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Tumor Necrosis Factor-β and Hypercalcemia a

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Kazuaki Ishibashi , Masahiko Kodama , Shuichi Hanada & Terukatsu Arima

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The Second Department of Internal Medicine, Faculty of Medicine, Kagoshima University, Kagoshima, Japan Published online: 01 Jun 2015.

To cite this article: Kazuaki Ishibashi, Masahiko Kodama, Shuichi Hanada & Terukatsu Arima (1992) Tumor Necrosis Factor-β and Hypercalcemia, Leukemia & Lymphoma, 7:5-6, 409-417, DOI: 10.3109/10428199209049796 To link to this article: http://dx.doi.org/10.3109/10428199209049796

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Leukemia and Lymphoma. Vol. I , pp. 409411 Reprints available directly from the publisher Photocopying permitted by license only

0 1992 Hanvood Academic Publishers GmbH Printed in the United Kingdom

Tumor Necrosis Factor-/? and Hypercalcemia KAZUAKI ISHIBASHI, MASAHIKO KODAMA, SHUICHI HANADA and TERUKATSU ARIMA The Second Department of Internal Medicine, Faculty of Medicine, Kagoshima University, Kagoshima, Japan

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(Received 17 December 1991 I. in final form I0 February, 1992)

Hypercalcemia in hematological malignancy is frequently encountered in lymphoid malignancies such as adult T-cell leukemia (ATL) and multiple myeloma and is difficult to manage. As a causative agent of hypercalcemia in ATL, tumor necrosis factor-/? (TNF-/?), previously known as lymphotoxin, has been carefully studied and reviewed here. Bone resorption studies showed the presence of activity in culture supernatants of HTLV-I infected cells. Enzyme linked immunosorbent assays (ELISA) for TNF-/? detected elevated TNF-/? in the sera of ATL patients with hypercalcemia. lmmunostaining by monoclonal anti-TNF-/? antibody demonstrated the presence of TNF-/? in both HTLV-I infected cell lines and freshly isolated ATL cells. Furthermore biological TNF-/? activity assay including inhibition of anti-TNF-/? antibody confirmed the conventional documentation of TNF-/? activity in the sera and culture supernatants of HTLV-I infected cell lines. These studies showed that the TNF-8 secreted from ATL cells might be one of the factors contributing to the hypercalcemia in patients with ATL functioning as a n osteoclast activating factor (OAF). K E Y WORDS:

Tumor necrosis factor

TNF-/?

INTRODUCTION Hypercalcemia due to malignancy is common, often severe, difficult to manage and sometimes lethal. Traditionally, hypercalcemia in malignancy was thought to be due to a local invasion and destruction of bone by tumor cells or humoral mediators. Prostaglandins (PG) were favoured as likely mediators of humoral hypercalcemia of malignancy in solid tumors, e.g. breast, kidney, lung and ovarian tumors"'. However, it is now less probable that P G mediate hypercalcemia in human cancer, because large amounts of PG must be infused or injected in order to cause hypercalcemia in uiuo in experimental animals3. Furthermore, treatment with drugs inhibiting PG synthesis has proven ineffective Address for correspondence: Kazuaki Ishibashi, M.D. The Second Department of Internal Medicine, Faculty of Medicine, Kagoshima University, Sakuragaoka 8-35-1, Kagoshima 890, Japan.

Hypercalcemia

in the treatment of hypercalcemia in malignancy4. In squamous cell carcinoma, for example, the tumor often produces polypeptides that are responsible for maintaining the transformed phenotype. The best known of these factors is transforming growth factor-a (TGF-a) and its effect on calcium and bone metabolism have been investigated in a number of earlier studies5-'. TGF-cr is thought to be a potent stimulator of osteoclastic bone resorption in uitro as well as in uiuo and is one of the candidates for causing hypercalcemia in solid tumors". Since TGF-a is often produced by tumors that also produce the parathyroid hormone-related protein (PTH-rp), these peptides probably affect calcium homeostasis in patients with solid tumors' '*". In hematological malignancy the principle interest in etiologic mechanisms of hypercalcemia has focused on the production of bone resorbing factors by activated normal lymphocytes and/or by malignant cells. In this manuscript we review previous reports regarding the hypercalcemia of hematological malignancies and

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K.ISHIBASHI et al.

discuss the relationship between cytokines possessing osteoclast activating factor (OAF), i.e. IL-l(s) and TNF(s), and these malignancies. Our recent data concerning TNF-j3 in the sera of a patient with adult T-cell leukemia (ATL) and the expression of TNF-P in cells from human T-cell leukemia virus (HTLV-I) infected cell lines are also summarized.

