Cyclic Nucleotide Changes in Murine Lymphocytes Following Thymosin Incubation In Vitro

Paul H. Naylor, Gary B. Thurman, and Allan L. Goldstein

Abstract: Thymosin fraction 5 and a more purified acidic fraction of thymosin at a 100-fold lower concen- tration elevated cGMP levels, but not cAMP levels, in murine thymocytes. When both thymus and spleen lympbocytes were fractionated via a BSA gradient procedure, both cAMP and cGMP basal values varied depending on the density of the subpopulations. Thymosin Fr5 did not elevate cAMP in any thymus subpopulafions of lymphocytes obtained from the BSA density gradients. The cGMP elevation due to thymosin Fr5 in thymocytes was maximal in the most buoyant thymocytes, and no elevation of cGMP was detected in nude mouse spleen lymphocytes. These results suggest that the cGMP elevation may be an early event in the thymosin-mediated differentiation of a more mature subpopulation of thymocytes. They also suggest that utilization of subpopulations may be necessary for the complete determination of the effects of agents on cyclic nucleotide values of lymphocytes.

Key Words: Thymosin; T-cell differentiation; Thymic hormones; Cyclic nucleotides

INTRODUCTION

The importance of the endocrine thymus in the control of immunity via secretion of a family of polypeptide hormones called thymosin has been established over the past 15 years (Goldstein et al., 1966; Trainin, 1974; White and Goldstein, 1975; Bach J-F, 1976). Recently it has been shown that thymosin is active in increasing T-cell numbers and responses in cancer patients and increasing cell-mediated immunity and resistance to infection in children with thymic- dependent primary immunodeficiency diseases (Goldstein et al., 1976).

Although the effects of thymosin on various cell populations both in vivo and in vitro have been repeatedly demonstrated, the mechanism by which thymosin activates T-cell populations is not known. In many cases the effects of thymosin are very rapid; e.g., in vitro thymosin-induced induction of surface T-cell markers ( I - 2 hr) such as Thy-1 or Ly (Kumuro and Boyse, 1973), or expression of azathioprine or anti-0 sensitivity as assessed by the mouse rosette assay (5 rain) (Bach J-F et al., 1971). In addition, in vivo injections of thymosin have been shown to increase the response of murine spleen cells to the T-ceU mitogen, Con A, within 2 hr (Thurman and Goldstein, 1977).

Received August 23, 1978; revised and accepted October 23, 1978. From the Department of Experimental Therapeutics, Grace Cancer Drug Center, Roswell Park Memorial

Institute, Buffalo, New York (P.H.N.); and the Department of Biochemistry, George Washington University Medical Center, Washington, D.C. (G.B.T. and A.L.G.).

Portions of this report have been previously presented as an abstract at the American Society for Cell Biology meeting in San Diego, November 1977 (Cell Biol 75: 100a).

Address reprint requests to: Dr. Paul H. Naylor, Department of Experimental Therapeutics, Roswell Park Memorial Institute, 666 Elm St., Buffalo, NY 14263 USA.

© Elsevier North Holland, Inc., 1979 lmmunopharmacology 1, 89-101 (1979) 0162-3109/79/01008913502.25 89

90 P.H. Naylor et al.

On the basis of the available experimental evidence, it has been proposed that thymosin binds to surface receptors on the lymphocytes and initiates the changes seen in the various assays via the generation of an intracellular signal (second messenger) (Scheid et al., 1975; Bach MA et al., 1975; Trainin et al., 1975; Naylor et al., 1976). Initial experiments in our laboratory suggested that cGMP levels rather than cAMP levels were elevated by in vitro incubation with thymosin (Naylor et al., 1976). This is in contrast to reports in the literature which suggest that cAMP can mimic the action of both thymosin and other thymic factors (prepared via different isolation procedures) in assays which involve changes in cell surface receptors and lymphocyte function (Scheid et al., 1975; Bach MA, 1973; Kook and Trainin, 1975).

In this paper we report the specifics of the assay procedure used for the determination of cGMP levels in murine lymphocytes. The assay was a modification of the acetylation procedure reported initially by Harper and Brooker (1975) and confirmed simultaneously in several other laboratories (Frandsen and Krishna, 1976; Zeilig and Goldberg, 1977). Sensitivity in the femtomole range, linearity over the range of cell concentrations utilized, phosphodiesterase susceptibility, and reliable assessment of values in the presence of other nucleotides and in nonextracted samples could all be demonstrated. The increase in cGMP induced by thymosin was mimicked by an acidic peptide-enriched fraction at concentrations of 10-100 ng/ml.

