Neuropeptides (1992) 23,203-207 0 Longman Group UK Ltd 1992
Neuropeptide Y lmmunoreactivity in the Spleen and Thymus of Normal Rats and Following Adjuvantinduced Arthritis D. JESSOP, S. BISWAS, L. D’SOUZA, H. CHOWDREY and S. LIGHTMAN Neuroendocrinology Unit, Charing Cross and Westminster Medical School, Fulham Palace Rd., London W6 8RF. (Reprint request to DJ)
Abstract-lmmunoreactive neuropeptide Y (irNPY) was detected by radioimmunoassay within the rat thymus and spleen. Total spleen and thymus irNPY contents in control animals were 77 f 3 ng and 23 f 1 ng respectively (means + S.E.M., n = IO). Total tissue contents of irNPY 14 days following bilateral adrenalectomy or induction of inflammatory arthritis were not significantly altered compared to controls. Most spleen irNPY coeluted with synthetic NPY after reversed-phase high-performance liquid chromatography, but two peaks of irNPY were detected in thymic extracts. This suggests that NPY may be differentially expressed in tissues of the immune system. Introduction Neuropeptide Y (NPY) is a 36 residue peptide widely distributed throughout the central (1,2) and peripheral nervous systems (3), with a considerable degree of co-localisation with catecholamines (4,5). NPY is also located within the neurointermediate lobe (6, 7), liver (8), adrenal medulla (9), pancreas (10) and blood platelets (11). In addition to its well-known vasoconstrictor effects, many functions have been ascribed to central NPY, including cardiovascular effects (12, 13) and control of food intake (14, 15). In addition, central NPY has been strongly impliDate received 10 June 1992 Date accepted 20 July 1992 Address correspondence to: Dr David S. Jessop, Neuroendocrinology Unit, Charing Cross Hospital, Fulham Palace Rd., London W6 8RF.
cated in the mechanisms controlling the release of the posterior pituitary peptide arginine vasopressin (AVP) in response to changes in plasma osmolality (6,7), andNPY can also modify the release of anterior pituitary hormones such as growth hormone and luteinising hormone (16). Recently there has been much interest in the interactions between the endocrine and immune systems (17, 18), with neuropeptides being located in tissues of the immune system, and cytokines within the hypothalamo-pituitary axis. NPY mRNA and peptide have been detected in rodent spleens ( 19) and a small population of cells containing NPY mRNA was located in the thymus (20). We have applied the techniques of radioimmunoassay @IA) and reversedphase high-performance liquid chromatography (HPLC) to further investigate NPY in the spleen and thymus, under normal conditions and in rats with
203
204
NEIJFCOPEPTIDES
inflammatory arthritis, a condition in which both the endocrine and immune systems are activated (2 l-23).
Table 1 Spleen weights (g) and neuropeptide Y content (ng total or pg/mg tissue) 14 days after adrenalectomy (ADX), ADX).
induction
Materials and methods
of arthritis
Weight
Animals
or both (ARTW
Total NPY w
Male Sprague-Dawley rats weighing ZOO-250 g were housed in a temperature and humidity controlled environment under 12 h light : 12 h darkness. They were allowed free access to food. To induce arthritis, rats were given an intradermal injection of 0.1 ml of a suspension of ground, heat-killed Mycobacterium butyricum (Difco Laboratories, UK) in paraffin oil at the base of the tail. Final dilution of the adjuvant solution was 10 mg/ml. Control groups were injected with vehicle alone. Bilateral adrenalectomies were performed under pentobarbitone anaesthesia and rats were given 0.9% saline to drink. After 14 d, the animals were decapitated and spleens and thymuses were removed immediately and frozen in dry ice. Tissue extraction Tissues were homogenised by hand in a glass dounce in 5 ml of ice cold phosphate buffered saline, and heated at 90°C for 10 min to eliminate proteolytic activity. After centrifkgation, supernatants were diluted in assay buffer prior to RIA for NPY. Radioimmunoassay for NPY Antiserum was raised in rabbits against synthetic NPY and used in the RIA at a final dilution of 1:25 000. Tracer was prepared using the Iodo-Gen method to iodinate NPY with lz51.Standard was synthetic human NPY (Sigma Pharmaceuticals Ltd., Poole, Dorset, UK), which shares identical sequence with rat NPY. Assay buffer was 0.05 M phosphate containing human serum albumin (0.25% w/v), Triton X-100 (0.1% v/v) and Z-mercaptoethanol (0.1% v/v). After 48 h incubation at 4”C, unbound tracer was separated from bound using a solution of second antibody assisted by 4% polyethylene glycol. Reversed-phase high-performance matography (HPLC)
(ARTH)
liquid
chro-
Reversed-phase HPLC was performed using a Nucleosil C-8 column (Macherey-Nagel, Duren,
Control ADX ARTH ARTWADX
0.73 f 0.04
77f3
0.86 f 0.06 1.05 * 0.07* 1.33 f 0.10*
75f2 7952 83f4
NPY @g/mg tissue)
110f 11 88f8 80 f 7* 64f4*
Values are means f S.E.M., n = 10. *p < 0.05 compared to controls
Germany) with a bead size of 5 p and pore size of 10 nm (0.45 cm internal diameter). Extracts of tissue were diluted in 0.1% trifluoroacetic acid and eluted on a shallow gradient of acetonitrile (40-60% in 60 min). Statistical analysis Data were analysed by two-way analysis of variance with Duncan’s allowance for multiple comparisons (p < 0.05%).
