Target organ expression and biomarker characterization of chemokine CCL21 in systemic sclerosis associated pulmonary arterial hypertension

Abstract

IntroductionSystemic sclerosis (SSc) is a heterogenous disorder that appears to result from interplay between vascular pathologies, tissue fibrosis and immune processes, with evidence for deregulation of chemokines, which normally control immune trafficking. We recently identified altered levels of chemokine CCL21 in SSc associated pulmonary arterial hypertension (PAH). Here, we aimed to define target organ expression and biomarker characteristics of CCL21.Materials and methodsTo investigate target organ expression of CCL21, we performed immunohistochemistry (IHC) on explanted lung tissues from SSc-PAH patients. We assessed serum levels of CCL21 by ELISA and Luminex in two well-characterized SSc cohorts from Oslo (OUH, n=552) and Zurich (n=93) University hospitals and in 168 healthy controls. For detection of anti-CCl21 antibodies, we performed protein array analysis applying serum samples from SSc patients (n=300) and healthy controls. To characterize circulating CCL21 in SSc, we applied immunoprecipitation (IP) with antibodies detecting both full length and tailless and a custom-made antibody detecting only the C-terminal of CCL21. IP products were analyzed by SDS-PAGE/western blot and Mass spectrometry (MS).ResultsBy IHC, we found that CCL21 was mainly expressed in the airway epithelial cells of SSc patients with PAH. In the analysis of serum levels of CCL21 we found weak correlation between Luminex and ELISA (r=0.515, p<0.001). Serum levels of anti-CCL21 antibodies were higher in SSc patients than in healthy controls (p<0.001), but only 5% of the SSc population were positive for anti-CCL21 antibodies in SSc, and we found no correlation between anti-CCl21 and serum levels of CCL21. By MS, we only identified peptides located within amino acid (aa) 23-102 of CCL21, indicating that CCL21 in SSc circulate as a truncated protein without the C-terminal tail.ConclusionThis study demonstrates expression of CCL21 in epithelial lung tissue from SSc patients with PAH, and indicate that CCL21 in SSc circulates as a truncated protein. We extend previous observations indicating biomarker potential of CCL21, but find that Luminex is not suitable as platform for biomarker analyses. Finally, in vivo generated anti-CCL21 antibodies exist in SSc, but do not appear to modify serum CCL21 levels in patients with SSc-PAH.

Current Trends in Vascular Biomarkers for Systemic Sclerosis: A Narrative Review

“…Moreover, CCL21 concentrations positively correlated with PAH development and the occurrence of PAH-related events, suggesting that this chemokine might be a promising marker for predicting the risk of SSc-related PAH and PAH progression [ 178 ]. CCL21 association with SSc-PAH and its predictive value of PAH development were recently confirmed by the same research group in two new independent cohorts [ 179 ].…”

Serum Biomarkers in Connective Tissue Disease-Associated Pulmonary Arterial Hypertension

“…In a cohort of SSc patients, C-C motif ligand 20 (CCL20) levels correlated directly with the mPAP values measured by RHC; a positive correlation was also found between CCL20 levels and the presence of primary biliary cholangitis [ 152 ], indicating a common pathogenesis between this rare manifestation and PAH in SSc. Moreover, the CCL21 serum levels were found to be elevated in the serum and lung tissue of patients with SSc-PAH [ 153 , 154 ].…”

Biomarkers in Systemic Sclerosis: An Overview

“…Recent studies have shown how vast and ever-expanding the field of biomarkers is in SSc. Biomarkers will become increasingly important in research and, consequently, in the diagnosis and therapeutic approach to SSc [30][31][32][33][34][35][36][59][60][61][62][63][64][65][66][67]. Several studies have demonstrated the involvement of at least 240 pathways and numerous dysregulated proteins in the pathogenesis of SSc, so the field of biomarkers in this disease is complex and evolving [30][31][32][33][34][35][36][59][60][61][62][63][64][65][66][67].…”

“…Biomarkers will become increasingly important in research and, consequently, in the diagnosis and therapeutic approach to SSc [30][31][32][33][34][35][36][59][60][61][62][63][64][65][66][67]. Several studies have demonstrated the involvement of at least 240 pathways and numerous dysregulated proteins in the pathogenesis of SSc, so the field of biomarkers in this disease is complex and evolving [30][31][32][33][34][35][36][59][60][61][62][63][64][65][66][67]. Cytokeratin 17 (CK17), marginal zone protein B1 (MZB1), and leucine-rich α2-glycoprotein-1 (LRG1) appear to be potential biomarkers for SSc, with CK17 negatively associated with disease severity and higher values of CK17 protective [37,[59][60][61][62][63][64][65][66][67].…”

“…Nail fold video capillaroscopy (NVC) plays an important role because it is a noninvasive and reliable method to detect microvascular involvement. NVC is now formally used to classify SSc because it plays a diagnostic role in recognizing microvascular involvement [3,4,26,[60][61][62][63][64][65][66][67][68]. Indeed, the presence of capillaroscopic abnormalities of the nail fold, together with the presence of ANA and recurrent Raynaud's phenomenon, are predictive factors for progression to definitive SSc.…”