Clinical significance of serum soluble IL-2R levels in patients with adult T cell leukaemia (ATL) and HTLV-1 carriers

Abstract

The levels of soluble IL-2Rα (sIL-2Rα) in serum were measured in HTLV-1 carriers and ATL patients in order to evaluate their possible correlation with clinical status. Mean sIL-2R levels in ATL patients were found to be 9704 U/ml for the acute/lymphoma type, 1961 U/ml for the chronic type and 788 U/ml for the smouldering type. The level for asymptomatic HTLV-1 carriers was 475 U/ml, and 165 U/ml for healthy young adult HTLV-1⁻ controls. The serial measurement of sIL-2R in ATL patients, healthy HTLV-1 carriers, and HTLV-1 carriers with diseases other than ATL showed a good correlation between serum levels of sIL-2R and the pathophysiological status of disease. Furthermore, an increase in the sIL-2Rα level in serum indicated the exacerbation of HTLV-1 infection and autoimmune diseases. The measurement of sIL-2Rα levels is therefore a very useful parameter for determining disease status.

INTRODUCTION

The retrovirus HTLV-1 is endemic in south-western Japan, especially in Okinawa, where there is a high incidence of ATL. Since the discovery in 1981 of HTLV-1 as the causative agent of ATL, the pathophysiology, virological and molecular biological bases of ATL have been elucidated [1–4]. However, the leukaemogenic mechanisms remain unclear. HTLV-1 does not have an oncogene, but it is thought that the tax gene of HTLV-1 may have a role in the proliferation of the retrovirus and may also stimulate proliferation of the lymphocytes that harbour the virus through the transactivation of endogenous genes such as c-fos, c-jun, IL-2 and the IL-2 receptor (IL-2R) [5,6].

The IL-2R consists of three genetically distinct subunits termed α, β, and γ. IL-2Rα (CD25, Tac antigen) [7,8] is a 55-kD protein that binds IL-2. IL-2Rβ (CD122) [9,10] is a 75-kD protein with a large intracellular segment and is known to play an important role in receptor-mediated signalling. IL-2Rγ is a 64-kD protein that also has structural homology to other cytokine receptors [11,12].

The serum level of soluble (s)IL-2R increases with an increase in the activity of immune defence mechanisms [13,14]. HTLV-1-infected cells are activated T cells, and the cellular immune responses against these cells are activated, leading to the release of sIL-2R which can be measured and may reflect the degree of infection.

In this study, we measured the level of sIL-2Rα in serum in order to evaluate its clinical significance in ATL patients, HTLV-1 carriers and patients with autoimmune diseases.

PATIENTS AND METHODS

Source of material

The controls in this study were 27 healthy HTLV-1⁻ young adults. Local residents (n = 724) were tested for HTLV-1 infection during medical examinations: 544 of them were HTLV-1⁺ and 180 were HTLV-1⁻ (Table 1). There were 185 patients with haematological diseases other than ATL such as myelodysplastic syndrome, aplastic anaemia, multiple myeloma, leukaemia, lymphoma, idiopathic thrombocytopenic purpura and so on. Of these 185 patients, 114 were HTLV-1⁺ and 71 were HTLV-1⁻. There were 34 patients with ATL (eight smouldering type, four chronic type and 22 acute/lymphoma type). The four ATL types (smouldering, chronic, lymphoma and acute) are characterized as follows: the smouldering type has at least 5% abnormal T lymphocytes, and the normal lymphocyte level in the peripheral blood is < 4 × 10⁹/l. Also skin and pulmonary lesions may be present. The chronic type has lymphocyte levels of at least 4 × 10⁹/l with at least 5% abnormal T lymphocytes, and the lactate dehydrogenase (LDH) value is up to twice the normal upper limit. The lymphoma type has no lymphocytosis. The lymphocyte level in the peripheral blood is < 4 × 10⁹/l with 1% or less abnormal T lymphocytes, and there is histologically proven lymphadenopathy. The acute type has both leukaemic manifestations and tumour lesions such as lymphadenopathy and extranodal lesions [15]. The sera were analysed after separating the subjects into two groups: one positive and one negative for the HTLV-1 antibody (Table 1).

Table 1.

Patients and controls

Soluble IL-2Rα chain measurement in serum

sIL-2R levels in the serum were measured by sandwich ELISA specific for IL-2Rα using two MoAbs (AM92.3 and 7G7/B6) which recognized two distinct epitopes on the α-chain of the IL-2 receptor [13,14,16]. Briefly, the serum or various concentrations of a standard preparation of soluble IL-2R were incubated with polystyrene beads coated with the antibody (AM92.3) to the first epitope, and the horseradish peroxidase-conjugated antibody (7G7/B6) to the second epitope of IL-2R. The non-reactive antibodies were then washed out and orthophenylenediamine (OPD) was added and allowed to react for 30 min. Sulphuric acid (1 n) was added to stop the reaction, and absorption at 492 nm was measured. The measurable levels of the soluble IL-2Rα concentration are 85–3000 U/ml. Samples containing levels higher than 3000 U/ml were diluted and measured. Finally, the concentration was calculated in terms of the dilution.