Previous studies have demonstrated that cAMP levels (and adenylate cyclase activity) vary with lymphocyte development (Bach MA, 1975; Makman, 1971; Naylor et al., 1978). These stud- ies show that cGMP values also vary with the source of the lymphocytes and suggest that more mature (e.g., "thymus influenced") lymphocytes may be the target of the thymosin effect. In order to determine whether these differences extend to subpopulations of lymphocytes, BSA gradients were utilized (Shortman, 1976). Such gradients separate cells on the basis of their density and the subpopulations obtained from such separations differ from each other in both function and antigenic markers (Droge and Zucher, 1975; Shortman et al., 1975). Using this procedure, differences between both cAMP and cGMP basal levels of subpopulations of lymphocytes from murine spleen and thymus were demonstrated. Although addition of thymosin Fr5 elevated cGMP in thymocytes from all layers of the gradients, the largest increase was localized in the most buoyant subpopulation of lymphocytes.

These studies suggest that the levels of cyclic nucleotides probably play an important role in lymphocyte differentiation, maturation, and function. The identification of the subsets of lymphocytes which respond to thymosin with changes in cyclic nucleotide levels is an important step towards the elucidation of the mechanism through which thymosin induces T-cell matura- tion in vitro. Studies such as these may more clearly define the subpopulations in the maturation sequence on which thymosin and other thymic factors exert their effects and hopefully will contribute to the understanding of the role of various thymic peptides in T-cell differentiation.

METHODS

Materials

Animals. The C57BI/6J mice (Jackson Laboratories, Bar Harbor, Maine) were used from 6-10 weeks of age. The nude mice were BALB/c background and from our own colony.

Thymosin. The thymosin Fr5 used in these studies was prepared for us by Hoffmann- La Roche Inc. (Nutley, New Jersey), and was identical to that used in our previous publications (Hooper et al., 1975). In addition, thymosin Fr5 was chromatographed on carboxymethyl- cellulose column in 10 mM NaOAc, 1.0 mM 2-mercaptoethanol, pH 5.0. The desalted (Sephadex G-25 in water) void volume was designated 67A and the eluted peaks pooled and

Abbreviations. BSA: bovine serum albumin; cAMP: adenosine cyclic monophosphate; cGMP: guanosine cyclic monophosphate; Con A: concanava]in A; Fr5: fraction 5.

Cyclic GMP and Thymosin 91

designated 67B. Elution was via a salt gradient utilizing equal volumes of buffer and 1.0 M NaCI in buffer (Goldstein et al., 1977).

Cell Preparation

Mice were sacrificed by cervical dislocation and the lymphoid organs removed. Bone marrow was obtained by aspiration of the femur marrow cavity with a fine gauge needle. Cells were dispersed through a fine mesh screen, aspirated through a 25-gauge needle, spun at 250 g for 10 min, and resuspended in Tris-buffered ammonium chloride (Boyle, 1968) to lyse the red cells. Following a 10-min incubation at room temperature and filtration through nylon mesh to remove debris, the cells were centrifuged at 250 g. Cells were then resuspended in HEPES-buffered RPMI-1640 (HRPMI) (GIBCO, Grand Island, N.Y.) at 25 × 106 cells/ml.

Gradient Separation

Pentex bovine serum albumin (35%, pH 7.1, mOsmole = 290, Pathocyte-4, Miles Laboratory, Elkhart, Ind.) was diluted with HRPMI to the percents indicated in the figures and tables. The refractive index was used to determine the exact concentration of BSA in the various layers. The early experiments utilized gradients with layers of 23, 27, 31, and 35% BSA. In later experiments, gradients were expanded to give an additional layer using 19, 23, 26, 29, and 33% BSA. The various layers were formed (with the cells being loaded first) in 8 × 1.5-cm polycarbonate centrifuge tubes (Sorvall, Norwalk, Conn.). The gradient was allowed to equilibrate at 4 ° for 15 min and then spun at 19,000 g for 30 min. The layers were carefully removed, the cells washed twice in HRPMI and then resuspended to the concentration desired for assay.

Determination of cAMP and cGMP via the Radioimmunoassay

Cells were prepared as described above and incubated with agents such that the final concentra- tion of cells was 10 -20 × 106 cells/ml. Unless otherwise noted, all experiments were performed in triplicate and data expressed as means _ SE. The reaction was stopped by immersion into a dry-ice ethanol bath for 1 min, followed by boiling for 4 rain to destroy the phosphodiesterase. Assays were performed on either the supematants of a 2200 g spin, or samples that had been extracted, lyophilized, and reconstituted in either HRPMI or 0.05 M sodium acetate.

Extraction and purification when utilized was via the method of Yamamoto and Webb (1975). After freezing and boiling the sample or standards, 0.2 ml of a Dowex with water slurry (1: I) was added. After vortexing three times over a 10-rain period, the samples were spun at 800g for 15 min to pellet the Dowex, the supematant removed by aspiration, and the pellet washed with water by the same spin-aspirate procedure. The cyclic nucleotides were extracted by adding 0.8 ml of 4 N formic acid (distilled), vortexing three times over a 10-min period, centrifuging, and decanting. Two more 0.8-ml extractions were performed, the supernatants pooled, and spun again (~85% recovery). Two milliliters were removed for lyophilization and subsequent resus- pension to 0.5 ml.

