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Research Article
A study of the correlation between phospholipase A2 enzyme activity and anti-complement antibodies in patients with systemic lupus erythematosus
expand article infoGinka Cholakova‡, Simona Stankova‡, Alexandra Kapogianni‡, Maria Gendzhova‡, Delina Ivanova§, Svetla Petrova‡, Ivanka Tsacheva‡
‡ Sofia University “St. Kliment Ohridski”, Sofia, Bulgaria
§ Clinic of Rheumatology, UMHAT “Sofiamed”, Sofia, Bulgaria

Corresponding author: Ginka Cholakova ( ginka.cholakova@biofac.uni-sofia.bg )

Academic editor: Magdalena Kondeva-Burdina
© 2024 Ginka Cholakova, Simona Stankova, Alexandra Kapogianni, Maria Gendzhova, Delina Ivanova, Svetla Petrova, Ivanka Tsacheva.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Cholakova G, Stankova S, Kapogianni A, Gendzhova M, Ivanova D, Petrova S, Tsacheva I (2024) A study of the correlation between phospholipase A2 enzyme activity and anti-complement antibodies in patients with systemic lupus erythematosus. Pharmacia 71: 1-6. https://doi.org/10.3897/pharmacia.71.e130210
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Abstract

Systemic lupus erythematosus (SLE) is an autoimmune disease that affects multiple organs or organ systems. SLE and one of its most common complications, lupus nephritis (LN), are characterized by chronic inflammation due to the secretion of pro-inflammatory molecules and a tissue deposition of immune complexes formed by autoantibodies and their target antigens, such as double-stranded DNA, other nuclear antigens, and complement proteins. Increased activity of phospholipase A2 enzymes (PLA2) like calcium-independent PLA2 (iPLA2), secretory PLA2 (sPLA2), and cellular PLA2 (cPLA2) has a crucial role in the maintenance of the inflammatory process during the course of SLE. This study aimed to evaluate PLA2 activity and identify the dominant type of PLA2 enzymes in a cohort of Bulgarian patients with SLE and LN. Additionally, we investigated whether the increased PLA2 activity was correlated with the presence of autoantibodies specific to the complement proteins C3 and factor H.

Keywords

phospholipase A2, SLE, autoantibodies, C3, factor H

Introduction

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by phases of remission and activation (Tsokos 2011). The pathogenesis of SLE includes multisystem tissue inflammation and the formation of a diverse set of autoantibodies that recognize double-stranded DNA fragments, nuclear proteins, ribosomal proteins, phospholipids, and complement proteins (Lou et al. 2022). A deposition of immune complexes in the kidneys leads to the development of lupus nephritis (LN) (Yu et al. 2017). Thus, many proteins of the complement system become autoantigens during the development of SLE and LN (Macedo and Isaac 2016; Weinstein et al. 2021; Matola et al. 2022). One of the well-established serum markers of the development of SLE and LN is the increased titer of autoantibodies against the complement component C1q (Stojan and Petri 2016; Trendelenburg 2021). The complement protein C3 and complement regulatory protein factor H have also been described as potent autoantigenic molecules in patients with SLE and LN (Vasilev et al. 2015; Birmingham et al. 2016; Li et al. 2020; Cai et al. 2022). The development of SLE and LN is characterized by acute or chronic inflammation that is maintained by a diverse family of phospholipase enzymes consisting of four types: A, B, C, and D (Vines and Bill 2015). The most studied are the phospholipase enzymes A2 (PLA2), which are distinguished by their diversity of structural and biological functions. PLA2 enzymes (EC 3.1.1.4) catalyze the hydrolysis of the sn-2 ester bond of membrane glycerophospholipids, releasing free fatty acids and lysophospholipids, which act as lipid mediators with proinflammatory activities and are involved in cell signaling, remodeling of membranes, lipid metabolism, cell growth and differentiation, inflammation, and apoptosis (Burke and Dennis 2008). The release of arachidonic acid is a rate-limiting and critical step in the generation of various lipid bioactive compounds such as prostaglandins, leukotrienes, and thromboxanes, with a key role in maintaining an inflammatory response. In humans, PLA2 enzymes are classified into six major groups: secreted PLA2 (sPLA2), cytosolic PLA2 (cPLA2), Ca2+-independent PLA2 (iPLA2), platelet activating factor acetyl hydrolases (PAF-AHs), lysosomal PLA2 (LPLA2), and adipose PLA2 (AdPLA2) (Dennis et al. 2011). PLA2 enzymes are secreted by immune cells that activate and maintain inflammation, such as macrophages, neutrophils, mast cells, and T cells (Khan and Ilies 2023). Increased levels of PLA2 enzymes are used as an early diagnostic marker to detect an active inflammatory process in patients with autoimmune diseases such as rheumatoid arthritis, ulcerative colitis, Crohn’s disease, and neurodegenerative diseases with autoimmune predisposition (Minami et al. 1994; Doody et al. 2015; Trotter et al. 2019; Duchez et al. 2019; Zuliani et al. 2023). Still, it is not well known whether increased PLA2 activity is linked to the activation and maintenance of the inflammatory process in patients who suffer from other autoimmune disorders such as SLE and LN. We studied whether an increased level of PLA2 activity was involved in SLE and LN pathogenesis and, if so, what was the current PLA2 isoform presented in their serum. We also analyzed whether the titers of anti-C3 and anti-factor H autoantibodies in patients with moderate to severe SLE were associated with PLA2 activity.

