selected human t cell lines respond to thymopoietin with intracellular cyclic gmp elevations

Immunopharmacology, 16 (1988) 115-122 Elsevier 115

IMO 00421

Selected human T cell lines respond to thymopoietin with intracellular cyclic GMP elevations

Bruce Baker , G e o r g e V i a m o n t e s , T a p a n A u d h y a and G i d e o n G o l d s t e i n

lmmunobiology Research Institute, Route 22 East, P.O. Box 999, Annandale, NJ 08801-0999, U.S.A.

(Received 18 December 1986; accepted 14 July 1988)

Abstract: Ten established human cell lines were tested for their responsiveness to thymopoietin by measuring their intracellular cyclic nucleotide levels. Three T cell lines (CCRF-CEM, MOLT-4 and CCRF-HSB-2) responded to thymopoietin with elevations of intracellular cGMP but not cAMP; seven other human cell lines did not respond to thymopoietin (three T cell lines, three B cell lines and one erythropoietic stern cell line). Interestingly, only one cell line (MOLT-4) was also responsive to the closely related polypeptide splenin, and this reactivity was restricted to human and not bovine splenin. The detection of human cell lines with distinctive patterns of response to immunoregulatory peptides should provide support for understanding the immunopharmacological mechanisms by which these molecules act.

Key words: Thymopoietin; Splenin; Cyclic GMP; Cyclic AMP; Immunoregulatory peptide

Introduction

Thymopoietin is a polypeptide hormone secreted by epithelial cells in the thymus (Audhya et al., 1981; Goldstein, 1974; Viamontes et al., 1986). It has pleiotropic effects in the body, with tissue targets including the nicotinic acetylcholine receptor at the neuromuscular junction (Goldstein, 1968; Gold- stein and Hoffman, 1969; Venkatasubramanian et al., 1986), prothymocytes (thymocyte precursors) (Basch and Goldstein, 1974; Ranges et al., 1982; Scheid et al., 1978) and mature T (thymus-derived) cells, both helper and suppressor T cells (Weksler et al., 1978; Lau et al., 1982; Goldberg et al., 1981, 1984). Complete amino acid sequences have been

Correspondence: G. Goldstein, Immunobiology Research Insti- tute, Route 22 East, P.O. Box 999, Annandale, NJ 08801-0999, U.S.A. Abbreviations. bSP, bovine splenin; cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanosine monophosphate; hSP, human splenin; PBS, phosphate-buffered saline; SP, splenin; SP5, splenopentin; Tpo, thymopoietin; TP5, thymopentin.

determined for bovine and human thymopoietin and for the closely related molecule splenin (Aud- hya et al., 1981, 1986). Amino acids 32-36 of bovine and human thymopoietin are identical (Arg-Lys- Asp-Val-Tyr) (Audhya et al., 1981) and the synthetic pentapeptide corresponding to this sequence (thymopentin, TP5) has been shown to have the biological activity of the full 48 (human) or 49 (bovine) amino acid native polypeptide (Goldstein et al., 1979). For splenin, the pentapeptide corresponding to amino acids 32-36 of bovine splenin (Arg-Lys-Glu-Val-Tyr) was also shown to be active in murine systems (Audhya et al., 1984b) but the corresponding sequence in human splenin is Arg- Lys-Ala-Val-Tyr.

The amino acid sequence of thymopoietin is distinct from that of the thymic hormone thymulin (Savino and Dardenne, 1984) and from that of the peptides which have been characterized in the thymus extract thymosin fraction V (Hooper et al., 1975). The functional relationship between these various thymic polypeptides remains unclear.

0162-3109/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

116

Polypeptide hormones usually interact with cell surface receptors, and transduction of the signal to the cell interior often involves alterations in intracellular cyclic nucleotide levels. For thymopoietin, there is evidence that interaction with a receptor on prothymocytes involves cAMP alterations with subsequent differentiation, as evidenced by cell surface phenotypic changes (Scheid et al., 1978), while interaction with receptors on mature T cells involves cGMP alterations with changes in functional activity (Sunshine et al., 1978; Audhya et al., 1984c).

