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Demonstration of Immunoreactive Vasoactive Intestinal Peptide (IR-VIP) and Somatostatin (IR-SOM) in Rat Thymus ROSA P. GOMARIZ, MARIA J. LORENZO, LUCINDA CACICEDO, ANGELES VICENTE, AND AGUSTIN G. ZAPATA Department of Cell Biology, Faculty of Biology, Complutense University, 28040 Madrid; and Department of Endocrinology, Ram& y Cajal Hospital, 28034 Madrid, Spain In the present work we demonstrate immunohistochemically the presence of both immunoreactive vasoactive intestinal peptide (IR-VIP) and immunoreactive somatostatin (IR-SOM) cells in the thymus of neonatal and adult rats. IR-VIP and IR-SOM from thymic tissue extracts were identified by gel chromatography, HPLC as VIP standard, and somatostatin S-28, respectively. IR-VIP (352.7 pg/thymus) amounts greater than those of IR-SOM (38.7 pg/thymus) detected by radioimmunoassay in the thymus of 3-month-old rats reflected the abundance of IR-VIP positive cells demonstrated by immunohistochemistry. Somatostatin-like immunoreactive cells were identified as epithelial or neuroendocrine-like cells arranged in the thymic cortico-medullary border, whereas IR-VIP positive cells appeared to be large lymphoid cells distributed along the connective tissue trabeculae. Furthermore, IR-VIP lymphoid cells occurred in the periarteriolar lymphoid tissue of the splenic white pulp where lymphoblasts accumulate. The results are discussed with respect to the mutual interactions between the neuroendocrine and immune systems and the possible role played by neuropeptides in these interactions.

6 1990 Academic

Press, Inc.

INTRODUCTION

There is increasing evidence that the neuroendocrine and immune systems do, in fact, communicate with each other (Ader, Cohen, & Felten, 1987). The immune system is perhaps capable of signalling to the nervous system through the action of cyto- or lymphokines, whereas in vivo and in vitro the immune response is influenced by hormones (Comsa, Leonhardt, & Wekerk, 1982), neurotransmitters-including neuropeptides-and biogenic amines (Johnson, Smith, Ton-es, & Blalock, 1982; Plotnikoff & Miller, 1983). Furthermore, peptinergic and adrenergic innervation of lymphoid tissues provides a structural basis for a local modulation of immunity (Felten, Felten, Carlson, Olschowka, & Livnat, 1985). Recently, presence of neuropeptides, including opioid peptides (Zozulya, Pschenichkin, Schehurin, Khomjakov, & Besvershenko, 1985), argininevasopressin, oxytocin, and neurophysins (Markwick, Lolait, & Funder, 1986; Geenen, Legros, Franchimont, Defresne, Boniver, Ivell, & Richter, 1987; Mol, Lane, Robert, Geenen, & Legros, 1988), met-enkephalin (von Gaudecker, Steinmann, Hausmann, Harpprecht, Milicevic, & Muller-Hermelink, 1986), and chromogranin (Nolan, Trojanowski, dz Hogue-Angeletti, 1985; Hogue-Angeletti & Hickey, 1987), has been observed in the mammalian thymus, a major lymphoid organ involved in T lymphocyte maturation and differentiation. The cell origin and intrathymic function of these neuropeptides are scarcely known. Moreover, strongly neurotensin-immunoreactive cells were found scattered throughout the 151 0889-1591/90 $3.00 Copyright 8 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

