Alterations of serum levels of plasminogen,TNF-α,and IDO in granulomatosis with polyangiitis patients

Abstract

Background

Granulomatosis with polyangiitis (GPA) is a representative of vasculitides associated with anti-neutrophil cytoplasmic autoantibodies. “Classical” antibodies directed against proteinase 3 are involved in the pathogenesis and are part of the GPA diagnosis at the same time. Along with them, however, antibodies against Lysosomal-Associated Membrane Protein-2 (LAMP-2) and antibodies directed against plasminogen have been described in GPA.

Objectives and methodology: We performed a cross-sectional study enrolling 34 patients diagnosed with GPA. Our study was aimed at looking for correlations between serum levels of LAMP-2 and plasminogen and the clinical manifestations of the GPA. Furthermore, we examined serum levels of tumor necrosis factor-alpha (TNF-α) and its associated indoleamine-pyrrole 2,3-dioxygenase (IDO), as well as we looked for a correlation between these cytokines and the clinical manifestations of GPA.

Results

The results showed that in GPA, serum plasminogen levels were negatively associated with renal involvement (receiver operating characteristic (ROC) area under the curve (AUC) of 0.78) (95% CI 0.53–0.91), p = 0.035, and the extent of proteinuria, Spearman’s Rho = –0.4, p = 0.015. Increased levels of TNF-α and IDO correlated with disease activity, Spearman’s Rho =0.62, p = 0.001 and Spearman’s Rho = 0.4, p = 0.022, respectively, whereas only TNF-α was increased in severe forms of GPA with lung involvement (ROC AUC of 0.8) (95% CI 0.66–0.94), p = 0.005.

Conclusions

In this study, we demonstrate the alteration of soluble factors, which play an important role in the pathogenesis of GPA and their relationship with the clinical manifestations of the disease. Our main results confirm the associations of increased secretory TNF-α and some clinical manifestations, and we describe for the first time decreased serum plasminogen levels and their association with renal involvement.

Keywords

Granulomatosis with polyangiitis granulomatosis serum levels of plasminogen serum levels of tumor necrosis factor-alpha serum levels of indoleamine 2,3-dioxygenase LAMP-2

Introduction

Granulomatosis with polyangiitis (GPA), previously known as Wegener’s granulomatosis (WG), is a rare autoimmune systemic disorder that belongs to the group of anti-neutrophil cytoplasmic autoantibodies (ANCAs)-associated vasculitis (AAV). This group of systemic autoimmune diseases was described in 1979 by Stilmant as pauci-immune vasculitides, in which there is no deposition of immunoglobulins.

ANCA plays a pivotal role in the pathogenesis of GPA, and in particular, antibodies directed against anti-proteinase 3 (PR3) and, according to some authors, anti-Lysosomal-Associated Membrane Protein-2 (LAMP-2) antibodies.¹ ANCA activates neutrophils and monocytes by binding to them via Fab and Fc fragments, thus leading to a change in the expression of their adhesive molecules, facilitating transendothelial migration.²,³ The role of ANCA in the immunopathogenesis of AAV has been demonstrated by both animal models and the efficacy of plasmapheresis as a therapeutic approach, but the correlation of ANCA with GPA activity has not yet been conclusively established.²,³ Both the data from animal models and the therapeutic efficacy of plasmapheresis demonstrated the role of ANCA in the immunopathogenesis of AAV; however, no correlation between ANCA and GPA activity has yet been conclusively established.