Antisera specific for cAMP and cGMP were purchased from Schwartz-Mann, Orangeburg, N.J. They were prepared via the procedure of Steiner et al. (1972) and their specificity and cross- reactivity similar to those reported for that procedure (Schwartz-Mann, cAMP and cGMP specification publication). The 12Sl-labeled tyrosine methyl ester of cAMP and cGMP were also purchased from Schwartz-Mann, and prepared by them utilizing the procedure of Steiner et al. (1972).

The cAMP present in the samples was assayed by placing 0.1 ml of sample in a glass tube with 0.2 ml of sodium acetate buffer (0.05 M, pH 5.4). To the tube was added [12Sl]cAMP derivative (0.5 ml) and after vortexing, an equal amount of cAMP specific antiserum was added. Samples were again vortexed, incubated overnight at 4°C, precipitated with 60% ammonium sulfate, the

92 P, H. Naylor et al.

supernatant aspirated and the ~251 content of the pellet analyzed in a gamma counter. Logit vs In pmole plots were used to determine the concentration of cAMP on the basis of a standard curve generated by the same procedure.

The cGMP was assayed by initially acetylating the samples (Harper and Brooker, 1975). To 0.4 ml of the supernatant from a 1000 g spin was added 0.010 ml of triethylamine, immediately followed by 0.005 ml of acetic anhydride. Recently we have switched to the use of 0.005 ml of a freshly prepared mixture of acetic anhydride and triethylamine (1:2). After vortexing for I mir~, I00 lambda was.removed for assay. To th~ ~ 0.1-ml aliquot was added 0.2 ml of buffer (sodium acetate 0.05 M), 0.05 ml of ]2~l-labeled cGMP and 0.05 ml of cGMP specific antisera. Samples were incubated as above, precipitated, counted, and cGMP determined.

The acetylated cGMP procedure allows the measurement of femtomole amounts of cGMP present in cells. Other nucleotides do not interfere in the assay at levels normally present in cells (Figure I and unpublished observations). Cyclic AMP, as reported by Harper and Brooker (1975) does not cross-react in the assay even at five times the amount normally present in cells (e.g., in a typical experiment, the percent binding for cGMP cells was 87.2 - 2.8%, with 5x addition of cAMP it was 83.6 -+ 1.0%).

Preparation of the Phosphodiesterase

The specificity of the cyclic nucleotide assays was verified by digestion of cAMP and cGMP by rat brain phosphodiesterase. Whole brain from adult male Charles River rats was homogenized for 1 rain in an ice bath using three volumes of 10 mM Tris buffer containing 1 mM MgCI2 (pH 7.5). The homogenate was centrifuged at 100,000 g for 60 min and aliquots of supematant stored at 0°C. Dilutions of l -6 - fo ld were made prior to use.

Figure 1 Comparison of acetylated and unacetylated cGMP standard curves: [Logit = in TrB/(IO0 -TrB)]. The acetylation procedure allows the measurement of fmole amounts of cGMP. This is lO0-fold more sensitive than when unacetylated cGMP is used.

a5

o

99

95

90

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7 0 -

6 0 -

5 0 -

4 0 -

3 0 -

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~ cGMP acetylated ~ ,

" ~ cGMP .~%~,~ I

" %%

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5.0 2 5 I00 2 0 0 4 0 0 I000

- 4 0

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frnoles cGMP

Cyclic GMP and Thymosin 93

_m

8

~E

~5

• = Thymosin Fr 5 o = MM 67A • = M M 6 7 B

(

!

,'0o io ', &, o'.0, Log Concentrofion (ug/ml)

Figure 2 The elevation of cGMP in thyrnocytes after 10 rnin incubation with thymosin Fr5 (100 I.u3/ml) is accomplished by 67A at 1~lOath the concentration (0.01 p~g/ml). An elevation by the eluate of the column (67B) was slightly present at 100 p.g/ml. The value forthe cells incubated with media alone (control) was 4.93 +_- 0.56 finales~106 cells. All points are the mean +_ SE of 3 independent triplicate samples.

R E S U L T S

As prev ious ly repor ted (Nay lor et al., 1976), thymos in Fr5 (but not a spleen control Fr5) induces an optimum stimulation of cGMP levels in thymocytes at a concentration of 100 /~g/ml (Figure 2). The increase is detectable at I min, with the optimal stimulation occurring between 5 and 15 rain (Naylor et al., 1976, unpublished observation). The separation of thymosin Fr5 into an acidic fraction (67A) and a basic fraction (67B) indicated that the elevation of cGMP was induced by the fraction which contained the acidic peptides. This acidic fraction was active at 10-100 ng/ml (Fig. 2) and represented 7% of the total protein in Fr5.

Table I Extraction of samples does not change thymosin elevation a

Exp. No. Extraction Medium control Thymosin SP

1 No 3.27 - 0.01 7.55 _+ 0.07 2.31 Yes 2.20 - 0.08 5.15 - 0.13 2.34

2 No 9.39 -+ 0.16 18.35 ± 2.30 1.95 Yes 11.30 ± 0.16 19.35 ± 0.55 1.74

a Thymosin was incubated with murine thymocytes for 10 rain at 100 /~g/ral. The values are expressed as fmoles/lO 8 cells. Extraction was via the procedure of Yamamoto and Webb utilizing Dowex-14ormate.

b Stimula~on index = thymosin value/medium control.

