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Multiparameter phospho-flow analysis of lymphocytes in early rheumatoid arthritis: implications for diagnosis and monitoring drug therapy.

Galligan CL, Siebert JC, Siminovitch KA, Keystone EC, Bykerk V, Perez OD, Fish EN - PLoS ONE (2009)

Bottom Line: Stratification by medications revealed that patients receiving leflunomide, systemic steroids or anti-TNF therapy had significant reductions in phospho-specific activation compared with patients not receiving these therapies.Correlative trends between medication-associated reductions in the levels of phosphorylation of specific signaling effectors and lower disease activity were observed.Notably, phosphorylation of these signaling effectors did not distinguish ERA from late RA, suggesting that the activation status of discrete cell populations is already established early in disease.

View Article: PubMed Central - PubMed

Affiliation: Toronto General Research Institute, University Health Network, Toronto, Ontario, Canada.

ABSTRACT

Background: The precise mechanisms involved in the initiation and progression of rheumatoid arthritis (RA) are not known. Early stages of RA often have non-specific symptoms, delaying diagnosis and therapy. Additionally, there are currently no established means to predict clinical responsiveness to therapy. Immune cell activation is a critical component therefore we examined the cellular activation of peripheral blood mononuclear cells (PBMCs) in the early stages of RA, in order to develop a novel diagnostic modality.

Methods and findings: PBMCs were isolated from individuals diagnosed with early RA (ERA) (n = 38), longstanding RA (n = 10), osteoarthritis (OA) (n = 19) and from healthy individuals (n = 10). PBMCs were examined for activation of 15 signaling effectors, using phosphorylation status as a measure of activation in immunophenotyped cells, by flow cytometry (phospho-flow). CD3+CD4+, CD3+CD8+ and CD20+ cells isolated from patients with ERA, RA and OA exhibited activation of multiple phospho-epitopes. ERA patient PBMCs showed a bias towards phosphorylation-activation in the CD4+ and CD20+ compartments compared to OA PBMCs, where phospho-activation was primarily observed in CD8+ cells. The ratio of phospho (p)-AKT/p-p38 was significantly elevated in patients with ERA and may have diagnostic potential. The mean fluorescent intensity (MFI) levels for p-AKT and p-H3 in CD4+, CD8+ and CD20+ T cells correlated directly with physician global assessment scores (MDGA) and DAS (disease activity score). Stratification by medications revealed that patients receiving leflunomide, systemic steroids or anti-TNF therapy had significant reductions in phospho-specific activation compared with patients not receiving these therapies. Correlative trends between medication-associated reductions in the levels of phosphorylation of specific signaling effectors and lower disease activity were observed.

Conclusions: Phospho-flow analysis identified phosphorylation-activation of specific signaling effectors in the PB from patients with ERA. Notably, phosphorylation of these signaling effectors did not distinguish ERA from late RA, suggesting that the activation status of discrete cell populations is already established early in disease. However, when the ratio of MFI values for p-AKT and p-p38 is >1.5, there is a high likelihood of having a diagnosis of RA. Our results suggest that longitudinal sampling of patients undergoing therapy may result in phospho-signatures that are predictive of drug responsiveness.

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Distinct phosphorylation signatures between ERA and OA PBMCs.PBMCs from patients with ERA (n = 10, closed circles) and OA (n = 9, open circles) were analyzed by multiparameter phospho-FACS, gating on CD3+CD4+, CD3+CD8+ and CD20+ cell populations, as indicated. Scatterplots of the MFI for 15 phospho-specific epitopes are shown. Significant differences were calculated by Student's t test (p<0.05).
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pone-0006703-g003: Distinct phosphorylation signatures between ERA and OA PBMCs.PBMCs from patients with ERA (n = 10, closed circles) and OA (n = 9, open circles) were analyzed by multiparameter phospho-FACS, gating on CD3+CD4+, CD3+CD8+ and CD20+ cell populations, as indicated. Scatterplots of the MFI for 15 phospho-specific epitopes are shown. Significant differences were calculated by Student's t test (p<0.05).

Mentions: Next, multiparameter phospho-FACS was employed to analyze the phosphorylation-activation status of specific signaling effectors in the PB of patients with ERA, established RA and OA, gating on CD3+CD4+, CD3+CD8+ and CD20+ cell populations. The 15 signaling effectors employed in this study were specifically chosen as they are critical signaling nodes activated by multiple pathways and are likely candidates as indicators of activation in multiple cell populations. Multiple phospho-epitopes were activated in the circulating CD4+ and CD8+ T cells and CD20+ B cells from ERA (Figure 2) and established RA patients (data not shown) compared with healthy individuals. Notably, p-AKT, p-CBL, p-JNK, p-PLC-γ, p-STAT1, p-STAT3, p-STAT6 and p-ZAP70 mean fluorescent intensity (MFI) levels were significantly elevated in ERA patient PB subsets in all three cellular compartments. The MFI values were not significantly different between the ERA and established RA patient PB subsets (Figure S1) for any of the 15 phospho-epitopes examined. Because this was an exploratory study investigating the general utility of phospho-flow in diagnosis and treatment, no correction for multiple comparisons was made in Figure 2, Figure S1, or other similar analyses (Please refer to the section on Statistical analysis). If we had corrected using the Bonferroni correction for 45 comparisons (as shown in Figures 2 and 3), a p-value of less than 0.0011 would be considered statistically significant. Rather than discourage the future study of potentially valuable phospho-epitopes by applying such a correction, we instead note that we have not adjusted for family-wise error.


