Results: We discovered that COAM (2 mg i.p. per mouse on days 0 and 7) protects significantly against hyperacute SCH-induced EAE in SJL/J mice and MOG₃₅₋₅₅-induced EAE in IFN-γ KO mice. COAM deviated leukocyte trafficking from the CNS into the periphery. In the CNS, COAM reduced four-fold the expression levels of the neutrophil CXC chemokines KC/CXCL1 and MIP-2/CXCL2. Whereas the effects of COAM on circulating blood and splenic leukocytes were limited, significant alterations were observed at the COAM injection site.

Conclusions: These results demonstrate novel actions of COAM as an anti-inflammatory agent with beneficial effects on EAE through cell deviation. Sequestration of leukocytes in the non-CNS periphery or draining of leukocytes out of the CNS with the use of the chemokine system may thus complement existing treatment options for acute and chronic neuroinflammatory diseases.

Show MeSH

Related in: MedlinePlus

COAM induces peritoneal alterations in leukocyte populations in animals with hyperacute EAE. Peritoneal cells were collected on days 0, 5, 9 and 14, 15 or 16 post immunization with SCH in SJL/J mice. (A) The percentages (morphologic examinations) of peritoneal macrophages and (B) neutrophils in the peritoneal exudates were compared between naive mice and EAE-induced mice, treated i.p. with saline or with COAM. Data are compiled from three experiments; bars represent averages ± SEM of three to nine mice. Statistical significance was determined using the Mann Whitney test. (C-E) Flow cytometric analysis of leukocytes in the peritoneal cavity of COAM-treated versus saline-treated EAE mice. Cells were stained for the presence of CD11b, Gr-1, CD4 and CD8. (C) Example of FACS analysis with indication of gating of double positive cells. The numbers within the gates indicate the percentages of Gr-1-positive cells present in the whole sorted peritoneal lavages. One representative analysis out of nine is shown. (D-E) The data of three experiments were pooled and the percentages of CD4+ and CD8+ cells on each of the examined days are shown. Bars represent averages ± SEM, the individual numbers of animals (n) for each condition are indicated below each of the histograms. Asterisks indicate * P <0.05; ** P <0.01 for comparison with naive mice (Mann Whitney test). Pairwise comparisons (P values) between saline- (control) and COAM-treated hyperacute EAE mice are indicated on top of the histograms. COAM, chlorite-oxidized oxyamylose; EAE, experimental autoimmune encephalomyelitis; SCH, spinal cord homogenate; SEM, standard error of the mean.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3526473&req=5

Figure 8: COAM induces peritoneal alterations in leukocyte populations in animals with hyperacute EAE. Peritoneal cells were collected on days 0, 5, 9 and 14, 15 or 16 post immunization with SCH in SJL/J mice. (A) The percentages (morphologic examinations) of peritoneal macrophages and (B) neutrophils in the peritoneal exudates were compared between naive mice and EAE-induced mice, treated i.p. with saline or with COAM. Data are compiled from three experiments; bars represent averages ± SEM of three to nine mice. Statistical significance was determined using the Mann Whitney test. (C-E) Flow cytometric analysis of leukocytes in the peritoneal cavity of COAM-treated versus saline-treated EAE mice. Cells were stained for the presence of CD11b, Gr-1, CD4 and CD8. (C) Example of FACS analysis with indication of gating of double positive cells. The numbers within the gates indicate the percentages of Gr-1-positive cells present in the whole sorted peritoneal lavages. One representative analysis out of nine is shown. (D-E) The data of three experiments were pooled and the percentages of CD4+ and CD8+ cells on each of the examined days are shown. Bars represent averages ± SEM, the individual numbers of animals (n) for each condition are indicated below each of the histograms. Asterisks indicate * P <0.05; ** P <0.01 for comparison with naive mice (Mann Whitney test). Pairwise comparisons (P values) between saline- (control) and COAM-treated hyperacute EAE mice are indicated on top of the histograms. COAM, chlorite-oxidized oxyamylose; EAE, experimental autoimmune encephalomyelitis; SCH, spinal cord homogenate; SEM, standard error of the mean.