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THE INCIDENCE OF HYPERCALCEMIA IN HEMATOLOGICAL MALIGNANCIES

BONE REMODELING AND OAF Bone is thought to freshly renew itself by remodeling cycles in which it is always replaced with newly produced bone tissue. The bone-construction-destruction cycle is regulated mainly by two typical cell lineages, osteoclasts and osteoblasts. Hypercalcemia is usually caused by the activation of the osteoclasts and the activating factors causing this osteoclast activity were termed OAF. OAF were defined initially as bone-resorptive cytokines present in the culture supernatants of antigen- or mitogen-stimulated human peripheral blood mononuclear cells2'. OAF represented the biological action of several different cytokines with bone resorbing activity and included interleukin-la ( I L - ~ U ) IL-I/?, ? ~ , tumor necrosis factora (TNF-a) and TNF-P. Stashenko et aL3' studied the bone resorption activity in uitro using the release of 4SCain co-culture of bone with various cytokines. According to their results, lo-'' to lo-' M of IL-lP induced about 16-20% of the release of 4sCa. A similar response was obtained by the addition of either to IL-la or lOP7TNFs. Moreover, they found a synergistic elevation of 4sCa release when IL-lP and TNF-a or TNF-P were added simultaneously in their culture system. However, to conclude that only monocytederived cytokine, IL-1P, affects the hypercalcemia in lymphoid malignancy is difficult to accept. Bertolini et aL3' reported that in a similar culture system, to M of TNFa or TNF-fl induced osteolytic bone resorption and inhibition of bone collagen synthesis. These contradictory results concerning the 4sCa release in the same culture system may result from the modified conditions of the original protocol used for assessing bone resorbing activity32, such as duration of the culture period required to incorporate 4sCa into the bone and the time period of culture with cytokines.

Multiple myeloma is a well recognized plasma cell dyscrasia and the yearly incidence is remarkably similar in a variety of countries throughout the world. Bone pain is the most common symptom and is present in nearly 70 percent of the patientsi3. The bone lesions are lytic in nature and rarely associated with new bone formation. Renal failure occurs in nearly 25 percent of myeloma patients and hypercalcemia is the most common cause of renal failurei3. On the other hand, ATL is the first example of human cancer associated with retrovirus infection and when the first reports were published, its geographic distribution, clustered in a southwestern district of Kyushyu in Japani4*", drew attention to a pathogenetic agent contributing to the occurrence of ATL. HTLV-I is a type C retrovirus isolated by Poiesz and coworkers from T-cell lines derived from patients with mycosis fungoides' and SCzary syndrome. After the nucleotide sequence of the virus was first reported, these patients with cutaneous lymphoma were re-diagnosed as ATLi7.It is well known that the virus participates in the occurrence of ATL'8-23. Within the category of T-cell lineage malignant lymphoma, ATL is regarded as one of the most aggressive lymphomas and 35.3% of the patients with ATL had hypercalcemia while 28.5% of the patients with ATL died of hypercalcemia and subsequent renal However, obvious bone lesions could only be found in a small proportion of patients with ATL and hypercalcemia, which is very different from the clinical THE EXPRESSION OF CYTOKINES IN findings found in multiple myeloma. It is the general MYELOMA CELL LINES consensus that hypercalcemia in ATL is caused by the destruction of bone and that the serum calcium Bataille et al.33 studied the biological activity of originates from bone lesions irrespective of whether cytokines secreted in the supernatant of cultured these are evident on X-ray examinations. To clarify myeloma cell lines. All of the cell lines did not secrete the cause of hypercalcemia in ATL, serum parathyroid IL-1 but ten of 11 myeloma cell lines produced hormone relating protein (PTHrp), serum levels of significant T N F activity, which was only blocked by 1,25-dihydroxyvitamin D and osteoclast activating specific anti-TNF-P antibody. The data from their factor (OAF)-like activities26s27were studied. How- study suggest that TNF-P was involved in myeloma ever, a clear relationship between the disease and bone resorption, but not IL-1. Garrett et al.34 also hypercalcemia was not evident. reported the implication of TNF-j? in the myeloma

TNF-/I AND HYPERCALCEMIA

cell lines by studying the biological activity in the culture supernatants and the expression of mRNA in their myeloma cell lines. In their study, IL-1 was neither secreted nor expressed in the studied myeloma cell lines. These studies suggested that IL-I, which was always regarded as one of the most potent OAF, was not secreted in the myeloma cell lines. The extent of TNF-fl expression and secretion was confirmed in these studies.

