Immunoreactive thymosin α1 is associated with murine T-cell lymphomas

Leukemia Research Vol. 8, No. 6, pp. 1003-1010, 1984. Printed in Great Britain.

0145-2126/8453.1~ + 0.~) :.~ 1984 Perg~,mon Press Ltd.

IMMUNOREACTIVE THYMOSIN WITH MURINE T-CELL

0tl IS A S S O C I A T E D LYMPHOMAS

MARION M. ZATZ, JOHN E. MCCLURE* a n d ALLAN L. GOLDSTEIN Department of Biochemistry, The George Washington University, School of Medicine and Health Sciences, Washington, D.C. 20037, U.S.A.

(Received 27 April 1984. Accepted 10 May 1984) Abstract--Growth of murine spontaneous and transplanted AKR T-cell lymphomas results in marked elevations of serum immunoreactive thymosin a,. Thymosin ct, is one of the peptide hormones believed to be secreted primarily by the thymic epithelium. This elevation, however, is not mediated by the thymus but rather, seems to be directly associated with the tumor cells. Growth of a B-cell lymphoma does not generate elevated immunoreactive thymosin ct, in the serum, thus, a thymosin ct, -like peptide is selectively associated with these T-cell lymphomas. The possible relationship between expression of T-leukemia viruses and ct, expression is discussed.

Key words: Thymosin a,, thymic hormones, T-cell leukemia, T-leukemia virus.

INTRODUCTION THYMOSlN a, is a peptide (MW 3,108) originally isolated from calf thymosin fraction 5, a partially purified preparation of bovine thymus [20]. The amino acid sequence of a, is known and it has been synthesized by solution and solid phase chemical procedures [37], and by recombinant DNA methodology [39]. Thymosin ¢t, is believed to be one of the thymic hormones secreted by the thymus epithelium [4, 15, 17, 18] and has known biological properties in inducing differentiation and maturation of T-lymphocytes in experimental animal models as well as in man [40]. Clinically, ~t~ has been tested as a biological response modifier in trials with lung cancer patients [35]. Immunoreactive serum a~ (IRa,) levels can be monitored by an established radioimmunoassay [23, 24]; levels have been reported to be low in certain primary immunodeficiencies [38], but are elevated in many individuals with the AIDS syndrome or at risk for AIDS [16, 19, 28]. Serum IRat levels also are elevated in individuals with T-cell leukemia and lymphoma [421. The thymic microenvironment has long been suspected to be one of the critical factors in the spontaneous development of thymic lymphomas and particularly those of leukemia prone-AKR mice [25, 43]. A second major factor in AKR leukemogenesis is the expression of T-lymphotropic murine leukemia viruses [13]. In the late 1950's, Metcalf reported that a circulating lymphocytosis stimulating factor appeared in the serum of leukemic AKR mice [26]. In order to investigate the relationship between leukemogenesis and thymic hormones in the AKR model, we sought to examine the serum thymosin a, levels in AKR mice bearing spontaneously arising or transplanted lymphomas. We took advantage of the existence of several in vivo AKR tumor cell lines with distinct T-cell or B-cell characteristics, respectively [21, 22, 41] to determine whether thymosin at might be

*Present address: Allergy-Immunology Service, Department of Pediatrics, Texas Children's Hospital, Houston, TX 77030, U.S.A. . Abbreviations: IRa,, immunoreactive thymosin a,; Tx, thymectomy or thymectomized; RIA, radioimmunoassay; HTL V, human T-cell leukemia virus; AIDS, acquired immunodeficiency syndrome. Correspondence to: Marion M. Zatz, Dept. of Biochemistry, George Washington University, 2300 Eye St., N.W., Washington. D.C. 20037, U.S.A. 1003