Materials and methods

Patients

Serum samples from 48 Bulgarian patients with SLE who attended the Clinic of Rheumatology, UMHAT “SofiaMed,” were analyzed. The patients participated in this study during a period from 10 December 2018 to 09 July 2019. The cohort consisted of 44 females (98.08%) and 4 males (1.92%) with a median age of 52 years (range 24–75 years) and a median SLE duration of 6 years (range 2–8 years). At the time of blood sampling, the SLE patients were categorized according to the EULAR/ACR SLE score as follows: moderate to severe systemic lupus erythematosus: all 48 patients; lupus nephritis: 10 patients; neuropsychiatric systemic lupus erythematosus: nine patients; musculoskeletal, mucocutaneous, and another SLE manifestation: 29 patients. All patients had a SLEDAI-2K score ≥ 6 with at least two points for musculoskeletal or mucocutaneus manifestation at the time point of sampling. Pooled sera from 24 healthy donors were used as a control. All serum samples were stored at - 30 °C until use. All patients have signed informed consent. The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of Sofia University (protocol code 93-È-115/14 June 2019).

Buffers

The following buffers were used: phosphate-buffered saline (PBS): 0.01 M Na2HPO4, 0.01 M NaH2PO4, 0.145 M NaCl, pH = 7.2; TPBS (PBS, containing 0.05% Tween-20); Carbonate buffer: 100 mМ NaHCO3, 100 mМ Na2CO3, pH = 9.6; AP buffer: 100 mM Tris, 100 mM NaCl, 5 mM MgCl2, pH = 9.6; Buffer for PLA2 activity: 50 mM Tris-HCl, 10 mM CaCl2, 50 mM NaCl, pH = 8.0; Transfer buffer (TB) for Western Blot: 25 mM Tris, 192 mM Glycine, pH = 8.3.

Anti-PLA antibodies

The following anti-PLA antibodies were used: anti-PLA2 single-chain fragment variable (scFv) antibodies, generated in the Department of Biochemistry (Stoyanova et al. 2012). Clone G15 specific for cellular PLA2 and clone H10 specific for secretory PLA2; rabbit anti-human PLA2G6 (iPLA2) antibody (Cusabio).