ANCA-activated neutrophils and monocytes adhere to the vascular wall, penetrate it, and release toxic oxygen metabolites that lead to apoptosis and necrosis of endothelial cells. In the kidney, they cause rupture of the glomerular basement membrane and accumulation of fibrin.⁴ As with many autoimmune diseases, defects in cell death are crucial for the development of GPA. Apoptotic neutrophils expressing PR3 are found in the glomeruli, and this expression interferes with their normal ordinary clearance by epherocytosis.⁵ Due to the retention of apoptotic neutrophils, they undergo a process of secondary necrosis, and, in parallel with the expression of PR3, they secrete HMGB1, a protein known as a potent endogenous adjuvant.⁴ The apoptotic pathways are also disrupted at the endothelial cell level, as antibodies against LAMP-2 expressed by endothelial cells lead to their apoptosis. The progression of GPA is closely related to the effect of tumor necrosis factor-alpha (TNF-α) in the lesions as well as in the circulation. The main function of TNF-α is to prime neutrophils, monocytes, and endothelial cells for ANCA activation.⁶,⁷ TNF-α enhances the expression of myeloperoxidase (MPO), PR3, LAMP-2, and CD11b/CD18 on neutrophils and monocytes, respectively, as well the expression of ICAM-1 on endothelial cells. As a consequence, activated neutrophils infiltrate the vascular wall.³,⁷,⁸ The immunopathogenic role of TNF-α has been well established in animal models of AAV, although the results of clinical trials with anti-TNF-α inhibitors are currently inconsistent.⁹,¹⁰

In summary, TNF-α, ANCA, C5a, neutrophils, and other immunocompetent cells, as well as alterations in cell death, form complex interactions in the context of the immunopathogenesis of GPA. This intricate system, along with endothelial cell-related factors, can be summarized by the term “endothelial dysfunction.”

In line with aforementioned and taking into consideration that LAMP-2 and plasminogen are two proteins against which antibodies are often formed in GPA, and which are involved in the process of endothelial dysfunction, we aimed to examine their serum levels. Furthermore, due to TNF-α importance in the process of neutrophil priming, another focus of our study was the investigation of TNF-α and the associated immunoregulatory factor indoleamine 2,3-dioxygenase (IDO). In our study, we aimed to examine the serum levels of LAMP-2 and plasminogen. We were driven by the fact that LAMP-2 and plasminogen are two proteins against which antibodies often are formed in GPA and which are involved in the process of endothelial dysfunction. We also aimed to test the serum levels of TNF-α and the associated immunoregulatory factor IDO due to the pivotal role of TNF-α in the process of neutrophil priming.

For study purposes, 34 patients with a proven GPA and 21 healthy controls were enrolled.

Materials and methods

Subjects and blood specimen collection and processing

Population studied

The selection and clinical evaluation of the patients were performed at the Clinic of Rheumatology of the University Hospital St. Ivan Rilski, Sofia, Bulgaria. Within the period from February 2017 to September 2018, we enrolled 34 eligible patients diagnosed with GPA at a mean age of 48 ± 14 years (Mean ± SD), and we performed a cross-sectional study. All patients met the 1990 American College of Rheumatology vasculitis classification and also the Update on the American College of Rheumatology (ACR)/European League Against Rheumatism (EULAR) Diagnosis and Classification of Vasculitis.¹¹,¹² We discarded patients based on the following exclusion criteria: an acute viral infection at the time of inclusion, presence of active or latent tuberculosis infection, concomitant malignant disease, and diagnosis of another autoimmune disease. All participants were aware of the objectives of the study and voluntarily signed informed written consent, according to the Declaration of Helsinki, and the study was approved by the Research Ethics Committee at the University Hospital St. Ivan Rilski, Sofia, Bulgaria.¹³

Along with the clinical and laboratory data, family history, and personal risk factors, data were collected on ANCA-associated implications of various organs and systems, including renal involvement, upper and/or lower respiratory tract, skin, musculoskeletal system, and central nervous system. The renal involvement was proven by biopsy and significant proteinuria and/or erythrocyturia, creatinine, and blood urea nitrogen. Reference ranges for blood urea nitrogen, serum creatinine levels, proteinuria and erythrocyturia were age and sex matched. The involvement of the upper/lower respiratory system was proven by a computed tomography scan. Disease activity was measured using the Birmingham Vasculitis Activity Score for WG (BVAS/WG).¹⁴ Patients with BVAS score < 8 were diagnosed with low activity, and patients with score ≥8 were diagnosed with high disease activity according to BVAS/WG.¹⁵ The disease stages of GPA patients were classified according to the 2007 EULAR recommendations for the treatment of localized, early systemic, generalized, severe, and refractory disease according to European Vasculitis Society (EUVAS) classification for stratification of AAV.¹⁶