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THE EXPRESSION OF CYTOKINES IN HTLV-I INFECTED CELL LINES Tschachler rr ~ 1reported . ~ that ~ high levels of TNFs were produced and secreted in both HTLV-I infected cell lines established by in oitro infection with HTLV-I and HTLV-I infected cell lines which had been directly established from ATL patients. They also studied the expression of IL-la and /I by HTLV-I infected cell lines. They reported that only three of ten HTLV-I infected T cell lines expressed IL-lx mRNA, and in none of the cell lines tested was IL-lfl mRNA expression detected. These results indicated that the cytokines involved in the HTLV-I infected cells were not IL-lx, but TNF-P. On the other hand, Wan0 rr ~ 1reported . ~ the~ expression of IL-la mRNA in four of six HTLV-I infected cell lines and the expression of IL-l/j mRNA in five out of six samples of freshly isolated leukemic cells obtained from patients with ATL. They implicated the IL-1 gene expression in the generation of some ATL-subpopulations, such as the chronic, smouldering and acute types. More recently, Paul rt d3’reported that TNF-/I transcription was regulated by the TNF-P promoter region which contains a site that appears similar to the immunoglobulin /,--chain NF-k-B-binding site found by the chloramphenicol acetyltransferase (CAT) assay of HTLV-I infected T-cell lines. Since the HTLV-I tax gene activates the nuclear form of NF-tiB, their finding suggested a possible means of HTLV-I activation of TNF-fl production.

EXPERIMENTS TO INVESTIGATE BONE RESORPTION ACTIVITY IN CULTURE SUPERNATANTS OF BOTH LYMPHOCYTES FROM PATIENTS WITH ATL AND HTLV-I INFECTED CELL LINES It would be rational to speculate that the presence of some substance produced and secreted from tumor cells would cause hypercalcemia by activating

41 I

osteoclasts and that subsequent Ca release from bone to serum would develop in ATL patients with hyper~alcemia~’. Bone resorption activity was studied in both short-term cultured supernatants of lymphocytes isolated from patients with ATL with or without hypercalcemia and HTLV-I infected cell lines. Lymphocytes from the peripheral blood of patients with ATL were fractionated by gradient centrifugation and washed three times with RPMI 1640 medium. HTLV-I infected cell lines, KUT-1 and 2, were established in our laboratory from the peripheral blood of patients with ATL. Detail of these cell lines was reported p r e v i o ~ s l yCells ~ ~ . from freshly isolated lymphocytes from patients and established HTLV-I infected cell lines were suspended in RPMI 1640 medium supplemented with 10% fetal calf serum at cell density of 5 x 106/ml and 2ml of the cell suspensions were seeded and cultured at 37”C, 5% CO, and 100% humidified conditions, independently. Seventy-two hours later, the culture-supernatants were passed through a 0.22 pm membrane filter and then used in the following assay. A n in vioo bone resorption assay was performed with minor modifications according to an original report described by Raisz3’ in 1965. Briefly, 300pCi of a “CaCI, saline solution were injected on day 18 of pregnancy in female rats and twenty-four hours later the embryos were removed. The other conditions different from the original work included the medium used in the embryonal bone culture and the duration of culture in the presence of culture-supernatant of ATL cells. BGJb medium (Gibco laboratory) supplemented with 10% heat inactivated fetal calf serum was used instead of modified Eagle’s medium and the culture period was 48 hours. In the bone culture system the aforementioned culture supernatants from ATL or HTLV-1 infected cell lines were added to the culture environment. The 45Ca released within the supernatants of the bone culture was measured by liquid scintillation counter. Bone cultures which were not subjected to exposure to culture supernatants were processed in the same system and counted as controls in each experiments. The 45Ca treated/control (T/C) ratios were then calculated. As shown in Figure 1, the T/C ratios were significantly elevated in patients with ATL and hypercalcemia compared to those in patients without hypercalcemia (p < 0.05). Moreover, in our bone culture system it was also evident that culture supernatants from the two HTLV-I infected cell lines also possessed bone resorption activity, as expected. According to these results we could propose that not only HTLV-I infected cell lines but also peripheral