94 P . H . Naylor et al.

Table 2 Thyrnosin-induced elevation of cGMP is cell concentration dependent a

Cell concentration Thymosin added (10 + ce#s/aliquot) Media Thymosin after freezing b

1.2 3.83 ± .07 14.08 __ .45 nd 2.4 4.00 ± .02 7.75 ± .02 4.57 ±_ .01 4.8 4.00 ± .02 8.13 ± .02 5.42 ± .05

a The assay conditions were as outlined in the paper with thymosin at 100 /~g/ml for 10 rain. The linearity of the cGMP assay (i.e., stability of the basal values) over the concentrations of cells utilized was demonstrated by comparing the cGMP/10 + cells for each cell concentration. Value expressed as fmole/10 + cells + SE for triplicate samples using 0. l-ml volumes for assay.

b The addition of the thymosin to the cells as they were being quick- frozen indicates that the elevation is not totally due to the cross-reacting material.

nd = not done.

The increase due to thymosin as assessed by the stimulation index was still present after extrac- tion of the cGMP (Table 1). There was also no consistent difference in the exper iments be tween the basal or stimulated values before and after extraction (Table 1). In addition, cGMP values were linear over the range of cell concentra t ions utilized, suggesting little interference from the cellular material present in the supernatants . The increase in cGMP due to 100 /~g /ml of Fr5 displayed an opt imum at cell concentra t ions of 1 0 - 2 5 x 106 cells/mE S u b s e q u e n t experiments were performed at these concentra t ions (Table 2).

At the concentrat ion present in the assay, thymosin FrS, when assayed a lone contains a small a m o u n t of material which cross-reacts in the assay (Naylor et al., 1976). However , as can be seen in Tables 2 and 3, the cross-reacting material, a l though contributing to the increase in cGMP, is not responsible for the total increase seen in the assay.

As indicated in three representat ive experiments , the a m o u n t of cross-reacting material measured in thymosin varied from assay to assay. This small con taminan t has not yet been

Table 3 The cross-reacting material in thyrnosin does not account for the total cGMP increase observed after in vitro incubation with thyrnocytes

Cells incubated Thymosin added Cells with thymosin Thymosin alone t' after lysis b

Experiment I" 3.58 ± 0.65 16.26 -*- 0.34 4.15 ± 0.68 nd

Experiment 2 2.47 ± 0.50 6.05 ± 0.08 2.46 ± 0.84 1.60 ± 0.66

Experiment 3 2.85 ± 0.34 8.97 ± 2.34 0.85 ± 0.26 4.36 ± 0.99

" The values in Exps. 1 and 3 are expressed as fmoles/assay aliquot. In Exp. 2 the values are converted to fmoles/106 cells.

b The amount of cross-reacting material was not consistent nor was the sum of the value of cross-reacting material plus media control always equal to the value when thymosin was added after lysing the cells. See text for discussion.

Cyclic GMP and Thymosin 95

Table 4 Thymosin- induced cGMP elevation is most pronounced in thymocytes a

Medium control Thymosin % Ce//origin (fmoles/lO 6 cells) ( fmoles/lO 8 cells) Increase b AcGMP c

Normal mice Bone marrow 8.26 ± 0.91 10.11 ± 0.66 22 (ns) 1.99

6.30 -+ 0.39 9.45 _+ 0.31 50 ~ 4.14

Thymus 4.80 -- 0.42 8.66 ± 0.28 80 s 4.86 3.41 -+ 0.08 8.39 ± 0.27 143 ~ 6.26

Spleen 6.18 ± 0.51 8.41 ± 0.23 35 f 2.35 7.13 -+ 1.22 11.30 ± 0.17 58 (ns) 4.83

Lymph nodes 7.73 ± 1.22 10.32 _+ 0.72 331 2.90 5.23 ± 2.64 8.27 ± 0.53 66 (ns) 4.53

Nude mice d Thymus 3.27 ± 0.42 6.56 _+ 0.81 1031 3.29

(nu/+) 6.31 ± 0.78 12.38 ± 1.23 96 f 6.07

Spleen 3.28 -+ 0.35 5.72 ± 0.48 74 s 2.44 (nu/+) 8.51 ± 0.74 7.18 _+ 0.84 - 1 6 (ns) -1 .33 e

Spleen 6.05 ± 0.29 6.20 _+ 0.76 2 (ns) 0.15 (nu/nu) 6.79 -+ 1.34 6.27 -+ 0.81 7 (ns) 0.52

a The results (mean _+ SE of triplicate samples) are for two separate experiments using whole populations of cells from the lymphoid organs of 3 - 2 0 mice.

b Percent increase = (thymosin value - medium control)/medium control.

c AcGMP represents the absolute increase in cGMP in the aliquot measured as a result of thymosin incubation and may suggest that the smaller increase measured in bone marrow, spleen, and lymph node lymphocytes may be due to the cross- reacting material in thymosin (see Tables 2 and 3 and Discussion in text).