The healthy control group consisted of 21 adults at mean age 40 ± 8 years, recruited among hospital employees of the University Hospital St. Ivan Rilski, Sofia, Bulgaria. They were classified by gender and had no systemic autoimmune diseases, oncological disorders, hypertension, or diabetes mellitus and no signs of infection at the time of blood sample collection. All routine immunological tests were performed in the Laboratory of Clinical Immunology at the University Hospital St. Ivan Rilski, Sofia, Bulgaria. The serum samples were processed according to the laboratory-approved standard operating procedures, and this portion of sera that was intended for testing of cytokines, plasminogen, and LAMP-2 was immediately stored at –80°C.

Methods

ANCA screening by indirect immunofluorescence

All samples were screened for ANCA by indirect immunofluorescence (IIF) on ethanol-fixed human neutrophils (Inova Diagnostics®, Inc., San Diego, CA) according to the manufacturer’s instructions. Diagnostically problematic sera were further examined on EUROPLUS Granulocyte Mosaic 22 (Euroimmun, Lübeck, Germany), a substrate arranged with four biochips per field: ethanol-fixed granulocytes, formaldehyde-fixed human granulocytes (HCHO), purified native antigens PR3, and MPO. ANCA titer ≥1:20 was reported positive.

Enzyme-linked immunosorbent assay

We examined all patients’ sera for the two main types of ANCA, PR3, and MPO by enzyme-linked immunosorbent assay (ELISA) using an automated Alegria® system (ORGENTEC Diagnostika GmbH, Mainz, Germany).

We determined the serum levels of TNF-α (Human TNF-α DY210-05, DuoSet® ELISA, R&D Systems, Inc.®, USA and Canada), Plasminogen (Plasminogen (PLG) Human ELISA Kit, ab108893, Abcam plc®, UK), LAMP-2 (Human Lysosomal-Associated Membrane Protein-2 (hLAMP-2) ELISA, CSB-E09701h, CUSABIO®, Houston, TX, USA), and IDO (Human indoleamine 2,3-dioxygenase/IDO, DY6030-05, DuoSet® ELISA, R&D Systems, Inc.®, USA and Canada).

Flow cytometric determination of the percentage and absolute number of B cells

Patients on Rituximab combined therapy were tested for the percentage and absolute number of B-lymphocytes in their peripheral blood using a standard protocol. We performed the specific fluorescent labeling with the BD Multitest TM IMK Kit (340503, Becton, Dickinson and Company, USA) according to the manufacturer’s instructions. We analyzed the samples on the BD FACSCalibur flow cytometer, where 10,000 lymphocytes were counted and analyzed using the software program MultiSET V3.0.2 of the same manufacturer.

Statistical processing

Statistical analyses and graphical processing of data were performed using Statistical Package for the Social Sciences (SPSS) version 21 for Windows (SPSS Inc., Chicago, IL) and GraphPad Prism 6. The normality of variables distribution was examined using Shapiro–Wilk test. Comparison of studied variables between GPA patients and healthy controls was performed using Mann–Whitney U test or Student’s t-test, as appropriate. Differences of studied variables within the group of GPA patients were investigated using the Kruskal–Wallis test or one-way analysis of variance with corrections for multiple testing. Correlational analysis was performed using Pearson and Spearman’s Rho analysis. Level of significance was set up for p < 0.05. Receiver operating characteristic (ROC) curve analysis was carried out to evaluate the potential diagnostic value of TNF-α for distinguishing between patients with or without pulmonary involvement and that of plasminogen for evaluating renal impairment, respectively.

