Serum leucine-rich alpha-2 glycoprotein levels in rheumatoid arthritis and spondyloarthritis: A promising biomarker

Ethics statement

This study has received approval from the Research Ethics Committee of Faculty of Medicine Universitas Indonesia and Dr. Cipto Mangunkusumo National Central Public Hospital (KET-197/UN2.F1/ETIK/PPM.00.02/2022) on 28 February 2022. Written informed consent for their participation in the study and publication of the patients’ details was obtained from each of the patients. The patients were anonymized and identified through the medical record number.

Selection and recruitment of subjects

This research was a cross-sectional study conducted at the Rheumatology Polyclinic of Dr. Cipto Mangunkusumo National Central Public Hospital (RSCM) (Jakarta, Indonesia) from March to May of 2022. This study is reported in line with the STROBE guidelines.²⁴ The research subjects were recruited by consecutive sampling. The inclusion criteria included: patients aged over 18 years old; confirmed seropositive active RA patients in accordance with the disease activity score-28 for RA (DAS28); active SpA patients in accordance with the classification criteria of each disease, namely AS disease activity score (ASDAS) for AS and peripheral SpA and disease activity index for PsA (DAPSA) for PsA; and (4) patients able to fill out the consent form and are willing to participate in the study. Patients with other systemic autoimmune diseases, acute infectious diseases, liver disorders, and malignancies were excluded from the study.

This study also divided the subjects to several subgroups. C-reactive protein (CRP) levels were measured and patients with elevated CRP, defined as greater than 5 mg/dL, were analyzed separately. The LRG levels of both RA and SpA patients were also analyzed separately based on their disease activity and disease onset. Naïve patients, defined as patients with no prior exposure to conventional or disease-modifying anti-rheumatic drugs (DMARD), were categorized as “new onset”. Meanwhile patients who have received DMARD were categorized as “old onset”. Appropriate criteria cut-off points were used according to the corresponding disease in determining disease activity. Determination of sample size can be found as Underlying data.²⁴

Measurement of leucine-rich alpha-2 glycoprotein and c-reactive protein levels

Blood samples collected from patients were analyzed for LRG and CRP levels in an ISO-certified laboratory at Universitas Indonesia. To extract the serum, 6 mL of whole blood was drawn and centrifuged (Heraeus Labofuge 200, Thermo Fisher Scientific, Waltham (MA), USA) at 3,000 rpm for 15 minutes and 2,000 rpm for 5 minutes for LRG and CRP, respectively. LRG measurement was performed with Human LRG ELISA Assay Kit (Immuno-Biological Laboratories Co., Ltd., Fujioka, Gunma, Japan) and read with spectrophotometry at 450 nm. Meanwhile, CRP measurement was performed with immunoturbidimetry using cobas c 311 analyzer (Roche Holding AG, Basel, Switzerland).

Statistical analysis

Statistical analyses of the collected data were processed using IBM SPSS Statistics (RRID:SCR_016479) 24.0 for Mac program (IBM Corp., Armonk (NY), USA). Comparison of LRG levels were analyzed using the parametric independent samples t-test or Mann-Whitney non-parametric test depending on the distribution of the data, which was determined via the Shapiro-Wilk test.

Leucine-rich alpha-2 glycoprotein levels in rheumatoid arthritis and spondyloarthritis based on age, sex, and onset

This study recruited 26 RA patients and 26 SpA patients who visited the RSCM Rheumatology Polyclinic from March to of May 2022 and met the inclusion and exclusion criteria of the study. The sex of the RA subjects was predominantly female, aligning with the theory that women have a 2–3 times higher risk of developing RA than men. However, it was reported that no meaningful difference exists in RA incidence between men and women men over 70 years old.⁵ There is strong evidence that autoimmunity is influenced by genetics and sexual chromosomes. For instance, estrogen has been shown to increase the secretion of pro-inflammatory cytokines and exert anti-apoptotic activity and its receptors are found on various immune cells. Hence, estrogen is associated with the development of RA.²⁵

