Romaldini, Ceres C.*; Barbieri, Dorina*; Okay, Thelma S.*; Raiz, Roberto Jr.*; Cançado, Eduardo L. R.†
Celiac disease (CD) is a small-bowel enteropathy precipitated by ingestion of cereal proteins, in particular gliadin of wheat gluten and similar prolamins in rye and barley, in individuals who are genetically predisposed to the disease. The characteristic lesion consists of villous flattening, crypt cell hyperplasia, and infiltration of the lamina propria by lymphocytes, macrophages, and plasma cells (1). Withdrawal of gluten from the diet usually results in clinical remission and normalization of the intestinal morphology. The gold standard for the diagnosis is the histologic demonstration of the intestinal changes. Serologic tests based on the determination of CD-associated antibodies, antigliadin, antiendomysial, and antitissue transglutaminase, are commonly used for screening high-risk individuals and to assess the adherence to a gluten-free diet (2–4).
It is now evident that the pathologic process of this disorder is the result of an inappropriate T-cell mediated immune response against ingested gluten. Abnormal cytokines release from T lymphocytes and macrophages plays an important role in histologic changes (5,6). Antigen-reactive T cells produce interleukin-2 (IL-2) and express specific cell surface receptors for this molecule (IL-2R). IL-2 acts as a growth factor for T and B lymphocytes, stimulates other cells, and plays a critical role in the immune system (7). A circulating soluble form of the receptor (sIL-2R) is detectable in healthy individuals indicating a baseline level of immune activation under normal physiologic stimuli (8). Increased serum levels have been reported in certain disease conditions characterized by increased immune activation, such as rheumatoid arthritis and systemic lupus erythematosus (9–11). Interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) are mainly secreted by activated macrophages, and both cytokines are capable of up-regulating T-cell functions, increasing cytokines production by other cells, and mediating tissue toxicity (12,13).
Gliadin responsive T cells have been cloned from the small intestinal mucosa of celiac patients (14), and the cytokine pattern described (15,16). Increased production of Th1-type cytokines, in particular interferon (IFN)-γ and IL-2, as well as macrophage-derived cytokines, TNF-α and IL-6, have been described in the intestinal mucosa of untreated celiac patients (16–19). Previous studies have also shown that circulating mononuclear cells in untreated and challenged CD patients secrete cytokines compatible with a Th1/Th0 profile (20–22). However, there are few reports concerning the evaluation of these cytokines in the serum of CD patients, particularly in children (16,23). Therefore, in this study we investigated the concentrations of sIL-2R, IL-6, and TNF-α in serum of celiac children before and after gluten exclusion, and evaluated whether variation in serum cytokines levels might be useful as an indicator of response to treatment.
PATIENTS AND METHODS
This study was approved by the ethics committee of Hospital das Clínicas, Faculdade de Medicina da Universidade de São Paulo, and conducted on 54 children after written informed consent of their parents. All children were also tested for serum immunoglobulin-A antiendomysium antibodies (IgA-EMA) and for serum IgA to avoid an antibody false-negative result. We examined 12 patients with untreated CD (4 boys and 8 girls aged 1.0–12.5 years, median age 1.8 years) taking a normal diet at the time of the study. Eight of these 12 patients, who claimed strict adherence to a gluten-free diet, were also investigated at 6 and 12 months after gluten exclusion. A group of 16 treated CD patients (8 boys and 8 girls aged 3.0–15.0 years, median age 8.3 years) were also studied. All had been on gluten-free diet for at least two years. The control group included 26 healthy children (13 boys and 13 girls aged 2.0–13.5 years, median age 8.7 years) seeking routine annual health examinations. They had no clinical signs or symptoms that suggested CD. None of the patients and controls had atopic dermatitis, psoriasis, or any other disease of known immunologic mechanism or any infectious disease.
CD diagnosis was established according to the revised European Society for Pediatric Gastroenterology and Nutrition criteria (24). The diagnostic intestinal biopsy was performed with a per oral suction jejunal biopsy capsule under a fluoroscopic control. Active CD was diagnosed when typical changes were observed on tissue stained with hematoxylin and eosin.