Results

Patients and clinical data

For the period of the study, we recruited 18 women and 16 men at a mean age of 48 ± 14 years (Mean ± SD). The average duration of the disease was 24 ± 20 months. According to the reference, 11 patients were with localized GPA, 8 with early systemic GPA, 10 with generalized GPA, and 5 with severe GPA. Of all patients studied, 24 had elevated neutrophil counts (70% and 6.4 × 10⁹ cells/L). Of the patients with renal involvement (n = 13), eight had histologically proven fibrinoid necrosis, nine patients had elevated serum creatinine, and four patients were within the sex-matched reference range. Regarding blood urea, of all patients with renal involvement, only two had elevated values above the reference range. All patients with renal involvement had varying degrees of proteinuria, and two of the patients had erythrocyturia at the time of blood sampling. Also, 11 of the patients with renal involvement had pulmonary involvement, and 7 of them had upper respiratory tract involvement. There were 23 patients with lung involvement. Eighteen of them have additional involvement of the upper respiratory tract, and 11 have additional involvement of the kidneys. Of the patients with pulmonary involvement, 14 had granulomatous changes, 2 had active pulmonary vasculitis, and 7 had fibrosis. The patients who had only upper respiratory tract involvement were 7.

The patients who received combined therapy with Rituximab monoclonal antibody were monitored for the percentage and absolute B-lymphocyte count, and both values were found below the lower limit of the reference range (total CD19⁺ B cells as percentage of all lymphocytes: 6.4%–23% and absolute count of CD19⁺ B cells: 100–430 cells/µL).

All patients had 0% of peripheral B cell blood count, and the mean absolute count was 2 cells/µL (SD ± 2.4).

By the time of blood sample collection, 14 of the patients had positive c- staining pattern of anti-neutrophil cytoplasmic antibodies (cANCA) on IIF. The patients’ epidemiological and clinical data are shown in Table 1. We observed a significant negative association between therapy with Rituximab and presence of specific antibodies (p = 0.002). We observed a trend toward significance—the patients with positive ANCA on IIF above titer of 1:80 had a high BVAS and were predominant with generalized form according to the treatment plan. A positive correlation was also found between BVAS/WG and C-reactive protein (CRP), Spearman’s Rho = 0.5, p = 0.004.

Table 1.

Epidemiological and clinical data of patients with GPA.

Variable	N (%)	Median (range)
Caucasian race	34 (100%)
Male/Female	16 (47.1 %)/18 (52.9 %)
Age (years)		48 ± 14
Disease duration (months)		24 ± 20
Disease localization:
Respiratory involvement^a	23 (67.6 %)
Upper respiratory tract	18 (53%)
Granulomas	14 (41 %)
Pulmonary vasculitis	2 (6%)
Pulmonary fibrosis	7 (20 %)
Renal involvement^a	13 (38.2 %)
Elevated serum creatinine levels	9 (26%)
Blood urea nitrogen	2 (6%)
Proteinuria	34 (100%)
Erythrocyturia	2 (6%)
Elevated neutrophils count	24 (70%)
Neutrophil count		9.95 ± 5.73
BVAS < 8	20 (58.8 %)
BVAS ≥ 8	14 (41.2 %)
Disease stages of GPA:
Localized	11 (32.4 %)
Early systemic	8 (23.5 %)
Generalized	10 (29.4 %)
Severe	5 (14.7 %)
Specific antibodies
cANCA (IIF) above 1:20	14
MPO	1
PR3	8
Therapy:
None	6 (17.6 %)
Corticosteroids + cyclophosphamide	11 (32.4 %)
Rituximab + corticosteroids	17 (50%)

BVAS: Birmingham Vasculitis Activity Score; cANCA: c- staining pattern of anti-neutrophil cytoplasmic antibodies; GPA: granulomatosis with polyangiitis; IIF: indirect immunofluorescence; MPO: myeloperoxidase; PR3: proteinase 3.

^aPatients with renal involvement without fibrinoid necrosis have histologically proven glomerulonephritis; upper respiratory tract involvement includes bloody nasal discharge (rhinitis), subglottic stenosis, sinusitis, saddle nose deformity, hearing impairment or deafness, otitis, and mastoiditis; pulmonary involvement refers to granulomas, active vasculitis, and fibrosis.

