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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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Related in: MedlinePlus

Phospho-specific epitopes are activated in RA synovial tissue lymphocytes.Cell lysates were prepared from ST lymphocytes isolated from the affected joints of 3 patients with late-stage RA, at the time of joint replacement, as described in Methods. Lysates were analyzed by a customized BD PowerBlot and data are shown as mean±SE. Values represent the average intensity of triplicate readings except for p-JNK, p-STAT5, p-cdc2 and p-src, which are averages of duplicate samples.
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pone-0006703-g001: Phospho-specific epitopes are activated in RA synovial tissue lymphocytes.Cell lysates were prepared from ST lymphocytes isolated from the affected joints of 3 patients with late-stage RA, at the time of joint replacement, as described in Methods. Lysates were analyzed by a customized BD PowerBlot and data are shown as mean±SE. Values represent the average intensity of triplicate readings except for p-JNK, p-STAT5, p-cdc2 and p-src, which are averages of duplicate samples.

Mentions: At the outset, to confirm the activation status of T cells in RA in the context of phosphorylation-activation, we employed a customized BD PowerBlot immunoarray as a high throughput Western blot screen for phosphorylated signaling effectors. 20 paired phospho-signaling effectors were evaluated. CD3+ T cells were isolated from the affected joints of 3 patients with established RA at the time of joint replacement and protein lysates prepared. We identified elevated levels of phosphorylated (p)-AKT, p-p38, p-JNK, p-STAT1, p-STAT3, p-STAT5, p-ATF2, p-cdc2, p-p120, p-PKARIIb and p-src in joint infiltrating CD3+ T cells (Figure 1). Notably, phosphorylation of src is consistent with T cell activation [27].


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)

Phospho-specific epitopes are activated in RA synovial tissue lymphocytes.Cell lysates were prepared from ST lymphocytes isolated from the affected joints of 3 patients with late-stage RA, at the time of joint replacement, as described in Methods. Lysates were analyzed by a customized BD PowerBlot and data are shown as mean±SE. Values represent the average intensity of triplicate readings except for p-JNK, p-STAT5, p-cdc2 and p-src, which are averages of duplicate samples.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0006703-g001: Phospho-specific epitopes are activated in RA synovial tissue lymphocytes.Cell lysates were prepared from ST lymphocytes isolated from the affected joints of 3 patients with late-stage RA, at the time of joint replacement, as described in Methods. Lysates were analyzed by a customized BD PowerBlot and data are shown as mean±SE. Values represent the average intensity of triplicate readings except for p-JNK, p-STAT5, p-cdc2 and p-src, which are averages of duplicate samples.
Mentions: At the outset, to confirm the activation status of T cells in RA in the context of phosphorylation-activation, we employed a customized BD PowerBlot immunoarray as a high throughput Western blot screen for phosphorylated signaling effectors. 20 paired phospho-signaling effectors were evaluated. CD3+ T cells were isolated from the affected joints of 3 patients with established RA at the time of joint replacement and protein lysates prepared. We identified elevated levels of phosphorylated (p)-AKT, p-p38, p-JNK, p-STAT1, p-STAT3, p-STAT5, p-ATF2, p-cdc2, p-p120, p-PKARIIb and p-src in joint infiltrating CD3+ T cells (Figure 1). Notably, phosphorylation of src is consistent with T cell activation [27].

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