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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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Quantitative differences in phospho MFI values distinguish ERA.The data from Figure 3 are plotted to (A) compare the CD8 MFI range/CD4 MFI range for each of p-AKT, p-CBL, p-H3, p-PLCγ and p-ZAP70 and (B) to compare the p-AKT/p-p38 and p-JNK/p-p38 ratios, between ERA (n = 10) and OA (n = 9) patients, in the indicated cell populations. Significant differences were determined by ANOVA followed by Tukey's multiple comparison (p<0.05).
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pone-0006703-g004: Quantitative differences in phospho MFI values distinguish ERA.The data from Figure 3 are plotted to (A) compare the CD8 MFI range/CD4 MFI range for each of p-AKT, p-CBL, p-H3, p-PLCγ and p-ZAP70 and (B) to compare the p-AKT/p-p38 and p-JNK/p-p38 ratios, between ERA (n = 10) and OA (n = 9) patients, in the indicated cell populations. Significant differences were determined by ANOVA followed by Tukey's multiple comparison (p<0.05).

Mentions: Next we examined whether phosphorylation of specific signaling effectors was disease specific, by comparing the phosphorylation profiles of PBMC from ERA and OA patients. The MFI levels for p-AKT, p-CBL and p-JNK were statistically higher (p<0.05) in ERA patient PBMCs compared with OA patient PBMCs, in the CD4+, CD8+ and CD20+ compartments (Figure 3). Additionally, the extent of phosphorylation-activation, as measured by the number of phosphorylated signaling effectors for which the MFI values were significantly different between ERA and OA samples, was greatest in the CD4+ T cell population (Figure 3). For each of the phospho-epitopes we next set an arbitrary threshold MFI level that was 10% higher than the maximum MFI level recorded amongst the healthy individuals (Table 1). Our subsequent analysis, based on this threshold, distinguished ERA patients with activated phospho-epitopes in all three cell compartments (CD4+, CD8+ and CD20+) (Table 1). In contrast, OA patients had fewer phospho-epitopes activated, predominantly in the CD8+ T cell compartment (Table 1). Further analysis directly comparing the ERA and OA patient groups, again using a threshold of 10% greater than the highest OA patient MFI value, provided evidence for a distinguishing activation profile in the CD4+ T cells and CD20+ B cells in patients with ERA (Table 2). The data in Figure 4A reveal a significant difference (p<0.001) in the ratio of the CD8 range/CD4 range for p-AKT, p-CBL, p-H3, p-PLCγ and p-ZAP70 between RA and OA patients.


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)

Quantitative differences in phospho MFI values distinguish ERA.The data from Figure 3 are plotted to (A) compare the CD8 MFI range/CD4 MFI range for each of p-AKT, p-CBL, p-H3, p-PLCγ and p-ZAP70 and (B) to compare the p-AKT/p-p38 and p-JNK/p-p38 ratios, between ERA (n = 10) and OA (n = 9) patients, in the indicated cell populations. Significant differences were determined by ANOVA followed by Tukey's multiple comparison (p<0.05).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0006703-g004: Quantitative differences in phospho MFI values distinguish ERA.The data from Figure 3 are plotted to (A) compare the CD8 MFI range/CD4 MFI range for each of p-AKT, p-CBL, p-H3, p-PLCγ and p-ZAP70 and (B) to compare the p-AKT/p-p38 and p-JNK/p-p38 ratios, between ERA (n = 10) and OA (n = 9) patients, in the indicated cell populations. Significant differences were determined by ANOVA followed by Tukey's multiple comparison (p<0.05).
Mentions: Next we examined whether phosphorylation of specific signaling effectors was disease specific, by comparing the phosphorylation profiles of PBMC from ERA and OA patients. The MFI levels for p-AKT, p-CBL and p-JNK were statistically higher (p<0.05) in ERA patient PBMCs compared with OA patient PBMCs, in the CD4+, CD8+ and CD20+ compartments (Figure 3). Additionally, the extent of phosphorylation-activation, as measured by the number of phosphorylated signaling effectors for which the MFI values were significantly different between ERA and OA samples, was greatest in the CD4+ T cell population (Figure 3). For each of the phospho-epitopes we next set an arbitrary threshold MFI level that was 10% higher than the maximum MFI level recorded amongst the healthy individuals (Table 1). Our subsequent analysis, based on this threshold, distinguished ERA patients with activated phospho-epitopes in all three cell compartments (CD4+, CD8+ and CD20+) (Table 1). In contrast, OA patients had fewer phospho-epitopes activated, predominantly in the CD8+ T cell compartment (Table 1). Further analysis directly comparing the ERA and OA patient groups, again using a threshold of 10% greater than the highest OA patient MFI value, provided evidence for a distinguishing activation profile in the CD4+ T cells and CD20+ B cells in patients with ERA (Table 2). The data in Figure 4A reveal a significant difference (p<0.001) in the ratio of the CD8 range/CD4 range for p-AKT, p-CBL, p-H3, p-PLCγ and p-ZAP70 between RA and OA patients.

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