Thymus and spleen from heterozygous litter mates (nu/+) of BALB/c nude mice were compared to homozygous (nu/nu) spleens. Values for two separate experiments as above.

e Decrease in spleen cGMP was not often observed (see Normal mice also).

fP ~ 0.05 using Student's t-test; ns = not significant.

identified (unpubl ished observations; H. Sheppard , personal communica t ion) but is not separable from Fr5 even at the "desal t ing" step in the isolation of thymosin. This cross-reacting material is not measurable in 67A at concentra t ions which elevated cGMP in thymocytes bu t at high concentra t ions (100 p,g/ml) some con taminan t can be measured in bo th 67A and 67B. Since variability existed be tween (a) the cells incubated with thymosin, (b) the addition of thymosin after freezing, and (c) the sum of the cells plus the cross-reacting material, corrections for the cross-reacting material were generally not m a d e (Tables 2 and 3).

As seen in Table 4, in two separate exper iments thymosin elevated cGMP in thymocytes to a greater degree than in lymphocytes ob ta ined from spleen, lymph nodes, or b o n e marrow. The cross-reactivity was not subtracted out since not only was it variable (see above) , but it was not detected in the spleen cells of nonrespond ing nude mice (Table 4). W h e n nude mouse spleens were compared to litter-mate spleens and thymus, no increase in cGMP due to thymosin was noted. The absolute increase in cGMP was calculated however, and does show the same results as the percent increase (Table 4).

Variations in the basal cyclic nucleotide levels of lymphocyte subpopula t ions w h e n separa ted

96 P.H. Naylor et aL

Basal Cyclic Nucleotide Levels BSA GRADIENT

cAMP cGMP =_. 5.0

THYMUS SPLEEN THYMUS SPLEEN ~z

j . o

tol ~ 2.0 _

} 1 ;r.ol

0''0 /iRn t,' l 11 ilml I 1I ~r ~ I Tr ~ T 3I ~r ~ I 3I ~ I ~ .

Figure 3 The separation of lymphocytes into subpopulations by density gradient centrifuga- tion indicates that variations in the basal levels of both cyclic nucleotides are present. The gradients were prepared as indicated in the text and represented in the insert. In the thymus and spleen, layer I is mean +- SE for duplicate samples. All other points were for triplicate samples. Note that the scale of cAMP and cGMP are different. The percentage of cells in each of the BSA gradient layers were as follows: Thymus I (4°/o), II (19%), I l l (58%), IV (19%); Spleen I (14%), II (43%), I l l (31%), IV (12%). Subsequent experiments expanded the gradients to remove an even lighter population (e.g., above 23% instead of pooling the 23 + 26% cells).

by discontinuous BSA gradient centrifugation are shown in Figure 3. In both the spleen and thymus, cAMP and cGMP levels decreased with increasing density of the cells. Also important may be the significant variations in ratios of the cyclic nucleotides (calculations not shown). Thymosin elevated the cGMP levels of all the subpopulations of the thymocytes but the largest increases were consistently found in the most buoyant population of thymocytes (Table 5). Cyclic AMP levels were not elevated by thymosin Fr5 in any of the thymus subpopulations (Table 6). While the spleen basal cAMP levels are significantly higher than thymocyte basal cAMP levels (Bach MA, 1975; Naylor et al., 1978), when the cells were separated on the BSA gradients, the summation of the measured cAMP values for the subpopulations (using the percent recovery to determine the contribution of each subpopulation to the whole) resulted in thymus values which were not significantly different from those of the spleen (Figure 3, un- published observations). Thymus and spleen lymphocytes incubated in BSA (under conditions similar to those of the separation) were also not significantly different from each other (data not shown). This may be a result of the depression of cAMP levels as a result of BSA incubation (e.g., media alone for 30 min at 4°C followed by wash and 1/2 hr incubation in HRPMI was 5.31 -- 0.21; 35% BSA at 4°C followed by HRPMI is 3.9 +- 0.15 (P < 0.005) pmole/107 cells. Cyclic GMP values were not significantly affected by BSA incubation at 4°C (HRPMI = 13.77 fmoles/106 cells - 1.14; 35% BSA = 11.83 -+ 0.78).

