Edited by: Mats Bemark, University of Gothenburg, Sweden
Reviewed by: Bodil Ohlsson, Lund University, Sweden; Ana Lleo, Humanitas Research Hospital, Italy
This article was submitted to Mucosal Immunity, a section of the journal Frontiers in Immunology
†Shared authorship
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Primary sclerosing cholangitis (PSC), a chronic immune-mediated, life threatening, genetically predisposed, cholestatic liver illness, is associated with the co-occurrence of inflammatory bowel disease (IBD) and in particular with the phenotype thereof (
Since the first report of GP2's over-expression in the inflamed intestine of CD patients (
Given the close association of PSC with IBD, the occurrence of aGP2 IgA in severe PSC (
In total, four human GP2 isoforms (GP21−4) were identified (
Patients with PSC were recruited from four European university hospitals specialized in autoimmune liver diseases (Table
Demographic and clinical data of patients and controls.
PSC | 212 | 43.0 (23.3) | 70 (33.0) | 136 (64.2) | 17 (8.0) | 119 (56.1) | 20 (9.4) | 86 (31.6) | 5 |
81 (38.2) |
Berlin | 23 | 52.5 (17.5) | 6 (26.1) | 19 (82.6) |
2 (8.7) | 17 (73.9) | 1 (4.3) | 19 (82.6) | 0 | 19 (82.6) |
Hamburg | 30 | 50.0 (17.3) | 18 (60.0) |
15 (50.0) | 4 (13.3) | 11 (36.7) |
5 (16.7) | 3 (10.0) |
0 | 0 |
London | 83 | 46.3 (18.7) | 23 (27.7) | 53 (63.9) | 1 (1.2) |
52 (62.7) | 5 (6.0) | 49 (59.0) |
5 (6.0) | 57 (68.8) |
Debrecen | 76 | 34.1 (21.6) |
23 (30.3) | 49 (64.5) | 10 (13.2) | 39 (51.3) | 9 (11.8) | 15 (19.7) |
0 | 6 (7.9) |
Controls | 145 | 26.9 (22.1) |
63 (43.4) | 0 |
0 |
0 |
0 |
0 |
0 | 0 |
CF | 95 | 15.6 (20.9) |
44 (46.3) |
0 |
0 |
0 |
0 |
0 |
0 | 0 |
HS | 50 | 36.0 (18.0) | 19 (38.0) | 0 |
0 |
0 |
0 |
0 |
0 | 0 |
Comparison of the prevalence in all patients with primary sclerosing cholangitis with control groups:
In total, 145 gender-matched controls were enrolled in this study comprising 95 patients with cystic fibrosis (CF) and 50 healthy subjects (HS). The patients with CF, a multi-systemic disorder with exocrine pancreatic insufficiency and biliary cirrhosis with a different pathogenesis, were included as disease controls with regard to PSC. The 50 apparently healthy subjects (HS) with no liver or intestinal pathology were both age- and gender-matched.
The study was approved by the ethics committees of the participating centers. Written informed consent was obtained from all patients included in this study. The study was conducted in accordance with the principles of the Declaration of Helsinki (World Medical Association Declaration of Helsinki 1989).
Stable HEp-2 cell lines expressing membrane GPI-anchored GP2 isoforms were generated through transduction with lentiviruses. Briefly, coding sequences of GP2 isoforms sequences were amplified with PCR and cloned into pLVX-IRES-puro plasmids each using T4 DNA ligase (ThermoFisher Scientific, Waltham, USA) in accordance with the manufacturer's protocol. To confirm successful cloning, plasmids were Sanger sequenced. For transduction, lentiviruses were produced using the Lenti-X Lentiviral Expression System (Clontech Laboratories, Mountain View, USA). Thus, an 80% confluent Lenti-X 293T cell line was co-transfected with the six plasmids containing GP2 isoforms or an “empty” vector. The harvested supernatants were concentrated by centrifugation in Amicon Ultra-15 centrifugal filter units (Merck Millipore, Darmstadt, Germany) and employed for transduction of HEp-2 cells. Selection of successfully transduced cells was performed by adding the antibiotic puromycin to cell culture.
Confirmation of GP2 transduction into HEp-2 cells was done with reverse transcription quantitative polymerase chain reaction (RT qPCR). In brief, RNA from transduced cells was isolated with RNeasy Mini Kit according to the manufacturer's protocol (Qiagen, Hilden, Germany). After RT reaction using Maxima First Strand Synthesis Kit (ThermoFisher Scientific), qPCR was performed in the CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories, München, Germany) with following primers: 5- ATCAACGTGATTCCACCATCC-3 and 5- TTGAGCAAGAAGGCTGGC-3 (for GP2 gene); 5-AAATGTTTCATTGTGGGAGC-3 and 5- ATATGAGGCAGCAGTTTCTC-3 (for RPLP0 gene). RPLP0 was used as reference gene. Obtained PCR products were analyzed by electrophoresis.