Results Intra-assay coefficient of variation was 7.6% (n = 18). Tissue extraction recovery of NPY was > 95%. Immunoreactive (ir) NPY was detected in the spleens and thymuses from all animals, with higher levels in the spleen. No differences in total spleen irNPY were observed in ADX or arthritic groups, or in those animals subjected to both treatments, although significant decreases occurred in both groups of arthritic animals when the data were expressed as amount of irNPY/weight of tissue Table 2 Thymus weights (g) and neuropeptide Y content (ng total or pg/mg tissue) 14 days after adrenalectomy (ADX), induction of arthritis (ARTH) or both (ARTW ADX). Weight
Total NPY (ns)
Control ADX ARTH AFCTI-VALIX
NPY @g/mg tissue)
0.65 + 0.04
23 f 1
37f2
0.76 f 0.04 0.44 f 0.05* 0.72 f 0.04
21* 1 23f2 22fl
28f2 5.5 f 5* 32f3
Values are means f S.E.M., n = 10. *p < 0.05 compared to controls
IMMUNE
SYSTEM
NEUROPEPTIDE
205
Y
detected which eluted in a position indicating a lower degree of hydrophobicity (Fig. B). a00
2
Discussion 600
i
E
400
200
0
A
B
0
5
10
15
20
25
30
FRACTION NUMBER
FRACTICN NUMBER
Fig.
Reversed-phase HF’LC profile of NPY immunoreactivity (IR-NPY) within extracts of spleen (A) and thymus (B) from control animals. Loading volume was 0.5 ml. Fractions were collected, desiccated and reconstituted in buffer for RIA. The elution position of synthetic rat NPY is arrowed. (Table 1). It was not possible to express the data as amount of irNPY/mg of protein, since the experimental protocol required tissue to be extracted in buffer containing BSA. No differences were observed between groups in total irNPY from thymus extracts, but a significant increase was apparent in arthritic animals subjected to sham ADX (Table 2). Reversed-phase HPLC profiles of irNPY in control extracts of spleen (Fig. A) revealed that almost all irNPY coeluted with the standard. However, in the thymus a second peak of irNPY was
In addition to NPY, several other peptides which have been previously more closely identified with a neuroendocrine role in the hypothalamus, such as corticotropin-releasing factor-4 1 (CRF-4 1) (24), gonadotropin releasing hormone (25), growth hormone releasing factor (26, 27), AVP and oxytocin (28-30), have also been detected in tissues of the immune system. The irNPY level of 110 pg/mg of tissue which we have measured in control spleens agrees well with that previously reported (19) and is roughly comparable to the 200 pg/mg level found in the neurointermediate lobe (NIL) of the pituitary (unpublished data). The thymus contains lower but still significant amounts of irNPY. These relatively high levels suggest that NPY may have a special function within the immune system. The chromatographic profile of irNPY in extracts of spleen revealed that 95% eluted in the position corresponding to that of the 36 residue peptide. This is a similar pattern to that observed in extracts of the neurointermediate lobe and paraventricular nucleus of the hypothalamus, in which irNPY also elutes as a single peak (unpublished data). However, irNPY in the thymus was separated into two peaks of similar size. The second peak of irNPY detected in the thymus may represent a fragment of NPY degraded during the extraction procedure, since the thymus contains high concentrations of proteases such as cathepsins. However, these enzymes are equally prolific in the spleen. Thus the appearance of only one form of irNPY in the latter tissue provides possible evidence for differential processing of NPY in tissues of the spleen and thymus. Removal of the source of circulating corticosteroids by ADX did not alter the content of irNPY in either the spleen or thymus. NPY mRNA andpeptide have been previously located in megakaryocytes of the spleen (19), which are involved in platelet production. Although it is not known whether these cells contain glucocorticoid receptors, such receptors are present in macrophages and Tlymphocytes (31) both of which are found in the spleen and thymus. Thus these organs are potential
