Thymosin α1 and thymosin β4 modulate human colonic lamina propria lymphocyte function

lmmunopharmacology, 20 (1990) 89-96 Elsevier

89

IMPHAR 00510

Thymosin (z1 and thymosin/?4 modulate human colonic lamina propria lymphocyte function Yoram Elitsur 1,2, Milton G. Mutchnick 2, Wael A. Sakr 3 and G o r d o n D. Luk 2 Departments of ~Pediatrics, 2Internal Medicine and 3pathology, Division of Gastroenterology, Children's Hospital of Michigan, Harper Hospital. Wayne State University, Detroit, MI, U.S.A. (Received 24 April 1990; accepted 12 June 1990)

Abstract: Thymosin cq and thymosin t4 are two thymosin fraction 5-derived peptides with the capacity to alter a variety of immune functions in human and animal models. In this study we investigated the effect of both thymosin cq and thymosin ~4 on human colonic lamina propria lymphocyte (LPL) proliferation and ornithine decarboxylase (ODC) activity. LPL from eighteen human colon specimens were cultured in the presence or absence of thymosin ct~ and thymosin f14.We found that both peptides suppressed thymidine incorporation into LPL. However, thymosin ~ and thymosin t4 did not alter thymidine incorporation into phorbol ester (PDB) and calcium ionophore (ionomycin) -stimulated LPL. Furthermore, thymosin ct1 and thymosin t4 also did not alter ODC activity in Con A-stimulated LPL. These results suggest that both peptides alter LPL proliferation, and that the mechanism for this inhibition may not involve the calcium fluxes or the ODC pathway but may involve protein kinase C. We postulate that thymosin ~ and thymosin f14 may participate in the modulation of the human mucosal immune system. Key words:

Lamina propria lymphocyte; Mucosal immune system; Thymosin ~ ; Thymosin/?4

Introduction

Thymosin ct1 (TA1) and thymosin t4 (TB4) a r e two thymosin fraction 5-derived peptides which have immune modulatory effects in both human and animal models. In vitro studies have deCorrespondence: Dr. Yoram Elitsur, Wayne State University, School of Medicine, 2136 Scott Hall, 540 E. Canfield, Detroit, MI 48202, U.S.A. Abbreviations: CMF-HB S S, calcium magnesium free-Hank's balanced salt solution; Con A, concanavalinA; DTT, dithiothreitol; 3H-Thd, [3H]thymidine; EDTA, ethylenediaminetetraacetic acid; HEPES, hydroxyethylpiperazine; IL-2, interleukin 2; LPL, lamina propria lymphocytes; ODC, ornithine decarboxylase; PDB, phorbol 12,13-dibutyrate; TPA, 12-O-tetradecanoyl phorbol 13-acetate; TA1, thymosin cq; TB4, thymosin f14.

monstrated that these thymic peptides can modulate graft-versus-host reaction, antibody formation, natural killer (NK) cell activity and lymphocyte proliferative responses (Goldstein etal., 1977; Kaufman etal., 1980; Baxevanis et al., 1985; Hu et al., 1981; Favalli et al., 1985; Bistoni et al., 1984; Schulofet al., 1985). TA 1has been shown to enhance the proliferative responses of human helper T (CD4) lymphocytes in mixed lymphocyte culture, while T B 4 suppresses their proliferative responses (Baxevanis et al., 1987). Other investigator s have shown increased interleukin 2 (IL-2) receptor expression and IL-2 production by human peripheral blood large lymphocytes after incubation with TA1 (Zatz et al., 1984; Sztein et al., 1986). Thymic peptides have also been reported to