Cyclic G M P a n d T h y m o s i n

Table 5 Thymosin consistently elevated the cGMP levels of the more buoyant thymocyte population

97

% BSA below % Recovered Basal level Increase with interface Exp. no. ceils ( frnoles/l O e) thymosin ~ Aver. SI b

1 2 9.70 ± 0.20 22.54 -+ 1.17 e 23 2 9 5.23 ± 0.36 10.77 ± 0.22 ~ 2.38 ± 0.20

3 5 7.94 ± 0.50 21.79 ± 1.09 ~

1 7 10.56 ± 0.84 15.85 ± 0.62 d 26 2 24 3.25 -+ 0.32 6.11 -+ 0.28 ~ 1.76 -+ 0.11

3 18 7.40 ± 0.26 13.34 ± 0.52 e

1 41 7.93 -+ 0.60 13.35 ± 1.04 d 29 2 18 2.66 ± 0.86 5.55 ± 0.68 (ns) 1.90 ± 0.16

3 53 7.67 -+ 2.31 14.96 -+ 1.26 e

1 24 6.80 ± 0.22 9.87 ± 0.54 d 33 2 11 nd - - 1.45 -- 0.11

3 13 nd - -

1 29 6.54 ± 0.59 11.38 + 2.01 (ns) pellet 2 36 nd - - 1.74 ± 0.31

3 12 nd - -

I - - 13.77 +- 1.18 29.87 -+ 1.14 d Media 2 - - 4.25 + 0.13 7.64 + 0.44 d

3 - - n d - -

2.06 -+ 0.18

1 - - 11.83 + 0.76 nd 35% BSA c 2 - - 4.46 ± 0.69 7.64 -- 0.44 d 2.12 -- 0.05

3 - - 5.25 ± 0.43 14.97 -+ 1.26 d

Incubation conditions were as described in Methods. This value represents the increase induced by thymosin in fmoles/10 e cells. Results are for three experiments.

b Average stimulation index is average of (value with thymosin/value for media alone) ± SE. All values except for media and 35% BSA were significantly ( P < 0.01) lower than upper layer cells.

c These cells were incubated in BSA at 4 ° for 1 hr and then treated identically to cells harvested from the gradients.

Significance based on Student 's t-test; ns = P > 0.05, dp < 0.05; ep < 0.01.

D I S C U S S I O N

It is clear f rom the da ta p r e s e n t e d a n d p rev ious repor t s (Makman, 1971; Bach MA, 1975; Naylor et al., 1978) that l y m p h o c y t e s vary cons ide rab ly in their basal cyclic nuc leo t ide levels a n d their s u b s e q u e n t c h a n g e s in cAMP a n d c G M P u n d e r the inf luence of var ious agents . Thymos in , a l though it is ab le to reconst i tu te the immuni ty of t h y m u s - d e p r i v e d an imals a n d is act ive in a lmos t all the assays for specific thymic h o r m o n a l effects, d o e s no t e levate cAMP levels in p o o l e d l y m p h o c y t e popu la t ions (Naylor et al., 1976; 1978) or in BSA dens i ty - sepa ra t ed sub- popu la t ions (Table 6). We have n o w used th ree different assays a n d var ied expe r imen ta l condi t ions such as media , cell concen t ra t ion , incuba t ion t imes (1 rain to 1 hr), d o s e s (1 r a g / r o l - l p,g/ml), a n d l y m p h o c y t e popu la t ions wi thout s ee ing significant e levat ion in cAMP (Naylor et al.,

98 P.H. Naylor et al.

Table 6 Thymosin Fr5 does not elevate cAMP values in any lymphocyte population present in the thymus a

Media control a Thymosin % BSA ~ % Recovery c (Thymus) (100 p~g/ml) ~

23 2 6.55 + 0.37 4.85 - 0.23 26 7 5.23 _+ 0.36 5.27 _+ 0.45 29 41 2.43 _+ 0.03 2.80 _+ 0.23 33 23 1.86 --- 0.I0 1.95 --- 0.09

pellet 29 2.38 _+ 0.10 2.38 +_ 0.12

a See Methods for procedures and previous tables for format.

b Percent BSA represents the density of the layer below the cells. See text for discussion of effect of BSA on basal cAMP values.

c Percent recovery is based on total cells recovered from the gradient.

d Values expressed as pmoles cAMP/107 cells. Data from Exp. 1 (Table 5), similar results for the other two experiments.

1978 and unpublished observations). This observation is in contrast to the reports of others that thymic extracts (prepared in different ways from thymosin) elevate cAMP levels in thymocytes (Scheid et al., 1975; Trainin et al., 1975). Still thymic extracts have been shown to be active in several assays where added cAMP has been shown to have a positive effect (Scheid et al., 1975; Bach MA and Bach J-F, 1973; Kook and Trainin, 1975). However, in these assays, any agent that elevated cAMP levels (such as epinephrine or endotoxin) would also be considered active (Scheid et al., 1975; Bach MA et al., 1975; Trainin et al., 1975). In our experiments, clinical quality thymosin was used. It is low in endotoxin (<5 ng/mg) as determined by limulus lysate assay, and was pyrogen-free as determined by the rabbit pyrogen assay. The endotoxin levels of the other factors which cause cAMP elevation has not been reported.

Although the measurement of cGMP levels in tissues has been difficult and controversial (Parker et al., 1974; Hadden, 1975; Goldberg et al., 1975; Wedner et al., 1975), the acetylation assay which was utilized in this study has proven to be both reproducible and accurate (Harper and Brooker, 1975; Frandsen and Krishna, 1976; Zeilig and Goldberg, 1977). Due to the assay sensitivity, the samples are not concentrated, and since the cyclic nucleotides are acetylated more readily than noncyclic compounds, the interference from the other cellular nudeotides is minimized (Harper and Brooker, 1975, unpublished observations).

