Pemphigus vulgaris (PV) is a life-threating organ-specific autoimmune blistering diseases of the mucous membranes and/or the skin. It is characterized by autoantibodies directed against desmogleins (Dsg) (transmembrane desmosomal cadherins) 1,2.
Patients with PV usually develop first mucous membrane erosions which are followed several months later by cutaneous flaccid blisters. Mucosal PV patients have anti-Dsg 3 autoantibodies while mucocutaneous PV patients develop autoantibodies against Dsg 1 in addition to Dsg 3 3–5.
Autoimmune diseases are characterized by loss of self-tolerance due to altered B-cell function. B cells contribute to the development of autoimmune diseases through autoantibody production, antigen presentation, in addition to cytokine production 6. A proliferation-inducing ligand (APRIL) and B-lymphocyte stimulator (BLyS) are members of the tumor necrosis factor superfamily. They bind to B-cell-expressed receptors, mediating B-cell survival, proliferation, and differentiation into both antibody-secreting plasma cells and long-lived memory B cells 7,8.
APRIL is an important factor modulating B-cell tolerance and homeostasis 9. It is expressed by a variety of cells including monocytes, macrophages, dendritic cells, and T cells 10,11. APRIL modulates the function and survival of antigen-experienced B cells and it is related to establishing the long-lived plasma cell pool in the bone marrow 12–15.
APRIL and BLys levels were found elevated in the sera and target organs of several autoimmune disease patients and even their levels correlate with disease severity and the level of autoantibodies in some of these autoimmune diseases 16–19. They are also considered as possible mediators of spinal cord injury-induced autoimmunity 20.
Treatment of PV is challenging, based on high-dose systemic corticosteroids and immunosuppressants. Although the currently available treatments are effective, they are associated with side effects 21. PV patients are in need for new drugs, more pathogenesis focused, exhibiting fewer side effects possibly replacing the use of broad immunosuppressive agents 3,22. The concept of targeting of B cells was tested and found effective in the treatment of autoimmune diseases. Anti-CD20 monoclonal antibody (rituximab) produced both rapid and long-term responses in a significant portion of pemphigus patients with a good safety profile. Therapeutic response to rituximab is associated with persistent B-cell depletion 23–25. Taking into consideration its important role in B-cell function, APRIL is to be considered therapeutic targets for novel therapies for B-cell-driven autoimmune diseases. Atacicept, the dual APRIL and BLyS inhibitor, was found effective in reducing total B cells, plasma cells, and serum immunoglobulin in systemic lupus erythematosus patients without increased risk of serious adverse events or severe infection 26,27.
Few studies have been conducted to evaluate the serum level of APRIL in patients with autoimmune blistering skin diseases and its relation to disease activity. The aim of this work was to evaluate the serum level of APRIL in patients with newly diagnosed active PV and compare it to both controlled PV patients on treatment and normal healthy participants.
Patients and methods
This case–control comparative study was conducted on 50 participants selected from the Outpatient Clinic of the Dermatology and Venereology Department of Alexandria Main University Hospital. They were divided into three groups:
Group A included 20 newly diagnosed PV patients who did not receive treatment with systemic steroids or immunosuppressant drugs; group B included 20 patients with PV in complete remission on therapy (absence of new or established lesions while the patient is receiving minimal therapy) 21; and group C included 10 normal healthy controls.
Informed consent was taken from the participants before the beginning of the study as well as the approval of the ethics committee of the Faculty of Medicine, Alexandria University.
Exclusion criteria for patients and control: history of diseases were associated with elevated serum level of APRIL either autoimmune disease (systemic autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis and organ-specific autoimmune diseases such as autoimmune hepatitis) or B-cell malignancy. For groups A and C, any participant who received any systemic steroid or immunosuppressive drugs during the past 3 months.
The patients were subjected to full history taking, general medical examination, and local dermatologic examination. All new PV patients were subjected to biopsy taken from a recent intact blister (lesional skin scalpel biopsies were formalin fixed and slides stained with hematoxylin and eosin were prepared and examined) to confirm the diagnosis, while controlled PV patients skin biopsies were reviewed to ensure the diagnosis.