Serial measurement of sIL-2Rα chain in serum

In a long-term study of patients with various diseases, sIL-2Rα levels were measured serially over a period of 4–6 years.

Soluble IL-2Rα chain measurement in culture supernatants

One million cells of established cell lines such as K562, Molt4, the HIV-infected cell line Molt4/HTLV-III, the HIV-2-infected cell line GH1, and the HTLV-1-infected cell lines MT1, MT2 and HUT102, were cultured in 2 ml of RPMI 1640 containing 10% fetal calf serum (FCS) in a 24-well culture plate [13]. After 3 days, the supernatants were harvested and the levels of sIL-2R were measured using the same method as described above for serum measurement.

The effect of mitogens on the release of sIL-2R from peripheral blood mononuclear cells (PBMC) was also examined. One million PBMC were cultured with phytohaemagglutinin (PHA-P) or concanavalin A (Con A) in 24-well culture plates; after 3 days the supernatants were harvested and the levels of sIL-2R were measured.

RESULTS

The geometric mean value of serum sIL-2R was found to be 165 U/ml for healthy young adults, 418 U/ml for HTLV-1⁺ local residents, and 750 U/ml for HTLV-1⁺ patients with diseases other than ATL (Table 2). The differences among these three groups were significant.

Table 2.

Serum levels of sIL-2R in patients and controls

The mean sIL-2R levels in ATL patients were 9431 U/ml for the acute/lymphoma type, 1961 U/ml for the chronic type and 868 U/ml for the smouldering type. These differences were also significant (Table 2).

The sIL-2R levels in serum were measured serially in healthy HTLV-1 carriers and in HTLV-1-infected patients with diseases other than ATL over a period of 4–6 years (Fig. 1). Patient CA1 only had HTLV-1 infection. Patient C-OD1 had chronic hepatitis B and HTLV-1 infection. Patient C-OD2 had liver cirrhosis and rheumatoid arthritis (RA) with HTLV-1 infection. Although no abnormal cells could be found in the peripheral blood of this patient, the levels of sIL-2R gradually increased along with the development of HTLV-1 infection and RA. Patient C-OD3 had haemophilia A with hepatitis C and HTLV-1 infection. Patient C-OD4 had HTLV-1-associated myelopathy (HAM). Patient C-OD5 had haemophilia B and AIDS with HTLV-1 infection. This patient (C-OD5) developed AIDS at the point indicated by the arrow. Patient C-OD6 had systemic lupus erythematosus (SLE) with HTLV-1 infection, and the sIL-2R levels of this patient changed depending on the activity of SLE.

Fig. 1.

Changes of serum sIL-2R levels in HTLV-1 carriers and patients with HTLV-1 infection.

The changes of sIL-2R levels in ATL patients were observed over a period of 4–6 years (Fig. 2). Patient S (TT) had the smouldering type of ATL with stationary sIL-2R levels and clinical status. Patient A1(AY) progressed to an acute crisis state from the chronic type of ATL at 3 years after our initial observation (arrow), but responded to combination chemotherapy which included deoxycoformycin (DCF), vincristine, adriamycin, etoposide, and prednisolone; after 2 years this patient (A1(AY)) was still alive in a chronic disease state. Patients A2(TY) and A3(CT) experienced acute crises at the points indicated by the arrows and died even though intensive chemotherapy was given. Patient C1(MK) had chronic type ATL and was given mild oral chemotherapy such as prednisolone and cyclophosphamide when sIL-2R levels began to increase. As shown in this Figure, there was a good correlation between the clinical status of ATL and the serum level of sIL-2R in all patients.

Fig. 2.

Changes in serum sIL-2R levels in ATL patients.

To understand better the secretion and the role of sIL-2R in vivo, in vitro experiments were performed to measure the sIL-2R released in culture supernatants. The level of sIL-2R in the supernatants of K562, Molt4, Molt4/HTLV-III and GH-1 (an HIV-2 cell line) cell cultures was found to be below the limit of detection. However, the HTLV-1-infected cell lines MT1, MT2 and HUT102 released high levels of sIL-2R: 1430 U/ml, 1920 U/ml and 8200 U/ml, respectively (Table 3). This shows that HTLV-1 infection has an important effect on the release of sIL-2R.