Circulating human lymphocytes contain hetero- geneous cellular populations. We therefore utilized human lymphocyte lines, which represent discrete differentiative stages, to determine whether we could identify cell lines that would respond to thymopoietin with alterations in levels of intracellular cyclic nucleotides. Clones of cells uniformly ex- pressing a given thymopoietin receptor would be of value in studying the structure of the receptor and the nature of thymopoietin receptor interactions.

The human T cell line CEM was previously shown to respond to thymopoietin with an elevation of intracellular cGMP and this responsiveness was shown to correlate with the appearance of a receptor molecule for thymopoietin in the cell membrane, as demonstrated by radio-receptor tech- niques (Audhya et al., 1984). We have now screened further human cell lines for their responsiveness to thymopoietin and splenin and now describe two additional T cell lines that respond to thymopoietin with elevations of cGMP, and three other T cell lines, three B cell lines and one erythropoietic stem cell line that did not respond to thymopoietin.

Materials and Methods

Human cell lines Human cell lines were obtained from the American Type Culture Collection or from the NIGMS Hu- man Genetic Mutant Cell Repository and grown in RPMI 1640 with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine and 50 #g/ml gentamycin.

CEM (ATCC TIB 195 CEM-CM3), MOLT-4

(ATCC CRL 1582 MOLT-4), HSB-2 (ATCC CCL 120.1 CCRF-HSB-2), MOLT-3 (ATTCC CRL 1552 MOLT-3) and GM3639 (MGMS T cell line 8402) were all derived from acute lymphatic leukemia cells in the peripherial blood. Hut-78 (ATCC TIB 161 Hut-78) was derived from cutanous T cell lymphoma cells, and both Daudi (ATCC CCL 213 Daudi) and Raji (ATCC CCL 86 Raji) were derived from Burkitt lymphomas. The origin of GM4680 (MGMS lines IM9) was derived from chronic lymphatic leukemia cells and K562 (ATCC CCL 243 K-562) was derived from chronic myeloid leukemia cells.

Cell surface phenotype

The cell lines were tested for cell surface marker expression with a panel of fluorescein-isothiocya- nate-conjugated monoclonal antibodies (OKT1, OKT3, OKT4, OKT6, OKT8, OKT9, OKT10 and OKT11) that react with cell surface antigens of human T cells and indicate their phenotypic differentiation (Goldstein et al., 1982). They were also tested for surface immunoglobulin using fluorescein-conjugated goat anti-human IgG (Pel-Freeze Biologicals, Rogers, AR). 1 × 106 cells were treated with 0.1 ml of antibody (10 /~g/ml in phosphate- buffered saline (PBS) with 5% fetal calf serum and 0.1% sodium azide) for 30 rain at 4°C, washed through a cushion of fetal calf serum, and resuspended in 1 ml of PBS with 0.1 percent sodium azide.

Treated cells were analysed by flow cytometry with an Ortho System 30 L Cytofluorograf.

Polyp~ti~s

Bovine thymopoietin (Tpo) and bovine and human splenin (SP) were prepared as described (Audhya et al., 1981). Synthetic thymopentin (Arg-Lys-Asp- Val-Tyr) (TP5) and splenopentin (Arg-Lys-Glu- Val-Tyr) (SP5) were synthesized using solid-phase methodology (Merrifield, 1963). Each had the cor- rect amino acid composition and was homogeneous by both thin-layer chromatography and high-per- formance liquid chromatography.

Assays for cyclic nucleotides

Cells were transferred into fresh medium at 5 × 105 cells/ml and incubated for 1 5 days prior to experimental treatments and assay. Cells to be assayed were washed 3 times with RPMI 1640, resuspended in the same medium at 1.0 x 10 7 cells/ml and aliquot- ted into glass test tubes (1 ml/tube). Tubes were

warmed in a 37°C water bath for 30 min. 25 pl of each test compound at 40 x final concentration were added to the appropriate tubes. After addition of compound, cells were incubated at 37°C for peri- ods ranging from 1 to 16 min. Reactions were termi- nated by the addition of 1 ml of cold 10% trichlo- roacetic acid. At this point, samples were frozen

and thawed 3 times, extracted with hydrated ether and lyophilized. Cyclic nucleotide levels were determined according to the procedure of Steiner et al. (1972) using commercial radioimmunoassay kits (New England Nuclear, Boston, MA).