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parenchyma of the chicken thymus (Sundler, Can-away, Hakanson, Alumets, & Dubois, 1978). In the present work, we demonstrate cells which contain immunoreactive vasoactive intestinal peptide (IR-VIP) and somatostatin (IR-SOM), two neuropeptides with a surprisingly large number of important biological activities, in the thymus of neonatal and adult rats. The presence of both neuropeptides was confirmed and quantified by radioimmunoassay and chromatographic characterization. Previously, Felten et al. (1985) had found VIP-ergic nerve terminals, but not IR-VIP cells, in the thymic cortex of several mammalian species. Somatostatincontaining cells have also been identified in mammalian (H&felt, Efendic, HellerStrom, Johansson, Luft, & Arimura, 1975) and chicken thymuses (Sundler et al., 1978); in patients with carcinoid tumors of the thymus, high concentrations of somatostatin occur in both plasma and the thymic tissue tumor itself (Mortimer, 1977; Penman, Wass, Besser, & Rees, 1980). Our results suggest that whereas IR-SOM containing cells resemble neuroendocrine-like or intrathymic epithelial cells, most IR-VIP cells may be large lymphocytes or lymphoblasts. MATERIAL Immunohistochemical

AND METHODS

Methods

Tissue specimens were obtained from neonatal and 3-month-old Wistar rats (five specimens for each stage) derived from animals purchased from a local supplier. Animals were killed by cervical dislocation under ether anesthesia. Samples of thymus and spleen were aseptically removed, in toto, fixed in Bouin’s solution for 1 h, dehydrated in ethanol, embedded in paraffin, and cut into 6-pm thick sections. Immunohistochemistry for the localization of either IR-VIP or IR-SOM was performed with a rabbit heterologous antiserum to porcine VIP (Serotec) or somatostatin (Milab), both used at a dilution of 1:lOO. The sensitivity of both antisera was established using control tissue from sections of rat spinal cord and pancreas, and the specificity was studied by absorbing the primary antiserum with either purified VIP or somatostatin. Specificity controls for each reaction with the used tissues were carried out by replacing the primary antiserum with nonimmune rabbit serum. A goat anti-rabbit IgG was used as second antibody. The reaction utilized the peroxidase-antiperoxidase complex, demonstrating the peroxidase activity by incubation with 3.3’-diaminobenzidine tetra HCL (DAB). Sections were counterstained with toluidine blue or examined without counterstain. Preparation of Samples for Immunoassay Chromatography Characterization

and

Seventeen rat thymuses were pooled. IR-VIP and IR-SOM from the thymuses were extracted by sonication in 5 vol of 1 M aqueous acetic acid. The mixture was then boiled for 5 min and spun down at 17000 rpm for 15 min. Recovery by this technique was -83.1 * 7 and 85.6 f 10% for SOM and VIP, respectively. After

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lyophilization, the extract was dissolved in 3 ml of 0.1 N HCl. Two milliliters of this extract was analyzed by Sephadex G-50 fine column and 0.5 ml by high pressure liquid chromatography. Production

of Antibodies

Highly purified porcine VIP (Bachem, San Diego, CA) was conjugated to BSA using the coupling reagent carbodiimide (Abraham & Grover, 1971). This complex, containing 12.2 mg VIP, was emulsified with 4 ml Freund’s complete adjuvant and 4 mg dried tubercle bacilii and injected intradermally in adult male rabbits (Vaitukatis, Robbins, & Nieschlag, 1971). Each animal also received 0.5 ml pertussis vaccine SC with the primary immunization. Booster injections were carried out at 2-week intervals. Most of the studies reported here were performed with antiserum at a final dilution of 2 x 10e6. Specificity of the VIP antisera was determined by the ability of pancreatic polypeptide, insulin, gastrin, glucagon, somatostatin, AVP, LH-RH, CCK, B-endorphin, secretin, and gastrin inhibitory peptide in concentrations from 10 to IO6 pg/ml to compete with “‘I-VIP for the binding sites of the antibodies. The VIP antisera did not cross-react with the peptides tested. S-14 antiserum was raised by immunizing rabbits with S-14 (Bachem, San Diego, CA) bound to bovine thyroglobulin with glutaraldehyde. A protocol similar to the one described for VIP was followed. Specificity of the antiserum was tested against rat GRF, porcine VIP, insulin, pancreatic polypeptide, ACTH, calcitonin, bradikinin, AVP, oxytocin, neurotensin, substance P, LH-RH, glucagon, and cholecystokinin. None of these peptides showed cross-reactivity with the antiserum. Immunoassay