Cytokines and other serological markers

We tested one time the serum levels of TNF-α, IDO, LAMP-2, and plasminogen in our patients with GPA (n = 34) and healthy controls (n = 21) and comparatively analyzed the results obtained in the two groups. We further evaluated the associations between the levels of TNF-α, IDO, LAMP-2, and plasminogen and the corresponding CRP levels, presence of anti-MPO/PR3, BVAS GW, granulomas, fibrinoid necrosis, internal organ involvement, and the disease categories of GPA. The BVAS was higher among patients with a severe GPA compared to those with localized form: 11 (4–14) versus 5 (0–11), p = 0.035 (Figure 1a).

Figure 1.

(a) Boxplot displaying BVAS/WG scores of different disease subtypes according to EUVAS. Data are presented as median, IQR, Min-Max. *Localized vs. Severe- p=0.035 (b) Boxplot displaying levels of TNF-α among different disease subtypes according to EUVAS. *Early systemic vs. Generalized- p=0.044; *Generalized vs. Severe- p=0.05. Data are presented as median, IQR, Min-Max. (c) Boxplot of TNF-α levels in GPA *high vs. low- p=0.042 patients with low and high disease activity based on GPA.

(1) TNF-α

TNF-α levels were below lower detectivity range in healthy control group. TNF-a levels were higher in patients with a severe GPA than in those with localized disease, median (Min–Max) 12.7 (3.25–990) versus 0.3 (0.1–57.1) pg/ml, p = 0.011 (Figure 1b). Patients with generalized GPA demonstrated higher levels of TNF-α compared to those with early systemic GPA, 1 (0.1–86.5) versus 0.8 (0.1–6.8) pg/ml, p = 0.044 (Figure 1b), and patients with a severe GPA had significantly higher levels than those with generalized GPA, 12.7 (3.25–990) versus 1 (0.1–86.5) pg/ml, p = 0.05 (Figure 1b). Furthermore, patients with active vasculitis and higher BVAS showed significantly increased levels of TNF-α, 3.2 (0–990) versus 0.1 (0–3.35 pg/ml, p = 0.042) (Figure 1c) and CRP, 16 (0–132) versus 0.5 (0–2), p = 0.001, compared to those with lower disease activity.

Significantly higher levels of TNF-α were found in patients with lung involvement compared to the levels of this cytokine in the rest of the group and the ROC area under the curve (AUC) was 0.8 (95% CI 0.66–0.94), p = 0.005, indicating that TNF-α e may be a potential marker for the presence of lung involvement (Figure 2a, b). The calculated threshold level of 2.8 pg/ml for serum TNF-a levels could distinguish GPA patients with pulmonary involvement with 60% sensitivity and 91% specificity.

Figure 2.

(a) Boxplot of TNF-α levels in GPA patients with or without pulmonary involvement. *p=0.005 (b) ROC curve analysis of serum TNF-α discriminating patients with pulmonary involvement.

(2) IDO

We found a positive correlation between serum levels of IDO and TNF-α, Spearman’s Rho = 0.62, p = 0.001, as well as between BVAS score and IDO, Spearman’s Rho = 0.4, p = 0.022 (Figure 3), and proteinuria and IDO, Spearman’s Rho = 0.4, p = 0.038.

Figure 3.

Scatterplot of IDO levels (pg/ml) against levels of TNF-α (pg/ml) and BVAS score.

(3) Plasminogen

GPA patients with renal impairment had significantly lower serum plasminogen levels than the rest of the GPA patients, 83 (4.6–870) versus 536 (387–890) µg/ml, p = 0.035. ROC AUC of 0.78 (95% CI 0.53–0.91), p = 0.035, showed a threshold value of 14.86% sensitivity and 70% specificity (Figure 4a, b). A negative correlation was found between proteinuria in GPA patients with renal impairment and serum plasminogen serum concentration, Spearman’s Rho=–0.4, p = 0.015, which identifies serum plasminogen as a potential prognostic marker for renal involvement.

Figure 4.

(a) Boxplot of plasminogen levels in GPA patients with or without renal involvement. *p=0.035; (b) ROC curve analysis of serum plasminogen levels discriminating patients without renal involvement.

