AIM: To study anti-Epstein-Barr virus (EBV) IgG antibodies in Crohn´s disease in relation to treatment, immune cells, and prior tonsillectomy/appendectomy.

METHODS: This study included 36 CD patients and 36 healthy individuals (controls), and evaluated different clinical scenarios (new patient, remission and active disease), previous mucosa-associated lymphoid tissue removal (tonsillectomy and appendectomy) and therapeutic regimens (5-aminosalicylic acid, azathioprine, anti-tumor necrosis factor, antibiotics, and corticosteroids). T and B cells subsets in peripheral blood were analyzed by flow cytometry (markers included: CD45, CD4, CD8, CD3, CD19, CD56, CD2, CD3, TCRαβ and TCRγδ) to relate with the levels of anti-EBV IgG antibodies, determined by enzyme-linked immunosorbent assay.

RESULTS: The lowest anti-EBV IgG levels were observed in the group of patients that were not in a specific treatment (95.4 ± 53.9 U/mL vs 131.5 ± 46.2 U/mL, P = 0.038). The patients that were treated with 5-aminosalicylic acid showed the highest anti-EBV IgG values (144.3 U/mL vs 102.6 U/mL, P = 0.045). CD19⁺ cells had the largest decrease in the group of CD patients that received treatment (138.6 vs 223.9, P = 0.022). The analysis of anti-EBV IgG with respect to the presence or absence of tonsillectomy showed the highest values in the tonsillectomy group of CD patients (169.2 ± 20.7 U/mL vs 106.1 ± 50.3 U/mL, P = 0.002). However, in the group of healthy controls, no differences were seen between those who had been tonsillectomized and subjects who had not been operated on (134.0 ± 52.5 U/mL vs 127.7 ± 48.1 U/mL, P = 0.523).

CONCLUSION: High anti-EBV IgG levels in CD are associated with 5-aminosalicylic acid treatment, tonsillectomy, and decrease of CD19⁺ cells.

Crohn’s disease (CD) is one of the main types of inflammatory bowel disease (IBD). Although its etiology remains has not been described, it is known as a relapsing-remitting or progressive inflammatory condition that affects the gastrointestinal tract[1]. In a previous study, we observed a decrease of lymphocytes in the peripheral blood of patients with CD[2]. Lymphocyte populations did not differ between treated and untreated patients, with the exception of CD8⁺ T and CD19⁺ lymphocytes. Further, in a case control study, we concluded that CD patients are at risk for microsporidiasis, so we proposed that microsporidia, a group of opportunistic pathogens, may be involved in the etiology of CD[3]. The above-mentioned immune- deficiency may predispose individuals to be vulnerable to opportunistic infectious agents responsible for the disease.

Currently, the most common drugs used to treat CD are 5-aminosalicylic acid (5-ASA) drugs, corticosteroids, immunosuppressive agents, biologic therapies, and antibiotics[4]. Wagner et al[5] summarized the current literature surrounding the association between viruses and CD, and suggested that Epstein-Barr virus (EBV), a ubiquitous gamma herpes virus, may be associated with CD.

Viral transmission through the oropharyngeal mucosal epithelium is crucial for the entrance of the virus into the body. Mucosa associated lymphoid tissue (MALT) is essential in EBV infection because viral particles colonize the tongue, oropharyngeal mucosa, and salivary glands; later, they infect B cells, establishing the latent infection[6,7]. It has been suggested that extirpation of MALT, such as of tonsils and the appendix, could be related with CD, although there are many discrepancies about this theory[8-11].

The aim of this work was to study the prevalence of anti-EBV IgG antibodies in patients with CD, and its relation with different clinical situations, treatments, T and B cells subsets, and MALTectomy (tonsillectomy and appendectomy).

We determined IgG by ELISA against VCA of EBV in sera from 36 CD patients and 36 healthy controls. The development of VCA IgG antibodies usually takes place at the beginning of the disease, reaching the peak titers after two or four weeks and remaining positive thereafter[16].

It is interesting to highlight that the patients who were in the initial stages of the disease showed the lowest anti-EBV IgG levels. IgG levels progressively increased throughout the evolution of the disease, showing similar levels in the active disease until reaching the highest values in patients in remission, who showed similar levels to those obtained in the control group. In a previous study, we showed that a complex alteration of immune responses that affects the total numbers and function of γδ T cells was present in CD[2]. We also observed that CD4⁺ and CD8⁺γδ T cells were lower in new patients, and CD4⁺ and CD8⁺αβ T cells were significantly lower than their controls.

5-ASA, also known as mesalazine, is a structural analogue of aspirin that, along with other non-steroidal anti-inflammatory drugs (NSAIDs), is among the most conventional drugs currently used to treat CD. Mesalazine negatively regulates the cyclooxygenase-2/prostaglandin E2 axis[4,17], and also inhibits the synthesis of 5-lipoxygenase products, including leukotriene B4[18]. 5-ASA inhibits production of thromboxane A2 and consequently decreases adhesion to the endothelium and the aggregation of platelets and polymorphonuclear cells[19]. The pharmacologic profile of mesalazine includes an ability to inhibit intestinal macrophage chemotaxis. 5-ASA can exert antioxidant actions in vitro and in vivo[20]. These mechanisms could facilitate the entry of a virus through the mucous membranes and therefore they could increase the production of antibodies in treated patients.