of VIP

VIP was iodinated by the chloramine T method (Greenwood, Hunter, & Glober, 1963). 1251was obtained from New England Nuclear (Boston, MA). The iodination mixture was fractionated in a Sephadex G-50 fine column eluted with 0.1 N acetic acid in 0.25% BSA. The specific activity of the label was -937 pCi/pg. Incubation tubes contained 100 ~1 sample of unknown or standard solutions, 300 l~,l buffer phosphate 0.05 M, pH 7.5 containing 0.25 M EDTA, 0.1% BSA, 100 p,l 1: 120 initial dilution normal rabbit serum, and 100 l,~l I:200000 initial dilution VIP antiserum. One hundred p,l i2’I-VIP (5000 cpm) was added 24 h later. The doubled antibody method of separation of bound from free was used. The sensitivity of the assay was 19-39 pg/ml. Intra-assay and inter-assay coefficients of variation were 4-6 and IO-15%, respectively. Immunoassay

of SOM

IR-SOM was quantified by RIA (Pate1 8z Reichlin, 1978) using an antiserum against S-14. S-14 was iodinated by chloramine T method. The specific activity of the label was -1000 @/pg. The initial dilution of the antiserum was 1:20,000 and the cross-reaction with S-28 was 20%. Assay sensitivity was 1 pg/tube, the intraassay and the inter-assay variations were 8 and 15%, respectively.

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Characterization

To achieve further characterization by gel chromatography, samples containing VIP and somatostatin were fractionated by gel filtration through a (1 x 68 cm) column of Sephadex G-50 fine (Pharmacia Fine Chemicals, Uppsala, Sweden), using a 0.1 N acetic acid, 0.25% BSA buffer, at a 4°C with constant gravity perfusion. The characteristics of the column were as follows: exclusion volume of blue dextran (V,) 24 ml; and total volume (V,) 54 ml (free lz51). One-milliliter fractions were collected; IR-SOM and IR-VIP were quantified by RIA. High Pressure Liquid Chromatography

IR-VIP and IR-SOM from thymic extracts, prepared as previously described, were further analyzed by HPLC. Reversed-phase analysis was carried out on a p,-Bondapack C-18 column (Waters Associates, Inc). The column was eluted with O-100% acetonitrile in 0.1% trifluoroacetic acid at a flow rate of 1 ml/min for 30 min; 2-ml fractions were collected, lyophilized, and then reconstituted in radioimmunoassay buffer. Aliquots of the fractions were analyzed for VIP and SOM by radioimmunoassay. RESULTS Intrathymic

Localization of IR-VIP

and IR-SOM

Numerous immunoreactive VIP cells occurred in the thymus of both neonatal (Fig. 1) and adult rats, arranged in parallel with the connective tissue trabeculae in the deep cortex. No differences in distribution, number, or reactivity were observed between neonatal and adult rats. Although some irregular ramified epithelial-like cells showed VIP-like reactivity, most IR-VIP elements seemed mainly to be large lymphocytes and lymphoblasts (Fig. 2). Metachromatic mast cells, which appeared scattered in the connective tissue trabeculae, did not exhibit VIP-like reactivity. The striking finding of the presence of IR-VIP-containing lymphoid-like cells in the rat thymus was confirmed in the splenic white pulp. Numerous IR-VIP positive cells appeared near the central arteries of the white pulp in the periarteriolar lymphoid tissue, a T-dependent splenic area recognized for its abundance of lymphoblasts (Fig. 3). Thymic IR-SOM positive cells appeared restricted to the cortico-medullary border (Fig. 4). They were fewer in number than the IR-VIP cells and showed a large size, round or polygonal shape and large cytoplasm, corresponding probably to isolated neuroendocrine-like or epithelial cells (Fig. 5). Neither IR-VIP nor IR-SOM nervous terminals were found in the rat thymus. IR-VIP and IR-SOM Content in Thymic Tissue

Amounts of both IR-VIP and IR-SOM were only evaluated month-old rats, and were 352.7 and 38.7 pg/thymus, respectively.