Expression of GP2 isoform proteins was confirmed by Western blotting. Briefly, transduced HEp-2 cells were lysed and the lysate run on SDS-PAGE. Separated bands transferred on the blotting membrane were developed with a rabbit polyclonal antibody to GP2 reactive with all isoforms (GA Generic Assays, Dahlewitz, Germany). Isoforms of GP2 were revealed by a secondary anti-rabbit antibody conjugated with horse radish peroxidase employing enhanced chemiluminescence.
Membrane expression of GPI-anchored GP2 isoforms was confirmed by flow cytometry analysis.
IgG and IgA to GP2 isoforms were determined by IFA employing stably transduced HEp-2 cells expressing membrane GPI-anchored GP2 isoforms 1-4. Briefly, cells were fixed on glass slides as described elsewhere (
Data were tested for normality by the Kolmogorov-Smirnov-test and non-normally distributed data were reported by median and quartile ranges. The two-tailed, Mann-Whitney and Kruskal–Wallis tests were used to test for statistically significant differences of independent samples in 2 and more groups, respectively. Prevalence comparison between groups was performed by two-tailed Fisher's exact test. Logistic regression analysis was employed to test for the influence of explanatory (independent) variables on a binomial response variable and to detect possible clinical confounders on such association (age, gender, concomitant IBD, concomitant overlap with AIH) by a backward exclusion strategy resulting in adjusted odds ratios.
GP2 isoforms were expressed stably in HEp-2 cells as GPI-anchored molecules in the membrane of these cells by lentiviruses transduction. As control, one cell line was transduced with an empty vector only. The presence of membrane-bound GP21 to GP24 in the respective lines and their absence in the empty vector cell line was confirmed by FACS analysis (Figure
Detection of the membrane expression of GP2 isoforms in HEp-2 cells by flow cytometry. GP2 expressed in HEp-2 cells was stained with polyclonal antibodies raised against full length human GP2 followed by FITC-conjugated anti-rabbit IgG:
For the detection of aGP21 to aGP24 by IFA, cells of each line were fixed to conventional glass slides and used as targets for specific autoAb analysis (Figure
Indirect immunofluorescence assay for the detection of IgA to GP2 isoforms: Exemplarily, two patient sera and one serum of a healthy subject as control were run on HEp-2 cells transduced with GP2 isoforms 1 (GP21) to 4 (GP24) with glycosylphosphatidylinositol anchor and an empty vector, respectively. Patient 1 demonstrated a strong specific binding to membrane-bound GP21 and a weak one to GP22, whereas patient 2 showed the typical binding pattern for a strong positive binding to GP24 and a weak one for GP23. The healthy subject did not reveal a positive membrane-reactive pattern on the respective transduced HEp-2 cells.
IgA and IgG against GP21−4 were determined in 212 patients with PSC of four European hospitals and 145 gender-matched controls. Of note, the 50 HS included as controls were gender- as well as aged-matched to all PSC patients (Table
Apart from aGP23, all other aGP2 demonstrated significantly elevated prevalences in PSC patients compared with controls including HS and patients with CF (
Frequency of IgA and IgG against GP2 isoforms 1 (aGP21) to 4 (aGP24) detected by indirect immunofluorescence assay on stabile isoform-transduced HEp2 cells in 212 patients with primary sclerosing cholangitis (PSC) from different hospitals and 145 controls.