The fact there was no difference between the increases before and after purification of cGMP implies that the values assessed for acetylated cGMP in debris-free lysates are valid. The initial observation that cGMP levels were elevated in thymocytes by thymosin has now been extended. The major population which is responding to the thymosin is in the thymus, but some responding cells may be present in other lymphoid organs. In addition, a purified biologically active prepara- tion containing the acidic peptides of thymosin Fr5 has been consistently active at 10-100 ng/ml.

The increase in cGMP is not due to the measurement of the cross-reactive material since 67A is active at concentrations where no contaminant is measurable and spleen cells from nude mice (nonresponding population) show no increase when incubated with thymosin Fr5. The contaminant is, however, present even in the 67A and 67B preparations when 100 mg/ml are assayed and was only partially sensitive to phosphodiesterase when large amounts of Fr5 (1000 mg/ml) were tested (data not shown). Spleen Fr5, as reported previously (Naylor et al., 1976), was not active and also contained a variable amount of cross-reacting material.

Cyclic GMP and Thymosin 99

Several investigators have pointed to the fact that lymphocyte cAMP levels vary among different populations and that this would seem to imply an important role for that cyclic nucleotide in lymphocyte development (Makman, 1971; Bach MA, 1975). Our observation that cGMP levels also vary points out that it is important to consider both cyclic nucleotides whenever possible. Of interest are our observations that density separation of lymphocytes using BSA gradients results in subpopulations which differ in their cyclic nucleotide content and their thymosin response.

The effect of BSA on the basal levels of the subpopulations is difficult to assess. In this study there was no statistical difference in cGMP between 35% treated whole populations and the media controls. The effect on cAMP levels, however, was significant and resulted in a depression of the basal levels. A complete study of the effects of BSA in the responses of these cells to cAMP elevating agents, was not done, nor was the effects of BSA on subpopulations obtained via different procedures evaluated.

These studies have pointed out the specificity and sensitivity of the acetylated cGMP assay, and extended the initial observations that cGMP levels were elevated by thymosin. We have also identified a subpopulation of lymphocytes (post-thymic since it is present in thymus but not nude spleen), which is especially sensitive to the elevating effects of thymosin. Thus it appears that cGMP but not cAMP levels are elevated in this population of thymocytes by thymosin. While this paper was in preparation, another thymic extract (thymopoietin) was reported to elevate cGMP but not cAMP in a post-thymic subpopulation of spleen and lymph node lymphocytes (Sunshine et al., 1978). Such a result is somewhat in agreement with our results (Naylor et al., in preparation) that the more dense spleen subpopulation but not lymph node lymphocytes are also responsive to thymosin with an increase in cGMP but not cAMP. Further studies utilizing the techniques presented in this paper and the thymosin peptides, which are being characterized in our laboratory, should contribute to the understanding of the mechanism whereby thymosin and the endocrine thymus controls T-cell development and maintenance.

The authors are grateful for the helpful advice of Dr. Herbert Sheppard during the course of the work and the preparation of the manuscript. The secretarial assistance of Helen Amato and Margie Watson is gratefully acknowledged. Research supported in part by NCI Grants CA-14108 and CA-16964, Hoffman- La Roche, Inc. (Nutley, New Jersey), and McLaughlin Predoctoral Fellowship (P.H.N.).

REFERENCES

Bach J-F, Dardenne M, Goldstein AL, Guha A, White A (1971) The appearance of T cell markers in bone marrow rosette-forming cells after incubation with thymosin, a thymic hormone. Proc Natl Acad Sci USA 68: 2734.

Bach J-F (1976) The mode of action of thymic hormones and its relevance to T-cell differentia- tion. Transplant Proc 8: 243.

Bach MA (1975) Differences in cyclic AMP changes after stimulation by prostaglandins and isoproterenol in lymphocyte subpopulations, d Clin Invest 55: 1074.

Bach MA, Foumier C, Bach JF (1975) Regulation of theta antigen expression by agents altering cyclic AMP levels and by thymic factors. Ann NY Acad Sci 249: 316.

Bach MA, Bach J-F (1973) Studies on thymus products VI. The effects of cyclic nucleotide and prostaglandins on rosette forming cells. Interaction with thymic factor. EurJ Immuno! 3- 778.

Boyle W (1968) An extension of the 5~Cr-release assay for the estimation of mouse cytotoxin. Transplantation 6: 761.

Droge W, Zucker R (1975) Lymphocyte subpopulations in the thymus. Transplant Rev 25: 3. Frandsen EK, Krishna G (1976) A simple ultrasensitive method for the assay of cyclic AMP and

cyclic GMP in tissues. Life Sci 18: 529. Goldstein AL, Slater FD, White A (1966) Preparation, assay and partial purification of a thymic

lymphocytopoietic factor (thymosin). Proc Nat/Acad Sci USA 56: 101.

