Inflammation ( # 2015) DOI: 10.1007/s10753-015-0132-2
Analysis of Programmed Death-1 in Patients with Psoriatic Arthritis Michael Peled,1 Marianne Strazza,1 Inbar Azoulay-Alfaguter,1 and Adam Mor1,2
Abstract—Programmed death-1 (PD-1) is an inhibitory co-receptor that is highly expressed in T lymphocytes that has been shown to downregulate inflammatory responses in several inflammatory diseases including systemic lupus erythematosus and rheumatoid arthritis. Yet, the role of PD-1 in psoriatic arthritis (PsA) has not been studied. In order to fill this gap, we measured the expression levels of PD-1 in peripheral T cells from patients with active disease. Twenty patients and fifteen age-matched healthy control subjects were recruited. The percentage of CD3+PD-1+ T cells was measured by flow cytometry. Despite normal concentration of peripheral T cells, the expression levels of PD-1 were significantly higher in patients compared to healthy controls. Interestingly, among the patients, the expression levels inversely correlated with disease activity measured by disease activity scores (DAS28). PD-1 expression levels strongly correlated with the number of tender and swollen joints, but not with Creactive protein (CRP) levels or psoriasis area and severity index (PASI). Functionally, in vitro ligation of PD-1 receptor in PsA T cells inhibited interleukin-2 (IL-2) secretion, Akt phosphorylation, and Rap1 activation. These findings suggest that PD-1 might serve as a biomarker for disease activity in PsA and highlight the need for additional studies in order to establish the role of PD-1 in PsA pathogenesis. KEY WORDS: psoriatic arthritis; PD-1; signaling.
INTRODUCTION Psoriatic arthritis (PsA) is an inflammatory arthritis that develops in 30 % of patients who have cutaneous psoriasis [1–3]. These patients suffer from synovitis, tendinitis, enthesitis, dactylitis, onycholysis, and spondylitis. Prolonged inflammation leads to extensive joint damage, and therefore, early diagnosis and treatment is mandatory. T lymphocytes (T cells) play a key role in the pathogenesis of both psoriasis and PsA [4]. Activated T cells infiltrate the skin and stimulate keratinocytes to proliferate and produce interleukin-2 (IL-2) and other growth factors. Activated T cells also overexpress CD80, a co-stimulatory receptor, suggesting that co-stimulation contributes to dermal T cell activity [5, 6]. T cells from synovial fluid of New York University Clinical and Translational Science Institute and the American College of Rheumatology supported this study. 1
The Department of Medicine, New York University School of Medicine, 450 E 29th Street, New York, NY 10016, USA 2 To whom correspondence should be addressed at The Department of Medicine, New York University School of Medicine, 450 E 29th Street, New York, NY 10016, USA. E-mail: [email protected]
patients with PsA have an activated phenotype and drugs that target T cells, such as cyclosporine and alefacept (LFA3/Fc), have demonstrated beneficial effects both in skin and joint manifestations [7]. During antigen presentation, T cells require at least two signals in order to become fully activated [8]. The first signal is antigen-specific and is delivered by the T cell receptor (TCR), upon interaction with antigenic peptides presented on major histocompatibility complex molecules on the antigen-presenting cell (APC) surface. The second signal, which could be either Bstimulatory^ or Binhibitory^, is antigen-independent and provided by the interaction between ligands on the APC and co-receptors on the T cell [9, 10]. One of the most important co-inhibitory receptors expressed by T cells is programmed death-1 (PD-1), which is engaged by the ligands PDL1 and PDL2 on the APC [11–13]. PD-1 is a homologue of CD28 and CTLA-4 and belongs to the immunoglobulin superfamily. PD-1 has two tyrosine residues in its cytoplasmic tail that form an immunoreceptor tyrosine-based inhibition motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM). Both PD-1 and CTLA-4, another important co-
0360-3997/15/0000-0001/0 # 2015 Springer Science+Business Media New York
