Immunobiol., vol. 180, pp. 419-430 (1990) Italian Neuroimmunology Group, UOl21 of General Physiology and 2 Human Anatomy, University of Siena, Sieria, Italy 1 Institutes
Somatostatin and Vasoactive Intestinal Peptide Reduce Interferon Gamma Production by Human Peripheral Blood Mononuclear Cells MICHELA MUSCE1TOLA 1 and GIOVANNI GRASS02 Received May 30, 1989 . Accepted in Revised Form February 9, 1990
Abstract There is increasing evidence that neuropeptide modulation of the immune response is an important physiological phenomenon which involves the interaction of peptidergic neuromodulators with specific neuropeptide receptors on the plasma membrane of immune effector cells. Many studies have examined the effect of neuropeptides on mitogen-induced lymphocyte proliferation and immunoglobulin synthesis but very little is known about specific lymphokine production. In this study, we describe the effect of somatostatin (SOM) and vasoactive intestinal peptide (VIP) on interferon gamma (IFN-y) production by normal human peripheral blood mononuclear cells (PBMC) stimulated in vitro with polyclonal T cell activator staphylococcal enterotoxin A (SEA). Our findings provide experimental evidence that both SOM and VIP reduce the IFN-y production by SEA-stimulated PBMe. This reduction was time- (with maximal effect at 72h) and dose-dependent (at doses as low as 10-11 M with maximal effect at concentrations between 10-9 and 10-8M of neuropeptides). This effect was absent in resting PBMe. The meaning of inhibitory effect of VIP and SOM on IFNy production and its role in immune response in vivo are discussed.
Introduction
The involvement of the nervous system in the direct and indirect regulation and modulation of immune response is now well recognized (1-4). Neuroendocrine and immune systems constitute a totally integrated circuit and share a common set of hormones, neurotransmitters and polypeptides, and their receptors (5, 6). Many newly identified neurotransmitters and neuromodulators are neuropeptides which produce receptor-mediated Abbreviations: SOM = somatostatin; VIP = vasoactive intestinal peptide; IFN-y = interferon gamma; PBMC = peripheral blood mononuclear cells; SEA = staphylococcal enterotoxin A; WISH = human amnionic cells; VSV = vesicular stomatitis virus; IL2 = interleukin-2;PFU = plaque-forming units; LGL = large granular lymphocytes; PBS = phosphate-buffered saline; EMEM = Eagle's Minimum Essential Medium. Part of this work was presented at the Annual Meeting of the International Society for Interferon Research, Kyoto, Japan, November 14-18, 1988.
420 . MICHELA MUSCETTOLA and GIOVANNI GRASSO
behavioral effects (7). In recent years, in vitro and in vivo studies have shown that neuropeptides, including vasoactive intestinal polypeptide (VIP), substance P and somatostatin (SOM), can modulate lymphocyte function (8-16). These studies have examined the effect of neuropeptides on mitogen-induced lymphocyte proliferation and immunoglobulin synthesis but very little is known about specific lymphokine production (17, 18). Because lymphokines (nonimmunoglobulin glycoproteins) are central in the regulation and modulation of immune and inflammatory responses, the possible effect of neuropeptides on lymphokine production is of interest. In this study we describe the inhibitory role of somatostatin (SOM) and vasoactive intestinal peptide (VIP) on interferon gamma (IFN-y) production by normal human peripheral blood mononuclear cells (PBMC) stimulated in vitro with polyclonal T cell activator staphylococcal enterotoxin A
(SEA).
Materials and Methods Chemicals Pure somatostatinl_14 and VIP I _28 (purchased from Boehringer and Sigma respectively) were initially dissolved in phosphate-buffered saline (PBS, pH 7.4) to 10-4 M and kept at -80 DC for up to two weeks. They were freshly diluted in RPMI-1640 medium before each experiment. Lymphocyte isolation PBMC were isolated from heparinized buffy coats of ten healthy donors (age range 28-45 years) by Ficoll-Paque (Pharmacia, Fine Chemicals) gradient centrifugation (19). The same donors were used for both neuropeptides. Blood samples were taken at 8 AM. The PBMC in the interface were washed twice with PBS and once with RPMI-1640 (Gibco) containing 20 mM L-glutamine (Gibco), 10 mM Hepes (Gibco) and 1 % penicillin-streptomycin solution (Labtek, Eurobio). Finally the cells were suspended in RPMI-1640 supplemented with 8 % heat-inactivated fetal-calf serum (Flow Laboratories). Cell numbers were determined by lightmicroscope count and viability was assayed by the trypan-blue dye exclusion technique. The technique routinely resulted in cell populations consisting of approximately 90 % lymphocytes and 10 % monocytes. Culture conditions and cell responsiveness The incubation was carried out in quadruplicate in 48-well plates (Costar) at 37 DC in a humidified atmosphere of 5 % CO 2 : 95 % air; each well contained 5 x 105 cells/0.5 m!. Cell responsiveness was checked by means of the potent mitogen staphylococcal enterotoxin A (Toxin Technology, Inc.) (20, 21) added at a dose of 0.1 [!g/106 cells at the start of the culture. Each plate contained unstimulated and stimulated cells with or without increasing concentrations of neuropeptides (10-12 M to 10-6 M) added at the beginning of the culture. At the end of incubation, the cultures were collected and centrifuged at 2,000 x g for 15 min at 4 DC. The supernatants were stored at -20 DC until IFN-y assay. Interferon assay The microplaque reduction assay (22) was used throughout for the titration of IFN activity using human amnionic cells (WISH) as indicator cells and vesicular stomatitis virus (VSV, Indiana strain) as challenge virus.