Phospholipase activity assay

Tested serum samples of SLE patients were centrifuged at 12000 rpm/4 °C for 10 min and then measured spectrophotometrically at 280 nm to determine the total protein concentration. Next, sera were diluted at a 1:100 ratio in PBS and incubated in a microtiter 96-flat bottom plate at 25 μl serum sample/well. Buffer for PLA2 activity was added at 220 μl/well. NOBA substrate (4-Nitro-3-(octanoyloxy) benzoic acid, Enzo Life Sciences, diluted to 1 mg/mL in acetonitrile) was added at 5 μl/well. The control wells without serum contained 5 μl of NOBA substrate solution and 220 μl of buffer for PLA2 enzyme activity. Another type of control well was coated with pooled sera from 24 healthy donors, and the wells were treated with the same reagents as the wells containing the SLE serum samples. The microtiter plate was incubated for 15 min at 37 °C and then measured at 450 nm with a microplate reader (DR-200B, Hiwell Diatek Instruments, Wuxi, China). All samples were analyzed in triplicate. The PLA2 activity in tested serum samples was expressed as specific activity (IU/protein, mg/mL). The relative PLA2 activity was presented in percentage following the equation:

PLA2 activity [%]= PLA2 specific activity of SLE serum sample  PLA2 specific activity of pooled sera from healthy donors ×100

ELISA analysis for the detection of autoantibodies against C3 and factor H

Microtiter 96-flat bottom plates were coated with C3 and factor H (both purchased from Quidel, Complement Technology, Inc.) at 1 μg/well in carbonate buffer for 1 h/37 °C and blocked with 0.5% Tween-20 (200 μl/well) for 1 h/37 °C. Tested SLE sera were diluted 1:100 in TPBS and incubated at 100 μl/well overnight/4 °C. The plates were washed three times with TPBS (200 μl/well), and then they were incubated with rabbit anti-human IgG-AP (Santa Cruz, 1:2000 dilution in TPBS, 100 μl/well) for 1 h/ 37 °C. The wells were washed three times with TPBS (200 μl/well), and bound complexes were detected by p-Nitrophenylphosphate (Acros Organics, working concentration: 0.5 mg/mL) in AP buffer at 100 μl/well. The absorbance was read at 405 nm with a microplate reader (DR-200B, Hiwell Diatek Instruments, Wuxi, China). Each serum sample had an individual control with an immobilized serum sample and an AP-conjugated antibody. All samples were analyzed in triplicate, and their standard deviation (S.D.) was calculated.

SDS-PAGE analysis

Tested SLE sera with increased PLA2 activity were diluted 1:100 in PBS and run in 5%–15% or 5%–12% non-reducing electrophoresis gel at a final protein concentration of 0.01 μg/mL per well. PageRuller™ Unstained Protein Ladder (Thermo Fisher Scientific™ Inc.) was used as a molecular weight standard. The gel was fixed in a buffer containing 25% i-propanol and 10% CH3COOH for 15 min, and then stained with 0.006% Coomassie Brilliant Blue G-250 in 10% CH3COOH for 15 min. The protein molecular weight was estimated with ImageJ software.

Western blot

Tested SLE sera with an estimated increased level of PLA2 activity were run on 5–15% or 5–12% non-reducing SDS-PAGE, then transferred to the nitrocellulose membrane by semi-dry electrotransfer in TB buffer for 1 h. The complete transfer of proteins was estimated with MemCode™ Reversible Protein Stain (Thermo Fisher Scientific), and then the signal was erased with MemCode™ Stain Eraser (Thermo Fisher Scientific). The membrane was blocked with 0.5% Tween-20 in PBS for 1 h/37 °C. Detection of cellular PLA2 enzymes and secretory PLA2 enzymes was performed by incubation of anti-PLA2 scFv clones G15 and H10, respectively (1:30 in TPBS, overnight/4 °C). The scFv/G15 and scFv/H10 were detected by incubation of mouse anti-c-Myc clone 9E10 (Merck Millipore, 1:2000 in TPBS) for 1 h/37 °C. Next, the membrane was incubated with goat anti-mouse IgG-AP (Agrisera, 1:2000 in TPBS).