in adult,

3-

VIP AND SOMATOSTATIN

FIG. 1. VIP immunoreactive

Characterization

IN RAT THYMUS

I55

cells (arrows) in the thymic cortex (C) of neonatal rat (x45).

of Thymic IR-VIP

Gel chromatographic characterization of the IR-VIP present in the thymic extracts revealed mainly one peak of immunoreactive material eluting in a similar retention volume as the authentic standard (Fig. 6). Another smaI1 component corresponding to a larger form that eluted close to the void volume was also present. Reversed-phase analvsis of the thvmic extracts. using a gradient of

FIG. 2. Lymphoblasts (X400).

(arrows) in the adult thymus showing VIP-immunoreactive

cytoplasm

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FIG. 3. VIP-positive immunoreactive cells (arrows) in the external zone of periarteriolar sheath (PALS) in the adult spleen (X80).

acetonitrile in aqueous TFA, rendered one peak of IR-VIP 22 min, corresponding to VIP standard (Fig. 7). Characterization

with retention

lymphoid

time of

of Thymic IR-SOM

Figure 8 shows elution profiles of extracts of thymic tissue using a Sephadex G-50 fine column. The somatostatin in the thymic extracts occurred mainly in a large form that coeluted with synthetic somatostatin S-28. HPLC analysis of thymic extracts in the above described conditions showed the presence of only one component with a retention time of 18 min corresponding to somatostatin S-28 (Fig. 9).

FIG. 4. Dispersed SOM-immunoreactive thymus (x200).

cells (arrows) in the cortico-medullary

border of adult

VIP

FIG.

5. Nonlymphoid

AND

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cell with SOM immunoreactive

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material in the cytoplasm (x400).

DISCUSSION

The present immunohistochemical study demonstrates the existence of both IR-VIP and IR-SOM containing cells in the thymus of neonatal and adult rats. Moreover, HPLC and gel filtration analysis indicates that VIP-like immunoreactive material in extracts of the rat thymus is identical to VIP,-,, standard. VIP characterization by gel chromatography, however, showed two peaks. One corresponds to VIP standard and the other represents a peptide of molecular weight greater than 10,000 Da. Different molecular weight variants of VIP have also been described in neural tissues (Dimaline & Dockray, 1979). Their existence in rat thymus may suggest a similar post-translational processing of VIP in both organs, thymus and brain. On the other hand, chromatographic characterization of IRSOM material shows only one peak corresponding to somatostatin S-28. vo

vs

VIP 1-28

300

A

200

i P i B

loo

0 0

10

20

30

40

50

FRACTION NQ FIG. 6. Characterization of IR-VIP in extract of thymic tissue by Sephadex G-50 Fine. The motility of VIP synthetic standard in the column is also represented. IR-VIP was found mainly in one fraction corresponding to VIP,.,,.

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ET AL. r150

VIP l-28

200

100

0 0

40

RETENTION2LlE

(min.)

FIG. 7. HPLC chromatography of IR-VIP from extracts of thymic tissue. Samples were eluted from u-Bondapack C-18 column with acetonitrile in aqueous trifluoroacetic acid (TFA 0.1%) for O-30 min at 0400% acetonitrile. Only one peak corresponding to VIP,,, was shown.

Penman et al. (1980) obtained the same results when analyzing the somatostatin content of thymic tumor tissue. In other rat tissues IR-SOM material elutes as three peaks, corresponding to somatostatin S-14, S-28, and a 12-kDa form referred to as pro-S. Interestingly, S-28 is the predominant form in somatostatin-containing gut mucosal D cells, whereas in the enteric plexuses, S-14 is the main immunoreactive species (Patel, Hans, & Srikant, 1985). Likewise, IR-SOM immunohistochemically identified in thymic cells but not in nerve terminals may reasonably be expected to occur as a S-28 form. Detectable amounts of IR-VIP and IR-SOM in rat thymus indicate greater values for the former than for somatostatin. This difference clearly reflects the number of positive cells immunohistochemically observed in each case, and also indicates that the high amount of IR-VIP found in rat thymus may be derived from the rich VIP-ergic innervation shown in this tissue (Felten et al. 1985). No information on the amount of IR-VIP in the thymus is available but, in any case,