Results

Cell lines responsive to thymopoietin and thymopentin Following incubation with thymopoietin or thymo-

pentin under optimal conditions (defined below),

CEM, MOLT-4 and HSB-2, three of the six T cell lines tested, showed significant elevations in

117

their intracellular cGMP levels (Tables I and II). In

contrast, cAMP levels were not affected in these lines. Three additional T cell lines (MOLT-3, HUT-78 and GM3639), the B cell lines Raji, Daudi

and GM4680, and a non-T, non-B cell line K562 were not affected by either Tpo or TP5. Bovine SP and SP5 did not induce cyclic nucleotide changes in any of the cell lines and MOLT-4 alone responded to human SP with a cGMP elevation.

Cell surface phenotype

The cell surface phenotypes of the cell lines are sum- marized in Table I. CEM, MOLT-4 and HSB-2 all displayed cell surface molecules indicative of their T cell origins.

Cyclic GMP response depends on time in culture

The growth characteristics of these three human T cell lines are displayed in Fig. 1, with CEM and

MOLT-4 reaching plateau conditions at 3 to 4 days. These lines became responsive to Tpo/TP5 as a function of time following transfer into fresh medi-

um. cGMP responses to TP5 were first detected on day 4 in CEM and on days 3~4 in MOLT-4 and

HSB-2 (Fig. 2). Response characteristics remained stable through at least 8 serial passages and similar results were obtained with frozen cells revived over several years.

TABLE I

List of human cell lines tested with their source, cell type, cell surface phenotype, and responsiveness to Tpo/TP5

Cell line Origin Type Surface markers cGMP responses to Tpo/TP5

CEM ATCC ALL (T) TI ,T4,T9,T 10 + MOLT-4 ATCC ALL (T) T1,T4,T6,TS,T9,TI0,T11 + HSB-2 ATCC ALL (T) T1,T3,T6,T9,TI0 + MOLT-3 ATCC ALL (T) T1,T3,T6,T9,TI0 - GM3639 NIGMS ALL (T) T1T4 - HUT-78 ATCC T lymphoma TI,T3,T4,T9,T10 - Daudi ATCC Burkitt lymphoma SIg Raji ATCC Burkitt lymphoma Slg - GM4680 NIGMS CML (B) SIg - K562 ATCC (CML stem cell) T9

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TABLE II

cGMP (A) and cAMP (B) levels of human cell lines after exposure to bovine thymopoietin (10-1 #g/ml), thymopoientin (102 #g/ml) and

bovine and human splenin (10 I #g/ml): results are expressed as pmol/ml, means 4- S.D., n = 4

Cell line Compound added

None Tpo TP5 bSP hSP

A. cGMP CEM 0.20 4- 0.02 8.50 4- 0.42 6.50 4- 1.40 0.20 4- 0.01 0.20 ± 0.03

MOLT-4 0.39 4- 0.02 5.40 4- 0.07 7.30 4- 0.14 0.28 4- 0.01 9.70 ± 0.13

HSB-2 0.124- 0.02 5.80 4- 0.57 6.30 4- 0.92 0.15 4- 0.05 0.11 4- 0.02

HUT-78 0.33 4- 0.02 0.34 4- 0.03 0.30 4- 0.01 0.25 4- 0.01 0.30 ± 0.03

MOLT-3 21.60 4- 5.20 16.80 + 0.40 25.00 4- 2.80 21.50 4- 0.70 28.00 4- 0.70

GM4680 0.35 4- 0.10 0.39 4- 0.01 0.38 4- 0.05 0.38 4- 0.02 0.37 4- 0.08

GM3639 4.40 4- 1.82 3.00 4- 0.2l 3.70 4- 0.42 3.00 4- 0.21 5.80 :L 1.53

Raji 0.I0 4- 0.03 0.08 4- 0.01 0.08 4- 0.01 0.15 ± 0.02 0.07 4- 0.01 Daudi 0.23 4- 0.02 0.20 4- 0.02 0.23 4- 0.03 0.21 4- 0.04 0.24 ± 0.01