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MARtON M. ZArZ et aL

associated with T- and/or B-cell subclasses of murine iymphomas. Experiments were performed in normal or Tx mice in order to determine the role of the thymus in these studies. The results show that an immunoreactive ct, (IRa,), as measured by radioimmunoassay [23], is elevated 3- to 4-fold in the serum of T-cell tumor bearing mice, and that the origin of this IRtx, is most likely the T-cell lymphoma rather than the endocrine thymus. The relevance of these results are discussed in relation to recent findings showing that serum ct, levels also are elevated in human T-cell leukemia patients, including those which are HTLV + [42], and individuals with AIDS, or at risk for AIDS, many of whom also show evidence of HTLV infection [16, 19, 28]. MATERIALS AND METHODS Mice Two-month-old AKR/J male mice were obtained from Jackson Memorial Laboratory, Bar Harbor, ME. Young AKR/J mice were used as recipients of in vivo transfer lymphoma lines; additional groups of mice were maintained for up to 12 months to permit development of spontaneously arising thymomas. Fhymectomie$ In selected experiments, 6-8-week-old AKR/J mice were thymectomized by a surgical suction technique and used 2-3 weeks later as recipients of tumor cell lines. Completeness of thymectomy was verified at autopsy. Tumor cell lines Mice were injected i.v. with l04 splenic tumor cell suspensions from AKR lymphoma cell lines maintained in passage or with 10' spontaneously arising iymphoma cells. The principal tumor cell lines used were AKTB-It and AKTB-Ih, which are well characterized T- and B-cell sublines, originally isolated from a spontaneously arising mixed tumor in an old thymectomized AKR mouse [21, 41]. These cells were cryopreserved at the fourth in vivo passage generation and subsequently thawed and transferred once into young mice prior to use in the experiments described. Two other tumor cell lines, AKX-3, and AKX-Ib, also were studied as additional examples of AKR T- and B-cell lymphomas, respectively [22]. Radioimmunoassay Serum samples, cell culture supernatants and soluble cell and tissue sonicates were evaluated for IRa, content by a radioimmunoassay (RIA) for thymosin a,, according to published procedures [23]. A highly specific heterologous rabbit antiserum raised against synthetic ct, coupled to keyhole limpet hemocyanin was used in this assay.

Serum Mice were bled by severing the blood vessels in the neck. The blood was collected in tubes, allowed to clot overnight at 4°C and then was separated by centrifugation and stored at -70oc until assayed.

RESULTS Elevated IRa, in serum o f old A K R mice with spontaneous T-cell leukemia In our initial studies, we observed that 6-10-month-old leukemic mice (defined as having a thymoma and enlarged peripheral lymphoid organs) had consistently elevated serum IRa, levels as compared to normal young and age-matched mice (Table 1). These data are consistent with earlier unpublished data (J. E. McClure, A. L. Goldstein and A. Barker). It also should be noted that the serum ct, levels of the normal old AKR mice were lower than those of the young mice. Similar age-related declines in IRct, have been noted in other mouse strains (data not shown). Elevated IRut in serum o f young A K R mice injected with lymphoma cells In an attempt to determine the source of the elevated serum IRa,, splenic lymphoma cells from two mice with spontaneously arising lymphomas were injected into normal young mice. Serum was collected from groups of three mice at intervals during the growth of the transferred lymphoma cells, which killed the recipients at 21-24 days. As shown in Table 2, serum IRet, levels increased in parallel with growth of the injected tumor cells, rising sharply after 14 days post injection, when the passaged tumor first became palpable in the spleens and lymph nodes and reaching levels similar to those found in the serum of donor mice.

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lmmunoreactive thymosin ct~ in T-cell iymphomas TABLE 1. THYMOSIN0tl IN SERUMOF NORMALAND LEUKEMIC AKR MICE Group

(n)

a~ (pg/ml)*

Normal, 2-month-old Normal, 6-10-month-old Leukemic, 6-10-month-old

(19) (20) (13)

937 :t: 146 395 + 70 5386 -I- 979t

*Mean -+- S.E.

"t'Significance at p <0.001 by Student's t-test compared to normal groups.

TABLE 2. KINETICSOF INCREASEDSERUMIRa, LEVELSIN MICEINJECTEDWITHSPONTANEOUSLYARISINGTHYMICLYMPHOMACELLS

Experiment*

l

2

Dayst

Uninjected control

10~ Thymoma cell recipients Ratio experimental/control

IRa,(pg/ml):~

IRttj(pg/ml)

7

1294 -I- 179

1208 ± 303

0.96

11

1494 "+- 258

1917 -I- 602

1.28

14

1588 "4" 50

2138 -t- 170

1.35

18

1724 ± 146

6138 "4" 688

3.59

6

1336 "4" 625

880 :l: 44

0.66

16

1039:1:66

2543 -I- 37

2.45

20

657 "4" 145

4015 -t- 142

6.11

*In experiment 1, the leukemic AKR donor of the spontaneous thymoma had a serum IRa, level of 5300 pg/ml; the donor for experiment 2 had a serum IRa, of 2900 pg/ml. tMice were bled on the days indicated after i.v. injection of 10~ thymoma cells into groups of 3 young AKR mice. ~:Data given as means :t: S.D.