10O P.H. Naylor et al.

Goldstein AL, Cohen GH, Rossio JL, Thurman GB, Brown CN, Ulrich JT (1976) Uses of thymosin in the treatment of primary immunodeficiency diseases and cancer. Med Clin North Am 60: 591.

Goldstein AL, Low TLK, McAdoo M, McClure J, Thurman GB, Rossio JL, Lai C-Y, Chang D, Wang S-S, Harvey C, Ramel AH, Meienhofer J (1977) Thymosin ~i: isolation and sequence analysis of an immunologically active thymic peptide. Proc Nail Acad Sci USA 74: 725.

Goldberg ND, Haddox NK, Nicol SE, Cass DB, Sanford FA, Kuehl FA, Estensen R (1975) Adv Cyclic Nucl Res 5: 307.

Harper JF, Brooker GB (1975) Femtomole sensitive radioimmunoassay for cyclic AMP and cyclic GMP after 2'0 acetylation by acetic anhydride in aqueous solution. J Cyclic Nucl Res 1: 207.

Hadden JW (1975) Molecular basis of cellular immunity. In Molecular Pathology. Eds., RA Good, SB Day, and JJ Yunis. Springfield: Charles C Thomas, p. 90.

Hooper J, McDaniel MC, Thurman GB, Cohen GN, Schulof RS, Goldstein AL (1975) The purification and properties of bovine thymosin. Ann NY Acad Sci 249: 125.

Kook AI, Trainin N (1975) The control exerted by thymic hormone on cellular cAMP levels and immunoreactivity. J Immunol 115: 8.

Kumuro K, Boyse EA (1973) Induction of T lymphocytes from precursor cells in vitro by a product of the thymus. J Exp Med 138: 479.

Makman MH (1971) Properties of adenylate cyclase of lymphoid cells. Proc Nail Acad Sci USA 68: 885.

Naylor PH, Sheppard H, Thurman GB, Goldstein AL (1976) Increase of cyclic GMP induced in murine thymocytes by thymosin fraction 5. Biochem Biophys Res Commun 73: 843.

Naylor PH, Camp CE, Phillips AC, Thurman GB, Goldstein AL (1978) Evaluation of three cyclic AMP assays utilized to measure the effects of thymosin and lypopolysaccharide on murine lymphocytes, d Immunol Methods 20: 143.

Parker CW, Sullivan TJ, Wedner HJ (1974) Cyclic AMP and the immune response. Adv Cyclic Nucl Res 4: I.

Scheid MP, Goldstein G, Hammerling A, Boyse EA (1975) Induction of T and B lymphocyte differentiation in vitro. In Membrane Receptors of Lymphocytes. Eds., M Seligrnan, JL Preudhomme, and FM Kourelsky. New York: Elsevier North-Holland, Inc., p. 353.

Shortman K, Von Boehmer H, Lipp J, Hopper K (1975) Subpopulations of T lymphocytes. Transplant lies 25: 163.

Shortman K (1976) Buoyant density separation of lymphocyte populations, by continuous albumin gradient analysis and simple albumin density cuts. In In vitro Methods in Cell- Mediated and Tumorlmmunity. Eds., BR Bloom and JR David. New York: Academic Press, p. 276.

Steiner AL, Parker CW, Kipniss DM (1972) Radioimmunoassay for cyclic nucleotides. J Biol Chern 247: 1106.

Sunshine GH, Basch RS, Coffey RG, Cohen KW, Goldstein G, Hadden JW (1978) Thymopoietin enhances the allogeneic response and cyclic GMP levels of mouse peripheral, thymus-derived lymphocytes. J Immunol 120: 1594.

Thurman GB, Goldstein AL (1977) Role of the spleen on thymosin induced lymphocyte maturation. In Immuno-aspects of the Spleen. Eds., JR Battisto and JW Strielein. New York: Elsevier North-Holland, Inc., p. 141.

Trainin N (1974) Thymic hormones and the immune response. Physiol Rev 54: 274. Trainin N, Kook AI, Umiel T, Albata M (1975) The nature and mechanism of stimulation of

immune responsiveness by thymic humoral factor. Ann NY Acad Sci 249: 349. Wedner HJ, Dankner R, Parker CW (1975) Cyclic GMP and lectin-induced lymphocyte activa-

tion. J Immunol 115: 1682. White A, Goldstein AL (1975) The endocrine role of the thymus and its hormone, thymosin,

Cyclic GMP and Thymosin 101

in the regulalion of the gro~.h and maturation of host immunological competence. In Advances in Metabolic Disorders, Vol. 8, Eds., R Levine and R Tuft. New York: Academic Press, p. 359.

Yamomoto I, Webb~DR (1975) Antigen stimulated changes in cyclic nucleotide levels in the mouse. Proc Natl Acad Sci USA 72: 2320.

Zeilig C, Goldberg ND (1977) Cell-cycle-related changes of 3':5'-cyclic GMP levels in Novikoff hepatoma cells. Proc Natl Acad Sci USA 74: 1052.