Department of Rheumatology, Oslo University Hospital, Rikshospitalet, Oslo, Norway.
Department of Immunology, University of Oslo and Oslo University Hospital, Oslo, Norway.
Front Immunol. 2022 Sep 23;13:991743. doi: 10.3389/fimmu.2022.991743. eCollection 2022.
Systemic 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.
To 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).
By 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.
This 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, generated anti-CCL21 antibodies exist in SSc, but do not appear to modify serum CCL21 levels in patients with SSc-PAH.
系统性硬化症（SSc）是一种异质性疾病，似乎是由血管病理学、组织纤维化和免疫过程相互作用引起的，有证据表明趋化因子失调，而趋化因子通常可控制免疫细胞的迁移。我们最近发现，在 SSc 相关肺动脉高压（PAH）患者中，趋化因子 CCL21 的水平发生了改变。在此，我们旨在确定 CCL21 在靶器官中的表达和生物标志物特征。
为了研究 CCL21 在靶器官中的表达，我们对 SSc-PAH 患者的肺组织进行了免疫组织化学（IHC）分析。我们通过 ELISA 和 Luminex 在来自奥斯陆（OUH）和苏黎世（n=93）大学医院的两个特征明确的 SSc 队列中以及在 168 名健康对照中评估了 CCL21 的血清水平。为了检测抗 CCL21 抗体，我们应用了 SSc 患者（n=300）和健康对照的血清样本进行了蛋白质阵列分析。为了表征 SSc 中的循环 CCL21，我们应用了针对全长和无尾 CCL21 的抗体进行免疫沉淀（IP），并应用了仅针对 CCL21 羧基末端的定制抗体。通过 SDS-PAGE/免疫印迹和质谱（MS）分析 IP 产物。
通过 IHC，我们发现 CCL21 主要在 SSc 合并 PAH 患者的气道上皮细胞中表达。在 CCL21 血清水平的分析中，我们发现 Luminex 和 ELISA 之间存在弱相关性（r=0.515，p<0.001）。与健康对照组相比，SSc 患者的血清抗 CCL21 抗体水平更高（p<0.001），但 SSc 患者中只有 5%为抗 CCL21 抗体阳性，并且我们没有发现抗 CCl21 与 CCL21 血清水平之间存在相关性。通过 MS，我们仅鉴定到 CCL21 中位于氨基酸（aa）23-102 内的肽，表明 SSc 中的 CCL21 作为没有羧基末端的截断蛋白循环。
这项研究表明 CCL21 在 SSc 合并 PAH 患者的肺上皮组织中表达，并表明 SSc 中的 CCL21 作为截断蛋白循环。我们扩展了先前表明 CCL21 具有生物标志物潜力的观察结果，但发现 Luminex 不适用于生物标志物分析。最后，在 SSc 中存在针对 CCL21 的自身抗体，但在 SSc-PAH 患者中似乎不改变血清 CCL21 水平。