Role o f the thymus in elevated serum 1Ra, In o r d e r to distinguish b e t w e e n the t w o possibilities t h a t (1) the s e r u m I R a , was d e r i v e d directly f r o m the g r o w i n g l y m p h o m a cells, or (2) was the indirect result o f a c t i v a t i o n o f the t h y m u s , l y m p h o m a cells were i n j e c t e d i n t o g r o u p s o f e i t h e r n o r m a l o r Tx A K R m i ce. T h e l y m p h o m a cells used were the A K T B - I t a n d A K T B - 1 b t r a n s f e r cell lines, b e c a u s e they p r o v i d e d the u n i q u e o p p o r t u n i t y to c o m p a r e the ef f ect s o f a T-cell a n d B-cell A K R l y m p h o m a . T h e results ( T a b l e 3) clearly d e m o n s t r a t e that the A K T B - l t T-cell l y m p h o m a , but n o t the A K T B - l b B-cell l y m p h o m a , gives rise to e l e v a t e d I R a , ser u m levels, regardless o f w h e t h e r or n o t the recipient a n i m a l possesses an intact t h y m u s . Since these t w o t u m o r cell lines h a v e been a d a p t e d to in vivo passage, o n l y 10' cells w e r e i n j e c t e d a n d a n i m a l s were s t u d i ed at day 14, at w h i c h time l y m p h o i d o r g a n s o f b o t h g r o u p s o f m i c e were c o m p ar ab l y e n l a r g e d 10-fold. B o t h the A K T B - I t an d A K T B - l b t u m o r s kill the m i c e 16-19

1006

MARION M. ZATZ et al. TABLE 3. SERUM IRa, LEVELSOF MICE INJECTED WITH T-CELL OR 8-CELL AKR L'fMPHOMA*

Cells injected

Normal

Recipients Thymectomized

IRa,(pg/ml)t

IRa,(pg/ml)

Control

1105 4. 351

1135 4. 305

AKTB-Ib

1292 4. 473

1753 4. 442

AKTB-lt

5298 4. 566

5775 4- 567

Ratio AKTB-lt/control

4.97

5.09

Ratio AKTB-It/AKTB-lb

4.10

3.29

*10 ~ AKTB-It or 10" AKTB-Ib splenic lymphoma cells were injected i.v. into groups of 5-7 young normal or thymectomized mice. Mice were bled at day 14 to determine serum IRa, levels. Controls consisted of uninjected animals. tData given are means 4- S.D. d a y s a f t e r t r a n s f e r . I t s h o u l d a l s o b e n o t e d t h a t n o c h a n g e i n s e r u m I R a , o c c u r s in t h e uninjected Tx controls, further suggesting that the thymus may not be the primary source of circulating IRa,.

Association of IRa, with T-iymphoma cells and tissue I n o r d e r t o t e s t w h e t h e r t h e s o u r c e o f t h e IRCtl i n s e r u m c o u l d b e t h e l y m p h o m a cells, t u m o r cell s u s p e n s i o n s a n d t i s s u e h o m o g e n a t e s c o n s i s t i n g o f >90°70 t u m o r cells w e r e s o n i c a t e d a n d t h e s o l u b i l i z e d s o n i c a t e s w e r e e v a l u a t e d f o r I R a , . L y m p h o m a cells (5 x 10'/ml) were also placed in short term tissue culture overnight at 37°C and the supern a t a n t s w e r e e v a l u a t e d f o r I R a , c o n t e n t . T h e r e s u l t s , s h o w n i n T a b l e 4, d e m o n s t r a t e t h a t TABLE 4. COMPARISON OF IRa, (pg/ml) ASSOCIATED WITH AKR T- AND B-CEI.L I YMPHOMAS Spontaneous thymoma

AKTB- I t

AKX-3

AKTB- I b

AKX- 1b

5586 4- 979 (n = 13)

5383 4- 316 (n = 16)

4295 4- 1500 (n = 2)

.1447 4- 219 (n = 21)

1270 4- 260 (n = 3)

4220 ___ 1358 (n = 2)

2635 (n = 1)

n.d.

816 4. 20 (n = 2)

564 -+" 221 (n = 2)

Cell Extract (per lO'/cells):l:

n.d.

1240

n.d.

<160

n.d.

Cell supernatant~:

590

510

n.d.

<160

n.d.