0162-3109/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

90 have antitumor activity in human and animal models (Umeda etal., 1983; Chirigos etal., 1977). TA t accelerated the rejection of tumors in immunosuppressed mice, possibly through enhancing NK cell activity (Umeda et al., 1983). TA t also increased survival of mice with lymphocytic leukemia (Chirigos et al., 1977). Thymic peptides were effective as adjuvants in antitumor therapy for patients with non-small-cell lung cancer (Schulof et al., 1985), early-stage melanoma (Bernengo et al., 1983), Hodgkin's disease or advanced metastatic gastrointestinal cancer (Schulof, 1985). Several investigators have reported that T B 4 is synthesized in non-lymphoid as well as lymphoid organs (Gondo etal., 1987; Hannappet etal., 1 9 8 2 ) . T B 4 is synthesized by human B cell lines (Hannappel et al., 1985), human fibroblast (FS7) cell lines and murine myoblasts (L6) (Goodall et al., 1983). Others have postulated that thymic peptides can serve as neurotransmitters by modulating the hypothalamic-adrenal and gonadal axes (Hall et al., 1985). Immunoreactive TA l has recently been described in crypt cells of rat ileum and in the IEC-6 rat crypt cell line (Conteas et al., 1989). These data suggest that TA 1 and T B 4 could be present in the gastrointestinal mucosa, in fibroblast secretions, peritoneal macrophages, or in the form of neurotransmitters. The reported antitumor effect and immunomodulatory capacity of T A 1 and TB4 on the circulatory immune system prompted the present investigation of the effect of these peptides on the human gut immune system as represented by colonic lamina propria lymphocytes (LPL). In this study we investigated the effect of TA~ and T B 4 o n normal human colonic LPL cell proliferation. Since other cellular signal transduction pathways such as activation of protein kinase C (Nishizuka et al., 1984), calcium fluxes (Nobrega etal., 1986) and ornithine decarboxylase (Fidelus et al., 1984) have also been shown to be important for DNA synthesis, we also studied the effect of TAt and T B 4 o n phorbol ester (PDB) and calcium ionophore (ionomycin)

-induced LPL proliferation and ornithine decarboxylase (ODC) activity. Our results show that in the presence of Con A both TA 1 and T B 4 significantly suppressed [3H]thymidine (3H-Thd) incorporation into LPL. However, this effect was abolished when LPL were stimulated with a combination of phorbol ester (PDB) and calcium ionophore (ionomycin). ODC levels were not affected by either peptide. These results suggest that TA l and TB 4 may modulate the gut immune system, and that the mechanism underlying this effect may involve calcium flux, but does not involve the ODC metabolic pathway.

Material and Methods

Materials Tissue culture media and antibiotic-antimycotic solution (penicillin 10,000 U/ml, amphotericin-B 25/~g/ml, streptomycin 10 mg/ml) were obtained from GIBCO Inc., Grand Island, NY. EDTA and DTT were purchased from Sigma Chemical Co., St. Louis, MO. Calcium ionophore (ionomycin) was purchased from CALBIOCHEM, La Jolla, CA. Con A was purchased from ICN ImmunoBiologicals, Lisle, IL. Radioactive materials [ 3H]thymidine (spec. activity 83 mCi/mmol) and L-[t4C]ornithine (50 mCi/mmol) were purchased from NEN DuPont, Boston, MA. Fluorescein-conjugated goat anti-mouse IgG, CD3, CD19 were purchased from Becton Dickinson, Mountain View, CA. CD4 and CD8 were purchased from Coulter Immunology, Hialeah, FL. T A 1 and T B 4 w e r e provided by Alpha-1 Biomedicals Inc., Foster City, CA, and serial dilutions were made in complete medium. Complete medium is RPMI-1640 containing 10~o human AB serum (Gibco), and antibiotic-antimycotic solution (1 ~o v/v). Memo& Human colonic specimens Eighteen human colon specimens were obtained from patients (mean age 60 years, range 32-82) who underwent colon

91 resection. Ten patients had colon cancer, eight had non-cancerous diseases. Histologically normal colon segments (at least 5 cm away from the diseased area) were used for our study. It has previously been shown that there is no difference between the LPL subpopulation in the mucosa of histologically normal, cancerous or inflammatory bowel disease patients (Hirata et al., 1986). The specimens were transferred and processed within 30 min after surgical resection. The project was approved by the Wayne State University Human Investigation Committee.

to established methods (Jackson et al., 1986) as previously described (Elitsur et al., 1990). Briefly, 0.05 ml of LPL suspension (5-10 million cells) was incubated with 0.02 ml of CD3 and CD19 and 0.005 ml of CD4 and CD8 at 4 °C for 30 min. After washing, the cells were incubated with 1 : 25 dilution of fluorescein-conjugated goat antimouse IgG at 4 ° C for an additional 30 min. After washing, the cells were analysed on a BectonDickinson FACS Analyzer using a 540 nm filter. The percentage of CD4 and CD8 positive cells was recorded.

