Evaluation of the expression of interleukin 1 beta(IL-1β) and interleukin 1 receptor antagonist (IL-1Ra) in leprosy patients

Abstracts

A hanseníase é uma doença infectocontagiosa espectral que acompanha-se por uma série de eventos imunológicos desencadeados pela resposta do hospedeiro frente ao agente etiológico, o Mycobacterium leprae. Evidências sugerem que a indução e manutenção da resposta imune/inflamatória na hanseníase estão vinculadas a interações de múltiplas células e fatores solúveis, particularmente através da ação de citocinas. Nesse estudo, foram mensurados níveis de IL-1β e IL-1Ra de 37 casos novos de hanseníase acompanhados ao longo do tratamento e 30 controles sadios pelo teste ELISA. A coleta de sangue periférico foi realizada em quatro tempos para os casos de hanseníase (pré-tratamento com PQT, 2ª dose, 6ª dose e pós-PQT) e em único momento para os controles. Na comparação dos níveis das moléculas de casos no pré-PQT e controles, houve diferença estatisticamente significativa somente para IL-1β. Nossos resultados sugerem a participação dessa citocina no processo imune/inflamatório.

Hanseníase; Interleucinas; IL-1β e IL-1Ra

ARTICLE

Evaluation of the expression of interleukin 1 beta(IL-1β) and interleukin 1 receptor antagonist (IL-1Ra) in leprosy patients

Rosane Dias Costa^I; Vanessa Amaral Mendonça^II; Sandra Lyon^III; Rachel Adriana Penido^IV; Ana Maria Duarte Dias Costa^V; Marina Dias Costa^I; Marina Pires Nishi^VI; Mauro Martins Teixeira^VII; Antônio Lúcio Teixeira^VI; Carlos Maurício de Figueiredo Antunes^VII

^ISanta Casa de Misericordia of Belo Horizonte, Belo Horizonte, MG, Brazil

^IIFederal University of Vales do Jequitinhonha and Mucuri, Diamantina, MG, Brazil

^IIIEduardo de Menezes Hospital, Minas Gerais State Foundation, Belo Horizonte, MG, Brazil

^IVSuperior Institute of Education Anisio Teixeira, Helena Antipoff Foundation, Ibirite, MG, Brazil

^VJose do Rosario Vellano University, UNIFENAS, Alfenas, MG, Brazil

^VIFaculty of Medicine, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil

^VIIInstitute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil

^{Address to}

ABSTRACT

Leprosy is an infectious and contagious spectral disease accompanied by a series of immunological events triggered by the host's response to the etiologic agent, Mycobacterium leprae. Evidence suggests that the induction and maintenance of the immune/inflammatory response in leprosy are linked to multiple cell interactions and soluble factors, mainly through the action of cytokines. The ELISA test was used to measure the levels of IL-1β and IL-1Ra in 37 new leprosy patients followed-up during treatment and 30 healthy controls. Peripheral blood was collected four times during the treatment of leprosy patients (MDT pretreatment, 2^nd dose, 6^th dose and post-MDT), and only once from the controls. The comparison of molecular levels in pre-MDT patients and controls showed a statistically significant difference for IL-1β. The results suggest the participation of this cytokine in the genesis of the immune/inflammatory process.

Key-words: Leprosy. Interleukins. IL-1β and IL-1Ra.

Leprosy is a chronic infectious and parasitic disease caused by Mycobacterium leprae, an atoxic, resistant, acid-alcohol-fast, obligate intracellular bacillus, which induces an extraordinary cell-mediated immune response in diseased individuals and affects mainly the skin and branches of peripheral nerves^{4 17 19 20}. Neural injury, recognized by many authors as the most serious complication of leprosy, is triggered by infection and accompanied by a series of immunological events, whose evolution sequelae often last many years after infection has ceased^{22 28}. It presents an important peculiarity to clinicians and immunologists: the host's diversity in responding to its etiologic agent imposes a diagnostic challenge and an excellent model for understanding cellular immunity in humans²³.

Leprosy is characterized by its high infectivity and low pathogenicity; moreover, more than 95% of the population are naturally immune to such an infection^{18 31 32 33}. This condition can be changed in function of the relationship between the agent, the environment and the host¹⁰. It is a spectrum disease characterized by contrasting clinical forms and the outcome of infection seems to depend on the predominant underpopulation of T lymphocytes, on macrophage activation and on when and how a certain cytokine is available at the site of the parasite^{9 21 24 26}.