K562 0.30 ± 0.05 0.32 4- 0.02 0.31 4- 0.02 0.27 4- 0.01 0.28 4- 0.01

B. cAMP CEM 12.6 4- 0.14 13.2 + 1.60 12.2 -4- 0.49 13.8 4- 0.70 12.8 + 0.27

MOLT-4 1.9 ± 0.06 1.7 + 0.07 1.4 4- 0.14 2.2 4- 0.15 1.6 + 0.18

HSB-2 4.3 + 0.28 5.8 ± 0.35 3.5 + 0.14 4.6 + 0.98 5.7 + 0.84

HUT-78 78.7 + 15.30 69.0 4- 5.60 55.2 4- 8.60 60.5 4- 4.90 61.5 4- 4.90

MOLT-3 10.0 4- 0.72 10.8 4- 1.10 11.0 4- 0.71 9.9 4- 0.88 10.6 4- 0.91

GM4680 26.5 4- 3.00 23.5 4- 0.71 29.5 4- 2.10 27.5 4- 0.71 26.0 4- 5.70

GM3639 2.6 ± 0.46 2.9 + 0.28 2.4 4- 0.28 3.0 4- 0.29 3.1 4- 0.35 Raji 1.9 4- 0.25 1.5 4- 0.41 2.1 4- 0.I0 2.0 4- 0.29 1.8 4- 0.32

Daudi 14.8 4- 2.31 13.5 4- 0.46 18.2 4- 1.61 19.1 4- 0.28 19.4 4- 1.71

K562 36.8 4- 8.50 32.1 4- 1.40 37.1 4- 4.50 35.5 4- 2.12 36.6 4- 5.20

Dose-response characteristics

The three responding T cell lines showed different dose-response patterns, with the threshold concentrations of Tpo evoking a cGMP response being 1 ng/ml for HSB-2, 1 ng/ml for CEM and 100 ng/ml

100

w

C E M

• • | • •

0 1 2 3 4 5 6

DAYS AFTER TRANSFER

Fig. 1. Cell growth curves for CEM, MOLT-4 and HSB-2 showing cell numbers as a function of time following cell transfer into

fresh medium. Growth of CEM and MOLT-4 cells shows a clear plateau phase after 3 days of culture.

for MOLT-4 (Fig. 3A). TP5 required considerably higher concentrations to evoke a response, the threshold concentrations being 0.1/~g/ml for HSB-2 and 1 #g/ml for CEM and MOLT-4 (Fig. 3B).

Time of cGMP response

The time of cGMP elevation with respect to exposure to TP or TP5 varied between the cell lines and was also dependent on the concentration of TP or TP5. The time courses of cGMP changes after exposure to various concentrations of TP5 are shown in Fig. 4. For CEM, cGMP elevations were evident at 2 min and declined by 8 min with 1 10 ktg/ml TP5, but the cGMP elevations remained sustained with higher concentrations of 100-1000/~g/ml TP5 (Fig. 4A). For MOLT-4, cGMP elevations were evident at 1 min with 1000 #g/ml TP5 but were first

lO A

CEM i •

~--¢," 10 0 101 1 ; 2 10 3

o • Day 1 "~ ~ . ~ • Day 2 0. • Day 3 (9 1 o Day 4 (J

i ~ IOLT-4 I i ,q- • 0 10 0 101 10 2 10 3

<C

HSB-2 |

' , o , , o , 0 10 0 101

TP5 CONCENTRATION (p.g/ml)

Fig. 2. Dose-response curves for C E M (A), M O L T - 4 (B) and

HSB-2 (C) cells, showing responsiveness to TP5 as a function of time following transfer into fresh medium. CEM cells become responsive on day 4, MOLT-4 cells on days 3 4 and HSB-2 ceils on days 3-4. Each point represents the mean + SD of at least 4

experiments.

seen at 2 min with lower concentrations; in this cell line the elevations persisted through 16 min with all effective concentrations (Fig. 4B). For HSB-2, cGMP elevations were first seen at 2 min with 100- 1000 #g/ml TP5 and at 4 min with 1-10 pg/ml TP5; at all effective concentrations the cGMP elevations persisted through 8 min (Fig. 4C).