Venous blood sample (3 ml) was withdrawn from all participants for quantitative determination of serum level of APRIL by enzyme-linked immunosorbent assay (ELISA) Kit (eBioscience, Germany), catalog number; 201-12-5350(48T) and prepared according to the manufacturer’s protocol. In brief, the kit uses a double-antibody sandwich ELISA to assay the level of human APRIL in samples. Add APRIL monoclonal antibody enzyme well which is precoated with APRIL monoclonal antibody, incubate and then add APRIL antibodies labeled with biotin and combined with streptavidin–horseradish peroxidase to form an immune complex; then carry out incubation and wash again to remove the uncombined enzyme. Then add chromogen solution A, B, and the color of the liquid changes into the blue. Owing to the effect of the acid, the color finally becomes yellow. The chroma of color and the concentration of the human substance APRIL of the sample were positively correlated.
Clinical and laboratory data were recorded and statistically analyzed using IBM SPSS Statistics for Windows, Version 20.0 (IBM Corp, Armonk, New York, USA). Comparisons between groups for categorical variables were done using the χ2-test. Student’s t-test was used to compare two groups for normally distributed quantitative variables, while analysis of variance was used for comparing multiple groups. Mann–Whitney test was used to compare two groups for abnormally distributed quantitative variables whereas Kruskal–Wallis test was used for comparing the four studied groups. Spearman’s coefficient was used to correlate between quantitative variables. Significance of the obtained results was judged at the 5% level.
Demographic data of the three studied groups are shown in Table 1. There was no significant difference between the three groups regarding age and sex distribution. The disease duration in group A ranged between 1 and 7 months with a mean duration of 2.78±2.16 months, while in group B it ranged between 12 and 60 months with a mean of 32.50±14.76 months. There was significantly longer mean disease duration in group B than in group A (P<0.001). All PV patients had clinically mucocutaneous affection (mucosal erosions and flaccid skin blisters) and the lesional skin biopsies showed suprabasal acantholysis in all of them. At the time of enrollment into the study, all group B patients were in complete remission on therapy. All of them were on systemic steroids in a dose range from 10 to 30 mg/day and five of them were on immunosuppressive drugs (azathioprine) in the dose range from 50 to 100 mg/day.
The mean serum APRIL level was 0.96±0.80, 0.53±0.15, and 0.49±0.08 ng/ml in group A, group B, and group C, respectively. Serum APRIL level was significantly higher in group A than in groups B and C. No statistically significant difference was found between groups B and C (Table 2, Fig. 1).
There was no significant correlation between serum APRIL level and duration of disease or the age of the patients (Table 3). There was no significant relation between serum APRIL level and sex in the three studied groups (Table 4).
APRIL is an important factor controlling B-cell function and allowing the survival of antigen-specific B cells. APRIL has been well studied in many autoimmune diseases and was found to be elevated. Its level also correlates in some of them with both disease severity and autoantibody levels. Few studied its role in dermatological diseases. When APRIL was studied in patients with atopic dermatitis, contradictory results were obtained 28,29. The concentration of APRIL was measured in patients with alopecia areata and was not significantly elevated when compared with controls 30.
Serum BLyS was studied in newly diagnosed PV and bullous pemphigoid (BP) patients and was compared with normal healthy participants by Asashima et al. 31. They found significant elevations of serum BAFF levels in patients with BP, but not with PV. According to their results, the BLyS levels appeared to increase when the skin lesions were actively spreading in the early stage of BP but not in PV.
Although BLyS is important for B-cell homeostasis it acts at early stages of B-cell development while APRIL seem to act later in maintaining both antigen-specific B cells and long-lived plasma cells 12–15. APRIL is expected to play a role in autoimmune blistering diseases as B cells are not only the precursors of antibody-secreting cells, but also have a regulatory role in immune responses. So we decided to study APRIL in one of the severe cutaneous autoimmune blistering diseases.