In tuberculoid (TT) lesions, predominance of Th1 response and type 1 cytokines occurs, including interleukin (IL) 1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α)^{13 29}. These molecules are among the most studied cytokines and evidence indicates that both can act synergically to prevent bacillary proliferation, but they can also become pathogens, causing skin and neural lesions in the absence of regulating factors^{2 5 7 8 25}. Thus, the decrease in the synthesis or blockage of the effects of a superproduction of such cytokines can particularly provide an option for the control of certain inflammatory disorders¹¹. IL-1β, for example, is one of the molecular forms of IL-1 produced by practically all nucleated cell types (mainly monocytes, macrophages and dendritic cells) and it is among the most important cytokine-induced markers of inflammatory response⁶. The mechanism of natural inhibition of this cytokine involves the blockage of receptor binding by cytokine receptor antagonists, such as IL-1 Ra, an IL-1 receptor antagonist. IL-1 Ra is a protein of the interleukin family, originally described as a molecule secreted by monocytes and macrophages, which modulates a variety of IL-1-related immune and inflammatory responses³.

In lepromatous (LL) lesions, on the other hand, predominance of Th2 response and type 2 cytokines occurs, including IL-4, IL-5 and IL-10. The borderline forms, in turn, represent a clinical and immunological pattern of intermediate response and the cytokines expressed both in vitro and in vivo cannot be related to any pattern already described^{2 5 7 8 25}.

Despite scientific advances in leprosy, many issues have yet to be investigated regarding immunology¹⁴ and it is important to highlight that the induction and maintenance of the immune/inflammatory response are linked to multiple cell interactions and soluble factors, particularly by cytokine action. However, T lymphocyte activation and cytokine production cannot be evaluated in isolation during infection²³. Therefore, the study of immunological processes is essential for understanding the mechanisms of the development and continuance of leprosy⁹.

The purpose of this paper was to study the expression of IL-1β inflammatory cytokine and the IL-1Ra anti-inflammatory molecule in leprosy patients assisted by the Reference Center of Sanitary Dermatology in the city of Belo Horizonte, State of Minas Gerais, Brazil.

MATERIAL AND METHODS

This is an exploratory, descriptive, longitudinal and analytical study involving 37 new leprosy cases followed-up during multidrug therapy (MDT) and 30 noninfected subjects from an endemic area, here considered as healthy controls, conducted at the Dermatology Ambulatory of the Eduardo de Menezes Hospital Sanitary Dermatology Reference Center of the Minas Gerais State Foundation (FHEMIG), in Belo Horizonte, Brazil, from May 2006 to December 2007.

Data collection was initiated after the project was approved by the Ethics in Research Committee of the Eduardo de Menezes Hospital and of the Santa Casa de Misericordia de Belo Horizonte. All the participants agreed to sign a term of free informed consent.

Patients' medical records were used to assess the following variables: sex, age, number of skin and nerve lesions, bacilloscopy, ML-Flow serological test, Madrid protocol and operational classifications. The subjects' peripheral blood (more specifically, their plasma) and the Sandwich ELISA technique were used to measure the levels of IL-1β and IL-1Ra. In cases of leprosy, blood collection occurred four times during treatment (pretreatment with MDT, 2^nd dose, 6^th dose and post-MDT); in healthy controls it occurred once. However, the number of patients submitted to peripheral blood collection on the 2^nd and 6^th dose was lower than patients in the pre and posttreatment, 15 and 19 patients, respectively. The samples were then sent to the Federal University of Minas Gerais, Institute of Biological Sciences, Immunopharmacology Laboratory, where the experiments were conducted according to a standard protocol. The levels of the molecules studied were expressed in pg/ml.

For data analyses, the following methods were used: measures of central tendency and variability; the Mann-Whitney test for comparing molecular levels of IL-1β and IL-1Ra of the pretreatment stage and healthy controls; the Wilcoxon¹ test for comparing the molecular levels studied in the leprosy cases between one another (pre-MDT and the other treatment stages) and the Generalized Linear Model for repeated measures with four factors (F test)¹⁵ for the longitudinal comparison of the IL-1β and IL-1Ra levels at the different treatment stages or phases. One limitation of the last analysis was the sample size, since the molecules were assessed at different stages (pre-MDT, 2^nd dose, 6^th dose and post-MDT) in only seven patients. In addition, for molecules that were significant in the comparison of leprosy cases in the pretreatment stage and healthy controls, a ROC (Receiver Operating Characteristic)³⁴ curve was used to characterize the best cutoff point of the measurements analyzed for the prediction of a positive leprosy case. The significance level adopted was 5%.