Responsiveness to SP

No cGMP responses were obtained in any cell line with bovine SP or SP5 but MOLT-4 did give a cGMP response with human SP (Fig. 5).

119

10

i -6 Eo o

o n" ~, 10

3 _J

er 1 z

A

~;" • • I I I 0 I 0 -4 I 0 - 2 10 0

TP CONCENTRATION (p,g/ml)

B T ~ l i

! i M O L T - 4

dl~:" • " I I I 0 10 -2 10 0 10 2

TP5 CONCENTRATION (#g/ml)

Fig. 3. Dose-response curve showing cGMP elevations in CEM, MOLT-4 and HSB-2 cells after a 4 min incubation with varying amounts o fTpo (A) or TP5 (B). Tpo is 10-1000 fold more potent than TP5 (I00-10,000 fold on a molar basis) in this system, with differences in the individual cell lines. Each point represents the

mean + SD of at least 4 experiments.

Discussion

The majority of polypeptide hormones deliver their message by interacting with cell surface receptors, this interaction in turn setting in motion intracellular biochemical cascades that trigger the appropriate responses by the cell. There is considerable het- erogeneity in the lymphocyte population and even in the T cell population, so that it has not been possible to isolate pure populations of normal cells which respond to thymopoietin in a uniform fash- ion. Another approach that has been used success- fully for other hormones is the use of hormone-responsive cell lines (Hedo et al., 1981; Hook et al., 1982), and we have previously reported that Tpo induces elevations of intracellular cGMP in cells of the human T cell line CEM (Audhya et al., 1984c). Furthermore, we have partially purified a Tpo receptor molecule from the membranes of CEM cells and have shown in radioreceptor studies that it binds two regions of the Tpo sequence corresponding to amino acids 32-36 and 3845 (Audhya et al., 1984a).

120

10 A O~i~. . . . . . =

CEM i l I • •

0.5 1 2 4 8 16

10 B 0 / 0 ~ o

M O L T - 4 I | • 1 2 4 8 16

10 C ~ / ~

H S B - 2 . = I

1 2 4 8

INCUBATION TIME (rnin)

-6 E o o "5.

(9 o n-

5

.J

Z

TP5 C o n c e n t r e t i o n

• 0 • 1 #g /m l • 10 #g /m l 0 100 # g / m l 0 1000/~g/ml

Fig. 4. Effects of incubation time on cGMP elevations in CEM (A), MOLT-4 (B) and HSB-2 (C) cells exposed to varying concentrations of TP5. At low doses of TP5, cGMP levels return to basal values in CEM cells (A), whereas they remain elevated over the time of the experiment at higher TP5 concentrations in CEM and at all concentrations tested in MOLT-4 and HSB-2. Each

point represents the mean ± SD of at least 4 experiments.

J 10 ¸

(9

°E

o o

I'- Z

bSP

M O L T - 4 q~l I I I

10-4 10-2 10 0

POLYPEPTIDE CONCENTRATION

(p.g/ml)

Fig. 5. Dose-response curve showing cGMP elevations in MOLT-4 cells after a 4 min incubation with varying amounts of human (hSP) or bovine (bSP) splenin. MOLT-4 cells were responsive to hSP but not to bSP. Each point represents the mean

i SD of at least 4 experiments.

Our present studies have extended this approach to determine the effects of Tpo on ten human cell lines (five T cell lines, four B cell lines and one erythropoietic stem cell line). A number of interesting findings have emerged. Only three cell lines (CEM, MOLT-4 and HSB-2) responded to Tpo and these all responded with elevations of intracellular cGMP, not cAMP. All three responsive lines were T cells, as evidenced by their cell surface phenotype. Thus CEM was positive for the T cell surface molecules, as detected by OKT1 and OKT4 monoclonal antibodies, MOLT-4 was positive for OKT1, 4, 6, 8 and 11 and HSB-2 was positive for OKT1, 3 and 6. These cell surface phenotypes suggest that the lines correspond to T cells at various stages of maturity although, as is commonly found with cell lines or malignant cells, these phenotypes do not correspond fully to the patterns seen in normal T cells.