206 targets for the well-known effects of steroids upon the immune system (17). NPY in the central nervous system is responsive to glucocorticoid manipulation. Hypothalamic NPY has been reported to increase following dexamethasone (32) while NPY mRNA in the arcuate nucleus decreases following ADX (33). However, no changes in peptide content in specific areas of the hypothalamus were observed following ADX (34), Thus, any effects of glucocorticoids upon NPY in tissues of the immune and endocrine systems remain undefined. Rats suffering from inflammatory arthritis, in addition to activation of the immune system, also have elevated levels of the stress hormones secreted by the pituitary-adrenal axis (21-23). If spleen or thymic NPY were involved in either the immune or endocrine responses during the course of this disease, then we might have observed changes in irNPY tissue content. Increases in NPY mRNA and tissue peptide contents have been reported in mice suffering from autoimmune disease (19). However, we could detect no change in total irNPY tissue content in arthritic animals. A decrease in the amount of irNPY/tissue weight occurred in the spleens from arthritic animals, but these spleens were enlarged, which might account for the effect. Likewise, the increased irNPY in thymuses from arthritic animals subjected to sham ADX may be due to the significant reduction in thymic weight in this group. However, our observations were made 14 days following the induction of arthritis, whereas immune and endocrine changes occurred within the first week. We are currently investigating irNPY tissue and plasma levels at earlier stages of this disease. In conclusion, we have located significant amounts of irNPY in the spleen and thymus of rats under normal conditions and after immune challenge. Chromatographic heterogeneity between spleen and thymic irNPY was observed. We intend to further investigate NPY mRNA and peptide levels in tissues of the immune system to determine if any changes occur during the onset of intlarnmatory arthritis.
References 1. Allen, Y. S., Adrian, T. E., Allen, J. M., Tatemoto, K., Crow, T., Bloom, S. and Polak, J. (1983). Neuropeptide Y distribution in the rat brain. Science 22 1: 877-879.
NEUROPEPTIDES 2. De Quidt, M. and Emson, P. (1986). Distribution of neuropeptide Y-like immunoreactivity in the rat central nervous system. Neuroscience 18: 527-543. 3. Lundberg, J., Terenius, L., Hokfelt, T. et al (1983). High levels of neuropeptide Y in peripheral noradrenergic neurons in various mammals including man. Neurosci. Lett. 42: 167-172. 4. Ever&t, B., Hokfelt, T., Terenius, L., Tatemoto, K., Mutt, V. and Goldstein, M. (1984). Differential coexistence of neuropeptide Y with catecholamines in the central nervous system of the rat. Neuroscience 11: 443462. 5. Sawchencko,P., Swanson,L.,Grzanna,R.,Howe, P.,Bloom, S. and Polak, J. (1985). Colocalization of neuropeptide Y immunoreactivity in brainstem catecholaminergic neurons that project to the paraventricular nucleus. J. Comp. Neurol. 241:138-153. 6. Hooi, S., Richardson, G., McDonald, J., Allen, J., Martin, J. and Koenig, J. (1989). Neuropeptide Y and vasopressin in the hypothalamo-neurohypophysial axis of salt loaded or Brattleboro rats. Brain Res. 486: 214-220. 7. Larsen, P., Sheikh, S. and Mikkelsen, J. (1992). Osmotic regulation of neuropeptide Y and its binding sites in the magnocellular hypothalamo-neurohypophysial pathway. Brain Res. 573: 181-189. 8. Goehler, L. and Stemini, C. (1991). Neuropeptide Y immunoreactivity in the mammalian liver: pattern of coexistence with tyrosine hydroxylase immunoreactivity. Cell Tissue Res. 265: 287-295. 9. Fried, G., Wikstrom, L., Franck, J. and Rokaeus, A. (1991). Galanin and neuropeptide Y in chromaffin granules from the guinea pig. Acta Physiol. Stand. 