Limits...
Possible association between expression of chemokine receptor-2 (CCR2) and amyotrophic lateral sclerosis (ALS) patients of North India.

Gupta PK, Prabhakar S, Sharma NK, Anand A - PLoS ONE (2012)

Bottom Line: Flow Cytometry revealed significantly reduced CCR2 expressing PBMCs in the ALS patients.We also found a significant decline in number of CCR2 expressing PBMCs in limb onset ALS when compared to bulbar onset ALS.CCR2 mRNA expression was found to be decreased among limb ALS patients as compared to bulbar onset ALS.

View Article: PubMed Central - PubMed

Affiliation: Neuroscience Research Laboratory, Department of Neurology, Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh, India.

ABSTRACT

Background and objectives: We earlier reported elevated chemokine ligand-2 (CCL2) in Indian amyotrophic lateral sclerosis (ALS) patients. We now analysed chemokine receptor-2 (CCR2), the receptor of CCL2, in these ALS patients.

Methods: Indian sporadic ALS patients (n=50) were included on the basis of El Escorial criteria. Percentage (%) of CCR2 expressing peripheral blood mononuclear cells (PBMCs) was evaluated using Flow Cytometry. Real Time Polymerase Chain Reaction (PCR) was used to quantitate CCR2 mRNA expression in PBMCs. Normal controls (n = 40) were also included for comparison.

Results: Flow Cytometry revealed significantly reduced CCR2 expressing PBMCs in the ALS patients. We also found a significant decline in number of CCR2 expressing PBMCs in limb onset ALS when compared to bulbar onset ALS. PBMCs from ALS patients showed substantial down-regulation of CCR2 mRNA. CCR2 mRNA expression was found to be decreased among limb ALS patients as compared to bulbar onset ALS. Further, the count of CCR2+ PBMCs and CCR2 mRNA transcript in PBMCs was significantly lower in severe and moderate ALS as compared to ALS patients with mild impairments.

Conclusions: Downregulation of PBMCs CCR2 may indicate its etio-pathological relevance in ALS pathogenesis. Reduced PBMCs CCR2 may result in decreased infiltration of leukocytes at the site of degeneration as a compensatory response to ALS. CCR2 levels measurements in hematopoietic stem cells and estimation of comparative PBMCs count among ALS, disease controls and normal controls can unveil its direct neuroprotective role. However, the conclusions are restricted by the absence of neurological/non-neurological disease controls in the study.

Show MeSH

Related in: MedlinePlus

Percentage (%) of CCR2+PBMCs in ALS patients and normal control subjects as measured by Flow Cytometry.(A) Dot plot showing side and forward scatter analysis of purified unlabeled PBMCs (large combined gate) from a normal control. PBMCs consists of two distinct populations namely lymphocytes and monocytes. Approximate lymphocytes and monocytes populations are indicated as smaller gates. Events outside the PBMCs gate represent cell debris and granulocytes. Same gating has been used for PBMCs from each ALS and normal control sample. ∼10,000 events have been acquired in each experiment. x-axis represents population cell size in forward scatter (FSC) and y-axis denotes population cell granularity in side scatter (SSC). (B,C) Single parameter representative histogram of flow cytometric expression pattern of CCR2 on gated PBMCs is showing decreased number of CCR2 expressing PBMCs in ALS (9%; C) as compared to normal control (26%; B). Number of cells is represented along y-axis and blue APC fluorescence along x-axis. Appropriate unlabeled PBMCs were used to set marker in histogram and measure background fluorescence. (D) Box plot compares CCR2 expressing PBMCs between ALS and normal subjects. Boxes include values from first quartile (25th percentile) to third quartile (75th percentile). Lower and upper error bar refers to 10th and 90th percentile respectively. The thick horizontal line in the box represents median for each dataset. Outliers and extreme values are shown in circles and asterisk respectively. Nonparametric Mann Whitney U test indicates significant difference betweesn the given conditions (p<0.05). ALS, amyotrophic lateral sclerosis; APC, allophycocyanin; CCR2, chemokine receptor 2; PBMCs, peripheral blood mononuclear cells.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3369904&req=5