Discussion

The main goal of our study was to examine the serum concentrations of TNF-α, IDO, LAMP-2, and plasminogen and their associations with the clinical course of GPA. In the immunopathogenesis of GPA, TNF-α plays a key role through its pro-inflammatory and pro-coagulant effect.⁸ Th1 cells are the most important major source of TNF-α in GPA.¹⁷ A specific subpopulation of T helpers with no expression of CD28 has been described in terms of its strong TNF-α secretion, and this subpopulation has been found increased in both granulomas and peripheral blood in GPA.¹⁷ Along with T helper lymphocytes, monocytes have been commented on as an alternative source of TNF-α.⁶ There are differing opinions as to whether serum TNF-α levels are elevated in GPA compared to healthy subjects. According to some studies, such an increase is observed, whereas other authors do not detect it.⁸,¹⁴,¹⁸ Our results also did not show statistically significant increases in TNF-α serum levels in patients with GPA. Possible reasons for the contradictory data could be the therapy of the patients, the stage of disease activity, as well as the characteristics of the healthy control subjects.

Our results showed positive associations between TNF-α levels and higher GPA activity, the severity of GPA, and lung involvement. We found a correlation between elevated levels of TNF-α in severe compared to localized stages of GPA as well as between generalized and early systemic GPA. Other studies have also shown the role of elevated TNF-α levels and a more severe GPA. Animal models of AAV have demonstrated that anti-TNF-α drugs reduce the severity of the induced disease, albuminuria, and crescent formation.³,⁹,¹⁰ Studies of TNF-α-secreting CD4⁺ CD28^– cells in GPA granulomas showed that their increase correlates with the number of organs affected as well as the severity of the disease.¹⁷ Other authors have revealed a link between TNF-α-secreting PBMCs that infiltrate the kidney and the activity of renal lesions in GPA.⁸ Furthermore, clinical trials have also shown also that anti-TNF-α monoclonal antibodies are associated with remission, beneficial effects on endothelial dysfunction, and the ability to treat patients with low doses of corticosteroids.⁷ In terms of specific organ involvement, our results display a correlation between TNF-α levels and lung involvement through granuloma formation.

As far as we are aware, the only single result in this direction has been obtained in a rat model of AAV, where the application of anti-TNF-α reduces pulmonary hemorrhage.⁷ Some authors have not found associations between plasma TNF-α levels and disease stages or with renal and pulmonary involvement.¹⁹ Despite our findings and that of many other scientific teams, numerous clinical trials using anti-TNF-α monoclonal antibodies have demonstrated that the role of the change in plasma TNF-α levels in the clinical course of GPA is still highly controversial and unclear.⁷,¹⁰

Regarding IDO, our results show that IDO, together with TNF-α, increases with higher GPA activity. This result is completely logical since the secretion of IDO is induced by the action of Interferon gamma (IFNγ) and TNF-α.^20–23 IDO exhibits immunosuppressive function by inhibiting T cell proliferation and B cell development. Besides, IDO realizes its immunosuppressive function by mediating the development of M2 macrophages, Tregs, and tolerogenic dendritic cells.²⁴ We suppose that IDO increases as a negative regulator of TNF-α in an attempt to compensate for the intense inflammatory processes in more severe forms of GPA. In parallel, another factor that is associated with an increased amount of IDO is the use of corticosteroids as therapeutic agents for GPA patients.²⁵

The presence of ANCA is a key feature of GPA, with anti-PR3 antibodies being the most typical of this AAV. However, several different antibodies have been described in GPA, including anti-LAMP-2 antibodies. In the serum of patients with AAV, anti-LAMP-2 antibodies have been found in varying percentages, becoming negative after therapy and recurring in a relapse of the disease.¹,²⁶ Anti-LAMP-2 antibodies have also been reported in rat models of cutaneous AAV, as well as in patients with Henoch–Schönlein disease and cutaneous polyarteritis nodosa.¹⁷ Anti-LAMP-2 antibodies, such as anti-PR3, activate neutrophils and induce endothelial cell apoptosis. They do not cross-react with MPO and PR3.²⁷ A peculiarity of the structure of these antibodies is that they recognize the epitope P41-49, which is common to the bacterial adhesin FimH, which gives the reason in the immunopathogenesis of GPA to comment on the mechanism of cross-reactivity.¹,³,²⁷ LAMP-2 is highly glycosylated and can be found in the lysosomal membranes of monocytes, neutrophils, and endothelial cells. Like PR3, LAMP-2 can be externalized on the cell membrane. It plays a major role in the antigenic presentation, autophagy, phagosome maturation, and clearance of intracellular pathogens.¹⁷,²⁷ All these properties suggest that LAMP-2 could play a role in the immunopathogenesis of GPA, but unfortunately, our results did not show correlations between changes in plasma LAMP-2 levels and any of the studied clinical parameters.