142: 487-493. 10. Jamal. H.. Jones. P.. Bvme. J.. Suda. K.. Ghatei. M.. Kanse. S. andBloom S. (1991;. Peptide contents ofneuropeptide Y; vasoactive intestinal polypeptide and beta-calcitonin generelated peptide and their messenger ribonucleic acids after dexamethasone treatment in the isolated rat islets of Langerhans. Endocrinol 129: 3372-3380. 11 Ericsson, A., Hemsen, A., Lundberg, J. and Persson, H. (1991). Detection of neuropeptide Y-like immunoreactivity and messenger RNA in rat platelets: the effects of vinblastine, resexpine and dexamethasone on NPY expression in blood cells. Exp. Cell Res. 192: 604611. 12. Carter, D., Vallejo, M. and Lightman, S. (1985). Cardiovascular effects of neuropeptide Y in the nucleus of the tractus solitarius of rats: relationship with noradrenalin and vasopressm. Peptides 6: 421-425. 13. Vallejo, M. and Lightman, S. (1986). Pressor effect of centrally administered neuropeptide Y in rata: role of the sympathetic nervous system and vasopressin. Life Sciences 38: 1859-1866. 14. Stanley, B. and Leibowitz, S. (1985). Neuropeptide Y injected in the paraventricular hypothalamus: a powerful stimulant of feeding behaviour. Proc. Natl. Acad. Sci. USA 82: 394&3943. 15. O’Shea, R. and Gundlach, A. (1991). Preproneuropeptide Y messenger ribonucleic acid in the hypothalamic arcuate nucleus of the rat is increased by food deprivation or dehydration. J. Neuroendocrinol. 3: 11-14. 16. McDonald, J., Lumpkin, M., Samson, W. and McCann, S. (1985). Neuropeptide Y affects secretion of luteinizing hormone and growth hormone in ovariectomized rats. Proc. Natl. Acad. Sci. USA 82: 561-564. 17 Bateman, A., Singh, A., Kral, T. and Soloman, S. (1989). The immune-hypothalamic-pituitary-adrenal axis. Endocrine Rev. 10: 92-l 12.
IMMUNE
SYSTEM NEUROPEPTIDE
Y
18. Buzzetti, R., McLaughlin, L., Scavo, D. and Rees, L. (1989). Acritical assessment ofthe interactions between the immune system and the hypothalamo-pituitary axis. J. Endocrinol. 120: 183-187. 19. Ericsson, A., Schalling, H., McIntyre, K. et al. (1987). Detection of neuropeptide Y and its mRNA in megakaryocytes: enhanced levels in autoimmune mice. Proc. Natl. Acad. Sci. USA 84: 5585-5589. 20. Ericsson, A., Geenan, V., Robert, F. et al. (1990). Expression of preprotachykinin-A and neuropeptide Y mRNA in the thymus. Mol. Endocrinol. 4: 121 I-1218. 21. Harbuz, M., Rees, R., E&land, D., Jessop, D., Brewerton, D. and Lightman, S. (1992). Paradoxical responses of hypothalamic corticotropin-releasing factor (CRF) messenger ribonucleic acid @RNA) and CRF-41 peptide and adrenohypophysial proopiomelanocortin mRNA during chronic stress. Endocrinol. 130: 1394-1400. 22. Sarlis, N., Chowdrey, H., Stephanou, A. and Lightman, S. (1992). Chronic activation of the hypothalamo-pituitaryadrenal axis and loss of circadian rhythm during adjuvantinduced arthritis in the rat. Endocrinol 130: 1775-1779. 23. Stephanou, A., Sarlis, N., Knight, R., Chowdrey, H. and Lightman, S. (1992). Response of pituitary and spleen proopiomelanocortin mRNA, and spleen and thymus interleukin-1S mRNA to adjuvant arthritis in the rat. J. Neuroimmunol. 37: 59-63. 24. Redei, E. (1992). Immunoreactive and bioactive corticotropin-releasing factor in rat thymus. Neuroendocrinol. 55: 115-118. 25. Emanuele, N., Emanuele, M., Tentler, J., Kirsteins, L., Azad, N. and Lawrence, A. (1990). Rat spleen lymphocytes contain an immunoreactive and bioactive luteinizing hormone-