Mentions: Finally, the effects of COAM on leukocyte populations at the injection site were studied. Mice were induced for hyperacute EAE, treated i.p. with 2 mg COAM on days 0 and 7 after immunization with SCH and sacrificed at different time points after immunization. Peritoneal lavage fluids were collected for morphologic examination on day 5, that is, the preclinical phase; day 9, that is, shortly before the expected onset of clinical symptoms of hyperacute EAE; and on days 14, 15 or 16, which corresponded with the peak of disease. Diseased mice were selected so that their mean clinical scores were comparable between COAM- and saline-treated groups. Peritoneal cells were cytospun onto slides, stained with Hemacolor, and leukocyte subsets were counted. Naive and saline-treated hyperacute EAE mice on day 5 after immunization yielded 1.47 to 1.93 x 106 cells/ml, comprising high numbers of macrophages (65.13% and 54.89% of total cells, respectively) and a small proportion of neutrophils (0.4% and 0.58% for naive and saline-treated hyperacute EAE mice, respectively). COAM induced a peritoneal leukocyte influx nine days after EAE induction (P <0.05 versus saline control). Macrophages (Figure 8A) and particularly neutrophils (Figure 8B) were found to be significantly increased in COAM-treated mice. These data are in line with previous findings in a virus infection model[19]. These effects of COAM were detectable in hyperacute EAE animals even on day 14, one week after the last COAM injection on day 7. FACS analyses reinforced these cytospin data. The peritoneal fluid of COAM-treated mice contained an excessive proportion of CD11b+Gr-1+ cells compared with saline-treated mice on each of the examined days (Figure 8C). When the numbers of CD11b+Gr-1+ cells from three independent experiments were expressed as percentages of total cell numbers collected per mouse, peritoneal fluids of hyperacute EAE-induced COAM-treated mice were shown to contain approximately four times higher numbers of neutrophils than those of saline-treated hyperacute EAE mice. For the analysis of defined lymphocyte subtypes in animals treated with COAM, peritoneal fluid cells were stained with anti-CD4-PE and anti-CD8-FITC antibodies. COAM injection in immunized mice significantly increased the percentage of peritoneal CD4+ and CD8+ cell numbers as compared to saline-treated EAE mice (Figure 8D and E). With the use of in vivo imaging, we could demonstrate that i.p. injected labeled COAM remained mainly in the peritoneal cavity for prolonged time intervals, whereas it disappeared quickly from the mice after intravenous injection (data not shown).


Rescue from acute neuroinflammation by pharmacological chemokine-mediated deviation of leukocytes.

Berghmans N, Heremans H, Li S, Martens E, Matthys P, Sorokin L, Van Damme J, Opdenakker G - J Neuroinflammation (2012)

COAM induces peritoneal alterations in leukocyte populations in animals with hyperacute EAE. Peritoneal cells were collected on days 0, 5, 9 and 14, 15 or 16 post immunization with SCH in SJL/J mice. (A) The percentages (morphologic examinations) of peritoneal macrophages and (B) neutrophils in the peritoneal exudates were compared between naive mice and EAE-induced mice, treated i.p. with saline or with COAM. Data are compiled from three experiments; bars represent averages ± SEM of three to nine mice. Statistical significance was determined using the Mann Whitney test. (C-E) Flow cytometric analysis of leukocytes in the peritoneal cavity of COAM-treated versus saline-treated EAE mice. Cells were stained for the presence of CD11b, Gr-1, CD4 and CD8. (C) Example of FACS analysis with indication of gating of double positive cells. The numbers within the gates indicate the percentages of Gr-1-positive cells present in the whole sorted peritoneal lavages. One representative analysis out of nine is shown. (D-E) The data of three experiments were pooled and the percentages of CD4+ and CD8+ cells on each of the examined days are shown. Bars represent averages ± SEM, the individual numbers of animals (n) for each condition are indicated below each of the histograms. Asterisks indicate * P <0.05; ** P <0.01 for comparison with naive mice (Mann Whitney test). Pairwise comparisons (P values) between saline- (control) and COAM-treated hyperacute EAE mice are indicated on top of the histograms. COAM, chlorite-oxidized oxyamylose; EAE, experimental autoimmune encephalomyelitis; SCH, spinal cord homogenate; SEM, standard error of the mean.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3526473&req=5