The main results of our study revealed reduced serum plasminogen in GPA patients compared to healthy subjects, as well as an inverse correlation between plasminogen levels and renal involvement. The role of plasminogen in the immunopathogenesis of GPA is closely related to the complementary peptide theory, which postulates that the initial immune response develops against a complementary PR3 peptide (cPR3). The resulting anti-cPR3 have an antigen-binding region that resembles PR3 so that anti-idiotypic antibodies against anti-cPR3 also attack PR3.^18,28,29 Homologs of cPR3 have been found in Staphylococcus aureus, Ross river virus, and others, but plasminogen is considered to be endogenous cPR3.²⁸,³⁰ This theory is supported by some studies showing that anti-plasminogen antibodies are found mainly in AAV with anti-PR3 antibodies, but not in healthy people or those with AAV with anti-MPO antibodies.²⁸,³⁰ However, other data challenge the complementary theory, suggesting that anti-plasminogen antibodies are also found in AAV with anti-MPO antibodies.³¹,³² In any case, anti-plasminogen antibodies are involved in the process of endothelial dysfunction and inflammatory reactions accompanying GPA.¹⁸,²⁹ They prevent its conversion to plasmin by blocking plasminogen and prolonging fibrinolysis, which causes a pro-thrombotic prothrombotic state.^18,28,29 Anti-plasminogen antibodies have been found to occur in approximately 18%–25% of patients with AAV and are associated with renal involvement.¹⁸,³¹

Our results, which reveal a negative association between serum plasminogen levels and renal involvement, as determined by both histological data and proteinuria, indirectly support the anti-plasminogen antibody and renal damage data cited in the literature. Our results for reduced serum plasminogen in GPA compared to healthy subjects could also be due to anti-plasminogen antibodies.

Conclusions

In this study, we demonstrate the changes of some soluble factors, which play an important role in the pathogenesis of GPA and their relationship with the clinical manifestations of the disease in 34 Bulgarian patients. Our main results confirm the associations of increased secretory TNF-α and some clinical manifestations, and we describe for the first time decreased serum plasminogen levels and their association with renal involvement.

Limitations of the study

We have no evidence and cannot claim that the decreased plasminogen is due to antibodies against it, because in our study, we did not test anti-plasminogen antibodies. In our opinion, this is the main limitation of our work—the lack of research on anti-plasminogen and anti-LAMP-2 antibodies. The main reasons for this are due to the fact that for these two indicators there is no commercial standardized test with accepted diagnostic value. The data for anti-LAMP-2 differ too much mainly due to various methodologies and impediments conected with the preparation of glycosylated native or recombinant LAMP. Concerning anti-plasminogen antibodies, there are also several challenges to the introduction of the diagnostic.³²

Footnotes

Author contributions

The authors contributed to the paper as follows: Research concept and design: DK and EI-T; methodology: EI-T and AY; data curation: AY, GV, EK, and TY; investigation: TY, YZ, and IS; data analysis and interpretation: GV, EK, TY, DK, EI-T, and RR; project administration: TY, EI-T, and IS; validation: DK and EI-T; writing the article: DK, EK, GV, and EI-T; critical revision and editing of the article: DK, EI-T, and RR.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ethical approval

All experiments performed in this study involving human participants were approved by the institutional and national research committee and complied with the Declaration of Helsinki of 1964 and its later amendments (2008). Voluntarily signed informed consent was obtained from all participants in accordance with the ethical recommendations of the Declaration of Helsinki before entering the study.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Ekaterina Ivanova-Todorova

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