Serum Levels of Soluble Interleukin-2 Receptor in Pulmonary Tuberculosis
Article information
Abstract
It is well known that the activation of cell mediated immunity has an important role in the pathogenesis of pulmonary tuberculosis and the production of the protective immunity against Mycobacterium tuberculosis. During the activation of T-cell by Interleukin-1 released from the macrophage, not only Interleukin-2 but also the soluble Interleukin-2 receptor (sIL-2R) molecule is released into the extracellular fluid. In vitro study reveals that the level of this sIL-2R is well correlated with the degree of activation of the T-cell. We therefore carried out this study to evaluate the significance of the serum IL-2R level in determining the disease activity of pulmonary tuberculosis.
The level of SIL-2R was measured by sandwich ELISA method.
The level of sIL-2R in 42 patients with bacteriologically -proven active pulmonary tuberculosis (29 far and moderately advanced pulmonary tuberculosis, age: 30.3 ±10.3 yrs; 13 minimal pulmonary tuberculosis, age: 34.4 ± 15.3 yrs) was 1111 ± 424 u/ml, which was significantly higher than the normal control group (age: 31.0±9.9 yrs) (365±143 u/ml) and inactive pulmonary tuberculosis group (age: 37.3 ± 16.9 yrs) (465 ± 131 u/ml).
But there was no significant difference between 29 patients with advanced pulmonary tuberculosis (1138 ± 405 u/ml) and 13 patients with minimal pulmonary tuberculosis (1051 ± 474 u/ml).
More than three months after the initiation of antituberculosis chemotherapy, the follow-up level of serum sIL-2R in 21 patients with active pulmonary tuberculosis (advanced pulmonary tuberculosis 15, minimal pulmonary tuberculosis six) was 533 ± 182 u/ml, which was markedly lower than the pretreatment level (1020 ± 323 u/ml).
In conclusion, the high level of sIL-2R (especially over 1000 u/ml) can be used as a marker of disease activity in pulmonary tuberculosis even though not diagnostic.
INTRODUCTION
According to the 5th National Tuberculosis Survey in Korea in 1985, the prevalence rate of pulmonary tuberculosis in Korea by chest X-ray was about 2.2%, with many new patients developing annually1). Detection of Mycobacterium tuberculosis is the most confirmative test in the assessment of disease activity of pulmonary tuberculosis. But AFB staining of sputum is less sensitive in paucibacillary tuberculosis and frequently impossible in cases of pediatric patients and sputum nonproductive patients with tuberculosis. The culture and sensitivity test of Mycobacterium tuberculosis takes two months to evaluate. Tissue biopsy reveals relatively specific findings of tuberculosis, but it is an invasive method and does not exclude other causes of granulomatous diseases, such as fungal diseases, pulmonary sarcoidosis, and berylliosis by itself. Radiological test (chest X-ray) is also easily available and can detect minor changes in tuberculous lesions but it is often difficult to differentiate between active and inactive lesions.
So many investigations have been carried out looking for better methods to detect early changes and asses disease activity of pulmonary tuberculosis easily and rapidly. Among these, the detection of antibodies against tuberculous antigens, specific antigens of Mycobacterium tuberculosis, specific chemical structures or DNA sequences of M. tuberculosis in serum or body fluids or sputum2–11) were tried. In addition, a Gallium scan of the lung12), the activity of leukocyte acid phosphatase13), urinary Neopterin14), the serum activity of angiotensin converting enzyme (ACE)15) and the serum level of adenosine deaminase (ADA)16) were studied for their usefulness in the assessment of disease activity of pulmonary tuberculosis.
We investigated the availability of the serum level of soluble IL-2R, which was known as the indicator of activiation of cell mediated immunity17), to determine the disease activity of pulmonary tuberculosis and follow up during antituberculosis chemotherapy.