O0

10

20

30

40

50

FRACTION NS FIG. 8. Elution profile of IR-SOM from thymic extract. Only 1 peak corresponding was shown.

to SOM S-28

VIP

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1.59

200

100

0 0

20

40

RETENTION TIME (min.) FIG. 9. Fractionation and identification of IR-SOM in thymic extracts by reverse phase HPLC. The retention time was 18 min corresponding to ‘251-SOM,,,.

according to our results it is remarkably lower than in other organs including gut and brain (Besson, Laburthe, Bataille, DuPont, & Rosselin, 1978; Emson, Gilbert, Loren, Eahrenkung, Sundler, & Schaffalitzky de Muckadell, 1979). No significant quantities of somatostatin have normally been reported in thymus (Arimura, Sato, DuPont, Nishi, & Schally, 1975; Kronheim, Berelowitz, & Pimstone, 1976; Pate1 & Reichlin, 1978), although they increase considerably in thymic tumors (Mortimer, 1977; Penman et al., 1980). In the mammalian thymus, VIP-like immunoreactive profiles had previously been associated with small varicose nerve terminals arranged preferably in the deep cortical parenchyma (Felten et al., 1985). VIP-containing nerves also present in murine Peyer’s patches (Ottaway, Lewis, & Asa, 1987). No IR-VIP containing cells like those reported in this study, however, have been reported. On the other hand, and in agreement with our observations, somatostatin-like presenting cells have been reported in the thymus of mammals (H&felt et al., 1975) and chickens (Sundler et al., 1978). Moreover they suggest an epithelial or neuroendocrine-like nature for thymic IR-SOM cells, in agreement with previous reports on somatostatin-containing cells found in the thymus (H&felt et al., 1975; Sundler et al., 1978). Thymic VIP immunoreactivity, however, is associated with the cytoplasm of free cells, most of which shows a lymphoid morphology. Furthermore, our immunohistochemical results demonstrate VIP immunoreactivity in lymphoblasts of the periarteriolar sheaths, a T-dependent area of rat spleen. The presence of VIP has been identified by radioimmunoassay in human neutrophils (O’Dorisio, O’Dorisio, Cotland, & Bakerzak, 1980; O’Dorisio, Wood, Wenger, & Vassalo, 1985) and by immunocytochemistry in rat mast cells (Cutz, Chan, Track, Goth, & Said, 1978). Reports show that leukocytes produce ACTH, endorphins, TSH, and chorionic gonadotropin (Smith & Blalock, 1986) and that mast cells contain somatostatin (Smith, Meyer, & Blalock, 1982). Recently, immunoreactive chromogranin positive cells were identified as null cells expressing a pan T lymphocyte marker W3/13 and a marker for class I antigens of the MHC (Hogue-Angeletti & Hickey, 1987). Moreover an S49, Thy-l negative lymphoma

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is strongly chromogranin-positive (Hogue-Angeletti & Hickey, 1987). Nevertheless, these results should be treated cautiously and further research will include immunodetection at electron microscopic level for a better characterization of involved cell types and/or isolation from lymphoid cells of specific VIP messenger RNAs. Besides the methodology used does not distinguish whether lymphoid cells synthetize neuropeptides or simply concentrate them in storage granules. On the other hand, a specific role for neuropeptides in the thymic functional activity is unknown. In fact, our understanding of the role of neuropeptides in the modulation of immune system is just beginning. ACKNOWLEDGMENTS This work was supported by CAYCIT grant number PB85-0045 from the Spanish Ministry of Education and Science. The secretarial support provided by M. J. Diaz Castellanos is highly appreciated. Miss Angeles Vicente holds a fellowship from the Spanish Ministry of Education and Science.

