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

Abstract

Introduction: 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.

Materials and methods: 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).

Results: 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.

Conclusion: 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, in vivo generated anti-CCL21 antibodies exist in SSc, but do not appear to modify serum CCL21 levels in patients with SSc-PAH.

Keywords: Anti-CCL21; CCL21; ELISA Enzyme-linked immunosorbent assay; Luminex (xMAP) method; pulmonary arteria hypertension; systemic sclerosis.

Figure 1

Schematic overview of the number of systemic sclerosis (SSc) patients included in ELISA 1, Luminex and ELISA 2. (A) In the previously published ELISA 1, 297 SSc patients and 100 random healthy controls were included. Of these 167 patients had undergone by RHC and 41 patients were diagnosed with PAH. In the Luminex analysis, serum samples from 458 SSc patients form Oslo University Hospital (OUH), 93 serum samples form SSc patients from Zurich University Hospital (USZ) and the random healthy controls were run, as well as 68 age and gender matched healthy controls. In total, 251/456 patients included in the Luminex analysis had undergone RHC and 72 were diagnosed with PAH. For the follow-up ELISA 2, 176 serum samples form OUS, 49 serum samples from USZ and 60 age and gender matched healthy controls were included. Of these patients, 152 had conducted RHC and 51 were diagnosed with PAH. (B) The samples overlapping in the three different runs were 243 samples between ELISA1 and Luminex, 80 in ELISA1 and ELISA2, and 218 in Luminex and ELISA 2. The number of samples overlapping in all three runs were 76 samples.

Figure 2

CCL21 expression in systemic sclerosis with associated pulmonary arterial hypertension from explanted lung tissue. CCL21 immunohistochemistry demonstrated protein expression (black arrows) from (A-C) airway epithelial cells (20x) and (D) alveolar macrophages (20x), which was confirmed by (E) CD68 macrophage marker in a serial cut of the same histopathology block.

Figure 3

Overlapping PAH patients in ELISA1, Luminex and ELISA2. The number of PAH patients overlapping in ELISA1 and Luminex were 26 patients. Serum samples from these 26 patients were also included in ELISA. The number of patients overlapping in Luminex and ELISA2 were 51 patients.

Figure 4

CCL21 serum levels and correlation analysis in ELISA 1, Luminex and ELISA 2. (A) The mean serum levels of CCL21 in ELISA 1 were higher in SSc patients compared to healthy controls (i) in Luminex (ii) and ELISA 2 (iii). (B) In ELISA 1, CCL21 serum levels were higher in patients diagnosed with PAH compared to patients with no PH (i) while similar in PAH patient and no PH (ii) in Luminex. The mean serum levels of CCL21 in ELISA 2 however were, like in ELISA 1, higher in PAH patients compared to those without PH (iii). (C) The correlation of CCL21 serum levels between ELISA 1 and Luminex (r=0.515, p<0.001) (i) and the correlation of CCL21 serum levels between ELISA 2 and Luminex (r=0.52, p<0.001) (ii) were poor. However, the correlation of CCL21 serum levels between ELISA 2 and ELISA 1 (r=0.788, p<0.001) (iii) were good.

Figure 5

ROC analysis of CCl21 levels from ELISA 1, Luminex and ELISA 2. (A) ROC analysis of CCL21 levels from ELISA 1 with AUC=0.843, 95% CI = 0.084-0.883 and p<0.001, (B) ROC analysis of CCL21 levels from Luminex with AUC=0.772, 95% CI = 0.711-0.833 and p<0.001, and (C) ROC analysis of CCL21 levels from ELISA 2 with AUC=0.770, 95% CI = 0.711-0.830 and p<0.001.

Figure 6

Serum anti-CCL21 antibody level in healthy controls and patients with different systemic connective tissue diseases. The dots in healthy controls (green), Systemic sclerosis (SSc; pink), primary Sjøgren syndrome (SS; purple), Systemic lupus erythematosus (SLE; blue) and Mixed connective tissue disease (MCTD; orange) shows the level of anti-CCL21 in the different patients. The dotted line represents the cut off value for considering the patients positive or negative for anti-CCL21, in which the patients above the dotted line are positive for anti-CCL21 antibodies. Levels of anti-CCL21 antibodies were significantly higher in patients with SSc, SS and SLE than in healthy controls (p-value).

Figure 7

CCL21 levels in SSc, SS and MCTD of anti-CCL21 antibody positive and negative patients. (A) Mean serum levels of CCL21 in anti-CCL21 antibody positive (green) and anti-CCL21 antibody negative (red) patients in SSc, SS and MCTD. (B) Mean serum levels of CCL21 of anti-CCL21 antibody positive (green) and anti-CCL21 antibody negative (red) patients in SSc patients diagnosed with PAH.

Figure 8

Test of CCL21-antibodies using Western blot analysis. The Western blot analysis showed that three of the CCL21 antibodies detected both full length and tailless CCL21 (four left lanes). The custom-made anti-C-terminal tail of CCL21 antibody from Sicgen only detected full length CCL21 (right lane).

Figure 9

Western blot of SSc serum samples. The four first lanes show the IP product of 1: costume made CCL21, 2: Tailless CCL21, 3: Patient serum 1, and 4: patient serum 2. The last two lanes CCL21 and Tailless CCL21 directly loaded on the gel.