MATERIALS AND METHODS
Table 1 summarizes the age and sex distribution of the subjects. Forty-two patients with active pulmonary tuberculosis (far and moderately advanced tuberculosis 29, minimal tuberculosis 13) were confirmed by AFB staining and mycobacterial culture of sputa.
Characteristics of the Subjects
The control groups included 16 patients with inactive pulmonary tuberculosis, eight patients with other lung diseases (pneumonia two, bronchiectasis three, lung cancer three), and 15 persons of a healthy control group who had no history of respiratory diseases.
The sera were obtained from venous blood and stored in −70°C till measurement of sIL-2R. In cases of active pulmonary tuberculosis, samplings were done before and during antituberculosis chemotherapy.
The level of sIL-2R was measured using Cell-free® ELISA kit prepared by T-cell Sciences Company (Cambridge, Mass, USA). This is a sandwich ELISA test using monoclonal antibodies against two different epitopes of IL-2R molecule, and processes of the test were as follows:
Anti-IL-2R was adsorbed onto the microtiter well.
Specimen IL-2R was bound to the antibody - coated microtiter well.
Enzyme (Horseradish peroxidase) conjugated anti-IL-2R was bound to a different IL-2R epitope.
Enzyme-substrate interaction resulted in color development proportional to IL-2R level, and the optical density of the specimen was measured by photometry at 490 nm.
The IL-2R level correlated with the optical density of the specimen gotten from standard curve, and when IL-2R level surpassed the reliable range of standard curve (1600 u/ml), the specimen was tested again after dilution two or three times. The serum level of sIL-2R was determined as average of the duplicate values on the same plate.
Results were expressed as Mean±Standard deviation, and statistical analysis was done by a two-tail Student t test.
RESULTS
The levels of sIL-2R of patients with active pulmonary tuberculosis and control groups are summarized in Table 2.
Soluble IL–2 Receptor Levels in Patients and Control Group
The level of sIL-2R of the normal control group was 365±143 u/ml, and of patients with inactive pulmonary tuberculosis it was 465±131 u/ml; there was no significant difference between these two groups (p>0.05). The level of sIL-2R of patients with other lung disease was 540±232 u/ml, in which the number of cases was small (n = 8), but the level of sIL-2R was similar to the control groups.
The level of sIL-2R in 42 patients with active pulmonary tuberculosis was 1111 ± 424 u/ml, which was significantly higher than the level of the normal control group and patients with other lung diseases (P<0.01, P<0.01 respectively). It also showed a significant difference compared with the level of patients with inactive pulmonary tuberculosis (P<0.01).
The level of sIL-2R of 29 patients with far and moderately advanced pulmonary tuberculosis was compared with that of 13 patients with minimal pulmonary tuberculosis in order to detect differences according to the severity of the tuberculous lesions, but there was no significant difference (P>0.5) between them (1138±405 u/ml vs 1051 ± 474 u/ml). Even the level of sIL-2R of patients with minimal pulmonary tuberculosis (1051 ±474 u/ml) showed a significant difference from the normal control group, patients with other lung diseases and patients with inactive pulmonary tuberculosis (p<0.01, p<0.02, p<0.01, respectively).
In 21 patients with active pulmonary tuberculosis (far and moderately advanced tuberculosis 15, minimal tuberculosis six), the level of sIL-2R could be followed up during antituberculosis chemotherapy (Fig. 2). The pretreatment level (1020 ± 303 u/ml) markedly decreased to 533±182 u/ml more than three months after the initiation of antituberculosis chemotherapy. After six or seven months of treatment, the level showed the tendency of recovery to the arbitrary normal range (mean±2SD of normal control group).
Serial observations of the serum sIL-2R levels in patients with active pulmonary tuberculosis during antituberculosis chemotherapy. Shaded area shows mean±2 SD of normal control group.