The majority of SpA subjects in this study were female. This predilection was also observed in the SpA subgroups, namely AS and PsA. Previous study by Baumberger et al., had shown that the majority of AS patients are male, with a ratio of 57:1 in 1980 and 1.03:1 in 2016.²⁶ This is also supported by a study conducted by Tsui et al., which had shown that the presence of the tissue non-specific alkaline phosphatase (TNAP) haplotype, which interacts with the progressive ankylosis protein homolog (ANKH) gene (a gene that is involved in osteogenesis and ankylosis in AS), is associated with AS in men.²⁷ Although AS is more dominant in men than women. Another subtype of SpA, namely PsA, affects men and women equally.^28–30 Sex distribution is related to disease presentation because men tend to have axial involvement, whereas peripheral joint involvement is more commonly seen in women. Kennedy et al., reported that the ratio of male to female sex in PsA subjects is 2.1–4.8:1. However, it should be noted that the study took many samples with axial involvement, which is more commonly seen in men.³¹ Meanwhile, Nishina et al., reported a different male-dominated result in peripheral SpA patients with a ratio of 2.3:1.6.³²

The mean age of RA subjects in this study was 49 years old. Other studies show that the highest incidence of RA cases is in the 50–54 year age group.³³ The incidence of RA tends to increase with age. The SpA subjects had a mean age of 41 years, which was consistent with the mean age of the SpA subjects reported by Kennedy et al., i.e., under 50 years old.³¹ The mean age of the AS subjects in this study was 38 years old. In one study, 92% of patients with AS were less than 45 years old at disease onset.³⁴ The mean age of the PsA subjects in this study was 41 years old. Research by Deike et al., showed that most cases of PsA began at 50–59 years old. The incidence of PsA increases with age, peaks before the age of 60 and declines aftewards.³⁵ The age of the sole female peripheral SpA patient in this study was 48 years old, consistent with the findings of de Winter et al., which stated that the median age of patients with peripheral SpA was 48 (36–55) years old.³⁶

Increased LRG levels were observed in the majority of both RA and SpA subjects. The highest median LRG levels were found in the RA group and the median CRP level was also higher in patients with RA. However, normal LRG results were found in six RA subjects (23.1%) and four SpA subjects (15.4%). The elevated LRG levels is consistent with findings by Birkelund et al., wherein joint fluid analysis in RA and SpA subjects showed striking differences in the number of proteins from the innate immune system, especially those from neutrophil granulocytes, including LRG. This protein was found in higher concentration in the RA patient group.²² Moreover, Nakajima et al., reported that LRG is able to differentiate RA from PsA, as shown by the significant increase in LRG levels in patients with RA compared to those with PsA. The increase in LRG concentration may be associated with higher levels of IL-6 in RA, thus increasing the activity of LRG expression.²³

LRG is a protein secreted during the inflammatory process, which like CRP, is also an acute phase protein. In patients with RA, serum LRG levels were correlated with DAS28-ESR score and CRP level.¹⁹ From the six RA subjects who had normal LRG levels in this study, two had normal CRP levels. In addition, all RA patients with normal LRG levels were old onset patients who have previously received DMARD. Methotrexate is a DMARD that lowers IL-6, an important cytokine in the formation of LRG.³² The same is true for SpA subjects; from the four subjects with normal LRG levels, there was one subject with normal CRP levels. All subjects classified as old onset had received DMARD therapy in the form of methotrexate and sulfasalazine. Hence, the observed normal CRP levels and the effect of DMARD treatment may explain the presence of subjects with normal LRG levels in this study.

Based on disease onset, five RA subjects were classified as new onset patients, while 21 subjects were classified as old onset patients. LRG levels showed higher results in RA subjects classified as new onset patients. As for the SpA subjects, one subject was categorized as new onset and 25 other subjects were categorized as old onset. Aside from being untreated, the higher level of LRG observed in subjects with new onset disease may be related to IL-6 expression in the early stages of the disease. As suggested by animal studies, synovial tissue samples of animals with antigen-induced arthritis (AIA) and adjuvant-induced arthritis (AA), revealed that the expression of IL-6 peaked in the early stages of the disease and decreased in the later stages of the disease.³⁷ Another study also found that IL-6 is expressed in the sacroiliac joints AS subjects of new disease onset.³⁸

The effect of comorbidity and treatment on leucine-rich alpha-2 glycoprotein levels

In this study, 17 RA subjects had comorbidities, among them were type 2 diabetes mellitus (T2DM), osteoarthritis (OA), fibromyalgia, controlled hypertension, and pregnancy of eight weeks. The highest concentrations of LRG were found in RA patients who also had OA and fibromyalgia. Of all the subjects with SpA, 14 had comorbidities, including controlled hypertension, IBD, dyslipidemia, T2DM, osteoporosis, OA, and hyper-IgE syndrome. The highest LRG levels were obtained in subjects without comorbidities as they happen to be new onset patients.