Peripheral blood samples were collected after an overnight fast, processed within 30 minutes, and serum samples were stored in aliquots at -70°C, until assayed (sIL-2R, IL-6, TNFα, IgA and IgA-EMA).
Serum sIL-2R, IL-6, and TNFα levels were evaluated by enzyme-linked immunosorbent assay (ELISA) with the use of reagents and the directions supplied by the manufacturer (R&D Systems, Minneapolis, MN, USA). All samples were assayed in duplicate. The cytokine concentrations were quantified by comparison with a standard curve generated using the appropriated recombinant cytokine. Results were expressed in picograms per milliliter (pg/mL). The detection limit was 6.0 pg/mL for sIL-2R, 0.7 pg/mL for IL-6, and 4.4 pg/mL for TNF-α. Intra- and inter-assay coefficients of variation were less than 7% for all cytokine assays.
Serum IgA was determined by immunonephelometry with antisera to human IgA (Behring Diagnostics, Marburg, Germany). In our laboratory, the reference interval varies from 36 to 490 mg/dL, according to the child age (1 to 14 years old).
Serum IgA-EMA was detected on cryostat sections of unfixed human umbilical cord by indirect immunofluorescence. Briefly, 4μm sections were incubated at 37°C for 30 minutes with serum samples at a 1:5 dilution with phosphate-buffered saline (PBS)-Tween 80, 0.2%. After washing two times for five minutes with PBS 10mM phosphate buffered saline, the sections were incubated at 37°C for 30 minutes with fluorescein isothiocyanate goat anti-human IgA conjugate (Sigma, Saint Louis, MO, USA). The slides were washed again two times for five minutes, mounted in glycerin buffer and examined by fluorescence microscopy by one observer. Serum samples were considered positive when a reticular fluorescent pattern was observed surrounding the vessel smooth muscle fibers. All positive samples were analyzed with titration at increasing (two-fold) dilution to the end point. The highest dilution yielding a positive reaction was reported as the result. Both positive and negative reference sera were used for each batch. The results were expressed as IgA-EMA titers.
Comparisons between groups were assessed by Kruskall-Wallis test or Mann-Whitney U test as appropriate. Correlations between variables were analyzed by Spearman's rank correlation test. The level of significance was set at 0.05. All statistics were performed using GraphPad InStat, version 3.01 for Windows (GraphPad Software Inc., San Diego, CA, USA).
Serum values of sIL-2R, IL-6, and TNF-α in untreated and treated CD patients and controls are shown in Table 1. Significantly higher values of sIL-2R and IL-6 were observed in sera from untreated CD patients than in controls. However, there was no significant difference between the sIL-2R and IL-6 values in treated CD and control patients. When TNF-α levels were analyzed, no difference was found between untreated and treated CD patients and controls.
Table 2 shows the serum sIL-2R, IL-6, and TNF-α values in 8 CD patients determined before treatment, and after 6 and 12 months on a gluten-free diet and in control group. Furthermore, individual serum sIL-2R levels before and after gluten exclusion are illustrated in Figure 1.
Despite the progressive decrease of sIL-2R values after treatment, we observed only a significant reduction only at 12 months from pretreatment levels. The 12-month levels were still significantly higher than those found in controls (Table 2). The serum IL-6 and TNF-α levels were similar before gluten exclusion and at 6 and 12 months on treatment; however, the IL-6 values at 12 months were significantly higher than those in controls (Table 2).
All patients and control children had normal serum IgA levels thus excluding IgA deficiency (data not shown). Serum IgA-EMA titers were positive in untreated CD patients, (median 1:5 120; range 1:320 to 1:10 240) and were negative in all 16 treated celiac patients on gluten-free diet for at least two years, as well as in the control cases, resulting in a sensitivity of 100%. In longitudinal studies, the 8 patients showed a decrease in IgA-EMA titers at six months on treatment (median 1:5 120 vs. 1:320; range 1:320 to 1:10 240 vs. 1:10 to 1:2 560), but this change was not significant. However, the IgA-EMA titers decreased significantly at 12 months (median 1:160; range <1:5 to 1:320) compared with those before gluten exclusion (P < 0.001). A significant positive correlation was found between IgA-EMA titers and sIL-2R levels (r = 0.84, P < 0.0001) and moderate positive relationship was observed between IgA-EMA titers and IL-6 levels (r = 0.47, P = 0.02) when the values before treatment, at 6 and 12 months on a gluten-free diet were considered (Fig. 2). In contrast, no correlation between IgA-EMA titers and TNF-α values was found (r = 0.08, P = 0.70).