Peled, Strazza, Azoulay-Alfaguter, and Mor inhibitory receptor, inhibit T cells, although through distinct mechanisms. PD-1 activation leads to decreases in the phosphorylated levels of Zap70, Akt (protein kinase B), and C3G by recruiting the phosphatase SHP-2 [14], whereas CTLA-4 inhibits the phosphorylation of signaling proteins by employing the phosphatase PP2A [12]. The role of PD-1 in maintenance of peripheral tolerance is highlighted by gene disruption studies in mice, demonstrating autoimmune phenotypes [14]. In humans, the role for PD-1 in tolerance and autoimmunity was first proposed by investigators in genome wide association studies reporting associations between PD-1 gene polymorphisms and development of autoimmunity [14]. Intervention at the level of the co-receptors and adhesion molecules represents an attractive target for treating autoimmune diseases. Recently, we have discovered that activation of PD-1 signaling in T cells leads to inhibition of cellular adhesion and migration [13]. In view of these findings, we hypothesis that PD-1 is a therapeutic target in PsA. To this end, we have examined the expression level of PD-1 in the peripheral T cells of patients with PsA and assessed PD-1 functions in the same cells. We discovered that activation of this receptor ex vivo inhibited IL-2 secretion, Akt phosphorylation, and Rap1 activation. To our knowledge, this data represents the first evaluation of the PD-1 in this disease and thus constitutes the first step in translating PD-1 biology to human disease.
MATERIAL AND METHODS General Reagents Roswell Park Memorial Institute (RPMI) 1640 medium, Dulbecco’s phosphate-buffered saline (DPBS), and fetal bovine serum (FBS) were purchased from Life Technologies. Ficoll-Paque was purchased from GE Healthcare. ACK lysing buffer was obtained from Lonza. Protein concentrations were measured with BCA assay (Pierce). Orthovanadate was obtained from Sigma-Aldrich. Antibodies and Flow Cytometry Anti-human PD-1 (J116) was obtained from Novus Biologicals. IgG isotype PE (12-4714) and anti-CD69 PE (E13987-102) were acquired from eBioscience. Antimouse PE (115-116-071) was from Jackson ImmunoResearch. Anti-pAkt (Tyr 308) and anti-Akt (9272S) were purchased from Cell Signaling. Anti-human CD28 (177020) was obtained from Ancell. Anti-human CD3 (MAB100), PDL2-Fc (1224-PL), and IgG1-Fc (110-HG)
were acquired from R & D. Anti-Rap1 (3/Rap1) was obtained from BD Biosciences. Non-permeabilized cells were re-suspended is wash buffer (PBS; 1 % FBS; 2 % formaldehyde) prior to analysis with FACS Caliber II (BD). Cell Culture and Stimulation Primary T cells were isolated from whole peripheral blood by isolation using RosetteSepTM human T cell enrichment cocktail (StemCell) followed by density gradient centrifugation. Cells were maintained in enriched media at 5 % CO2 and at 37 °C. Cells were serum-starved for 2 h prior to indicated stimulation. M-450 tosylactivated beads (Life Technology) were coated with anti-CD3 antibodies (20 % of binding capacity), anti-CD28 antibodies (20 % of binding capacity), and either isotype control IgG or PDL2-Fc (60 % of binding capacity). Enzyme-Linked Immunosorbent Assay Cytokines in supernatants were assayed by enzymelinked immunosorbent assay (ELISA) using antibodies to human IL-2, according to the manufacturer’s instructions (Aviva Systems). Western Blotting and Rap1 Activation Assay Activated Rap1 was detected by the glutathione Stransferase (GST) pull-down assay, as previously described [15]. Primary T cells were stimulated with immobilized anti-CD3 and anti-CD28 antibodies±PDL2-Fc for 15 min prior to cell lysis. Cell lysates were incubated with GSTRBD-RalGSD coupled to glutathione beads to pull-down activated Rap1. Pull-down lysates were then separated by Tris-Glycine polyacrylamide gel electrophoresis and transferred to nitrocellulose filters. Blots were blocked, and incubated with the primary antibodies at 4 °C overnight. Immunoreactive bands were visualized using the Odyssey Imaging System (Li-COR Biosciences). To calculate the percentage of activated Rap1, we used the ratio of GTPbound Rap1 over corrected total Rap1 intensity detected (100 %). Patient and Control Populations A case-control study including 20 PsA patients and 15 age-, gender-, and ethnicity-matched healthy blood donors was conducted to test the association between PD1 expression levels and disease activity. PsA patients were diagnosed using the CASPAR criteria [16] and recruited from the Bellevue Hospital of New York University School
PD-1 is Psoriatic Arthritis of Medicine (Manhattan, New York). The Institutional Review Committee approved the study, and all subjects gave written informed consent. Statistics Values are reported as means ± SEM. Statistical analyses were performed using Student’s t test and Pearson correlation coefficient (R). Statistical significance was defined as a two-tailed P value of less than 0.05. Statistical analyses were performed using the Microsoft Excel 2011 program and Social Science Statistics (http://www.socscistatistics.com).