Isolation of human colonic lam&a propria lymphocytes Isolation of LPL was done according to the method of Bull and Bookman (1977) as previously described from our laboratory (Elitsur et al., 1990). Briefly, the mucosa was minced into 0.5 x 0.5 cm pieces. To remove mucus, tissue was treated for 10 min with calcium-magnesiumfree Hanks' balanced salt solution (CMF-HB S S) containing dithiothreitol (DTT). The mucosal fragments were then treated with ethylenediaminetetraacetate (EDTA) for 90 min. Collagenase digestion was performed with RPMI-1640 containing 15~o fetal calf serum, collagenase type lA (50U/ml) and antibiotic-antimycotic solution for 14-16 h at 37 °C. The resulting cell suspension was centrifuged on Ficoll-Hypaque gradient and suspended in complete cell culture medium (RPMI-1640, 10~o human AB serum and antibiotic-antimycotic solution (1~o v/v), penicillin 10,000 U/ml, amphotericin-B 25 #g/ml, and fungizone-streptomycin 10 mg/ml), for thymidine incorporation studies. Viability and purity were determined by light microscopy and by trypan blue exclusion or ethidium bromide/acridine orange staining. Purity and viability were found to be always above 90~o.

LPL studies Con A (5 #g/ml)-stimulated LPL (0.2 ml, 1 × 106 cells/ml) in complete culture medium were incubated in fiat-bottom multiwell culture plates in triplicates with varying concentrations of TA~ (2.5-25#g/ml) and TB 4 (5-100 ng/ml). Control LPL cultures did not contain TA~ or TB 4. Cultures were incubated in a humidified incubator (5~o CO2, 37 °C) for 5 days. Our previous results showed that Con Astimulated LPL had maximal increases in DNA synthesis on days 4 and 5. Six hours before cell harvesting the cultures were pulsed with 0.5/~Ci/well of 3H-Thd and thymidine incorporation was assayed (Elitsur, 1990). Cells were harvested on glass filter paper (MASH-2) and radioactivity was quantitated in a Packard 1500 liquid scintillation counter. Data are expressed as a stimulation index according to the following formula: stimulation index = cpm(test - background)/cpm(control - background), as previously described (Elitsur, 1990). Concentrations o f T A 1 at or above 0.5/~g/ml and T B 4 a t or above 1 ng/ml were found to significantly inhibit LPL D N A synthesis. The inhibitory concentrations of TA 1 (12.5/lg/ml) and T B 4 ( 5 0 ng/ml) were used for subsequent experiments. In subsequent experiments, Con A-stimulated LPL were exposed to TAI (12.5/~g/ml) o r T B 4 (50 ng/ml) for 1 h. Cells were washed to remove the thymic peptides and were resuspended in complete cell culture medium containing Con A (0.5/~g/ml) to complete a total of 5 days incubation. Cultures were pulsed with [3H]thymidine

Flow cytometry characterization To characterize the helper/suppressor ratio of the LPL cell population, the ratio of helper/inducer (CD4) and suppressor/cytotoxic (CD8) lymphocytes was assayed by flow cytometry in each experiment. The flow cytometry analysis was performed according