PV is an example of an autoimmune blistering skin disease where B cells play a central role. In the current study, serum APRIL levels were significantly elevated in patients with new active PV than both controlled PV cases on treatment and normal healthy participants. This is the first study to demonstrate difference in serum APRIL level between new active and controlled PV patients that might indicate role of APRIL in early development of PV as well as disease activity. In spite of the higher serum APRIL level in controlled PV on treatment than the normal healthy control the difference was not significant. This could be partially explained by the few number of normal healthy controls in the present study. The difference in serum APRIL level between controlled PV patients and normal healthy controls, in spite of being insignificant, may implicate that the serum APRIL level might decrease with treatment but still remaining higher than in normal healthy participants. This suggests a possible role of APRIL in recurrence or reactivation of the disease upon stopping the treatment which needs to be confirmed by further follow-up studies on relapsed PV patients.
In the present study, there was no association observed between age or sex and serum APRIL level in both PV groups. This is expected as the APRIL level was not previously reported to differ between men and women or to show change with aging. Serum APRIL level did not correlate with the duration of the disease in each of groups A and B, yet this could be explained by the significant difference in disease duration between new active PV and controlled PV cases. The mean disease duration in active PV cases in the present study was very short as this group included only newly diagnosed cases and excluding relapsed cases, whereas the duration of the disease was significantly higher in controlled PV patients on treatment. Yet we expect that if the relapsed active PV patients were also included and not just newly diagnosed cases, the results would be different.
In the study of Watanabe et al. 32, serum APRIL levels were measured using human APRIL EIA kit in 15 PV patients, 43 BP patients, and 15 normal controls. Serum APRIL levels in BP patients were significantly higher than those in the control group. PV patients tended to have higher levels of APRIL than normal control but the difference was not significant. No correlation was found between serum APRIL levels and anti-Dsg3 antibody levels in PV patients; also no correlation was found between serum anti-BP180 antibody levels with APRIL levels in BP patients. They also found a significant negative correlation between serum APRIL levels and disease duration in BP patients not in PV patients. Their explanation for the difference was that in their study PV patients had longer disease duration than BP patients. Unfortunately they did not show this data for comparison with ours. Serum APRIL levels were longitudinally assessed in three BP patients treated with oral corticosteroid and was found to decrease with treatment early before the level of anti-BP180 antibody started to decrease; yet they did not follow up the APRIL level in PV patients to see the change with treatment. Also they did not measure the level of APRIL during the disease-free period nor in PV patients controlled in remission.
Torkamani et al. 33 conducted a study on 22 active PV patients and 22 sex-matched and age-matched healthy controls. BLyS and APRIL levels were evaluated in patient and control groups by the use of ELISA. BLyS levels were undetectable in both PV patients and control sera. Serum APRIL level was higher in PV patients than the control yet the difference in APRIL levels between patient and control groups was not significant. They concluded that although BLyS and APRIL may play a role in autoimmune diseases, their role in PV is doubtable.
The contradiction between the results of the present study and the previous studies could be explained by the difference in the patient selection criteria and also by the difference in the number of PV cases. In the present study in the active PV patients group, we included only newly diagnosed untreated patients, but in previous studies the active PV group includes also relapsed cases of PV after discontinuation of the treatment that could affect the serum APRIL level which might be elevated during the early stage of the disease and may not return to the same high level with the relapse. Unfortunately, there is only few available data on APRIL in PV for comparison as most of the studies conducted concentrate more on BP.
From the present study, we could conclude that APRIL may play a role in the early initial active stage of PV development, and that active PV patients had significantly higher APRIL serum level than both controlled PV patients on treatment and normal healthy participants. APRIL could be considered as a potential marker of early initial attack of PV. Targeting the APRIL system may be a potential therapeutic goal in the management of PV.
Further longitudinal follow-up studies are required to detect the change in serum APRIL level in PV patients with treatment and correlate it with the change in the level of anti-Dsg antibodies and to detect whether the APRIL level will show further elevation with relapse of the disease or not. Comparative studies between a larger number of normal healthy controls and controlled PV patients are needed to show possible significant differences in serum APRIL level.
Further therapeutic trials are also needed to study the possible effectiveness of inhibiting APRIL pathway using atacicept on active PV patients and for detecting the change in serum APRIL level in them.
Conflicts of interest
There are no conflicts of interest.