Three other T cell lines, three B cell lines and an erythropoietic stem cell line failed to respond to Tpo with cyclic nucleotide changes, establishing the distinctive and specific nature of the response to Tpo in the three responsive T cell lines. Furthermore, none of the ten cell lines responded to bovine SP, which dif- fers from Tpo by a substitution of glutamic acid for aspartic acid at position 34 in the active-site region (Audhya et al., 1981), and none responded to synthetic SP5 (Arg-Lys-Glu-Val-Tyr) (Audhya et al., 1984b), again establishing the exquisite specificity of the responses to Tpo of CEM, MOLT-4 and HSB-2.

Freshly cultured cells of the responsive lines were initially unresponsive, with Tpo responsiveness de- veloping after several days in culture. Typically, when cells are cultured at low density, undifferen- tiated cells proliferate and predominate, with differentiative features appearing as cells reach higher densities or confluence and proliferation decreases (Akeson and Herschman, 1974). We have previously shown that, for CEM, the amount of thymopoietin binding protein that can be extracted from the cell surface membrane rises at 100 h when a plateau in cell density has occurred and proliferation has decreased (Audhya et al., 1984a,c). We hypothesize that Tpo receptors are absent from less differentiat- ed proliferating cells but appear as differentiation

antigens on the cell surface as the cells reach higher

densities and stop proliferating; these cells are now responsive to thymopoietin, as evidenced by changes in intracellular cGMP.

Our present findings suggest that the three Tpo- responsive human T cell lines identified, CEM, MOLT-4 and HSB-2, correspond to relatively mature human T cells as judged by their cell surface phenotype and cGMP responsiveness (as opposed to the cAMP responsiveness to Tpo found in prothymocytes). Each cell line responded to significantly lower concentrations of Tpo than of TP5. This may be attributable to the more effective presentation of the active-site region in Tpo consequent to the sec-

ondary folding of the entire 49 amino acid sequence compared to the configuration of the small synthetic pentapeptide Alternatively it may be related to our finding that native Tpo binds the CEM receptor by an additional binding site corresponding to re- sidues 38~45. This second binding site does not trigger the biological changes effected by binding of TP5 but would facilitate the binding of Tpo and thus increase the probability that the TP5 region of Tpo engaged its corresponding region of the recep-

tor. CEM and HSB-2 both had similar dose-response

curves, with Tpo thresholds of 1 ng/ml and TP5 thresholds of 0.1-1 ktg/ml. In contrast, MOLT-4 consistently needed 0.1 /~g/ml TP to trigger cGMP

responses although it also responded to TP5 at a threshold dose of 1 /~g/ml. These differing dose-response characteristics could suggest a difference in the Tpo receptors of these cell lines. Furthermore, MOLT-4 alone also gave a cGMP response with human SP. The fact that MOLT-4 was responsive to human but not to bovine SP led us to predict a change in the active site between bovine and human

SP, a prediction that has now been directly con- firmed from amino acid sequence (Audhya et al., 1986). Further studies will be needed to determine whether MOLT-4 has distinct receptors for Tpo and SP or whether Tpo receptors in general are het- erogeneous, with SP interacting with the particular Tpo receptor represented on MOLT-4 cells.

These studies establish that human lymphocyte lines which are biologically responsive to Tpo can

121

be detected by a screening process. These cell lines

should prove invaluable in studying the nature of the Tpo receptors on the cells and the nature of Tpo-receptor interactions.

Acknowledgements

We thank Alexander Collins for expert technical as-

sistance, Nancy Lawery for typing the manuscript and Marilyn Sanders for editorial assistance.

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selected human t cell lines respond to thymopoietin with intracellular cyclic gmp elevations

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