RESULTS

The results showed that most patients were male, with a mean age of approximately 50 years-old, with more than five skin lesions and more than one nerve affected, classified as borderline by the Madrid classification and as multibacillary (MB) by the operational classification; 37.8% of the cases were bacilloscopy positive and 64.9% were positive for the ML-Flow serological test.

Comparison of the molecular levels in leprosy cases in the pretreatment stage and in a healthy controls showed statistically significant difference (p<0.05) for IL-1β only (Figure 1), while comparison of molecular levels in leprosy cases at the pretreatment stage and the other stages (2^nd dose, 6^th dose and post-MDT) showed no statistically significant difference for any molecule. The F test also revealed no statistically significant difference and the graphic record showed a tendency for increase of the marginal mean of IL-1β levels starting from the 6^th dose (referring to the 3^rd phase, as observed in Figure 2). In addition, according to the ROC curve, patients with IL-1β levels greater than the established cutoff point (0.015) would be classified as probable leprosy cases with 75.7% sensitivity and 70% specificity (Figure 3).

The levels of IL-1Ra were undetectable in cases of leprosy and controls.

DISCUSSION

In this paper, the IL-1β levels revealed a statistically significant difference when the pretreatment leprosy cases and control group were compared. This finding agrees with that observed by Moubasher et al¹⁶, in which nontreated leprosy patients exhibited significantly higher IL-1β serum levels and other molecules (IFN -γ, IL-2R, IL-10 and TNF-α) detected by the ELISA test compared to healthy controls. Therefore, we may deduce that IL-1β release in leprosy cases is an indication of the activation of a cell-mediated immune response to the bacillus, according to the observation of Watson et al³⁵, who quantified the production of IL-1β and IL-2 by mononuclear adhering cells from the peripheral blood of patients with different forms of leprosy.

The present study showed no statistically significant difference in IL-1β levels when comparing leprosy cases in pretreatment and other stages (2^nd dose, 6^th dose and post-MDT). These findings show that the respective cytokine levels were already altered before the onset of treatment in function of the bacillary load and no significant serum alterations were identified during multidrug therapy. The massive destruction and reduction of viable bacilli probably initiates the release of antigenic fractions, which would cause a persistent stimulus with consequent immune and inflammatory hyperreactivity^{5 12 29 30}. Sarno et al²⁷ identified significantly higher IL-1β serum levels in all patients with borderline tuberculoid forms who showed no reaction, independent of the chemotherapy duration. They also detected high levels of the cytokine in the serum of leprosy patients with erythema nodosum leprosum (ENL).

In relation to the longitudinal comparison of IL-1β levels, no statistically significant difference was observed at the different stages of the study (pre-MDT, 2^nd dose, 6^th dose and post-MDT), and the graphic representation showed a tendency for an increase in the marginal mean of IL-1β levels starting from the 6^th dose. This finding disagrees with that of Moubascher et al¹⁶, who demonstrated a significant reduction in IL-1β serum levels and other molecules (IL-2R and IL-10) after one year of treatment in 36 leprosy cases. However, for this analysis, the present sample consisted of only seven patients, being considered too small for the researchers to make inferences. Nevertheless, the present results suggest that new investigations are still required to determine the effect of prolonged antimicrobial therapy on IL-1β production capability, an issue previously discussed³⁵.

Regarding IL-1β levels, the ROC curve showed a moderate prediction power to distinguish a positive leprosy case, with 75.7% sensitivity and 70% specificity.

The dosage of IL-1Ra, an anti-inflammatory molecule that selectively inhibits the effects of IL-1 by competing with the cell surface IL-1 receptor, the negative results obtained for both the controls and leprosy cases at the various stages of treatment could be justified by the fact that the levels were lower than the concentrations detected by the ELISA test.

Concerning the general limitations of this study, it is worth highlighting the loss of patients during follow-up, which prevented the group from collecting peripheral blood at all the stages of the study, and patient-related factors, such as genetic factors, nutritional condition and parallel use of other drugs.

Despite the existence of evidence concerning the role of these molecules in the course of leprosy in this study, we conclude that new investigations are necessary the evaluate the expression and the role of agonists and/or antagonists in either the proinflammatory or antiinflammatory effects, in order to confirm the presence of such molecules as disease-predicting markers and to serve as parameters for following-up the patient's immunological condition in future biological assays. A clearer understanding of the highly complex biological mechanisms involved will contribute to new therapeutic approaches.

ACKNOWLEDGMENTS

The authors are grateful to Dr. Maria Aparecida de Faria Grossi for her professionalism and constant encouragement in our search for knowledge.

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Publication Dates

• Publication in this collection
  17 July 2009
• Date of issue
  2008