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(previously termed lymphotoxin) had 28% homology with TNF-a in the amino acid sequence and 46% homology in the nucleotide sequence. It displays some of the same biological activities as TNF-a, including a cytotoxic/cytostatic action on tumor cells synergistic 0 with I F N - Y ~direct ~ , anti-viral activity44,chemotactic activity for phagocytes4', activation of osteoclastic bone resorption3' and induction of the transcription and production of granulocyte-macrophage-CSF, macrophage-CSF and IL- 146. In our preliminary study, a TNF-P bioassay using a mouse fibroblast cell line L929 was insufficient to analyze the in uiuo secretion of the cytokines because the serum samples were not always sterile and the limitation of the sensitivity of the assay was more than 1 u/ml, i.e 500 pg/ml as recombinant human TNF-P. Therefore, we developed an enzyme linked immunosorbent assay (ELISA) using biotinylated monoclonal anti-TNF-P and avidin labeled ALP to titrate TNF-P I in the serum and studied the relationship between Ca level and TNF-P in the serum of patients with ATL. ATL ATL KUT1 KUT2 We also immunostained TNF-P within the cytoplasm E Ca I Ca of both freshly isolated ATL cells and cells from Figure 1 Bone resorption activity present in the supernatant of short term cultured lymphocytes from patients with ATL or HTLV-I infected cell lines. I

I

I

I

HTLV-I infected cell lines. Cells were obtained from ATL patients with hypercalcemia, ATL patients without hypercalcemia, KUT- 1 cell line and KUT-2 cell line. Statistically significant differences were confirmed between the former two groups. (*:p > 0.05).

PATIENTS, SERUM SAMPLES AND REAGENTS

Thirty-six sera from thirty-four patients with acute ATL admitted to the Second Department of Internal Medicine, Kagoshima University Hospital and related hospitals during 1988-1989 were collected. They were stored at -20°C. The sources of materials used in this work were as follows: mouse monoclonal anti-human EXPERIMENTS TO INVESTIGATE THE ROLE TNF-P antibody from Boehringer Co. (Mannheim, OF TNF-8 IN ACL W.G); recombinant TNF-P from R & D Systems Inc. (Minneapolis, MN); bovine serum albumin Lymphokines or cytokines with bone resorbing (BSA) and Tween 20 from Nakarai Chemicals Co. activity or osteoclast activating OAF include IL-la, (Kyoto, Japan); biotinyl-N-hydroxy-succinimideester IL-1P, TNF-a and TNF-P29.Among these cytokines, (BNHS), alkaline phosphatase (ALP)-conjugated IL-lP has been thought to be the most potent3', avidin biotin complex, biotin-conjugated anti-mouse however, studies on the spontaneous in uitro secretion IgG and VectorR Red for the ALP substrate in the of cytokines in multiple myeloma cell lines33*34and immuno-staining from Vector Laboratories (BurHTLV-I infected cell lines35 reported that the lingame, CA): P and actinomycin D and phosphatase substance measured by in uitro secretion and/or by substrate (p-nitrophenyl phosphate) in the ELISA expression of mRNA in these cell lines was not IL-lB, from Sigma Chemical Co. (St. Louis, MO). but TNF-P. It has been suggested that TNF-P is a product of both activated B and T lymphocytes, whereas TNF-a has been considered until now to be TNF-P BIOASSAY a protein produced and secreted mainly by monocytes/ma~rophages~~~~'. Studies on the nucleotide To investigate the actual bioactivity of TNF-fl in sequence of TNF-P4'*42 reported that TNF-fi serum of ATL patients with hypercalcemia, a

blood lymphocytes from patients with ATL and hypercalcemia produce and secrete factors promoting bone

