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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.

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Real Time PCR analysis of relative mRNA expression of CCR2 in PBMCs of subjects.(A) Agarose gel electrophoresis of Real Time PCR products of target gene CCR2 and endogenous control β-actin. Reactions were performed with mRNA isolated form PBMCs of ALS patient and normal control. (B) Representative amplification curves depicting increase in fluorescence of CCR2 and β-actin from cDNA of same sample. No increment in fluorescence of negative controls was observed. The x-axis indicates cycle number and the y-axis shows intensity of relative fluorescence in linear scale. Ct is threshold cycle where normalized fluorescent signal of SYBR green intersect with threshold line. (C) Melt curve profile of Real Time PCR products of CCR2 and β actin clearly indicates the absence of any non specific amplification. The x-axis indicates negative derivative of change in amount of fluorescence per unit change in temperature (dF/dT) and the y-axis represents temperature in Celsius (°C). (D) Bar diagram showing fold change in CCR2 mRNA expression PBMCs from ALS and normal subjects. Values are plotted as mean ± SE (Standard error). Data was analyzed by unpaired, independent 2-tailed student t test with equal variance. # indicates significant difference among the groups (p<0.05). Expression of CCR2 were normalized to expression of endogenous control β-actin. ALS, amyotrophic lateral sclerosis; CCR2, chemokine receptor 2; PBMCs, peripheral blood mononuclear cells.
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pone-0038382-g003: Real Time PCR analysis of relative mRNA expression of CCR2 in PBMCs of subjects.(A) Agarose gel electrophoresis of Real Time PCR products of target gene CCR2 and endogenous control β-actin. Reactions were performed with mRNA isolated form PBMCs of ALS patient and normal control. (B) Representative amplification curves depicting increase in fluorescence of CCR2 and β-actin from cDNA of same sample. No increment in fluorescence of negative controls was observed. The x-axis indicates cycle number and the y-axis shows intensity of relative fluorescence in linear scale. Ct is threshold cycle where normalized fluorescent signal of SYBR green intersect with threshold line. (C) Melt curve profile of Real Time PCR products of CCR2 and β actin clearly indicates the absence of any non specific amplification. The x-axis indicates negative derivative of change in amount of fluorescence per unit change in temperature (dF/dT) and the y-axis represents temperature in Celsius (°C). (D) Bar diagram showing fold change in CCR2 mRNA expression PBMCs from ALS and normal subjects. Values are plotted as mean ± SE (Standard error). Data was analyzed by unpaired, independent 2-tailed student t test with equal variance. # indicates significant difference among the groups (p<0.05). Expression of CCR2 were normalized to expression of endogenous control β-actin. ALS, amyotrophic lateral sclerosis; CCR2, chemokine receptor 2; PBMCs, peripheral blood mononuclear cells.

Mentions: Real Time PCR analysis also indicated a 7.1-fold down-regulation of CCR2 mRNA expression in PBMCs of ALS patients as compared to normal subjects (Figure 3A–3D; p = 0.032). A 27-fold and 20-fold reduction was observed in severe ALS as compared to normal and mild ALS respectively (Figure 4A; p = 0.009 and p = 0.023 respectively), however, mRNA levels were comparable across moderate and severe ALS patients (Figure 4A; p>0.05). In addition, there was no significant reduction of CCR2 mRNA in PBMCs from ALS patients with disease duration >19 months in comparison to ALS patients with ≤19 months (Figure 4B; p>0.05). There was a significant decrease of 8.0-fold in PBMCs CCR2 mRNA in limb onset ALS when compared with bulbar onset ALS patients (Figure 4C; p = 0.009). PBMCs CCR2 transcript expression was comparable between ALS patients with respiratory dysfunction and without respiratory problems (Figure 4D; 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)

Real Time PCR analysis of relative mRNA expression of CCR2 in PBMCs of subjects.(A) Agarose gel electrophoresis of Real Time PCR products of target gene CCR2 and endogenous control β-actin. Reactions were performed with mRNA isolated form PBMCs of ALS patient and normal control. (B) Representative amplification curves depicting increase in fluorescence of CCR2 and β-actin from cDNA of same sample. No increment in fluorescence of negative controls was observed. The x-axis indicates cycle number and the y-axis shows intensity of relative fluorescence in linear scale. Ct is threshold cycle where normalized fluorescent signal of SYBR green intersect with threshold line. (C) Melt curve profile of Real Time PCR products of CCR2 and β actin clearly indicates the absence of any non specific amplification. The x-axis indicates negative derivative of change in amount of fluorescence per unit change in temperature (dF/dT) and the y-axis represents temperature in Celsius (°C). (D) Bar diagram showing fold change in CCR2 mRNA expression PBMCs from ALS and normal subjects. Values are plotted as mean ± SE (Standard error). Data was analyzed by unpaired, independent 2-tailed student t test with equal variance. # indicates significant difference among the groups (p<0.05). Expression of CCR2 were normalized to expression of endogenous control β-actin. ALS, amyotrophic lateral sclerosis; CCR2, chemokine receptor 2; PBMCs, peripheral blood mononuclear cells.
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Related In: Results  -  Collection

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pone-0038382-g003: Real Time PCR analysis of relative mRNA expression of CCR2 in PBMCs of subjects.(A) Agarose gel electrophoresis of Real Time PCR products of target gene CCR2 and endogenous control β-actin. Reactions were performed with mRNA isolated form PBMCs of ALS patient and normal control. (B) Representative amplification curves depicting increase in fluorescence of CCR2 and β-actin from cDNA of same sample. No increment in fluorescence of negative controls was observed. The x-axis indicates cycle number and the y-axis shows intensity of relative fluorescence in linear scale. Ct is threshold cycle where normalized fluorescent signal of SYBR green intersect with threshold line. (C) Melt curve profile of Real Time PCR products of CCR2 and β actin clearly indicates the absence of any non specific amplification. The x-axis indicates negative derivative of change in amount of fluorescence per unit change in temperature (dF/dT) and the y-axis represents temperature in Celsius (°C). (D) Bar diagram showing fold change in CCR2 mRNA expression PBMCs from ALS and normal subjects. Values are plotted as mean ± SE (Standard error). Data was analyzed by unpaired, independent 2-tailed student t test with equal variance. # indicates significant difference among the groups (p<0.05). Expression of CCR2 were normalized to expression of endogenous control β-actin. ALS, amyotrophic lateral sclerosis; CCR2, chemokine receptor 2; PBMCs, peripheral blood mononuclear cells.
Mentions: Real Time PCR analysis also indicated a 7.1-fold down-regulation of CCR2 mRNA expression in PBMCs of ALS patients as compared to normal subjects (Figure 3A–3D; p = 0.032). A 27-fold and 20-fold reduction was observed in severe ALS as compared to normal and mild ALS respectively (Figure 4A; p = 0.009 and p = 0.023 respectively), however, mRNA levels were comparable across moderate and severe ALS patients (Figure 4A; p>0.05). In addition, there was no significant reduction of CCR2 mRNA in PBMCs from ALS patients with disease duration >19 months in comparison to ALS patients with ≤19 months (Figure 4B; p>0.05). There was a significant decrease of 8.0-fold in PBMCs CCR2 mRNA in limb onset ALS when compared with bulbar onset ALS patients (Figure 4C; p = 0.009). PBMCs CCR2 transcript expression was comparable between ALS patients with respiratory dysfunction and without respiratory problems (Figure 4D; 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