Several studies support the prevalence of DM in patients with RA. TNF-α and IL-6 are associated with the pathogenesis of DM, insulin resistance and RA. Long-term exposure to elevated levels of IL-6 triggers insulin resistance, which may play a role in the incidence of DM in patients with RA.³⁹ Plasma LRG levels can predict the progression of kidney disease and the incidence of albuminuria in patients with DM. LRG levels in subjects with albuminuria were significantly higher than subjects with stable albuminuria or those in regression. LRG levels in DM subjects with chronic kidney disease (CKD) progression were also much higher than subjects with normal kidney function.⁴⁰ In another study, LRG levels were twice as high in subjects with end-stage renal disease (ESRD) who had been treated with hemodialysis than in patients with stage 2/3 CKD.⁴¹ Prospective study in Southeast Asia in subjects with T2DM stated that high plasma LRG levels were associated with an increased risk of heart failure events.⁴² LRG is a novel factor in the pathogenesis of heart failure in patients with T2DM. Elevated serum LRG is also associated with peripheral arterial disease and several risk factors for cardiovascular diseases, such as arterial stiffness and endothelial dysfunction.⁴³ Furthermore, LRG levels were found to be elevated in subjects with IBD, as shown by the elevated levels of IL-6 in the colonic mucosal tissue of patients with IBD.⁴⁴

LRG levels are also elevated in other inflammatory diseases such as OA. In a murine model of OA, increased LRG expression was found in the subchondral bone and articular cartilage.⁴⁵ The increase in LRG is also associated with IL-6 as it is one of the pro-inflammatory cytokines that is elevated in OA synovial fluid samples.⁴⁶ In addition, LRG is also a biomarker of fibromyalgia.^23,47,48 As serum IL-6 levels are elevated in subjects with fibromyalgia, it is suggested that fibromyalgia may affect the assessment of inflammatory activity in patients with RA.⁴⁹ This is in line with the results of this study, which showed that RA subjects with OA and fibromyalgia had the highest concentration of serum LRG.

The highest levels of LRG in RA subjects were found in subjects who had not received DMARD therapy, aligning with previous studies that showed that DMARD therapy reduced IL-6 concentration in patients with RA.⁵⁰ As for AS subjects, the highest LRG levels were found in subjects with a combination of DMARD and steroid treatment, while the lowest was in the combination of DMARD and paracetamol. In PsA subjects, the highest LRG levels were found in the DMARD monotherapy group and the lowest in the DMARD and steroid combination group. It can be inferred that DMARDs and steroids play a role in regulating IL-6 and blocking NF-κB as promoters of LRG synthesis.^32,50,51

Leucine-rich alpha-2 glycoprotein levels in rheumatoid arthritis

LRG levels were found to be increased in 76.9% of subjects with RA, with the highest median LRG level observed in subjects with RA (77.03 vs. 68.67 μg/mL). These results align with the studies of Fujimoto et al., Naka et al., and Serada et al., which reported the elevation of serum LRG in various autoimmune diseases, including RA.^19,52,53 Ha et al., also showed that LRG can differentiate RA patients from normal controls as serum LRG concentrations were significantly increased in patients from the RA group compared with patients in the control group.⁵⁴ LRG stimulation is influenced by pro-inflammatory cytokines, one of which is IL-6, an inflammatory cytokine that is critically involved in the pathogenesis of RA and significantly correlated with serum LRG levels.⁵⁵ Serum and synovial IL-6 levels are elevated in RA patients and are associated with disease activity.⁵⁶ Previous study conducted by Madhok et al., showed that RA patients had higher levels of IL-6 (55 (28–139) IU/mL) compared with the control group (10 (7–12) IU/mL).⁵⁷