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TNF-/I AND HYPERCALCEMIA

conventional TNF-/J bioassay was performed on serum which was fractionated and stored in a sterile condition. TNF-D bioactivity was measured by using a modified cytotoxic assay against the L929 fibroblast cell line33*35.47. L929 cells were seeded at a density of 3 x 104cells in 96-well flat-bottom microtiter plates and cultured in 100 p1 of RPMI-1640 culture medium supplemented with 5% fetal calf serum. After 24 hours, the culture supernatant of the wells was removed and replaced by 100 pl of serial dilutions of the samples to be tested. Actinomycin D was added to the wells at a final concentration of 2pg/ml. The microplates were then incubated at 37"C, in 5% CO,. Twenty-four hours later, the supernatants were removed and the wells were stained with 0.5% (w/v) crystal violet, dissolved in 20% methanol-water. After ten minutes, the wells were gently washed four times with double-distilled water (DDW) and finally the wells were filled with 100 pl of DDW, then the optical density (OD) was read at 570 nm by an ELISA reader (MPR-A4, Toyo Soda, Co., Tokyo, Japan). The serum showed dose-dependent cytotoxicity against L929 cells in the presence of Actinomycin D. Moreover, the addition of monoclonal anti-TNF-P antibody (1 ng/ml) partially inhibited the cytotoxicity in the serum. Addition of the antibody (5ng/ml) completely inhibited the cytotoxic activity against L929 cells (Figure 2).

2

4

8

16

SAMPLE

32

64

128 256 512

DILUTIONS

Figure 2 Bioactivity of serum TNF-S in an ATL patient with from an ATL patient with hypercalcemia hypercalcemia. Serum (A) stocked in a sterile condition was used in a cytotoxic bioassay against L929 cells in the presence of actinomycin D as described in the text. Culture supernatant of MT2 cells was used as a positive control (0).An addition of 1 ng/ml (B) or 5 ng/ml (*) of monoclonal anti-TNF-p antibody to the serum decreased or inhibited the cytotoxicity in the bioassay, respectively.

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SANDWICH ELISA FOR TNF-/J A modification of the method of Vilja et was used for biotinylation of monoclonal antibody. The coating buffer, rinse buffer, diluent buffer and substrate buffer used in the ELISA were as follows49: 100 mM sodium carbonate-bicarbonate buffer (pH 9.6); phosphatebuffered saline (pH 7.4) containing 0.05% Tween 20; 100 mM Tris-HCI saline (pH 7.6) containing 1% BSA; and 50 m M sodium carbonate-bicarbonate buffer (pH 9.8). Monoclonal anti-TNF-/3 antibody was diluted with the coating buffer to make an antibody concentration of lpg/ml and the wells of a polystyrene microtitration plate (Nunc, No. 439454, 96F) were coated with the antibody by incubating overnight at 4°C. They were then washed three times using the rinse buffer and were filled with 100 pl of either serially twofold diluted recombinant TNF-/3 or 1/10 diluted serum samples in triplicate. After incubation for 90 minutes at room temperature, the wells were washed and 1/500 diluted biotinylated anti-human TNF-P antibody was added to the wells. The plates were incubated for 90 minutes at room temperature, the wells were washed and the 1/1000 diluted ALP-conjugated avidin was added. Ninety minutes later, the wells were washed and the substrate solution containing 1 mg/ml of p-nitrophenyl phosphate was added. Afterwards, color development OD was measured at 405 nm using an ELISA reader. A well of the microplates, which contained neither serum nor ALP-conjugated avidin, but only diluent buffer, served as a blank and the O D of this well was subtracted by an ELISA reader. For the determination of TNF-/J, a standard curve made by ELISA in triplicate using recombinant TNF-P and standard deviations of the results is also shown in Figure 3. TNF-P was measured in a dose dependent manner and the reproducibility of the standard curve was confirmed in three independent experiments (data not shown). The level of serum TNF-/J was calculated according to the standard curve. Sera from eight ATL patients with hypercalcemia and twenty-eight ATL patients without hypercalcemia were tested in triplicate by the ELISA to determine the level of TNF-/J. In sera with hypercalcemia, TNF-/3 was elevated in all cases except one (Figure 4), and the mean titer of TNF-/J in these eight patients was 515 f 393 pg/ml. However, in all sera without hypercalcemia the TNF-/3 level was less than 100 pg/m. Serum TNF-P levels were studied on two occasions in two patients with ATL and hypercalcemia during

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Table 1 Serum TNF-j and Ca levels in ATL

g

1

case KI date serum Ca (mEq/l) serum TNF-B (pg/mU

0.100

3120 1560 780

390

TNF

recombinant

0

195

97.5

(pg/ml)

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Figure 3 Standard curve of the OD in twofold reciprocal diluted recombinant TNF-B. ELISA described in the text were performed in triplicate and mean f 1 SD of the OD values at 405 nm were plotted in each standard sample.

case Y T date serum Ca (mEq/l) serum TNF-/3 (pg/ml)