The interaction of IL-2 and IL-2R critically regulates the T-cell immune response following antigen activation. The release of the IL-2R (8), which occurs in proportion to their cell surface expression, the use of the serum sIL-2R measurement as an index of the immune activation.
In the present study untreated celiac patients have significantly elevated serum sIL-2R concentrations. These results are in accordance with the ones described in another pediatric population (23), as well as those reported in adults with untreated CD by Crabtree et al. (25) and Srivastava et al. (26). Our results also showed that sIL-2R values in CD patients on gluten-free diet for at least two years are comparable to those found in controls. Blanco et al. (23) have described no differences in sIL-2R levels between treated celiac patients with normal jejunal biopsies and controls, although the authors did not discuss how long the patients had been on treatment.
We found that the serum sIL-2R levels of celiac patients examined after a gluten-free diet for one year were lower than the pre-treatment levels, though they were still higher than those of controls. Crabtree et al. (25) also reported a significant difference in serum sIL-2R levels between untreated and treated celiac patients following gluten exclusion for a variable period (2 to 14 months). They also pointed out that the levels were higher in comparison with controls. These reports, as well as the results of our study showed that the clinical remission of the disease activity occurs before the reduction of serum sIL-2R levels to basal values (control group). The continued production of the serum sIL-2R, even after gluten exclusion for one year, suggests a long-term immunostimulation before treatment and the persistence of an active immune process, which might be explained either by the persistence of T lymphocytes auto-reactivity even after stimulus elimination, or by a slow and gradual return of lymphocytes to a quiescent state.
In in vivo studies, Kontakou et al. (27) have shown that the number of cells expressing mRNA for IL-2 increased significantly in the mucosa of treated celiac patients four hours after challenge with gliadin, and returned to pre-challenged values after six hours. Some investigators have also reported an increase in serum sIL-2R after only one-week of gluten ingestion and a significant decrease following four weeks on a gluten-free diet (25) in treated celiac patients. The serum sIL-2R rapid increase following gluten challenge suggests the involvement of circulating antigen-primed memory T cells bearing the CD45RO marker, identified among the peripheral blood mononuclear cells from CD patients (28).
Our study showed markedly higher serum IL-6 levels in untreated CD patients in comparison with controls and celiac patients on gluten-free diet for at least two years. These findings are in agreement with Fornari et al. (29) who demonstrated elevated serum IL-6 in untreated CD women and a significant decrease after three years on treatment. On the contrary, we have not found a significant difference between pre-treatment IL-6 values and those determined one-year after treatment, but these values were still higher in comparison with controls, suggesting a long-term immunostimulation before treatment. Furthermore, significant expression of IL-6 in small-intestinal mucosa of patients with untreated CD (16–18) and in peripheral T cells culture supernatants of celiac patients after gluten challenge have been already reported (20–22). These data and our findings demonstrated the relevance of IL-6 in the immune pathogenesis of the CD, knowing its important effect inducing the final maturation step of B cells into antibody-producing cells (12).
In contrast with sIL-2R and IL-6, we found no differences in serum TNF-α levels between the groups. To our knowledge, it is the first report on serum TNF-α in celiac patients. Our results suggest that the serum TNF-α concentrations did not reflect the inflammatory reactions in the small intestinal mucosa of the untreated celiac patients. One possible explanation for the negative finding could be the low sensitivity of the TNF-α assay used. However, increased TNF-α expression has been reported in intestinal (16–18) and peripheral samples (16) taken from untreated CD patients when compared with treated CD patients and controls. TNF-α in combination with TNF-γ induces increased epithelial HLA class-II molecules expression in jejunal mucosa (30). In addition, studies suggest that the tissue injury (loss of absorptive villi and crypt cell hyperplasia) can be mediated by cytokines (TNF-α and IL-1β)-induced changes in epithelial growth factors and matrix metalloproteinase production by the myofibroblasts of the lamina propria of the intestinal mucosa (31).