DISCUSSION
It is well known that the activation of cell mediated imunity has an important role in the pathogenesis of pulmonary tuberculosis and the production of protective immunity against Mycobacterium tuberculosis18–20). Several years or decades after primary infection of M. tuberculosis the remaining mycobacteria proliferate and make a hypersensitivity reaction with sensitized T-cells and develop tubercule or cavity formations when defense mechanisms of the host become weak19). In addition, upon reintroduction of antigens to a primed cell population, IL-2receptors are expressed more synchronously21). In such a cell mediated immune reaction, macrophages have a very important role because activated macrophages are the most important cells that can kill mycobacteria22). Thus macrophages phagocytize and process the antigens, which can be presented to T-cells, and when highly specific receptors of T-cells recognize the antigens in the context of HLA-DR glycoproteins on the membranes of macrophages, then full activation of T-cells can occur18). But activation of T-cells requires synthesis and the release of Interleukin-1 from macrophages, and activated T-cells produce IL-2, and IL-2 stimulates proliferation of other T-cells.
Once T-cells are activated, IL-2 receptors are expressed on the surface of activated T-cells and also those are released into the extracellular fluid in vitro, and the level of sIL-2R is known as an indicator of the activation of T-cells17). Also sIL-2R was released and detected in vivo23).
Among IL-2R, high affinity receptors and low affinity receptors are known24), and high afinity receptors are supposed to have an affinity with IL-2R about 1000 times more than low affinity receptors25), while IL-2R is composed of alpha chain (p55, Tac) and beta chain (p70–75). High affinity receptor is composed of both alpha and beta chain and binding to its ligand, IL-2 is rapid, but dissociation from IL-2 is slow. The low affinity receptor is composed of the alpha chain only, and binding to and dissociation from IL-2 are rapid. The intermediate affinity receptor is known to be composed of beta chain only, and binding to IL-2 is slow, but dissociation from IL-2 is rapid26–30). The high affinity receptor and low affinity receptor are known to be encoded in the genes of the human chromosome 1031). Two kinds of sIL-2R are known to exist: 55 kDa sIL-2R, which is the same size with cell surface low affinity IL-2R and smaller 45 kDa sIL-2R29). Three hypotheses have been supposed as mechanisms of release of sIL-2R: 1) release of molecules lacking transmembrane domain derived from alternate sliced mRNA, 2) release by proteolytical cleavage of the surface expressed IL-2R and 3) degradation product of released 55-kDa sIL-2R. This is still controversial29,30,32). In addition, released sIL-2R is known to bind to IL-2R with low affinity (KD = 30 nM)30,33), and so sIL-2R has been supposed to have an immunoregulatory role by competing with cell surface IL-2R17).
The range of sIL-2R in the serum of the normal individual is between 100 and 500 u/ml, which reflects the lymphocyte activation normally occurring upon physiological stimuli34). Abnormally increased sIL-2R may result either from an increased release rate by activated cells or from release by neoplastic cells constitutionally expressing the IL-2R molecule34).
Diseases which show increased levels of sIL-2R are as follows: lymphoreticular malignancies34,35), such as hairy cell leukemia, Hodgkin’s disease, non Hodgkin’s lymphoma, B-chronic lymphocytic leukemia, acute myeloid leukemia, acute lymphocytic leukemia, angioimmunoblastic lymphadenopathy, adult T-cell leukemia and Sezary’s syndrome; autoimmune diseases36–39), such as rheumatoid arthritis, systemic lupus erythematosis, primary Sjögren’s syndrome, atopic dermatitis, psoriasis, chronic progressive multiple sclerosis and type I diabetes; and viral infections, such as measles40), HIV infection and infectious mononucleosis34); and viral hepatitis41), rejection reaction of transplantation42), burn patient43), administration of IL-244) and systemic or local granulomatous diseases45,46), such as sarcoidosis and fungal infections. In tuberculosis, increased levels of sIL-2R were reported in pulmonary or extrapulmonry tuberculosis47) and in the fluid of suction-induced blisters in vivo after injection of PPD48).