RESULTS Increased PD-1 Expression in T Cells from Patients with Psoriatic Arthritis T lymphocytes play a critical role in PsA pathogenesis. PsA T cells have been extensively characterized, and demonstrate activated phenotypes, but PD-1 expression levels have never been studied [17]. Our first objective was to study whether PD-1 expression levels were aberrant in patients with PsA. We recruited PsA patients (n=20) from the arthritis clinic of Bellevue Hospital (Manhattan, New York). Special attention was given to newly diagnosed patients with active disease who have not yet received systemic drug therapy. Age-, gender-, and ethnicity-matched healthy volunteers (n=15) served as controls (Table 1). We measured articular disease activity using the disease activity score 28 (DAS28) system and cutaneous involvement using the psoriasis area and severity index (PASI). Twenty cubic centimeter of peripheral blood were collected in anti-coagulated tubes and delivered to the laboratory within 2 h. CD3+ T cells were isolated, and serum was saved for later analysis.
The expression levels of PD-1 on the plasma membrane of T cells were documented by FACS analysis. Notably, the percentage of the cells expressing PD-1 was 11±2.1 % among the patients compared to 1.2±0.3 % in the control group (P
Analysis of Programmed Death-1 in Patients with Psoriatic Arthritis Michael Peled,1 Marianne Strazza,1 Inbar Azoulay-Alfaguter,1 and Adam Mor1,2
Abstract—Programmed death-1 (PD-1) is an inhibitory co-receptor that is highly expressed in T lymphocytes that has been shown to downregulate inflammatory responses in several inflammatory diseases including systemic lupus erythematosus and rheumatoid arthritis. Yet, the role of PD-1 in psoriatic arthritis (PsA) has not been studied. In order to fill this gap, we measured the expression levels of PD-1 in peripheral T cells from patients with active disease. Twenty patients and fifteen age-matched healthy control subjects were recruited. The percentage of CD3+PD-1+ T cells was measured by flow cytometry. Despite normal concentration of peripheral T cells, the expression levels of PD-1 were significantly higher in patients compared to healthy controls. Interestingly, among the patients, the expression levels inversely correlated with disease activity measured by disease activity scores (DAS28). PD-1 expression levels strongly correlated with the number of tender and swollen joints, but not with Creactive protein (CRP) levels or psoriasis area and severity index (PASI). Functionally, in vitro ligation of PD-1 receptor in PsA T cells inhibited interleukin-2 (IL-2) secretion, Akt phosphorylation, and Rap1 activation. These findings suggest that PD-1 might serve as a biomarker for disease activity in PsA and highlight the need for additional studies in order to establish the role of PD-1 in PsA pathogenesis. KEY WORDS: psoriatic arthritis; PD-1; signaling.