pone-0038382-g001: Percentage (%) of CCR2+PBMCs in ALS patients and normal control subjects as measured by Flow Cytometry.(A) Dot plot showing side and forward scatter analysis of purified unlabeled PBMCs (large combined gate) from a normal control. PBMCs consists of two distinct populations namely lymphocytes and monocytes. Approximate lymphocytes and monocytes populations are indicated as smaller gates. Events outside the PBMCs gate represent cell debris and granulocytes. Same gating has been used for PBMCs from each ALS and normal control sample. ∼10,000 events have been acquired in each experiment. x-axis represents population cell size in forward scatter (FSC) and y-axis denotes population cell granularity in side scatter (SSC). (B,C) Single parameter representative histogram of flow cytometric expression pattern of CCR2 on gated PBMCs is showing decreased number of CCR2 expressing PBMCs in ALS (9%; C) as compared to normal control (26%; B). Number of cells is represented along y-axis and blue APC fluorescence along x-axis. Appropriate unlabeled PBMCs were used to set marker in histogram and measure background fluorescence. (D) Box plot compares CCR2 expressing PBMCs between ALS and normal subjects. Boxes include values from first quartile (25th percentile) to third quartile (75th percentile). Lower and upper error bar refers to 10th and 90th percentile respectively. The thick horizontal line in the box represents median for each dataset. Outliers and extreme values are shown in circles and asterisk respectively. Nonparametric Mann Whitney U test indicates significant difference betweesn the given conditions (p<0.05). ALS, amyotrophic lateral sclerosis; APC, allophycocyanin; CCR2, chemokine receptor 2; PBMCs, peripheral blood mononuclear cells.

Mentions: Flow Cytometry analysis of PBMCs of ALS and normal controls indicates a significant decrease in proportion of CCR2 expressing PBMCs in ALS patients than normal controls (Figure 1A–1D; p = 0.0001). CCR2 expressing PBMCs were found to be lower in severe ALS than ALS patients with mild and moderate neurological impairment (Figure 2A; p = 0.006 and p = 0.032 respectively). No such difference was observed in ALS patients with varying duration (Figure 2B; p>0.05). Reduced CCR2+ PBMCs was observed in bulbar and limb variants of ALS as compared to control group (Figure 2C; p = 0.005 and p = 0.0001 respectively). Moreover, CCR2+ PBMCs were found to be lower in limb onset ALS than bulbar onset ALS patients (Figure 2C; p = 0.048). In order to examine any possible association with hypoxia the CCR2 levels were also analysed in ALS patients with respiratory dysfunction and those without respiratory dysfunction, however, no difference in CCR2 was been observed between these ALS groups (Figure 2D; p>0.05).


Possible association between expression of chemokine receptor-2 (CCR2) and amyotrophic lateral sclerosis (ALS) patients of North India.

Gupta PK, Prabhakar S, Sharma NK, Anand A - PLoS ONE (2012)

Percentage (%) of CCR2+PBMCs in ALS patients and normal control subjects as measured by Flow Cytometry.(A) Dot plot showing side and forward scatter analysis of purified unlabeled PBMCs (large combined gate) from a normal control. PBMCs consists of two distinct populations namely lymphocytes and monocytes. Approximate lymphocytes and monocytes populations are indicated as smaller gates. Events outside the PBMCs gate represent cell debris and granulocytes. Same gating has been used for PBMCs from each ALS and normal control sample. ∼10,000 events have been acquired in each experiment. x-axis represents population cell size in forward scatter (FSC) and y-axis denotes population cell granularity in side scatter (SSC). (B,C) Single parameter representative histogram of flow cytometric expression pattern of CCR2 on gated PBMCs is showing decreased number of CCR2 expressing PBMCs in ALS (9%; C) as compared to normal control (26%; B). Number of cells is represented along y-axis and blue APC fluorescence along x-axis. Appropriate unlabeled PBMCs were used to set marker in histogram and measure background fluorescence. (D) Box plot compares CCR2 expressing PBMCs between ALS and normal subjects. Boxes include values from first quartile (25th percentile) to third quartile (75th percentile). Lower and upper error bar refers to 10th and 90th percentile respectively. The thick horizontal line in the box represents median for each dataset. Outliers and extreme values are shown in circles and asterisk respectively. Nonparametric Mann Whitney U test indicates significant difference betweesn the given conditions (p<0.05). ALS, amyotrophic lateral sclerosis; APC, allophycocyanin; CCR2, chemokine receptor 2; PBMCs, peripheral blood mononuclear cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3369904&req=5