Both cell-mediated (17–19,28) and humoral (32) immune activation has been reported in the pathogenesis of CD. IgA-EMA is considered a specific autoantibody for active CD. It is direct against the intermyofibril substance of smooth muscle and it has been suggested that this antibody is a specific marker for the disease. The positive correlation between IgA-EMA titers and serum sIL-2R and IL-6 values suggests a specific immune response of the small intestinal mucosa to gluten ingestion.
Our study showed that untreated celiac patients have significantly elevated serum sIL-2R and IL-6 concentrations. The raised concentrations in untreated patients clearly differentiated such patients from treated CD patients and controls. In conclusion, the serum sIL-2R and IL-6 levels may be used as a noninvasive measure of CD activity and response to treatment.
This study was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo, São Paulo, Brazil. Grant 1998/7333–3.
We are grateful to Clarice P. A. Lemos and Terezita M. A. Montersino for technical assistance.
1. Marsh MN. Gluten, major histocompatibility complex, and the small intestine. A molecular and immunobiologic approach to the spectrum of gluten sensitivity (“Celiac sprue”). Gastroenterology 1992; 102:330–54.
2. Romaldini C, Barbieri D. Serum antigliadin immunoglobulin-A class antibodies in celiac disease. Arq Gastroenterol 1997; 34:254–61.
3. Rossi TM, Kumar V, Lerner A, et al. Relationship of endomysial antibodies to jejunal mucosal pathology: specificity towards both symptomatic and asymptomatic celiacs. J Pediatr Gastroenterol Nutr 1988; 7:858–63.
4. Troncone R, Maurano F, Rossi M, et al. IgA antibodies to tissue transglutaminase: an effective diagnostic test for celiac disease. J Pediatr 1999; 134:166–71.
5. Halstensen TS, Scott H, Fausa O, et al. Gluten stimulation of coeliac mucosa in vitro induces activation (CD25) of lamina propria CD4+ T cells and macrophages but no crypt-cell hyperplasia. Scand J Immunol 1993; 38:581–90.
6. MacDonald TT. T cell-mediated intestinal injury. In: Marsh MN, ed. Coeliac Disease. Oxford: Blackwell Scientific Publications; 1992:283–304.
7. Goldsmith MA, Greene WC. Interleukin-2 (IL-2). In: Nicola NA, ed. Guidebook to cytokines and their receptors. Oxford: Oxford University Press; 1997:27–30.
8. Rubin LA, Kurman CC, Fritz ME, et al. Soluble interleukin-2 receptors are released from activated human lymphoid cells in vitro. J Immunol 1985; 135:3172–7.
9. Smenzato G, Bambara LM, Biasi D, et al. Increased serum levels of soluble interleukin-2 receptor in patients with systemic lupus erythematosus and rheumatoid arthritis. J Clin Immunol 1988; 8:447–52.
10. Manoussakis MN, Papadopoulos GK, Drosos AA, et al. Soluble interleukin 2 receptor molecules in the serum of patients with autoimmune diseases. Clin Immunol Immunopathol 1989; 50:321–32.
11. Rubin LA, Nelson DL. The soluble interleukin-2 receptor: biology, function, and clinical application. Ann Intern Med 1990; 113:619–27.
12. Kishimoto T, Akira S, Taga T. Interleukin-6 and its receptor: a paradigm for cytokines. Science 1992; 258:593–7.
13. Bemelmans MHA, Van Tits LJH, Buurman WA. Tumor necrosis factor: function, release and clearance. Crit Rev Immunol 1996; 16:1–11.
14. Lundin KEA, Scott H, Hansen T, et al. Gliadin-specific, HLA-DQ (α1*0501, β1*0201) restricted T cells isolated from the small intestinal mucosa of celiac disease patients. J Exp Med 1993; 178:187–96.