INTRODUCTION Psoriatic arthritis (PsA) is an inflammatory arthritis that develops in 30 % of patients who have cutaneous psoriasis [1–3]. These patients suffer from synovitis, tendinitis, enthesitis, dactylitis, onycholysis, and spondylitis. Prolonged inflammation leads to extensive joint damage, and therefore, early diagnosis and treatment is mandatory. T lymphocytes (T cells) play a key role in the pathogenesis of both psoriasis and PsA [4]. Activated T cells infiltrate the skin and stimulate keratinocytes to proliferate and produce interleukin-2 (IL-2) and other growth factors. Activated T cells also overexpress CD80, a co-stimulatory receptor, suggesting that co-stimulation contributes to dermal T cell activity [5, 6]. T cells from synovial fluid of New York University Clinical and Translational Science Institute and the American College of Rheumatology supported this study. 1
The Department of Medicine, New York University School of Medicine, 450 E 29th Street, New York, NY 10016, USA 2 To whom correspondence should be addressed at The Department of Medicine, New York University School of Medicine, 450 E 29th Street, New York, NY 10016, USA. E-mail: [email protected]
patients with PsA have an activated phenotype and drugs that target T cells, such as cyclosporine and alefacept (LFA3/Fc), have demonstrated beneficial effects both in skin and joint manifestations [7]. During antigen presentation, T cells require at least two signals in order to become fully activated [8]. The first signal is antigen-specific and is delivered by the T cell receptor (TCR), upon interaction with antigenic peptides presented on major histocompatibility complex molecules on the antigen-presenting cell (APC) surface. The second signal, which could be either Bstimulatory^ or Binhibitory^, is antigen-independent and provided by the interaction between ligands on the APC and co-receptors on the T cell [9, 10]. One of the most important co-inhibitory receptors expressed by T cells is programmed death-1 (PD-1), which is engaged by the ligands PDL1 and PDL2 on the APC [11–13]. PD-1 is a homologue of CD28 and CTLA-4 and belongs to the immunoglobulin superfamily. PD-1 has two tyrosine residues in its cytoplasmic tail that form an immunoreceptor tyrosine-based inhibition motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM). Both PD-1 and CTLA-4, another important co-
0360-3997/15/0000-0001/0 # 2015 Springer Science+Business Media New York
Peled, Strazza, Azoulay-Alfaguter, and Mor inhibitory receptor, inhibit T cells, although through distinct mechanisms. PD-1 activation leads to decreases in the phosphorylated levels of Zap70, Akt (protein kinase B), and C3G by recruiting the phosphatase SHP-2 [14], whereas CTLA-4 inhibits the phosphorylation of signaling proteins by employing the phosphatase PP2A [12]. The role of PD-1 in maintenance of peripheral tolerance is highlighted by gene disruption studies in mice, demonstrating autoimmune phenotypes [14]. In humans, the role for PD-1 in tolerance and autoimmunity was first proposed by investigators in genome wide association studies reporting associations between PD-1 gene polymorphisms and development of autoimmunity [14]. Intervention at the level of the co-receptors and adhesion molecules represents an attractive target for treating autoimmune diseases. Recently, we have discovered that activation of PD-1 signaling in T cells leads to inhibition of cellular adhesion and migration [13]. In view of these findings, we hypothesis that PD-1 is a therapeutic target in PsA. To this end, we have examined the expression level of PD-1 in the peripheral T cells of patients with PsA and assessed PD-1 functions in the same cells. We discovered that activation of this receptor ex vivo inhibited IL-2 secretion, Akt phosphorylation, and Rap1 activation. To our knowledge, this data represents the first evaluation of the PD-1 in this disease and thus constitutes the first step in translating PD-1 biology to human disease.