92 and radioactivity was quantitated as described above. In parallel experiments, a time-course study was carried out. Specifically, Con Astimulated LPL were incubated in the presence or absence of TA1 (12.5 ~g/ml) and TB 4 (50 ng/ml). Cells were harvested after 2, 3 and 5 days of incubation (5 ~o CO2, 37 ° C). Radioactivity was quantitated as previously described. To elucidate further the intracellular effect of TA1 and T B 4 o n LPL proliferation, intracellular mitogens (PDB and ionomycin) were used. Previous results from our laboratory have shown that thymidine incorporation of PDB (10 8 M) and ionomycin (0.5 #M) -stimulated LPL peaked after 24 h (unpublished results). Accordingly, LPL were exposed to PDB (10-8 M) and ionomycin (0.5 #M) for 24 h. Cells were washed ( x 3) and were resuspended in complete cell culture medium in the presence or absence of TA~ (12.5/~g/ml) or TB4 (50 ng/ml). Cultures were incubated (5 ~o COz, 37 ° C) in multi-well titer plates (2 x 105 cells/well) for an additional 4 days to complete 5 days in culture. Cells were harvested (MASH-2) and radioactivity was quantitated as described above.

Accordingly, Con A-stimulated LPL (2 x 106 cells/tube) containing TA1 (12.5 pg/ml) o r T B 4 (50 ng/ml) were incubated in 5-ml culture tubes (Gibco) for 22 h. Control tubes did not contain TA~ or TB4. Tubes were centrifuged and the cell pellet was harvested. ODC activity was determined using the method of Beaven et al. (1978) as previously described from our laboratory (Elitsur et al., 1990). Briefly, cells were collected by centrifugation and the pellet was homogenized in a buffer containing 50 mM Tris (pH 7.5), 0.25 M sucrose, 0.1 mM EDTA, 0.4 mM pyridoxal 5'phosphate and 1 mM DTT. Homogenates were centrifuged at 100,000 x g for 60 min and the supernatants were assayed for ODC activity. The incubation mixture contained 50 mM HEPES, 1 mM EDTA, 0.25 mM pyridoxal 5'-phosphate, 1 mM DTT, 130/~M ornithine and 0.144/~M [~4C]ornithine. Bradford's protein-dye method (197-6) was used to determine protein concentration in the assay mixture.

Results

Flow cytometry characterization of LPL Ornithine decarboxyta6e I,-'DC) assay Previously we showed that Con A-stimulated LPL ODC activity peaked after 22 h (Elitsur et al., 1990).

Tile LPL CD4/CD8 mean ratio was 2.6 + 0.3 (Table I). This was similar to previous results in

TABLE I Flow cytometry studies on human colon LPL: Pt.Dx.

(%:)

Colon cancer Mean SEM Non cancer Mean SEM Total Mean SEM

(10)

CD19

CD3

CD4

CD8

CD4/CD8

Viability

11.8 2.6

66.5 4.0

40.7 4.4

20.9 2.4

2.4 0.5

93.0 1.3

11.3 1.7

69.4 3.9

47.6 1.4

19.1 2.6

2.8 0.4

92.7 1.9

11.3 1.5

69.1 3.0

43.8 2.6

20.1 1.7

2.6 0.3

92.8 1.1

(8)

(18)

LPL from 18 specimens were isolated by EDTA/collagenase digestion. LPL subpopulations were quantitated by immunofluorescent antibodies. CD19, Pan B; CD3, Pan T; CD4, helper T cell; CD8, suppressor T cells. Viability was determined by the trypan blue exclusion test and/or by ethidium bromide and acridine orange staining.

93 normal LPL (Elitsur et al., 1990). There was no significant difference between the cell population profile and proliferative functions of LPL from histologically normal colon specimens obtained from patients with colon cancer compared with the specimens obtained from patients with nonmalignant diseases. Accordingly, flow cytometry data and the results of the thymidine incorporation assay as well as ODC activity from both groups were combined. The effect of TA 1 and DNA synthesis

TB 4 on

Con A-induced LPL

Previous experiments have shown that T A 1 (2.5-25 #g/ml) and TB4 (5-100 ng/ml) did not alter [ 3H]Thd incorporation into non-stimulated LPL (data not shown). The effects of TA 1 and TB4 on 3H-Thd incorporation into Con Ainduced LPL are seen in Figs. 1 and 2. TA~ (conc. 0.5-25#g/ml) and T B 4 ( c o n c . 1-100ng/ml) significantly suppressed Con A-induced LPL DNA synthesis. Time-course study showed that