Serum* Tissue extract (per 0.1 mg protein)t

*Serum was collected from mice with spontaneously arising thymoma (see Table 1) or from mice injected with 10~ splenic lymphoma cells 14 days earlier. tSpleens from mice bearing spontaneous or passaged lymphomas were weighed, and sonicated (see Methods). Results are expressed as pg/ml/0.16 mg protein. :~106 Splenic lymphoma cells were either pelleted and sonicated or cultured overnight. After overnight culture, supernatant was collected and assayed for IRct~ content. Results are expressed either as lRct, pg/ml/106 cells or pg/ml supernatant. n.d., not done.

lmmunoreactive thymosin ¢~, in T-cell lymphomas

1007

IRa, can be released into the medium by both T-cell lymphomas and recovered from the tissues or cells, in much greater amounts than those for the two B-cell lymphomas. These results are consistent with the in vivo data showing that IRa, is directly associated with, and can be released by, T-cell lymphomas. DISCUSSION AND CONCLUSIONS These results demonstrate that a thymosin o,-like material, which reacts with a highly specific rabbit anti a, antibody, is elevated in serum of AKR mice bearing spontaneous or transplanted T-cell lymphomas. The studies reveal that this IRa, is associated directly with the T-iymphoma cells and is not dependent upon the thymus for expression since the elevation is comparable in Tx animals. The present studies do not distinguish between the two possibilities that the IRa, released by the T-lymphoma cells is passively acquired or actively synthesized. Further studies in humans have shown that sera from patients with T-cell leukemias, particularly those with acute T-cell lymphocytic leukemia, also have elevated a, levels [42]. One of these patients with a leukemia classified as Japanese adult T-cell form, also was positive for HTLV. Thus thymosin IRa, may be associated with T-cell lymphomas in both experimental animals and man. These studies are of importance in several respects: first, it has long been suspected that the thymus epithelium may be the primary, but not the only source, of thymic hormones. Thus, detectable a, levels are found in sera of nude (athymic) mice, and as well as in adult Tx mice (see Table 3, also ref. 24). Although thymic epithelial cells can be most strongly stained with the same antibody to a, [4, 15, 17, 18] as was used in these studies, epithelial cells in other organs also can be weakly labeled [18]. It should be noted that the antiserum to thymosin a, used in the RIA does not cross-react with a-fetal proteins, carcinoembryonic antigen, or a spectrum of other well defined thymic and non-thymic hormones [23], but does stain subcapsular cortical and medullary thymic epithelial cells [4, 15, 17, 18]. A second thymosin peptide, 13, which also stains thymic cortical epithelial cells [17] was tested in these studies and found not to be elevated in the serum of the T-cell tumor bearing mice (data not presented). However, Goodall et al. have reported [10] that thymosin [3, also can be isolated from non thymic tissue, i.e. macrophages, and is synthesized by several non lymphoid tumor cell lines. The biochemical analyses now in progress will enable us to determine whether or not the immunoreactive a, associated with the T-cell lymphomas is produced by the T-cell tumor cells, and is biochemically and functionally identical to the thymic peptide, or an inactive and/or cross-reactive product. Recent studies have established that a number of hormone and hormone-like factors, termed 'tissue hormones', including insulin, ACTH, glucagon, gastrin and bombe~in, are produced by tissues far removed from the primary endocrine glands associated with these molecules [34]. In addition, there is a considerable body of evidence documenting the production of biologically active hormones by dedifferentiated tumor cells [27, 30, 32]. An a,-like material that immunologically and chemically appears to be identical with the a, produced by the thymus gland [29] has been isolated from normal rat and pig brain. A thymopoietin-like material, termed thymopoietin III, has been isolated from calf spleen [1]. A second important aspect of this work is in the potential for detection and classification of human tumors. In at least one case of a patient with thymic lymphoma, two-fold elevated serum IRa, levels were detected; this level decreased to the normal range following thymectomy (unpublished data). Tissue extracts of the thymoma contained twice as much IRa, as did thymus from an age and sex matched normal individual. Earlier studies have shown that some leukemia patients have elevated serum a~ levels which decline during chemotherapy and remission (A. L. Goldstein, J. E. McClure and E. M. Hersh et al., unpublished).

1008

MARION M. ZATZet al.