MATERIAL AND METHODS General Reagents Roswell Park Memorial Institute (RPMI) 1640 medium, Dulbecco’s phosphate-buffered saline (DPBS), and fetal bovine serum (FBS) were purchased from Life Technologies. Ficoll-Paque was purchased from GE Healthcare. ACK lysing buffer was obtained from Lonza. Protein concentrations were measured with BCA assay (Pierce). Orthovanadate was obtained from Sigma-Aldrich. Antibodies and Flow Cytometry Anti-human PD-1 (J116) was obtained from Novus Biologicals. IgG isotype PE (12-4714) and anti-CD69 PE (E13987-102) were acquired from eBioscience. Antimouse PE (115-116-071) was from Jackson ImmunoResearch. Anti-pAkt (Tyr 308) and anti-Akt (9272S) were purchased from Cell Signaling. Anti-human CD28 (177020) was obtained from Ancell. Anti-human CD3 (MAB100), PDL2-Fc (1224-PL), and IgG1-Fc (110-HG)
were acquired from R & D. Anti-Rap1 (3/Rap1) was obtained from BD Biosciences. Non-permeabilized cells were re-suspended is wash buffer (PBS; 1 % FBS; 2 % formaldehyde) prior to analysis with FACS Caliber II (BD). Cell Culture and Stimulation Primary T cells were isolated from whole peripheral blood by isolation using RosetteSepTM human T cell enrichment cocktail (StemCell) followed by density gradient centrifugation. Cells were maintained in enriched media at 5 % CO2 and at 37 °C. Cells were serum-starved for 2 h prior to indicated stimulation. M-450 tosylactivated beads (Life Technology) were coated with anti-CD3 antibodies (20 % of binding capacity), anti-CD28 antibodies (20 % of binding capacity), and either isotype control IgG or PDL2-Fc (60 % of binding capacity). Enzyme-Linked Immunosorbent Assay Cytokines in supernatants were assayed by enzymelinked immunosorbent assay (ELISA) using antibodies to human IL-2, according to the manufacturer’s instructions (Aviva Systems). Western Blotting and Rap1 Activation Assay Activated Rap1 was detected by the glutathione Stransferase (GST) pull-down assay, as previously described [15]. Primary T cells were stimulated with immobilized anti-CD3 and anti-CD28 antibodies±PDL2-Fc for 15 min prior to cell lysis. Cell lysates were incubated with GSTRBD-RalGSD coupled to glutathione beads to pull-down activated Rap1. Pull-down lysates were then separated by Tris-Glycine polyacrylamide gel electrophoresis and transferred to nitrocellulose filters. Blots were blocked, and incubated with the primary antibodies at 4 °C overnight. Immunoreactive bands were visualized using the Odyssey Imaging System (Li-COR Biosciences). To calculate the percentage of activated Rap1, we used the ratio of GTPbound Rap1 over corrected total Rap1 intensity detected (100 %). Patient and Control Populations A case-control study including 20 PsA patients and 15 age-, gender-, and ethnicity-matched healthy blood donors was conducted to test the association between PD1 expression levels and disease activity. PsA patients were diagnosed using the CASPAR criteria [16] and recruited from the Bellevue Hospital of New York University School
PD-1 is Psoriatic Arthritis of Medicine (Manhattan, New York). The Institutional Review Committee approved the study, and all subjects gave written informed consent. Statistics Values are reported as means ± SEM. Statistical analyses were performed using Student’s t test and Pearson correlation coefficient (R). Statistical significance was defined as a two-tailed P value of less than 0.05. Statistical analyses were performed using the Microsoft Excel 2011 program and Social Science Statistics (http://www.socscistatistics.com).
RESULTS Increased PD-1 Expression in T Cells from Patients with Psoriatic Arthritis T lymphocytes play a critical role in PsA pathogenesis. PsA T cells have been extensively characterized, and demonstrate activated phenotypes, but PD-1 expression levels have never been studied [17]. Our first objective was to study whether PD-1 expression levels were aberrant in patients with PsA. We recruited PsA patients (n=20) from the arthritis clinic of Bellevue Hospital (Manhattan, New York). Special attention was given to newly diagnosed patients with active disease who have not yet received systemic drug therapy. Age-, gender-, and ethnicity-matched healthy volunteers (n=15) served as controls (Table 1). We measured articular disease activity using the disease activity score 28 (DAS28) system and cutaneous involvement using the psoriasis area and severity index (PASI). Twenty cubic centimeter of peripheral blood were collected in anti-coagulated tubes and delivered to the laboratory within 2 h. CD3+ T cells were isolated, and serum was saved for later analysis.
The expression levels of PD-1 on the plasma membrane of T cells were documented by FACS analysis. Notably, the percentage of the cells expressing PD-1 was 11±2.1 % among the patients compared to 1.2±0.3 % in the control group (P