1. Amagai M, Klaus-Kovtun V, Stanley JR. Autoantibodies against a novel epithelial cadherin in pemphigus vulgaris, a disease of cell adhesion. Cell 1991; 67:869–877.
2. Harman KE, Brown D, Exton LS, Groves RW, Hampton PJ, Mohd Mustapa MF, et al. British Association of Dermatologists’ guidelines for the management of pemphigus vulgaris 2017. Br J Dermatol 2017; 177:1170–1201.
3. Di Zenzo G, Amber KT, Sayar BS, Müller EJ, Borradori L. Immune response in pemphigus and beyond: progresses and emerging concepts. Semin Immunopathol 2016; 38:57–74.
4. Koga H, Ohyama B, Tsuruta D, Ishii N, Hamada T, Dainichi T, et al. Five Japanese cases of antidesmoglein 1 antibody-positive and antidesmoglein 3 antibody-negative pemphigus with oral lesions. Br J Dermatol 2012; 166:976–980.
5. Kamiya K, Aoyama Y, Yamaguchi M, Ukida A, Mizuno-Ikeda K, Fujii K, et al. Clues to diagnosis for unusual mucosal pemphigus demonstrating undetectable anti-desmoglein 3 serum antibodies by routine tests. J Dermatol 2015; 42:572–579.
6. Blüml S, McKeever K, Ettinger R, Smolen J, Herbst R. B-cell targeted therapeutics in clinical development. Arthritis Res Ther 2013; 15 (Suppl 1):S4.
7. Rickert RC, Jellusova J, Miletic AV. Signaling by the tumor necrosis factor receptor superfamily in B-cell biology and disease. Immunol Rev 2011; 244:115–133.
8. Cancro MP, D’Cruz DP, Khamashta MA. The role of B lymphocyte stimulator (BLyS) in systemic lupus erythematosus. J Clin Invest 2009; 119:1066–1073.
9. Mackay F, Silveira PA, Brink R. B cells and the BAFF/APRIL axis: fast-forward on autoimmunity and signaling. Curr Opin Immunol 2007; 19:327–336.
10. Mackay F, Schneider P, Rennert P, Browning JL. BAFF and APRIL: a tutorial on B cell survival. Annu Rev Immunol 2003; 21:231–264.
11. Litinskiy MB, Nardelli B, Hilbert BM, Bing H, Schaffer A, Casali P, Cerutti A. DCs induce CD40-independent immunoglobulin class switching through BLyS and APRIL. Nat Immunol 2002; 3:822–829.
12. Thompson JS, Bixler SA, Qian F, Vora K, Scott ML, Cachero TG, et al. BAFF-R, a newly identified TNF receptor that specifically interacts with BAFF. Science 2001; 293:2108–2111.
13. Schiemann B, Gommerman JL, Vora K, Cachero TG, Shulga-Morskaya S, Dobles M, et al. An essential role for BAFF in the normal development of B cells through a BCMA-independent pathway. Sci 2001; 293:2111–2114.
14. Belnoue E, Pihlgren M, McGaha TL, Tougne C, Rochat AF, Bossen C, et al. APRIL is critical for plasmablast survival in the bone marrow and poorly expressed by early-life bonemarrow stromal cells. Blood 2008; 111:2755–2764.
15. O’Connor BP, Raman VS, Erickson LD, Cook WJ, Weaver LK, Ahonen C, et al. BCMA is essential for the survival of long-lived bone marrow plasma cells. J Exp Med 2004; 199:91–98.
16. Mackay F, Sierro F, Grey ST, Gordon TP. The BAFF/APRIL system: an important player in systemic rheumatic diseases. Curr Dir Autoimmun 2005; 8:243–265.
17. Thangarajh M, Masterman T, Rot U, Duvefelt K, Brynedal B, Karrenbauer VD, et al. Increased levels of APRIL (a proliferation-inducing ligand) mRNA in multiple sclerosis. J Neuroimmunol 2005; 167:210–214.
18. Koyama T, Tsukamoto H, Miyagi Y, Himeji D, Otsuka J, Miyagawa H, et al. Raised serum APRIL levels in patients with systemic lupus erythematosus. Ann Rheum Dis 2005; 64:1065–1067.