207 releasing hormone. Endocrinol. 126: 2482-2486. 26. Weigent, D. and Blalock, J. (1990). Immunoreactive growth hormone-releasing hormone in rat leukocytes. J. Neuroimmunol. 29: 1-13. 27. Stephanou, A., Knight, R. and Lightman, S. (1991). Producton of a growth hormone releasing-hormone-like peptide and its mRNA by human lymphocytes. Neuroendocrinol 53: 628- 633. 28. Geenan, V., Legros, J., Franchimont, P., Defresne, M., Boniver, J., Ivell, R. and Ritcher, D. (1987). The thymus as a neuroendocrine organ: synthesis of vasopressin and oxytocin in humanthymic epithelium. Ann. NY Acad. Sci. 496: 56-66. 29. Geenan, V., Legros, J., Franchimont, P., Baudrihaye, M., Defresne, M. and Boniver, J. (1986). The neuroendocrine thymus: coexistence of oxytocin and neurophysin in the human thymus. Science 232: 508-5 11. 30. Markwick, A., Iolait, S. and Funder, J. (1986). Immunoreactive arginine vasopressin in the rat thymus. Endocrinol. 119: 1690-1696. 3 1. Werb, Z., Foley, R. and Munck, A. (1978). Interaction of glucocorticoids withmacrophages. J. Exp. Med. 147: 1684-1686. 32. Corder, R., Pralong, F., Turnill, D., Saudan, P., Muller, A. and Gaillard, R. (1988). Dexametbasone treatment increases neuropeptide Y levels in rat hypothalamic neurons. Life Sciences 43: 1879-1886. 33. White, B., Dean, R. and Martin, R. (1990). Adrenalectomy decreases neuropeptide Y mRNA levels in the arcuate nucleus. Brain Res. Bull. 25: 711-715. 34. Rivet, J-M., Castagne, V., Corder, R., Gaillard, R. and Mormede, P. (1989). Study of the inthtence of stress and adrenalectomy on central and peripheral neuropeptide Y levels. Neuroendocrinol. 50: 413-420.
Neuropeptide Y lmmunoreactivity in the Spleen and Thymus of Normal Rats and Following Adjuvantinduced Arthritis D. JESSOP, S. BISWAS, L. D’SOUZA, H. CHOWDREY and S. LIGHTMAN Neuroendocrinology Unit, Charing Cross and Westminster Medical School, Fulham Palace Rd., London W6 8RF. (Reprint request to DJ)
Abstract-lmmunoreactive neuropeptide Y (irNPY) was detected by radioimmunoassay within the rat thymus and spleen. Total spleen and thymus irNPY contents in control animals were 77 f 3 ng and 23 f 1 ng respectively (means + S.E.M., n = IO). Total tissue contents of irNPY 14 days following bilateral adrenalectomy or induction of inflammatory arthritis were not significantly altered compared to controls. Most spleen irNPY coeluted with synthetic NPY after reversed-phase high-performance liquid chromatography, but two peaks of irNPY were detected in thymic extracts. This suggests that NPY may be differentially expressed in tissues of the immune system. Introduction Neuropeptide Y (NPY) is a 36 residue peptide widely distributed throughout the central (1,2) and peripheral nervous systems (3), with a considerable degree of co-localisation with catecholamines (4,5). NPY is also located within the neurointermediate lobe (6, 7), liver (8), adrenal medulla (9), pancreas (10) and blood platelets (11). In addition to its well-known vasoconstrictor effects, many functions have been ascribed to central NPY, including cardiovascular effects (12, 13) and control of food intake (14, 15). In addition, central NPY has been strongly impliDate received 10 June 1992 Date accepted 20 July 1992 Address correspondence to: Dr David S. Jessop, Neuroendocrinology Unit, Charing Cross Hospital, Fulham Palace Rd., London W6 8RF.
cated in the mechanisms controlling the release of the posterior pituitary peptide arginine vasopressin (AVP) in response to changes in plasma osmolality (6,7), andNPY can also modify the release of anterior pituitary hormones such as growth hormone and luteinising hormone (16). Recently there has been much interest in the interactions between the endocrine and immune systems (17, 18), with neuropeptides being located in tissues of the immune system, and cytokines within the hypothalamo-pituitary axis. NPY mRNA and peptide have been detected in rodent spleens ( 19) and a small population of cells containing NPY mRNA was located in the thymus (20). We have applied the techniques of radioimmunoassay @IA) and reversedphase high-performance liquid chromatography (HPLC) to further investigate NPY in the spleen and thymus, under normal conditions and in rats with
203
204
NEIJFCOPEPTIDES
inflammatory arthritis, a condition in which both the endocrine and immune systems are activated (2 l-23).