We have previously demonstrated that levels of CD3⁺, CD4⁺, CD8⁺, and CD19⁺ lymphocytes were decreased in CD patients compared to the control group[2]. However, no correlations were observed among anti-EBV IgG levels and different lymphocyte subsets.

The lowest levels of anti-EBV IgG were observed in the new CD patients group and, according to the treatment, the lowest IgG levels were observed in the group of patients that were not receiving specific treatment. However, it is a well-known fact that the anti-inflammatory properties of 5-ASA reflect an inhibition of antibody synthesis[19]. On the other hand, it is interesting to know that other NSAIDs such as aspirin induce cell killing of EBV-positive B lymphocytes by reactivating EBV into lytic replication[21]. The induction of EBV lytic replication in host cells may produce the increase in anti-EBV IgG levels observed in the sera of 5-ASA-treated patients. Besides, the decrease of CD19⁺ cells in those patients may be the result of a cytotoxic effect of the drug on EBV-positive cells, which resulted in their death. Komatsuda et al[22] reported a case of sulfasalazine-induced hypersensitivity syndrome associated with reactivation of EBV with progressive cytopenia. It is important to note that not one of the new patients were in treatment, and as it was reported before, lymphocyte populations did not differ between treated and untreated patients, except in CD8⁺ T and CD19⁺ cells[2]. Consequently, there was an inverse relationship between CD19⁺ cells rates and anti-EBV IgG levels. In a study carried out by Magro et al[23] with IBD patients, the prevalence of EBV DNA was significantly higher in patients treated with 5-ASA compared to controls, though IgG-VCA positivity was not relevant for EBV DNA presence or load. In addition, the 5-ASA-treated group had a significantly higher percentage of patients with > 500 copies/mL compared to controls. This is in accordance with the induction of cell killing of EBV-positive B lymphocytes by reactivating EBV into lytic replication exerted by NSAIDs[21], which is reflected in the CD19⁺ decrease observed by us in the group of CD patients that had received treatment compared to the untreated ones.

Following a review of the literature, the relationship between appendectomy and CD is unclear. For example, Kurina et al[8] observed an association of prior appendectomy with a significantly increased risk of CD that was strongest in the year after the appendectomy. They concluded that CD could have been first misdiagnosed as appendicitis in these patients because they observed a reduction in risk of CD (considered as non-significant) when five or more years separated appendectomy and CD. Furthermore, Radford-Smith et al[9] showed an inverse association between appendectomy and CD and, conversely, Duggan et al[10] found no association with appendectomy for CD. In our study, only 9.8% of the subjects were appendectomized, and this number is too small to draw conclusions.

Our results indicate that tonsillectomy could act as a protective factor for CD. Although Kurina et al[8] did not observe an association between prior tonsillectomy and any increase or decrease of risk of CD, Duggan et al[10] reported that tonsillectomy showed a weak positive association with CD. However, Hansen et al[11] showed that tonsillectomy was negatively associated with the development of CD, which is in accordance with the results obtained in the present study, though they need to be analyzed with caution because of the small group sizes.

It is interesting to note that in the CD patient group, levels of anti-EBV IgG were significantly higher in the tonsillectomized group. In a previous work, we found that both specific antibody levels against Kudoa (a frequent fish parasite) and the number of patients with recognized Kudoa antigens were higher in tonsillectomy patients when compared to the control group[24]. This fact shows the role of tonsils as the first MALT defense line, suggesting that tonsils may play an important role in avoiding parasites and other infectious agents travelling along the digestive tract and preventing potential pathogenicity.

EBV has a tropism for the oral and nasopharyngeal tissues, and is one of the main viruses involved in the pathogenesis of recurrent tonsillitis and tonsillar hypertrophy[25]. We have previously demonstrated that MALTectomy significantly decreases secretory IgA levels in serum. The decrease of this IgG that plays a critical role in mucosal immunity was more pronounced when both surgeries (tonsillectomy and appendectomy) have been performed in the same patient[26].

Taking into account that tonsils exposed to EBV play an important role in controlling infection by EBV in the oropharynx it is not surprising that, in tonsillectomized patients, the dissemination of viruses takes place easily and, consequently, the antibody levels to EBV were higher in the sera from these patients.

In summary, high anti-EBV IgG levels in CD were associated with 5-ASA treatment, tonsillectomy, and decrease of CD19⁺ cells.

We would like to thank Anselmo Villar-Grimalt, MD, PhD, Head of Internal Medicine Department, Arnau de Vilanova Hospital and President of Association for Medical-Oncology Research, Valencia Spain, José Mayans, MD, Head of Hematology Department, and Miguel Bixquert-Jiménez, MD, PhD, for their unconditional support for our work.