Dec./2/88

two patients with

6.8

Jan./30/89 4.8

865

< 100

Mar./9/89

Apr./3/89

7.0

5.0

1756

< 100

CYTOPLASMIC EXPRESSION OF TNF-P IN HTLV-I INFECTED CELLS BY chemotherapy in order to assess the effect of treatment IMMUNOSTAINING USING AVIDIN-BIOTIN (Table 1). They were given various chemotherapeutic COMPLEX regimens in addition to corticosteroids and inIn order to study the production of TNF-/l by ATL travenous injections of calcitonin derivatives in order cells, cytoplasmic TNF-P was stained with a to reduce the disease activity of ATL and the levels biotinylated anti-TNF-P antibody and avidin-biotin of hypercalcemia. Both the serum Ca and TNF-P complexSo in freshly isolated ATL cells or in the levels decreased following therapy. HTLV-I infected cell line. Poly-L-lysine was used to make cells adherent followed by fixation with glutaraldehyde on the multiwell glass slide”. The glass slide was then blocked with horse serum, mounted first with appropriately diluted biotinylated 1600 anti-TNF-P antibody and then with avidin-biotin complex. The reacted sites were visualized by Vector RedTM solution according to the manufacturer’s instruction. In three HTLV-I infected cell lines, 800 KUT-1, KUT-2 and MT-2, the cytoplasm stained with this antibody, and the presence of TNF-P was I confirmed by the presence of red granules in their E 400 cytoplasm as shown in Figure 5, A, B and C, 0 Q respectively. In freshly isolated lymphocytes from two ATL patients with hypercalcemia, TNF-P was (2 identified in a small number of ATL cells which g 200 showed typical nuclear abnormalities (Figure 5, D Iand E). However, lymphocytes from ATL patients without hypercalcemia did not stain with the same 100 procedure.

-

h

-

v

I

st’

*) !$&

3.6mna5,

4

5

serum

6

7

8

Ca (mEq/l)

Figure 4 Ca level and TNF-j in the serum of patients with ATL. TNF-/I increased in seven sera out of eight patients with ATL and hypercalcemia. TNF-fl was less than 100 pg/ml in all sera without hypercalcemia.

DISCUSSION Serum TNF-P from patients with ATL was studied by a sandwich ELISA technique developed in our laboratory using biotinylated monoclonal anti-TNF/?and recombinant TNF-8. Seven out of eight patients

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TNF-p AND HYPERCALCEMIA

Figure 5 lmmunostaining of cytoplasmic TNF-/l using an avidin-biotin complex and a biotinylated monoclonal anti-TNF-P antibody. Cells adhered by poly L-lysine and glutaraldehyde onto glass slides so as not to damage the morphological appearance of ATL cells. Then Brij-35R-treatedcells were stained with biotinylated monoclonal anti-TNF-8 antibody and subsequent avidin-biotin complexes as described in the text. A: MT-2 cells, B: KUT-I cells, C: KUT-2 cells, D and E: freshly isolated lymphocytes from two different ATL patients with hypercalcemia. (See Colour Plate X at the back of this publication.)

with hypercalcemia showed elevated serum TNF-/I. On the other hand, TNF-/I was not detected by the same ELISA methodology in twenty-eight patients without hypercalcemia. The lower limit for detection of TNF-/I in this assay was 100pg/ml which corresponded to 500 pg/ml using the conventional method. In two patients, both the serum TNF-/I level and the serum calcium levels decreased after chemotherapy. Furthermore, immuno-staining using anti-TNF-/I and avidin-biotin complex showed the presence of cytoplasmic TNF-P not only in HTLV-I infected cell lines but also in freshly isolated cells from ATL patients with hypercalcemia. The true biologic activity of TNF-/I in serum was confirmed by a conventional bioassay in a patient with hypercalcemia and its cytotoxic activity was inhibited by the addition of anti-TNF-/I antibody in the assay. These results suggested that serum TNF-/I might be one of factors contributing to hypercalcemia, at least in patients with ATL. In the ELISA system, serum TNF-/I can be detected using a monoclonal antibody both as a capture antibody and as a secondary labeled antibody.