Table 1 Spleen weights (g) and neuropeptide Y content (ng total or pg/mg tissue) 14 days after adrenalectomy (ADX), ADX).
induction
Materials and methods
of arthritis
Weight
Animals
or both (ARTW
Total NPY w
Male Sprague-Dawley rats weighing ZOO-250 g were housed in a temperature and humidity controlled environment under 12 h light : 12 h darkness. They were allowed free access to food. To induce arthritis, rats were given an intradermal injection of 0.1 ml of a suspension of ground, heat-killed Mycobacterium butyricum (Difco Laboratories, UK) in paraffin oil at the base of the tail. Final dilution of the adjuvant solution was 10 mg/ml. Control groups were injected with vehicle alone. Bilateral adrenalectomies were performed under pentobarbitone anaesthesia and rats were given 0.9% saline to drink. After 14 d, the animals were decapitated and spleens and thymuses were removed immediately and frozen in dry ice. Tissue extraction Tissues were homogenised by hand in a glass dounce in 5 ml of ice cold phosphate buffered saline, and heated at 90°C for 10 min to eliminate proteolytic activity. After centrifkgation, supernatants were diluted in assay buffer prior to RIA for NPY. Radioimmunoassay for NPY Antiserum was raised in rabbits against synthetic NPY and used in the RIA at a final dilution of 1:25 000. Tracer was prepared using the Iodo-Gen method to iodinate NPY with lz51.Standard was synthetic human NPY (Sigma Pharmaceuticals Ltd., Poole, Dorset, UK), which shares identical sequence with rat NPY. Assay buffer was 0.05 M phosphate containing human serum albumin (0.25% w/v), Triton X-100 (0.1% v/v) and Z-mercaptoethanol (0.1% v/v). After 48 h incubation at 4”C, unbound tracer was separated from bound using a solution of second antibody assisted by 4% polyethylene glycol. Reversed-phase high-performance matography (HPLC)
(ARTH)
liquid
chro-
Reversed-phase HPLC was performed using a Nucleosil C-8 column (Macherey-Nagel, Duren,
Control ADX ARTH ARTWADX
0.73 f 0.04
77f3
0.86 f 0.06 1.05 * 0.07* 1.33 f 0.10*
75f2 7952 83f4
NPY @g/mg tissue)
110f 11 88f8 80 f 7* 64f4*
Values are means f S.E.M., n = 10. *p < 0.05 compared to controls
Germany) with a bead size of 5 p and pore size of 10 nm (0.45 cm internal diameter). Extracts of tissue were diluted in 0.1% trifluoroacetic acid and eluted on a shallow gradient of acetonitrile (40-60% in 60 min). Statistical analysis Data were analysed by two-way analysis of variance with Duncan’s allowance for multiple comparisons (p < 0.05%).
Results Intra-assay coefficient of variation was 7.6% (n = 18). Tissue extraction recovery of NPY was > 95%. Immunoreactive (ir) NPY was detected in the spleens and thymuses from all animals, with higher levels in the spleen. No differences in total spleen irNPY were observed in ADX or arthritic groups, or in those animals subjected to both treatments, although significant decreases occurred in both groups of arthritic animals when the data were expressed as amount of irNPY/weight of tissue Table 2 Thymus weights (g) and neuropeptide Y content (ng total or pg/mg tissue) 14 days after adrenalectomy (ADX), induction of arthritis (ARTH) or both (ARTW ADX). Weight
Total NPY (ns)
Control ADX ARTH AFCTI-VALIX
NPY @g/mg tissue)
0.65 + 0.04
23 f 1
37f2
0.76 f 0.04 0.44 f 0.05* 0.72 f 0.04
21* 1 23f2 22fl
28f2 5.5 f 5* 32f3
Values are means f S.E.M., n = 10. *p < 0.05 compared to controls
IMMUNE
SYSTEM
NEUROPEPTIDE
205
Y
detected which eluted in a position indicating a lower degree of hydrophobicity (Fig. B). a00
2
Discussion 600
i
E
400
200
0
A
B
0
5
10
15
20
25
30
FRACTION NUMBER
FRACTICN NUMBER
Fig.