Multiparameter phospho-flow analysis of lymphocytes in early rheumatoid arthritis: implications for diagnosis and monitoring drug therapy.

Galligan CL, Siebert JC, Siminovitch KA, Keystone EC, Bykerk V, Perez OD, Fish EN - PLoS ONE (2009)

Distinct phosphorylation signatures between ERA and OA PBMCs.PBMCs from patients with ERA (n = 10, closed circles) and OA (n = 9, open circles) were analyzed by multiparameter phospho-FACS, gating on CD3+CD4+, CD3+CD8+ and CD20+ cell populations, as indicated. Scatterplots of the MFI for 15 phospho-specific epitopes are shown. Significant differences were calculated by Student's t test (p<0.05).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2724743&req=5

pone-0006703-g003: Distinct phosphorylation signatures between ERA and OA PBMCs.PBMCs from patients with ERA (n = 10, closed circles) and OA (n = 9, open circles) were analyzed by multiparameter phospho-FACS, gating on CD3+CD4+, CD3+CD8+ and CD20+ cell populations, as indicated. Scatterplots of the MFI for 15 phospho-specific epitopes are shown. Significant differences were calculated by Student's t test (p<0.05).
Mentions: Next, multiparameter phospho-FACS was employed to analyze the phosphorylation-activation status of specific signaling effectors in the PB of patients with ERA, established RA and OA, gating on CD3+CD4+, CD3+CD8+ and CD20+ cell populations. The 15 signaling effectors employed in this study were specifically chosen as they are critical signaling nodes activated by multiple pathways and are likely candidates as indicators of activation in multiple cell populations. Multiple phospho-epitopes were activated in the circulating CD4+ and CD8+ T cells and CD20+ B cells from ERA (Figure 2) and established RA patients (data not shown) compared with healthy individuals. Notably, p-AKT, p-CBL, p-JNK, p-PLC-γ, p-STAT1, p-STAT3, p-STAT6 and p-ZAP70 mean fluorescent intensity (MFI) levels were significantly elevated in ERA patient PB subsets in all three cellular compartments. The MFI values were not significantly different between the ERA and established RA patient PB subsets (Figure S1) for any of the 15 phospho-epitopes examined. Because this was an exploratory study investigating the general utility of phospho-flow in diagnosis and treatment, no correction for multiple comparisons was made in Figure 2, Figure S1, or other similar analyses (Please refer to the section on Statistical analysis). If we had corrected using the Bonferroni correction for 45 comparisons (as shown in Figures 2 and 3), a p-value of less than 0.0011 would be considered statistically significant. Rather than discourage the future study of potentially valuable phospho-epitopes by applying such a correction, we instead note that we have not adjusted for family-wise error.

Bottom Line: Stratification by medications revealed that patients receiving leflunomide, systemic steroids or anti-TNF therapy had significant reductions in phospho-specific activation compared with patients not receiving these therapies.Correlative trends between medication-associated reductions in the levels of phosphorylation of specific signaling effectors and lower disease activity were observed.Notably, phosphorylation of these signaling effectors did not distinguish ERA from late RA, suggesting that the activation status of discrete cell populations is already established early in disease.

View Article: PubMed Central - PubMed

Affiliation: Toronto General Research Institute, University Health Network, Toronto, Ontario, Canada.

ABSTRACT

Background: The precise mechanisms involved in the initiation and progression of rheumatoid arthritis (RA) are not known. Early stages of RA often have non-specific symptoms, delaying diagnosis and therapy. Additionally, there are currently no established means to predict clinical responsiveness to therapy. Immune cell activation is a critical component therefore we examined the cellular activation of peripheral blood mononuclear cells (PBMCs) in the early stages of RA, in order to develop a novel diagnostic modality.

Methods and findings: PBMCs were isolated from individuals diagnosed with early RA (ERA) (n = 38), longstanding RA (n = 10), osteoarthritis (OA) (n = 19) and from healthy individuals (n = 10). PBMCs were examined for activation of 15 signaling effectors, using phosphorylation status as a measure of activation in immunophenotyped cells, by flow cytometry (phospho-flow). CD3+CD4+, CD3+CD8+ and CD20+ cells isolated from patients with ERA, RA and OA exhibited activation of multiple phospho-epitopes. ERA patient PBMCs showed a bias towards phosphorylation-activation in the CD4+ and CD20+ compartments compared to OA PBMCs, where phospho-activation was primarily observed in CD8+ cells. The ratio of phospho (p)-AKT/p-p38 was significantly elevated in patients with ERA and may have diagnostic potential. The mean fluorescent intensity (MFI) levels for p-AKT and p-H3 in CD4+, CD8+ and CD20+ T cells correlated directly with physician global assessment scores (MDGA) and DAS (disease activity score). Stratification by medications revealed that patients receiving leflunomide, systemic steroids or anti-TNF therapy had significant reductions in phospho-specific activation compared with patients not receiving these therapies. Correlative trends between medication-associated reductions in the levels of phosphorylation of specific signaling effectors and lower disease activity were observed.

Conclusions: Phospho-flow analysis identified phosphorylation-activation of specific signaling effectors in the PB from patients with ERA. Notably, phosphorylation of these signaling effectors did not distinguish ERA from late RA, suggesting that the activation status of discrete cell populations is already established early in disease. However, when the ratio of MFI values for p-AKT and p-p38 is >1.5, there is a high likelihood of having a diagnosis of RA. Our results suggest that longitudinal sampling of patients undergoing therapy may result in phospho-signatures that are predictive of drug responsiveness.

Show MeSH
Related in: MedlinePlus