TA~ and TB 4 significantly suppressed thymidine incorporation after 3 days of incubation when compared with controls (Fig. 3). The effect of TA 1 and T B 4 o n LPL induced by a combination of phorbol ester (PDB) and calcium ionophore (ionomycin)

To investigate further the mechanism underlying the suppressive effect of the thymic peptides on LPL induction, the 'intracellular mitogens' phorbol ester and calcium ionophore were used. TA1 and T B 4 did not alter thymidine incorporation into PDB + ionomycin-stimulated LPL (Fig. 4). The effect of TA1 and

1.2

1.0

1.0

0.8

o.8 8

0.6

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0.6

0.4

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0.4

+j o

,(_ h_

ODC activity

ODC has been shown to be critical for cell proliferation. A series of experiments were performed to evaluate the effects of TA1 and T B 4 o n LPL ODC activity. Our results showed that peak ODC activity at 22 h was not significantly altered by TA 1(12.5 #g/ml) or by T B 4 ( 5 0 ng/ml) (Fig. 5).

1.2

O

u '5

T B 4 on

u o [_

0.2 0.0

0.2 0.0

0

0.5

1

2.5

5

12.5

25

TA 1 ( p g / m l )

Fig. l. Effect of increasing concentrations of TA~ on Con Astimulated LPL D N A synthesis: Con A (5 #g/ml)-induced LPL were incubated in the presence or absence of TA~ for 5 days. In each experiment results were calculated according to the following formula: c p m ( T A l ) - cpm(background)/ c p m ( c o n t r o l ) - cpm(background), and were expressed as fractions of control. Results are the means +_ SEM of 4 - 8 experiments. The average counts for unstimulated (background) and for Con A-stimulated (control) LPL were 541 cpm and 24003 cpm, respectively. *p < 0.05.

0

1

2.5

5

10

50

100

TA4. ( n g / m I )

Fig. 2. Effect of increasing concentrations ofTB 4 on Con Astimulated LPL D N A synthesis: Con A (5 #g/ml)-induced LPL were incubated in the presence or absence of TB 4 for 5 days. In each experiment results were calculated according to the following formula: c p m ( T A 1 ) - cpm(background)/ c p m ( c o n t r o l ) - cpm(background), and were expressed as fractions of control. Results are the means _+ SEM. of 4 - 8 experiments. The average counts for unstimulated (background) and for Con A-stimulated (control) LPL were 541 cpm and 24003 cpm, respectively. *p < 0.05.

94

I .2"

L

160

140

1.0-

120 ou

0.8-

'~

0,6"

100 E

5 d

o

0.4-

0 L

w

E £). v

0.2-

£9

c~ ©

0,0. 1 hr

2 days

3 days

8O

60 40 20 0

5 days

Time

Fig. 3. Effect o f T A l and TB 4 on Con A-induced LPL DNA

synthesis over time: Con A (5 #g/ml)-stimulated LPL were incubated with TA] (12.5/~g/ml) and TB 4 (50 ng/ml) for 2 h, 2, 3 and 5 days. LPL were harvested and Thd incorporation was quantitated. Results arc the means + SEM of three different experiments done in triplicate determinations. *p < 0.05.

Control

TA 1

TB&

(12.Spg/ml)

(50 n g / m ] )

Fig. 5. Effect of TA~ and TB 4 on ODC activity. Con A (5/~g/ml)-stimulated LPL were incubated in the presence or absence o f T A I (12.5/~g/ml) or TB 4 (50 ng/ml) for 22 h. LPL were then pelleted and ODC activity was measured as described in Methods. Results are the means + SEM of four experiments in triplicate.

Discussion

TA I & T B 4 e f f e c t on P D B + i o n o m y c i n s t i m u l a t e d LPL 1.2 1 .©J o U

0.80.6-

S

g

L

h

0.40.20.0Control

TA1 (12.Spg/ml)

TB~ (50ng/ml)

Fig. 4. Effect of TA~ and TB 4 on thymidine incorporation into PDB + ionomycin-stimulated LPL: PDB + ionomycinstimulated LPL were incubated for 24 h and subsequently were incubated in the presence or absence of TA~ and TB 4. Thymidine incorporation was quantitated after a total of additional 4days of incubation. Results are the means _+ S.D. of three different experiments done in triplicate determinations. Thymidine incorporation into PDB + ionomycin-stimulated LPL was not altered by TA, or TB4. The average counts for unstimulated (background) and for PDB + ionomycin-stimulated (control) LPL were 702 cpm and 40 760 cpm, respectively. *p < 0.05.