Detection of iPLA enzymes was performed by incubation of the blotted serum proteins with rabbit anti-human iPLA2 (1:500 in TPBS overnight/4 °C), followed by incubation with goat anti-rabbit IgG-AP (Agrisera, 1:2000 in TPBS for 1 h/37 °C). The colorimetric detection was carried out with the substrate solution: 33 μl 5-bromo-4-chloro-3-indolyl phosphate (BCIP, 50 mg/mL in 100% DMSO) mixed with 66 μl nitro-blue tetrazolium (NBT, 50 mg/mL in 70% DMSO) in 10 mL AP buffer. After each incubation, the membranes were washed three times with TPBS. Before incubation with the substrate solution, the membranes were washed once more with PBS. The molecular weight of detected proteins was estimated with PageRuler™ Prestained Protein Ladder (Thermo Fisher Scientific) using ImageJ software.

Statistical analysis

Statistical analysis was performed in GraphPad Prism software version 8.0.1. A Wilcoxon matched-pairs signed rank-sum test was used for nonnormally distributed differences between the pairs. The Pearson correlation was used to analyze the correlation. Statistical significance was considered to be p < 0.05.

Results

We analyzed the PLA2 activity in serum samples from 48 patients with active SLE. The measured triplicate values for each tested serum were averaged, and the respective S.D. values were subtracted. The cut-off value was determined by calculating the sum of the averaged triplicate values for the healthy donors and the respective S.D. The analyzed sera were considered positive for increased PLA2 activity if they exceeded the cut-off. The screening revealed that 26 of 48 SLE patients (54.16%) were positive for increased PLA2 activity, and 22 of 48 SLE patients (45.84%) were below the cut-off of the control group (Fig. 1).

Figure 1. 

Measurement of PLA2-specific activity in serum samples of SLE patients. 1 dot = 1 serum sample; healthy donors: pooled sera from healthy donors (n= 24). The horizontal dotted line indicates the cut-off value.

We performed an ELISA analysis to measure the level of anti-C3 antibodies and anti-FH antibodies in the tested serum samples. The cut-off value was calculated likewise as with the PLA2 activity. In the cohort of SLE patients, 15 of 48 (31.25%) were positive for anti-C3 autoantibodies, and 11 of 48 (22.92%) were positive for anti-FH antibodies (Fig. 2A). The anti-FH antibodies tended to occur more rarely but to be of higher affinity compared to the anti-C3 antibodies (Fig. 2B). Interestingly, the individuals with the highest titers of anti-FH were negative for anti-C3.

Figure 2. 

ELISA assay for the presence of autoantibodies against C3 and factor H in SLE patients. Panel A:1 dot = 1 serum sample; healthy donors: pooled sera sample from healthy donors (n = 24). The horizontal dotted line indicates the cut-off value. Wilcoxon matched-pairs signed rank test (panel B).

The frequency of anti-C3 antibodies in the tested sera of SLE patients weakly correlated with PLA2 activity (r = 0.232, p = 0.113) (Fig. 3A), whereas there was no significant correlation between the frequency of anti-FH antibodies and PLA2 activity (r = 0.057, p = 0.698) (Fig. 3B). However, there was a moderate correlation between anti-C3 autoantibodies and anti-FH antibodies in tested SLE serum samples (r = 0.345, p = 0.016) (Fig. 3C).

Figure 3. 

Correlation analysis between PLA2 activity and the presence of autoantibodies against C3 and factor H in sera of SLE patients. 1 dot = 1 serum sample, Pearson correlation.

Our next aim was to determine the PLA2 isoform in tested serum samples of SLE patients. We selected sera that showed increased PLA2 activity, and we performed a western blot assay using antibodies against 3 types of PLA2 enzymes: secretory PLA2 (sPLA2), cellular PLA2 (cPLA2), and calcium-independent PLA2 (iPLA2). All analyzed SLE patients were positive for the iPLA2 enzyme, and some of them were positive for all 3 types of tested isoforms of PLA2 enzymes. The estimated molecular weight of the detected proteins was 250 kDa (Fig. 4). In some serum samples, an additional protein band at 220 kDa was detected.