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Markwick, A. J., Lolait, S. T., & Funder, J. W. (1986). Immunoreactive arginine-vasopressin in the rat thymus. Endocrinology 119, 1690-1696. Mol, U. M., Lane, B. L., Robert, F., Geenen, V., & Legros, J. J. (1988). The neuroendocrine thymus. Abundant occurrence of oxytocin, vasopressin and neurophysin-like peptides in epithelial cells. Histochemistry 89, 385-390. Mortimer, C. H. (1977). Clinical application of the gonadotrophin releasing hormone. Clinics in Endocrinol. and Metabolism 6, 167-179. Nolan, J. A., Trojanowski, J. Q., & Hogue-Angeletti, R. (1985). Neurons and neuroendocrine cells contain chromogranin: Detection of the molecule in normal bovine tissues by immunochemical and.immunohistochemical methods. J. Histochem. Cytochem. 33, 791-798. O’Dorisio, M. S., O’Dorisio, T., Cotland, S., & Bakerzak, S. P. (1980). Vasoactive intestinal polypeptide as a biochemical marker for polymorphonuclear leukocytes. J. Lab. Clin. Med. 96, 666-672. O’Dorisio, M. S., Wood, C. L., Wenger, G. D., & Vassalo, L. M. (1985). Cyclic AMP-dependent protein kinase in Molt 4b lymphoblasts: Identification by photoaffinity labelling and activation of intact cells by vasoactive intestinal polypeptide and peptide histidine isoleucine. J. Immunol. 134, 4078-4086. Ottaway, C. A., Lewis, D. L., & Asa, S. L. (1987). Vasoactive intestinal peptide-containing nerves in Peyer’s patches. Brain Behav. Immun. 1, 148-158. Patel, Y. C., Hans, H. Z., & Srikant, C. B. (1985). Somatostatin-14 like immunoreactive forms in the rat: Characterization, distribution and biosynthesis. In Y. C. Pate1 & G. S. Tannenbaum (Eds.), Advances in experimental medicine and biology (Vol. 188). New York and London: Plenum Press. Patel, Y. C., & Reichlin, S. (1978). Somatostatin in hypothalamus, extrahypothalamus and peripheral tissues of the rat. Endocrinology 102, 523-530. Penman, E., Wass, J. A. H., Besser, G. M., & Rees, L. H. (1980). Somatostatin secretion by lung and thymic tumours. Clin. Endocrinol. 13, 613-620. Plotnikoff, N., & Miller, G. C. (1983). Enkephalins as immunomodulators. Int. J. Immunopharmacol. 5, 437-442. Smith, E. M., & Blalock, J. E. (1986). A complete regulatory loop between the immune and neuroendocrine systems operates through common signal molecules (hormones) and receptors. In N. P. Plotnikoff, R. E. Faith, A. J. Murgo, & R. A. Good (Eds.), Enkephalins and endorphins. Stress and immune system. New York and London: Plenum Press. Smith, E. M., Meyer, W. J., & Blalock, J. E. (1982). Virus-induced increases in corticosterone in hypophysectomized mice: A possible lymphoid-axis. Science 218, 1311-1312. Sundler, F., Carraway, R. E., Hakanson, R., Alumets, J., & Dubois, M. P. (1978). Immunoreactive neurotensin and somatostatin in the chicken thymus. A chemical and histochemical study. Cell Tissue Res. 194, 367-376. Vaitukatis, J., Robbins, J. B., & Nieschlag, E. (1971). A method for producing specific antisera with small doses of immunogen. J. C/in. Endocr. and Merab. 33, 988-991. von Gaudecker, B., Steinmann, C. G., Hausmann, M. L., Harpprecht, J., Milicevic, N. M., & Muller-Herrnelink, H. K. (1986). Immunohistochemical characterization of the thymic microenvironment. A light microscopic and ultrastructural immunocytochemical study. Cell Tissue Res. 244, 403-412. Zozulya, A. A., Pschenichkin, S. P., Schehurin, M. R., Khomjakov, J. N., & Besvershenko, I. A. (1985). Thymus peptides interacting with opiate receptors. Acta Endocrinologica 110, 284-288. Received April 6, 1989