TA~ and TB 4 have previously been identified in lymphoid tissue (Gondo et al., 1987). Moreover, these peptides have been found in mammalian myoblast and fibroblast cell lines (Goodall et al., 1983). Studies from our laboratory have identified immunoreactive TA~ and TB4 in lamina propria lymphocytes (LPL). The subsets of these lymphocytes have not been identified (unpublished data). TA~ has been identified in a rat intestinal crypt cell line (IEC-6), as well as in the intestinal crypts of developing rat small intestine (Conteas et al., 1989). We found that TA1 and T B 4 suppressed thymidine incorporation in human colonic LPL. Concurring with previous results (Zatz et al., 1984), neither peptide suppressed 3H-Thd incorporation into unstimulated LPL, suggesting that TA~ and TB4 can affect only cells which are beyond the G o / G 1 stage. These data suggest that these thymic peptides which are present in the gastrointestinal mucosa may participate in the modulation of the gut immune system. However, the intracellular metabolic pathway by which these thymic peptides affect cell division is not known. One intracellular metabolic pathway for cell proliferation involves the activation of the poly-

95 phosphoinositol system (Marx et al., 1984). Following receptor-ligand binding, diacylglycerol and inositol triphosphate (PIP3) are formed. These second messengers influence cell proliferation by activating protein kinase C (PKC) and by increasing intracellular calcium levels. In this study we investigated these second messengers by looking at the effect of TA1 and T B 4 on cell proliferation after induction of LPL with a combination of phorbol ester and calcium ionophore. These mitogens induce cell proliferation by activating P K C (phorbol ester) or by increasing intracellular calcium levels (ionomycin) (Marx etal., 1984; Nishizuka, 1984). Our studies showed that TA1 and T B 4 suppressed thymidine incorporation into LPL in the presence of Con A but not in the presence of PDB and ionomycin. On the other hand, the antiproliferative effect of TA~ and T B 4 w a s abolished when LPL were stimulated with PDB + ionomycin. These results, when taken together, suggest that TA~ and T B 4 may affect D N A synthesis in LPL at a site proximal to calcium fluxes. Alternatively, since phorbol ester and calcium ionophore are known for their synergistic effect on lymphocyte proliferation and IL-2 or gamma-INF secretion (Kumagai et al., 1987; Nobrega et al., 1986), it is possible that the inhibitory effect o f T A 1 and T B 4 o n LPL may be mediated via specific lymphokines. Ornithine decarboxylase (ODC) is considered to be critical for cell proliferation in various tissues, including human lymphocytes (Scott et al., 1985a,b). To elaborate further on the mechanism of action of TA1 and T B 4 o n LPL cell proliferation, peak O D C levels after Con A stimulation were determined. Our results show that neither thymic peptide altered peak O D C levels. Our results suggest that O D C probably does not play a major role in the suppressive effect of LPL D N A incorporation produced by these peptides, and that another metabolic pathway may be responsible for this effect. Further investigations to explore the intracellular mechanisms by which TA~ and TB4 modulate cell proliferation are needed. We conclude that TAI and T B 4 suppress

human LPL cell proliferation. This effect may involve protein kinase C, but is probably not mediated via calcium fluxes or the O D C metabolic pathway.

Acknowledgements This work was supported in part by Grant CA 43280 from the National Institute of Health and by a grant from Harper Hospital and the HudsonWebber Foundation. G D L is a recipient of an American Cancer Society Faculty Research Award. We acknowledge the assistance of the Department of Pathology in Harper Hospital, Detroit, MI, for their help in obtaining the human intestinal specimens, and Mrs. Susan Ottosen and Mrs. Gretta VanBree for their assistance in the flow cytometry studies.

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