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Tschachler and coworkers35 reported that TNF-/I bioactivity in the supernatant of MT-2, HTLV-I infected T-cell line, was not completely blocked by the anti-TNF-/I antibody suggesting the presence of additional TNF-/I-like substances produced by the cell line. Regarding the biochemical properties of this ~ y t o k i n e ~ ' .it~ ~seems , that the difference in the molecular weight depends on the glycosylation of TNF-/135 and the results of this study suggests that the epitopes of TNF-j? which reacted with monoclonal antibody are plural and not saturated by a single reaction. In respect to the specificity of the ELISA method it will be necessary in the future to confirm whether the assay is in fact titrating and detecting true TNF-/I or also reacting with non-specific substances in the sera. Confirmation may be achieved by a comparison of TNF-P level in the ELISA with that recorded in the bioassay in larger patient populations, which must include ATL or non-ATL patients with or without hypercalcemia. In the present study, at the very least we may presume that the specificity of the assay was partially confirmed by comparison of the ELISA to the bioassay results. Serum TNF-/I was less than 100pg/ml in twenty-eight ATL patients without hypercalcemia. While levels were elevated in 7 out of 8 ATL cases with hypercalcemia. In two cases with hypercalcemia, the serum TNF-/I level was examined on different occasions during treatment and serum TNF-/I was seen to decrease after the therapy in association with a decrease in hypercalcemia. These results indicate that TNF-/I may induce hypercalcemia in patients with ATL. Very recently, we reported the correlation between serum Ca and serum soluble IL-2 receptor (Tac)(sIL-2R) levels in patients with ATL54*55.In the present study, although we did not investigate the immunocytological property of cells secreting TNF-/I either in uiuo or in vitro, it is indeed possible that the cells secreting TNF-/I are in fact activated lymphocytes expressing such the Tac antigen, which may be released as sIL-2R from the cells carrying cell-surface IL-2R. However, ATL cells d o not always produce TNF-/I, as the TNF-/I is within the normal range in some patients whose disease activity is acute but whose cell proliferation is still aggressive. This may indirectly indicate that the presence and the absence of hypercalcemia is not necessarily due to the proliferative state of the cells. In a preliminary study (data not shown), a conventional TNF-/I bioassay was insufficient to determine the serum levels of TNF-fl because of the sensitivity of the technique and the sterility of the

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K. ISHIBASHI er al.

samples. Serum samples are sterile but usually stored in non-sterile conditions, thus one cannot use these samples in a sterile culture system. Bataille et a l l 4 reported that the lower imitation of the TNF-fi bioassay using the L929 cell line was 1 u/ml in a study of T N F - j secreted by myeloma cell lines. According to the manufacturer's specification, 500 pg/ml of the recombinant TNF-P is equivalent to 1 u/ml of the bioactivity of TNF-P. Thus an improvement in the determination of TNF-P was achieved by using biotinlavidin ELISA as described in this paper. The large family of OAF includes IL-1, which is the most potent OAF secreted by monocytes or activated T-cells. Recently, it was reported that IL-lP produced by ATL cells might generate hypercalcemia in ATL36 and Stashenko et aL3' reported the synergistic interactions between IL-1 and TNF-P in normal rat-bone resorption. However, more recently, Tschachler et aI.35 showed that the mRNA of IL-lP was undetectable in the HTLV-I-infected T-cell lines tested. It is indeed possible that accessory cells immunoreactive to the leukemic cells are in fact responsible for producing materials contributing to the hypercalcemia. For instance, an in uiuo in patients with ATL showed that IL-1 is not only produced and secreted by tumor cells but also by non-tumor cells. So there is a possibility that IL-1 secreted by cells other than tumor cells have an effect on T N F - j during hypercalcemia in ATL patients. Accordingly, TNF-B may be one of the factors contributing to the presence of hypercalcemia in patients with ATL. Acknowledgements We would like to thank fellows of the Blood group of the Second Department of Internal Medicine, Kagoshima University Hospital for fractionating and collecting serum of patients with ATL.

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Colour Plate X (see p. 415 Figure 5) lmmunostaining of cytoplasmic TNF-P using an avidin-biotin complex and a biotinylated monoclonal anti-TNF-8 antibody. Cells adhered by poly L-lysine and glutaraldehyde onto glass slides so as not to damage the morphological appearance of ATL cells. Then Brij-35R-treatedcells were stained with biotinylated monoclonal anti-TNF-B antibody and subsequent avidin-biotin complexes as described in the text. A : MT-2 cells, B: KUT-1 cells, C: KUT-2 cells, D and E: freshly isolated lymphocytes from two different ATL patients with hy percalcemia.