15. Nilsen EM, Lundin KE, Krajci P, et al. Gluten specific, HLA-DQ restricted T cells from coeliac mucosa produce cytokines with Th1 or Th0 profile dominated by interferon gamma. Gut 1995; 37:766–76.
16. Lahat N, Shapiro S, Karban A, et al. Cytokine profile in coeliac disease. Scand J Immunol 1999; 49:441–6.
17. Przemioslo RT, Kontakou M, Nobili V, et al. Raised pro-inflammatory cytokines interleukin 6 and tumor necrosis factor alpha in coeliac disease mucosa detected by immunohistochemistry. Gut 1994; 35:1398–403.
18. Kontakou M, Przemioslo RT, Sturgess RP, et al. Expression of tumor necrosis factor-alpha, interleukin-6, and interleukin-2 mRNA in the jejunum of patients with coeliac disease. Scand J Gastroenterol 1995; 30:456–63.
19. Nilsen EM, Jahnsen FL, Lundin KE, et al. Gluten induces an intestinal cytokine response strongly dominated by interferon gamma in patients with celiac disease. Gastroenterology 1998; 115:551–63.
20. Nilsen EM, Jensen K, et al. Gluten activation of peripheral blood T cells induces a Th0-like cytokine pattern in both coeliac patients and controls. Clin Exp Immunol 1996; 103:295–303.
21. O'Keeffe J, Mills K, Jackson J, et al. T cell proliferation, MHC class II restriction and cytokine products of gliadin-stimulated peripheral blood mononuclear cells (PBMC). Clin Exp Immunol 1999; 117:269–76.
22. Hansson T, Dannæus A, Klareskog L. Cytokine-producing cells in peripheral blood of children with coeliac disease secrete cytokines with a type 1 profile. Clin Exp Immunol 1999; 116:246–50.
23. Blanco A, Garrote JA, Arranz E, et al. Increased serum IL-2R levels in coeliac disease are related to CD4 but not CD8 antigens. J Pediatr Gastroenterol Nutr 1992; 15:413–7.
24. Walker-Smith JA, Guandalini S, Schmitz J, et al. Revised criteria for diagnosis of coeliac disease. Arch Dis Child 1990; 65:909–11.
25. Crabtree JE, Heatley RV, Juby LD, et al. Serum interleukin-2-receptor in coeliac disease: response to treatment and gluten challenge. Clin Exp Immunol 1989; 77:345–8.
26. Srivastava MD, RossI TM, Lebenthal E. Serum soluble interleukin-2 receptor, soluble CD8 and soluble intercellular adhesion molecule-1 levels in Crohn's disease, celiac disease, and systemic lupus erythematosus. Res Commun Mol Pathol Pharmacol 1995; 87:21–6.
27. Kontakou M, Przemioslo RT, Sturgess RP, et al. Cytokine mRNA expression in the mucosa of treated coeliac patients after wheat peptide challenge. Gut 1995; 37:52–7.
28. Kerttula TO, Hällström O, Maki M. Phenotypical characterization of peripherical blood T cells in patients with coeliac disease: elevation of antigen-primed CD45RO+
T lymphocytes. Immunology 1995; 86:104–9.
29. Fornari MC, Pedreira S, Niveloni S, et al. Pre- and post-treatment serum levels of cytokines IL-1β, IL-6, and IL-1 receptor antagonist in celiac disease. Are they related to the associated osteopenia? Am J Gastroenterol 1998; 93:413–8.
30. Sturgess RP, Hooper L, Spencer, J, et al. Effects of interferon-γ and tumor necrosis factor-α on epithelial HLA class-II expression on jejunal mucosal biopsy specimens cultured in vitro. Scand J Gastroenterol 1992; 27:907–11.
31. MacDonald TT, Bajaj-Elliott M, Pender SLF. T cells orchestrate intestinal mucosal shape and integrity. Immunol Today 1999; 20:505–10.
32. Scott H, Kett K, Halstensen TS, et al. The humoral immune system in coeliac disease. In: Marsh MN, ed. Coeliac disease. Oxford: Blackwell Scientific Publications; 1992:239–82.
© 2002 Lippincott Williams & Wilkins, Inc.