Leucine-rich alpha-2 glycoprotein levels in spondyloarthritis

Increased LRG levels were seen in 84.6% of subjects with SpA, with a median of 68.67 (33.15–115.18) μg/ml. Nakajima et al., previously reported that LRG levels were increased in patients with PsA (51.1 ± 32.9 μg/mL) and psoriasis vulgaris (37.8 ± 25.1 μg/mL) compared with controls (25.5 ± 11.5 μg/mL).²³ A multicenter retrospective observational study showed that serum levels of IL-6 are elevated in AS and PsA subjects.³⁸ A previous study also reported that the mean IL-6 level was significantly higher in AS subjects compared with controls.³⁸ The highest median LRG levels in SpA subjects were found in the PsA subset in the present study.

Differences in levels of leucine-rich alpha-2 glycoprotein in rheumatoid arthritis and spondyloarthritis

This study found no significant difference between LRG levels in subjects with RA and SpA. This result is different from a previous study by Lee et al., LRG was also previously identified as a protein that can differentiate RA from PsA as shown by the significantly increased LRG levels in MRM analysis of patients with RA.⁵⁸ In another study of 32 synovial fluid samples from RA patients and 24 synovial fluid controls, it was found that LRG in RA synovial fluid and SpA correlated significantly with synovial fluid circulating free DNA (cfDNA), and it was known that LRG was closely related to plasma CRP.⁵⁹

The insignificant difference in this study may be due to other factors affecting LRG levels, including disease onset and treatment received. However, the effects of DMARD treatment on LRG levels in RA and SpA subjects have not yet been studied. Old onset subjects predominated this study. Disease onset is related to IL-6 concentration. IL-6, a known precursor of LRG, increases in both mild and severe inflammatory conditions. Higher LRG levels in subjects with new-onset may be related to IL-6 expression in the early stages of the disease, as suggested in a study by Ferraccioli et al., which showed that IL-6 is a pro-inflammatory cytokine that is expressed in the early stages of the disease and plays a role in pathogenesis.³⁷ Expression of IL-6 in sacroiliac joint biopsies of AS patients is also associated with new disease onset.³⁸ Besides IL-6 activity, disease onset is also related to the patient's initiation of the treatment regimen. Old onset patients in the present study were defined as patients who had been diagnosed before the time of sampling and had received DMARD. Studies have shown that RA subjects receiving DMARD therapy have decreased serum concentrations of IL-6, a known precursor for LRG formation.⁶⁰ Most of the research subjects also received DMARD treatment in the form of sulfasalazine; from the results of previous studies, it was known that sulfasalazine caused a decrease in LRG levels in four female albino rats suffering from ulcerative colitis.⁵¹ The effect on subjects with SpA and RA is still unknown.

Because this study initially did not consider the level of disease activity, there was a possibility for the patients’ disease activity to be distributed unevenly. The results of this study found differences in the predominant disease activity between the two groups, wherein the RA group was predominated by moderate disease activity, while the SpA group was predominated by high to very high disease activity. The insignificant difference in LRG levels between the two groups in this study may be due to this distribution of disease activity. The difference in LRG levels between RA and SpA subjects may be more apparent if the disease activity between these two groups were controlled. A previous study conducted by Ha et al., only compared LRG levels between active and inactive RA patients, where serum LRG concentrations were found to be significantly higher in patients with active RA compared with patients with RA in remission.⁵⁴ No previous study has compared LRG levels in moderate and high disease activity; the said study did not distinguish between disease activity groups. Meanwhile, we found different levels of LRG in the moderate and high to very high activity groups.

Leucine-rich alpha-2 glycoprotein levels in rheumatoid arthritis and spondyloarthritis subjects with increased c-reactive protein levels

Median LRG levels based on increased CRP levels showed higher results in RA subjects than in SpA. The Mann-Whitney test results showed no significant difference in LRG levels in RA and SpA subjects with increased CRP levels. Previous studies have stated that LRG is correlated with CRP levels, as such a positive correlation between the two variables were expected.⁵⁵ Future research should confirm this finding.

Research strengths and limitations

To the knowledge of the researchers, this is the first study to compare LRG levels of patients with RA and generalized SpA. The limitation of this study was the different predominant disease activity in the two groups. Future studies with a larger subject population along with control of DMARD status and disease activity is warranted.