pone-0038382-g001: Percentage (%) of CCR2+PBMCs in ALS patients and normal control subjects as measured by Flow Cytometry.(A) Dot plot showing side and forward scatter analysis of purified unlabeled PBMCs (large combined gate) from a normal control. PBMCs consists of two distinct populations namely lymphocytes and monocytes. Approximate lymphocytes and monocytes populations are indicated as smaller gates. Events outside the PBMCs gate represent cell debris and granulocytes. Same gating has been used for PBMCs from each ALS and normal control sample. ∼10,000 events have been acquired in each experiment. x-axis represents population cell size in forward scatter (FSC) and y-axis denotes population cell granularity in side scatter (SSC). (B,C) Single parameter representative histogram of flow cytometric expression pattern of CCR2 on gated PBMCs is showing decreased number of CCR2 expressing PBMCs in ALS (9%; C) as compared to normal control (26%; B). Number of cells is represented along y-axis and blue APC fluorescence along x-axis. Appropriate unlabeled PBMCs were used to set marker in histogram and measure background fluorescence. (D) Box plot compares CCR2 expressing PBMCs between ALS and normal subjects. Boxes include values from first quartile (25th percentile) to third quartile (75th percentile). Lower and upper error bar refers to 10th and 90th percentile respectively. The thick horizontal line in the box represents median for each dataset. Outliers and extreme values are shown in circles and asterisk respectively. Nonparametric Mann Whitney U test indicates significant difference betweesn the given conditions (p<0.05). ALS, amyotrophic lateral sclerosis; APC, allophycocyanin; CCR2, chemokine receptor 2; PBMCs, peripheral blood mononuclear cells.
Mentions: Flow Cytometry analysis of PBMCs of ALS and normal controls indicates a significant decrease in proportion of CCR2 expressing PBMCs in ALS patients than normal controls (Figure 1A–1D; p = 0.0001). CCR2 expressing PBMCs were found to be lower in severe ALS than ALS patients with mild and moderate neurological impairment (Figure 2A; p = 0.006 and p = 0.032 respectively). No such difference was observed in ALS patients with varying duration (Figure 2B; p>0.05). Reduced CCR2+ PBMCs was observed in bulbar and limb variants of ALS as compared to control group (Figure 2C; p = 0.005 and p = 0.0001 respectively). Moreover, CCR2+ PBMCs were found to be lower in limb onset ALS than bulbar onset ALS patients (Figure 2C; p = 0.048). In order to examine any possible association with hypoxia the CCR2 levels were also analysed in ALS patients with respiratory dysfunction and those without respiratory dysfunction, however, no difference in CCR2 was been observed between these ALS groups (Figure 2D; p>0.05).

Bottom Line: Flow Cytometry revealed significantly reduced CCR2 expressing PBMCs in the ALS patients.We also found a significant decline in number of CCR2 expressing PBMCs in limb onset ALS when compared to bulbar onset ALS.CCR2 mRNA expression was found to be decreased among limb ALS patients as compared to bulbar onset ALS.

View Article: PubMed Central - PubMed

Affiliation: Neuroscience Research Laboratory, Department of Neurology, Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh, India.

ABSTRACT

Background and objectives: We earlier reported elevated chemokine ligand-2 (CCL2) in Indian amyotrophic lateral sclerosis (ALS) patients. We now analysed chemokine receptor-2 (CCR2), the receptor of CCL2, in these ALS patients.

Methods: Indian sporadic ALS patients (n=50) were included on the basis of El Escorial criteria. Percentage (%) of CCR2 expressing peripheral blood mononuclear cells (PBMCs) was evaluated using Flow Cytometry. Real Time Polymerase Chain Reaction (PCR) was used to quantitate CCR2 mRNA expression in PBMCs. Normal controls (n = 40) were also included for comparison.

Results: Flow Cytometry revealed significantly reduced CCR2 expressing PBMCs in the ALS patients. We also found a significant decline in number of CCR2 expressing PBMCs in limb onset ALS when compared to bulbar onset ALS. PBMCs from ALS patients showed substantial down-regulation of CCR2 mRNA. CCR2 mRNA expression was found to be decreased among limb ALS patients as compared to bulbar onset ALS. Further, the count of CCR2+ PBMCs and CCR2 mRNA transcript in PBMCs was significantly lower in severe and moderate ALS as compared to ALS patients with mild impairments.

Conclusions: Downregulation of PBMCs CCR2 may indicate its etio-pathological relevance in ALS pathogenesis. Reduced PBMCs CCR2 may result in decreased infiltration of leukocytes at the site of degeneration as a compensatory response to ALS. CCR2 levels measurements in hematopoietic stem cells and estimation of comparative PBMCs count among ALS, disease controls and normal controls can unveil its direct neuroprotective role. However, the conclusions are restricted by the absence of neurological/non-neurological disease controls in the study.

Show MeSH
Related in: MedlinePlus