Finally, the elevated IRct, may be related to the activation an/or expression of T-lymphotropic leukemia viruses. In recent studies of homosexual populations with acquired immunodeficiency syndrome (AIDS) shown to be at high risk for development of rare malignancies such as undifferentiated Hodgkins and Kaposi's sarcoma [5, l l, 12, 36], serum IRa, levels also were consistently elevated [16, 28]. The source of the elevated ct, in the AIDS patients is not yet known, however, several studies have shown an association of the human T-cell leukemia virus [31, 33] with AIDS [2, 6, 8, 9]. Another study has shown that hemophiliacs who are HTLV ÷ also have elevated serum levels of IRa, [19]. Haynes et aL have reported [14] that human thymic epithelial cells which are positively stained for thymosin ct, using the same antibody used in the present study, also are stained with a monoclonal antibody to the pl9 protein of HTLV. Interestingly, human thymic epithelium is the only normal tissue which reacts with this anti HTLV antibody. Thus it is possible that infection with murine or human T-lymphotropic viruses, may result in production of a thymosin ct,-like peptide, or alternatively, that the HTLV and ct, genes share homologous sequences. Additional studies are underway to determine, in both experimental and clinical situations, which lymphomas are associated with elevated thymosin ~t, levels and what the source, biochemical characteristics and significance of this molecule are in the development of these malignancies. The association of elevated serum thymosin ct, in certain tumor-bearing individuals may provide a valuable diagnostic test to monitor the growth and regression of the tumor, as well as clues to the biology of lymphoma development. The data clearly demonstrate that a serum product or products, serologically cross reactive with thymosin ~t,, can be produced by an extrathymic source.

Acknowledgements--This work was supported in part by grants from the National Institutes of Health, U.S.P.H.S., CA 29943 and CA 24974 and by funds from Hoffman-LaRoehe, Inc. We wish to thank John Hallam for excellent technical assistance and Rachel Quynn for preparation of the manuscript. This work was presented in preliminary form at the 15th International Leukocyte Culture Conference, held at Asilomar, CA, December 1982. REFERENCES I. AUDHYA T., SCHLESIN(iER O. H. & GOLDSTEIN G. (1981) Complete amino acid sequence of bovine thympoietins l, II and Ill. Closely homologous peptides. Biochemistry 30, 6195. 2. BARRE-SINoUSSIF., CHERMANNJ. C., REv F., NUGEYRE M. TR., CHAMERETS., GRUEST J., DAUGUETC., AXLER-BLIN C., VEXINET-BRUNF., Rouzoux C., ROZENBAUMW. & MONTAGNIER L. (1983) Isolation of a T-lymphocytropic retrovirus from a patient at risk for acquired immunodeficiency syndrome AIDS. Science 220, 868. 3. BIRr C. & STOt.t.ENWERK U. (1979) Synthesis of thymosin ct,, a polypeptide of the thymus, Angew. Chem. EngL 18, 394. 4. DAL.AKAS M. C., EN(iLE W. K., McCLURE J. E., GOI.DSTEIN A. L. & ASKANAS V. (1981) Immunocytochemical localization of thymosin ct, in thymic epithelial cells of normal and myasthenia gravis patients and culture. J. neurol. Sci. 50, 239. 5. DURACK D. T. (1981) Opportunistic infections and Kaposi's sarcoma in homosexual men. New. Engl. J. Med. 305. 1439. 6. Essex M., McLANE M. F., LEE T. H., FAt.K L., HOWe C.W.S., MULLINSJ. 1., CABRADILLAC. & FRANCIS D. P. (1983) Antibodies associated with human T-cell leukemia virus in patients with AIDS. Science 220, 865. 7. Fol KErs K. (1980) Current advances in biologically active synthetic peptides. In Polypeptide Hormones (BEERS R. F. & BASSETT E. G., Eds.), p. 149. Raven Press, New York. 8. GAI.lO R. C., SARIN P. S., GEl.MANN E. O., RonErt-Guroe~ M., RICHARDSONE., KALYANARAYMANV. S., MANN D., SIDHU G. D., STAHL. R., ZOLLA-PAZNERS., LEIBOWlTCH J. & PoPOVlC M. (1983) Isolation of human T-cell leukemia virus in acquired immune deficiency syndrome (AIDS). Science 220, 86. 9. GEl.MANN E. P., PoPovlc M., Bt.AYNEY D., MAsur H., StDHU G., StAHt. R. & GALLO R. C. (1983"1 Provirual DNA of a retrovirus, human T-cell leukemia virus in two patients with AIDS. Science 220, 862. 10. GOODAt I G. J., MOR~AN J. I. & HorECKER B. L. (1983) Thymosin ct, in cultured mammalian cell lines. Archs Biochem~ Biophy. 221,598. II. GoorENm-r(; J. E., Rus¢'tiTXl F. W. & GAttO R. C. 11982) A biochemical variant of human T-ceil growth factor produced by a cutaneous T-cell lymphoma cell line. J. lmmun. 129, 1499.

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