Reversed-phase HF’LC profile of NPY immunoreactivity (IR-NPY) within extracts of spleen (A) and thymus (B) from control animals. Loading volume was 0.5 ml. Fractions were collected, desiccated and reconstituted in buffer for RIA. The elution position of synthetic rat NPY is arrowed. (Table 1). It was not possible to express the data as amount of irNPY/mg of protein, since the experimental protocol required tissue to be extracted in buffer containing BSA. No differences were observed between groups in total irNPY from thymus extracts, but a significant increase was apparent in arthritic animals subjected to sham ADX (Table 2). Reversed-phase HPLC profiles of irNPY in control extracts of spleen (Fig. A) revealed that almost all irNPY coeluted with the standard. However, in the thymus a second peak of irNPY was
In addition to NPY, several other peptides which have been previously more closely identified with a neuroendocrine role in the hypothalamus, such as corticotropin-releasing factor-4 1 (CRF-4 1) (24), gonadotropin releasing hormone (25), growth hormone releasing factor (26, 27), AVP and oxytocin (28-30), have also been detected in tissues of the immune system. The irNPY level of 110 pg/mg of tissue which we have measured in control spleens agrees well with that previously reported (19) and is roughly comparable to the 200 pg/mg level found in the neurointermediate lobe (NIL) of the pituitary (unpublished data). The thymus contains lower but still significant amounts of irNPY. These relatively high levels suggest that NPY may have a special function within the immune system. The chromatographic profile of irNPY in extracts of spleen revealed that 95% eluted in the position corresponding to that of the 36 residue peptide. This is a similar pattern to that observed in extracts of the neurointermediate lobe and paraventricular nucleus of the hypothalamus, in which irNPY also elutes as a single peak (unpublished data). However, irNPY in the thymus was separated into two peaks of similar size. The second peak of irNPY detected in the thymus may represent a fragment of NPY degraded during the extraction procedure, since the thymus contains high concentrations of proteases such as cathepsins. However, these enzymes are equally prolific in the spleen. Thus the appearance of only one form of irNPY in the latter tissue provides possible evidence for differential processing of NPY in tissues of the spleen and thymus. Removal of the source of circulating corticosteroids by ADX did not alter the content of irNPY in either the spleen or thymus. NPY mRNA andpeptide have been previously located in megakaryocytes of the spleen (19), which are involved in platelet production. Although it is not known whether these cells contain glucocorticoid receptors, such receptors are present in macrophages and Tlymphocytes (31) both of which are found in the spleen and thymus. Thus these organs are potential
206 targets for the well-known effects of steroids upon the immune system (17). NPY in the central nervous system is responsive to glucocorticoid manipulation. Hypothalamic NPY has been reported to increase following dexamethasone (32) while NPY mRNA in the arcuate nucleus decreases following ADX (33). However, no changes in peptide content in specific areas of the hypothalamus were observed following ADX (34), Thus, any effects of glucocorticoids upon NPY in tissues of the immune and endocrine systems remain undefined. Rats suffering from inflammatory arthritis, in addition to activation of the immune system, also have elevated levels of the stress hormones secreted by the pituitary-adrenal axis (21-23). If spleen or thymic NPY were involved in either the immune or endocrine responses during the course of this disease, then we might have observed changes in irNPY tissue content. Increases in NPY mRNA and tissue peptide contents have been reported in mice suffering from autoimmune disease (19). However, we could detect no change in total irNPY tissue content in arthritic animals. A decrease in the amount of irNPY/tissue weight occurred in the spleens from arthritic animals, but these spleens were enlarged, which might account for the effect. Likewise, the increased irNPY in thymuses from arthritic animals subjected to sham ADX may be due to the significant reduction in thymic weight in this group. However, our observations were made 14 days following the induction of arthritis, whereas immune and endocrine changes occurred within the first week. We are currently investigating irNPY tissue and plasma levels at earlier stages of this disease. In conclusion, we have located significant amounts of irNPY in the spleen and thymus of rats under normal conditions and after immune challenge. Chromatographic heterogeneity between spleen and thymic irNPY was observed. We intend to further investigate NPY mRNA and peptide levels in tissues of the immune system to determine if any changes occur during the onset of intlarnmatory arthritis.
References 1. Allen, Y. S., Adrian, T. E., Allen, J. M., Tatemoto, K., Crow, T., Bloom, S. and Polak, J. (1983). Neuropeptide Y distribution in the rat brain. Science 22 1: 877-879.
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