19. Tan SM, Xu D, Roschke V, Perry JW, Arkfeld DG, Ehresmann GR, et al. Local production of B lymphocyte stimulator protein and APRIL in arthritic joints of patients with inflammatory arthritis. Arthritis Rheum 2003; 48:982–992.
20. Saltzman JW, Battaglino RA, Salles L, Jha P, Sudhakar S, Garshick E, et al. B-cell maturation antigen, a proliferation-inducing ligand, and B-cell activating factor are candidate mediators of spinal cord injury-induced autoimmunity. J Neurotrauma 2013; 30:434–440.
21. Hertl M, Jedlickova H, Karpati S, Marinovic B, Uzun S, Yayli S, et al. Pemphigus. S2 guideline for diagnosis and treatment – guided by the European dermatology forum (EDF) in cooperation with the European Academy of Dermatology and Venereology (EADV). JEADV 2015; 29:405–414.
22. Lotti R, Shu E, Petrachi T, Marconi A, Palazzo E, Quadri M, et al. Soluble fas ligand is essential for blister formation in pemphigus. Front Immunol 2018; 9:370.
23. Ahmed AR, Spigelman Z, Cavacini LA, Posner MR. Treatment of pemphigus vulgaris with rituximab and intravenous immune globulin. N Engl J Med 2006; 355:1772–1779.
24. Colliou N, Picard D, Caillot F, Calbo S, Le Corre S, Lim A, et al. Long-term remissions of severe pemphigus after rituximab therapy are associated with prolonged failure of desmoglein B cell response. Sci Transl Med 2013; 5:175ra30.
25. Leshem YA, Hodak E, David M, Anhalt GJ, Mimouni D. Successful treatment of pemphigus with biweekly 1-g infusions of rituximab: a retrospective study of 47 patients. J Am Acad Dermatol 2013; 68:404–411.
26. Dall’Era M, Chakravarty E, Wallace D, Genovese M, Weisman M, Kavanaugh A, et al. Reduced B lymphocyte and immunoglobulin levels after atacicept treatment in patients with systemic lupus erythematosus: results of a multicenter, phase Ib, double-blind, placebo-controlled, dose-escalating trial. Arthritis Rheum 2007; 56:4142–4150.
27. Pena-Rossi C, Nasonov E, Stanislav M, Yakusevich V, Ershova O, Lomareva N, et al. An exploratory dose-escalating study investigating the safety, tolerability, pharmacokinetics and pharmacodynamics of intravenous atacicept in patients with systemic lupus erythematosus. Lupus 2009; 18:547–555.
28. Chen Y, Lind Enoksson S, Johansson C, Karlsson MA, Lundeberg L, Nilsson G, et al. The expression of BAFF, APRIL and TWEAK is altered in eczema skin but not in the circulation of atopic and seborrheic eczema patients. PLoS One 2011; 6:e22202.
29. Matsushita T, Fujimoto M, Echigo T, Matsushita Y, Shimada Y, Hasegawa M, et al. Elevated serum levels of APRIL, but not BAFF, in patients with atopic dermatitis. Exp Dermatol 2008; 17:197–202.
30. Kuwano Y, Fujimoto M, Watanabe R, Ishiura N, Nakashima H, Ohno Y, et al. Serum BAFF and APRIL levels in patients with alopecia areata. J Dermatol Sci 2008; 50:236–239.
31. Asashima N, Fujimoto M, Watanabe R, Nakashima H, Yazawa N, Okochi H, et al. Serum levels of BAFF are increased in bullous pemphigoid but not in pemphigus vulgaris. Br J Dermatol 2006; 155:330–336.
32. Watanabe R, Fujimoto M, Yazawa N, Nakashima H, Asashima N, Kuwano Y, et al. Increased serum levels of a proliferation-inducing ligand in patients with bullous pemphigoid. J Dermatol Sci 2007; 46:53–60.
33. Torkamani N, Sarvestani EK, Naseri M. Investigation of serum APRIL and BAFF levels in pemphigus Vulgaris patients in Southern Iran. Iran J Dermatol 2011; 14:16–19.