Table 3.

sIL-2R levels in the culture supernatants of established cell lines

The effect of mitogens on the release of sIL-2R from PBMC was also examined. In a control culture without mitogen, the level of sIL-2R was almost undetectable even in the PBMC of ATL patients (Table 4). Soluble IL-2R levels from PBMC cultured with PHA-P were, however, detectable and the PBMC of some subjects showed high levels of sIL-2R: the levels in non-HTLV-1 carriers were 1000–1920 U/ml, HTLV-1 carriers showed either high (2700–3120 U/ml) or low (145–290 U/ml) levels, and levels in chronic ATL subjects were 950–2800 U/ml in spite of oral chemotherapy. PBMC responses to Con A varied from undetectable to high levels of sIL-2R (Table 4).

Table 4.

Effect of mitogens in the release of sIL-2R of peripheral blood mononuclear cells (PBMC) in vitro

DISCUSSION

Many of the patients in Okinawa with ATL who were treated with combination chemotherapy subsequently died despite such intervention. In 1981, the pathogenic agent human T-lymphotropic virus type-1 (HTLV-1) was discovered and many features relating to ATL were reported, but ATL still remains a severe and fatal disease. The median survival time is 6.2 months for the acute type, 10.2 months for the lymphoma type, and 24.3 months for the chronic type, though survival has not yet been determined for the smouldering type. The projected 2-year and 4-year survival rates were established as 16.7% and 5.3%, respectively, for the acute type, and 26.3% and 5.7%, respectively, for the lymphoma type [15,17].

The role of HTLV-1 in the development of ATL and its effect on IL-2 and sIL-2R levels are unclear. We measured serum sIL-2R levels in ATL patients and HTLV-1 carriers. In the case of ATL, the level in the smouldering type was 868 U/ml, in the chronic type 1961 U/ml and in the acute/lymphoma type 9431 U/ml (Table 2). In addition, the levels of sIL-2R in the serum of ATL patients might reflect tumour load and prognosis [16]. The sIL-2R levels of each subtype at the time of diagnosis appear to be correlated with tumour burden, malignant behaviour, and prognosis. This correlation is better than that observed with LDH [18].

Serial measurements of sIL-2R levels in sera were performed over a period of 4–6 years in HTLV-1 carriers and in patients who had HTLV-1 infection in addition to other diseases. In an SLE patient with HTLV-1 infection, sIL-2R levels changed depending on the activity of SLE. In a patient who had liver cirrhosis and RA as well as HTLV-1 infection, although abnormal lymphoid cells could not be found in the peripheral blood, the levels of sIL-2R gradually increased. The sIL-2R levels of these two autoimmune disease cases were dependent on the disease status and activity. In haemophilia B, in spite of the changes in the pathophysiological state, sIL-2R levels in the sera did not change at all, even after the HIV-1 infection progressed and T cells decreased to the point of immunodeficiency (Fig. 1).

The changes of sIL-2R levels in ATL patients were observed over a period of 4–6 years. There was a good correlation between clinical status and sIL-2R levels in the serum (Fig. 2). According to Wagner et al. [19], sIL-2R is a more specific marker of lymphoid malignancy than LDH, and it is also the best prognostic indicator among the factors assessed.

The sIL-2R levels were measured in the culture supernatants of established cell lines: K562, Molt4, Molt4/HTLV-III, and GH1 (HIV-2 cell line) were all negative for sIL-2R; whereas the HTLV-1-infected cell lines MT1, MT2 and HUT102 produced sIL-2R levels of 1430 U/ml, 1920 U/ml and 8200 U/ml, respectively (Table 3). The HTLV-1-infected cells released sIL-2Rα into the culture supernatant without any stimulant and so it is clear that HTLV-1 is a strong stimulus for sIL-2R production. However, this abnormality in production of IL-2 and/or expression of IL-2 receptors might not be essential or sufficient for leukaemogenesis by HTLV-1 [20].

The effect of mitogens on the release of sIL-2R in vitro was examined (Table 4). HTLV-1-infected and established cells invariably released sIL-2Rα spontaneously into the supernatant (Table 3), but no such release was seen with the PBMC of non-carriers, HTLV-1 carriers and chronic ATL patients during chemotherapy. It remains unknown why lymphocytes of HTLV-1-infected patients failed to produce sIL-2Rα spontaneously in vitro as in vivo. The mitogens PHA-P and Con A, however, caused release of sIL-2Rα into the culture supernatant of almost all samples. In HTLV-1 carriers, high and low response groups were observed in the presence of mitogen but the meaning of this is unknown. The release of soluble IL-2R appears to be a characteristic marker of T lymphocytes and might serve an immunoregulatory function during both normal and abnormal cell growth and differentiation [7,14].

In conclusion, our data show that the increase of sIL-2R levels reflects the exacerbation of the disease state and that measurements of sIL-2R levels are useful in assessing disease status.