In this study we can observe a markedly increased level of sIL-2R in patients with far and moderately advanced pulmonary tuberculosis (1138±405 u/ml) and minimal pulmonary tuberculosis (1051±474 u/ml). But levels of sIL-2R in patients with inactive pulmonary tuberculosis (465±131 u/ml) was not significantly different from that of the normal control group (365±143 u/ml) and patients with other lung diseases (540±232 u/ml). These results could be interpreted as the reflection of disease activity of pulmonary tuberculosis. But there was a broad overlapping range in the distribution between the level of patients with active pulmonary tuberculosis and the control groups (Fig. 1). which did not guarantee the diagnostic value of sIL-2R. So if we would determine 1000 u/ml as the cut-off value in the assessment of disease activity of pulmonary tuberculosis, the sensitivity and specificity were 55% and 100%, respectively. Therefore, a higher level of sIL-2R (1000 u/ml) could be used as an indicator of disease activity in patients with pulmonary tuberculosis. However, the level of sIL-2R increases in patients with many other diseases, such as lymphoreticular malignancies, viral infections, autoimmune diseases, sarcoidosis and fungal diseases. So the level of sIL-2R cannot be suspected to be valuable in the differential diagnosis with those diseases. Concerning the level of sIL-2R in extrapulmonary tuberculosis, Brown et al47), reported 921 u/ml in eight patients of extrapulmonary tuberculosis (tuberculous lymphadenitis six, genitourinary tuberculosis one, spine tuberculosis one), which were slightly lower than 1192 u/ml in 12 patients with pulmonary tuberculosis, but showed significant differences from the level of the control group. We observed an increased level of sIL-2R in three patients with tuberculous lymphadenitis, 1440, 1221, 736 u/ml, respectively. These observations suggest the possible value of sIL-2R in assessing the disease activity of extrapulmonary tuberculosis. Furthermore, a higher increase of sIL-2R can be expected in tuberculous pleural effusion, which is immunologically more active than pulmonary tuberculosis. Further study in this field will be necessary.
Levels of serum soluble interleukin-2 receptor (sIL-2R) in patients and control groups. NC: Normal control: FA & MA TB: Far advanced and moderately advanced pulmonary tuberculosis: Min TB: Minimal pulmonary tuberculosis: Inac TB: Inactive pulmonary tuberculosis: OLD: Other lung diseases (pneumonia, bronchiectasis, cancer)
Another known indicator of activation of cell mediated immunity, adenosine deaminase (ADA), was reported to be valuable in the diagnosis of miliary tuberculosis and tuberculous pleural effusion16), but in the assessment of disease activity of pulmonary tuberculosis, ADA had a wider overlapping range with other lung diseases than sIL-2R. So sIL-2R may be a more valuable indicator of disease activity of pulmonary tuberculosis than ADA, but this will require control study.
In addition, the follow-up level of sIL-2R in 21 patients with active pulmonary tuberculosis, three or more months after initiation of antituberculosis chemotherapy showed a marked decrease of the level (533±182 u/ml) compared with the pretreatment level (1020±323 u/ml) (Fig. 2). which could be interpreted as the result of loss of mycobacterial burden and a subsequent decrease of T-cell activation due to lack of persistent antigen stimulation.
But Brown et al.47) reported a persistent high level of sIL-2R after two or three months of antituberculosis chemotherapy, which could be due to the short period of observation. If they could have been followed longer, they might have been able to recognize recovery to nearly normal range as in our study. Rapid decline of sIL-2R during the first three months of antituberculosis chemotherapy (Fig. 2) showed a better reflection of the course of treatment than the gradual decline of other reports49–51).
In tuberculosis, the level of serum sIL-2R is the reflection of antigen nonspecific T-cell activation at the disease sites and may be a useful auxilliary method to the conventional bacteriological and radiological methods in diagnosis of pulmonary tuberculosis and its role in immunoregulation was suggested by Rubin et al,17) but still is not clear.
Notes
This study was supported by a grant from the Inje Research Foundation.