PSC | 212 | 100 (47.2) |
92 (43.4) |
21 (9.9) |
40 (18.9) |
10 (4.7) | 31 (14.6) | 103 (48.6) |
81 (38.2) |
140 (66.0) |
119 (56.1) |
154 (72.6) |
140 (66.0) |
Berlin | 23 | 6 (26.1) |
7 (30.4) |
0 | 0 | 0 | 0 | 3 (13.0) |
2 (8.7) | 8 (34.8) |
8 (34.8) | 10 (43.5) | 8 (34.8) |
Hamburg | 30 | 11 (36.7) |
12 (40.0) |
5 (16.7) |
1 (3.3) | 1 (3.3) | 1 (3.3) | 9 (30.0) |
8 (26.7) |
16 (53.3) |
16 (53.3) |
17 (56.7) |
16 (53.3) |
London | 83 | 42 (50.6) |
39 (47.0) |
2 (2.4) | 28 (33.7) |
4 (4.8) | 26 (31.3) |
52 (62.7) |
40 (48.2) |
59 (71.1) |
50 (60.2) |
68 (81.9) |
59 (71.1) |
Debrecen | 76 | 41 (53.9) |
34 (44.7) |
14 (18.4) |
11 (14.5) | 5 (6.6) | 4 (5.3) | 39 (51.3) |
31 (40.8) |
57 (75.0) |
45 (59.2) |
59 (77.6) |
57 (75.0) |
Controls | 145 | 2 (1.4) | 11 (7.6) | 1 (0.7) | 12 (8.3) | 4 (2.8) | 20 (13.8) | 1 (0.7) | 11 (7.6) | 6 (4.1) | 33 (22.8) | 34 (23.4) | 3 (2.1) |
CF | 95 | 1 (1.1) | 8 (8.4) | 1 (1.1) | 9 (9.5) | 4 (4.2) | 18 (18.9) |
1 (1.1) | 9 (9.5) | 5 (5.3) | 26 (27.4) |
26 (27.4) | 2 (2.1) |
HS | 50 | 1 (2.0) | 3 (6.0) | 0 | 3 (6.0) | 0 | 2 (4.0) | 0 | 2 (4.0) | 1 (2.0) | 7 (14.0) | 8 (16.0) | 1 (2.0) |
YI (PSC vs. controls) | 0.46 | 0.36 | 0.09 | 0.11 | 0.02 | 0.00 | 0.48 | 0.31 | 0.62 | 0.33 | 0.49 | 0.64 |
Regarding IgA reactivity, aGP21 (47.2%) and aGP24 positivity (48.6%) revealed the highest frequencies in PSC patients resulting in an even significantly elevated combined positive rate of 66.0% (aGP21and/or4 IgA) compared with both rates of single aGP2 isoform IgA testing (
In terms of IgG, aGP21, and aGP24 testing revealed the highest positive rates in PSC patients, too. However, their prevalences were lower in contrast to the corresponding IgA, but only the difference for aGP24 reached significance (
The possible association of the presence of IgA and IgG to GP21−4 in PSC patients with performed liver transplantation (LTx) and concomitant occurrence of autoimmune hepatitis, cirrhosis; cholangiocarcinoma, CD, UC, IBD (CD or UC) was investigated by Fisher's exact test (Table
Positive and negative (italic) significant associations of IgA and IgG against GP2 isoforms 1 (aGP21) to 4 (aGP24) with the clinical phenotype in 212 patients with primary sclerosing cholangitis (PSC) by Fisher's exact test.
PSC | 212 | aGP24 IgA |
aGP23 IgG |
|||||
Berlin | 23 | |||||||
Hamburg | 30 | |||||||
London | 83 | aGP24 IgA |
aGP23 IgG |
|||||
Debrecen | 76 | aGP22 IgA |
A significantly positive association of aGP2 isoform IgA and IgG positivity in PSC was established for the concomitant occurrence of cirrhosis. Thus, aGP21 and aGP24 IgA as well as aGP22 and aGP24 IgG were more prevalent in PSC patients with cirrhosis than in those without (
Logistic regression analysis of independent variables for the risk prediction of liver transplantation (LTx) and the occurrence of cirrhosis in 212 patients with primary sclerosing cholangitis (PSC).
cirrhosis | aGP21 IgA | 0.3243 | 0.1514 | 1.3831 | 1.0279–1.8609 | 0.0322 |
aGP24 IgA | 0.1729 | 0.1771 | 1.5178 | 1.0727–2.1478 | 0.0185 | |
age | 0.0273 | 0.0100 | 1.0277 | 1.0077–1.0480 | 0.0063 | |
gender | −0.6693 | 0.3235 | 0.5121 | 0.2716–0.9654 | 0.0385 | |
LTx | aGP22 IgA | −3.02104 | 1.27092 | 0.0488 | 0.0040–0.5886 | 0.0175 |
cirrhosis | 3.70772 | 0.49727 | 40.7608 | 15.3800–108.0262 | < 0.0001 | |
AIH overlap | −2.63423 | 1.00286 | 0.0718 | 0.0101–0.5124 | 0.0086 | |
UC | 1.82313 | 0.49012 | 6.1912 | 2.3691–16.1799 | 0.0002 |
A significantly negative association of aGP22 IgA was revealed for LTx performed in PSC patients of all cohorts. This significantly more prevalent aGP22 IgA occurrence in PSC patients without LTx was not detected in the single PSC cohorts but confirmed by logistic regression analysis as independent predictor in PCS patients without LTx demonstrating concomitant cirrhosis, UC and no AIH overlap (Table
Patients with PSC and concomitant CD demonstrated a significantly lower prevalence of aGP21/4 and aGP21/2/3/4 IgA. However, this association was neither found in the single PSC cohorts nor confirmed by logistic regression analysis. There was no further association of all PSC patients with and without IBD, UC or CD regarding the concomitant presence of aGP2 isoform autoAbs by Fisher's exact test. Only the London cohort demonstrated significantly less frequent aGP23 IgG in PSC patients with concomitant IBD and the Hamburg cohort significantly less frequent aGP22 IgA in patients with UC (
Logistic regression analysis revealed the concomitant occurrence of UC as an independent risk factor for liver transplantation along with cirrhosis. The presence of GP22 IgA and AIH overlap were negative predictors in this regard (Table
The recent reports of the occurrence of aGP2 IgA in patients with large biliary duct disorders including PSC and of aGP2 IgA as marker of severe disease, as well as cholangiocarcinoma ushered in a new era in PSC serology (
In contrast to recent reports demonstrating preferentially aGP2 IgA in PSC (
The IgG to GP2 isoforms determined in this study demonstrated both lower sensitivities and specificities in contrast to IgA against the corresponding GP2 isoforms which resulted in poorer assay performances of the former. Of note, apart from significantly higher positive rates of IgG to GP2 isoforms in PSC, we detected a significantly elevated prevalence of IgG GP23Ab in control patients with CF (18.9%) vs. healthy controls (4.0%). This finding warrants further investigation and could indicate a different loss of tolerance to GP2 isoforms in CF.