Figure 8: COAM induces peritoneal alterations in leukocyte populations in animals with hyperacute EAE. Peritoneal cells were collected on days 0, 5, 9 and 14, 15 or 16 post immunization with SCH in SJL/J mice. (A) The percentages (morphologic examinations) of peritoneal macrophages and (B) neutrophils in the peritoneal exudates were compared between naive mice and EAE-induced mice, treated i.p. with saline or with COAM. Data are compiled from three experiments; bars represent averages ± SEM of three to nine mice. Statistical significance was determined using the Mann Whitney test. (C-E) Flow cytometric analysis of leukocytes in the peritoneal cavity of COAM-treated versus saline-treated EAE mice. Cells were stained for the presence of CD11b, Gr-1, CD4 and CD8. (C) Example of FACS analysis with indication of gating of double positive cells. The numbers within the gates indicate the percentages of Gr-1-positive cells present in the whole sorted peritoneal lavages. One representative analysis out of nine is shown. (D-E) The data of three experiments were pooled and the percentages of CD4+ and CD8+ cells on each of the examined days are shown. Bars represent averages ± SEM, the individual numbers of animals (n) for each condition are indicated below each of the histograms. Asterisks indicate * P <0.05; ** P <0.01 for comparison with naive mice (Mann Whitney test). Pairwise comparisons (P values) between saline- (control) and COAM-treated hyperacute EAE mice are indicated on top of the histograms. COAM, chlorite-oxidized oxyamylose; EAE, experimental autoimmune encephalomyelitis; SCH, spinal cord homogenate; SEM, standard error of the mean.
Mentions: Finally, the effects of COAM on leukocyte populations at the injection site were studied. Mice were induced for hyperacute EAE, treated i.p. with 2 mg COAM on days 0 and 7 after immunization with SCH and sacrificed at different time points after immunization. Peritoneal lavage fluids were collected for morphologic examination on day 5, that is, the preclinical phase; day 9, that is, shortly before the expected onset of clinical symptoms of hyperacute EAE; and on days 14, 15 or 16, which corresponded with the peak of disease. Diseased mice were selected so that their mean clinical scores were comparable between COAM- and saline-treated groups. Peritoneal cells were cytospun onto slides, stained with Hemacolor, and leukocyte subsets were counted. Naive and saline-treated hyperacute EAE mice on day 5 after immunization yielded 1.47 to 1.93 x 106 cells/ml, comprising high numbers of macrophages (65.13% and 54.89% of total cells, respectively) and a small proportion of neutrophils (0.4% and 0.58% for naive and saline-treated hyperacute EAE mice, respectively). COAM induced a peritoneal leukocyte influx nine days after EAE induction (P <0.05 versus saline control). Macrophages (Figure 8A) and particularly neutrophils (Figure 8B) were found to be significantly increased in COAM-treated mice. These data are in line with previous findings in a virus infection model[19]. These effects of COAM were detectable in hyperacute EAE animals even on day 14, one week after the last COAM injection on day 7. FACS analyses reinforced these cytospin data. The peritoneal fluid of COAM-treated mice contained an excessive proportion of CD11b+Gr-1+ cells compared with saline-treated mice on each of the examined days (Figure 8C). When the numbers of CD11b+Gr-1+ cells from three independent experiments were expressed as percentages of total cell numbers collected per mouse, peritoneal fluids of hyperacute EAE-induced COAM-treated mice were shown to contain approximately four times higher numbers of neutrophils than those of saline-treated hyperacute EAE mice. For the analysis of defined lymphocyte subtypes in animals treated with COAM, peritoneal fluid cells were stained with anti-CD4-PE and anti-CD8-FITC antibodies. COAM injection in immunized mice significantly increased the percentage of peritoneal CD4+ and CD8+ cell numbers as compared to saline-treated EAE mice (Figure 8D and E). With the use of in vivo imaging, we could demonstrate that i.p. injected labeled COAM remained mainly in the peritoneal cavity for prolonged time intervals, whereas it disappeared quickly from the mice after intravenous injection (data not shown).

Bottom Line: However, in view of unsatisfactory results and severe side effects, complementary therapies are needed.We have examined the effect of chlorite-oxidized oxyamylose (COAM), a potent antiviral polycarboxylic acid on EAE.These results demonstrate novel actions of COAM as an anti-inflammatory agent with beneficial effects on EAE through cell deviation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Rega Institute for Medical Research, Department of Microbiology and Immunology, University of Leuven, Leuven, Belgium.

ABSTRACT

Background: Neutrophil influx is an important sign of hyperacute neuroinflammation, whereas the entry of activated lymphocytes into the brain parenchyma is a hallmark of chronic inflammatory processes, as observed in multiple sclerosis (MS) and its animal models of experimental autoimmune encephalomyelitis (EAE). Clinically approved or experimental therapies for neuroinflammation act by blocking leukocyte penetration of the blood brain barrier. However, in view of unsatisfactory results and severe side effects, complementary therapies are needed. We have examined the effect of chlorite-oxidized oxyamylose (COAM), a potent antiviral polycarboxylic acid on EAE.

Methods: EAE was induced in SJL/J mice by immunization with spinal cord homogenate (SCH) or in IFN-γ-deficient BALB/c (KO) mice with myelin oligodendrocyte glycoprotein peptide (MOG₃₅₋₅₅). Mice were treated intraperitoneally (i.p.) with COAM or saline at different time points after immunization. Clinical disease and histopathology were compared between both groups. IFN expression was analyzed in COAM-treated MEF cell cultures and in sera and peritoneal fluids of COAM-treated animals by quantitative PCR, ELISA and a bioassay on L929 cells. Populations of immune cell subsets in the periphery and the central nervous system (CNS) were quantified at different stages of disease development by flow cytometry and differential cell count analysis. Expression levels of selected chemokine genes in the CNS were determined by quantitative PCR.

Results: We discovered that COAM (2 mg i.p. per mouse on days 0 and 7) protects significantly against hyperacute SCH-induced EAE in SJL/J mice and MOG₃₅₋₅₅-induced EAE in IFN-γ KO mice. COAM deviated leukocyte trafficking from the CNS into the periphery. In the CNS, COAM reduced four-fold the expression levels of the neutrophil CXC chemokines KC/CXCL1 and MIP-2/CXCL2. Whereas the effects of COAM on circulating blood and splenic leukocytes were limited, significant alterations were observed at the COAM injection site.

Conclusions: These results demonstrate novel actions of COAM as an anti-inflammatory agent with beneficial effects on EAE through cell deviation. Sequestration of leukocytes in the non-CNS periphery or draining of leukocytes out of the CNS with the use of the chemokine system may thus complement existing treatment options for acute and chronic neuroinflammatory diseases.

Show MeSH
Related in: MedlinePlus