Figure 4. 

Western blot analysis of PLA2 enzyme isoform in SLE patients with increased PLA2 activity. 1–11: serum samples of SLE patients with increased PLA2 activity. The estimated molecular mass of proteins is expressed in kilodaltons (kDa).

Discussion

In the last few years, phospholipase enzymes have been intensively studied for their pro-inflammatory characteristics and their involvement in neurodegenerative and autoimmune diseases. In the current research, we analyzed the relationship between increased phospholipase levels in SLE patients and the maintenance of inflammation as a prerequisite for the appearance of autoantibodies specific for complement proteins. Our data showed a weak correlation between increased PLA2 activity and the presence of anti-C3 antibodies and no correlation between increased PLA2 and anti-factor H antibodies in tested SLE patients. This result suggested that the PLA2 contribution to the inflammatory process is weakly influenced by the generation of autoreactive antibodies against C3 and factor H. SLE is characterized by a wide spectrum of autoantibodies of different specificities, and clearly, anti-C3 and anti-FH are not the primary triggers for the inflammatory basis of SLE. Besides, the induction and maintenance of inflammation in SLE depend on multiple factors, such as the action of other pro-inflammatory molecules generated by the innate immune cells, which could explain the weak correlation between increased PLA2 activity and the presence of analyzed autoantibodies against the complement proteins C3 and factor H.

The observed frequency of 31.25% (15 of 48) positive SLE patients for anti-C3 autoantibodies is within the range of 30%–32% estimated in previous studies with SLE patients (Durand and Burge 1984; Kenyon et al. 2011). Moreover, a similar frequency of 30% of the anti-C3 antibodies was observed in a studied cohort of patients with LN (Vasilev et al. 2015). While the anti-C3 antibodies are typical for lupus and lupus-associated diseases, the autoantibodies against regulatory complement proteins like factor H antibodies are occasionally observed in patients with SLE and LN (Hristova and Stoyanova 2017; Foltyn Zadura et al. 2012; Li et al. 2020). In our study, we found that 11 of 48 (22.92%) SLE patients had increased FH autoantibody frequency compared to the control group. In another study analyzing LN patients, it was found that 8.3% (10/120) of them were positive for anti-FH antibodies (Li et al. 2020). These results showed that complement C3 is a more common autoantigen in SLE and LN than factor H, and the molecular mechanisms of its autoantigenicity have to be studied in detail in further experiments.

The western blot assay showed a PLA2 fraction that is recognized by three different antibodies specifically binding sPLA2, cPLA2, and iPLA2 enzyme isoforms, respectively. These results could be related to antibody cross-reactivity due to similar binding epitopes in these three types of PLA2 enzymes. We have detected total PLA2 enzyme activity, which is due to the cell relationship and interconnection of all phospholipase isoforms as well as their possibilities of forming oligomeric complexes with molecular masses of 250 kDa or even larger (Murakami et al. 1997). Another study found that in the initial phase of inflammation, iPLA2 played a major role in the generation of pro-inflammatory molecules, such as prostaglandins, leukotrienes, and interleukin-1, whereas the cellular and serum phospholipases were activated in later stages of the inflammatory process (Gilroy et al. 2004), which could explain the lack of signal that corresponds to the molecular mass of sPLA2 and iPLA2 enzymes.

Conclusion

We found that the serum level of PLA2 enzyme activity was increased in a small group of analyzed patients, and the PLA2 activity in SLE is weakly influenced by the presence of anti-complement autoantibodies. Other factors contribute as well to the increase in PLA2 activity during the development of SLE. A possible presence of iPLA2 enzymes or an oligomeric form of sPLA2/cPLA2 enzymes was detected in the analyzed patients.

Acknowledgments

This study was financed by grant KP-06-M51/1 of the Bulgarian NSF and by the European Union-NextGenerationEU, through the National Recovery and Resilience Plan of the Republic of Bulgaria, project № BG-RRP-2.004-0008-C01.

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