In terms of discrimination of PSC from controls, aGP21 IgA (47.2%) and aGP24 IgA-positives (48.6%) revealed the highest frequencies amongst all aGP2. Interestingly, combination of both led to a significantly elevated sensitivity of 66.0%. This was a significantly higher prevalence than the corresponding total prevalence reported by Jendrek et al. (66.0 vs. 48.7%,
As GP21 and GP24 represent long (537 amino acids) and short GP2 isoforms (387 amino acids), respectively, they could bear differing epitopes (
IgG and in particular IgA against GP2 isoforms revealed different associations with the PSC phenotype. In fact, aGP21 and aGP24 IgA were demonstrated as positive predictors of cirrhosis in older males with PSC. The cirrhosis in PSC is mainly characterized by extensive fibrosis around the larger bile ducts (
There is clearly a need for risk stratification in PSC (
Interestingly, CD appears to confer prognostic favor in PSC and a lower risk to develop adverse effects (
All 5 PSC patients with cholangiocarcinoma demonstrated aGP21 and/or aGP24 IgA. This might be a hint that aGP2 IgA occurs earlier in the disease course and that the tolerance break is linked with the mucosal microbiota interaction of GP2. In this context, the elevation of aGP23 IgG in patients with CF is of interest since approximately 30% of patients with CF have clinically significant liver disease (
The finding that aGP21/4 and aGP21/2/3/4 IgA demonstrated significantly lower prevalences in PSC patients with CD was surprising. We speculate that PSC with concomitant CD might be different from CD without obvious liver involvement regarding the loss of tolerance to GP2. However, this notion needs to be treated with caution since it could be confirmed neither in the single cohorts nor by logistic regression analysis.
As most multi-center studies of this kind, our study lacks perfection. The controls were not age matched to patients with PSC due to the younger age of the controls with CF. Further, there was a high level of diversity in the distinct PSC cohorts of the four European hospitals regarding number of patients and their phenotype. The established independent predictors of cirrhosis aGP21 and aGP24 IgA could not be associated in all single cohorts with the concomitant occurrence of cirrhosis.
Altogether, combined aGP21 and aGP24 analysis is required for sensitive PSC-specific autoAb testing and should be preferred to the autoAb analysis against single aGP2 isoforms only. aGP21 and aGP24 IgA might be predictors of cirrhosis in PSC and, thus, an useful alternative for risk prediction. Elevated aGP2 IgA may demonstrate a link to fibrotic changes observed in IBD in particular in CD. Prospective studies including patients tested pre- and post-liver transplantation will provide excellent hints regarding the pathophysiological role of these autoAbs.
DR and PS conceived of the study and participated in its design and data evaluation. RK and JS conducted the transduction experiments and participated in the development of the indirect fluorescence assay. MS participated in the development of the indirect fluorescence assay and carried out the assays. MP, TT, DCB, JP, MM, DB, JH, ML, KC, AF, and CS recruited patients, provided patient data and participated in the statistical evaluation thereof. All authors read and approved the final manuscript.
DR is a shareholder and employee of GA Generic Assays GmbH and Medipan GmbH. MS receives a grant from GA Generic Assays GmbH. Both have no further personal or professional interests to declare.
The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
zymogen glycoprotein 2
primary sclerosing cholangitis
GP2 isoform 1
indirect immunofluorescence assay
odds ratio
confidence interval
inflammatory bowel disease
Crohn's disease
ulcerative colitis
autoantibody
enzyme-linked immunosorbent assay
glycosylphosphatidylinositol
autoimmune hepatitis
cystic fibrosis
healthy subjects
interquartile range
liver transplantation
Youden index