Neuropeptides and IFN-y production· 421 Briefly, two-fold dilutions of samples in Eagle's Minimum Essential Medium (EMEM, Gibco) supplemented with 2 % heat~inactivated fetal calf ,syr~m . (Flow Laboratories) were added to cell monolayers in flat-boti:om tnicrotiter wells (Costar): After 16-18 h incubation, the medium was removed by inversion and the monolayers carefully washed three times with EMEM without serum to remove interfering substances such as lipoproteins and immunoglobulins. The monolayers were then infected with about 60 plaque-forming units (PFU) of VSV in 1 % fetal-calf serum-EMEM. The samples were tested at least in quadruplicate and twice. The highest dilution of a sample causing a 50 % reduction in virus microplaque was determined to be the endpoint, and the reciprocal of this dilution was recorded as the IFN titer. Each assay included a human IFN-y WHO International standard (obtained from NIH, Bethesda, MD, USA) to check the sensitivity and reproducibility of our test system according to the Expert Committee on Biological Standardization of Interferons (23). The IFN activity was expressed in International Units (IU) and the results are presented as IFN-y production per 106 cells (IU/l0 6 cells) and percentage change in IFN-y production calculated as follows: IFN -y produced in the presence of neuropeptide IFN-y produced in the absence of neuropeptide IFN -y produced in the absence of neuropeptide
x 100
Statistical analysis The data were processed by one-way analysis of variance (ANOVA) for repeated samples.
Results
The cell viability with all the concentrations of SOM and VIP tested was not significantly different from that of control cultures as quantified by similar uptake of about 4 % of the trypan blue after 72 h culture. The geometric mean titer of the human IFN-y NIH reference preparation (Gg23-901-530) with a defined potency of 3.61 Log lO IV/vial when reconstituted in 1 ml of sterile distilled water, in our laboratory conditions, was 3.4834 Log lO IV/ml (with a standard deviation of 0.2128 Log lO corresponding to about a 1.6322-fold variation; total number of titrations = 25). The antiviral activity induced by SEA was characterized as IFN-y (20, 24). This IFN was labile at pH 2, neutralized by anti-IFN-y antibodies (monoclonal antibody B3, specific for human IFN-y, kindly provided by Professor Jan Vilcek) and was not affected by antibodies to human IFN-a or B(Boehringer Biochemia Robin). SEA-unstimulated cells produced only negligible amounts of IFN-y (16 ± 4.7 IV/106 cells). On the other hand, when neuropeptides alone were added to PBMC cultures, no effect on IFN-y production was observed. Maximal IFN-y levels were found 72h after stimulation with SEA (Figs. 2, 4) and therefore, in our experiments on dose response, the effect of neuropeptides on IFN-y production was assessed after 3 days of exposure. It has been demonstrated that the levels of SOM in the T-Iymphocyte cultures diminished as a function of time and after 24 hover 95 % of SOM was degraded (10). Furthermore, in the medium of human transformed
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colonic epithelial cells, after 20 min there were no more intact VIP molecules (25). Nevertheless, we determined what effect if any, neuropeptides had on the IFN assay. We found that over a wide range of concentrations the neuropeptides. 1) were not toxic to WISH cells, 2) did not protect Table 1. Range (expressed in %) of inhibition of IFN-y production by SEA-stimulated peripheral blood mononuclear cells in presence of increasing concentrations of SOM Stimulus
SEA SEA + 10-12 M SOM SEA + 10-11 M SOM SEA+ 10-10 M SOM SEA + 10-9 M SOM SEA + 10-8 M SOM SEA.+ 10-7 M SOM SEA + 10-6 M SOM
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Neuropeptides and IFN-y production . 423
cells from viral plaque formation and 3) did not inhibit the antiviral activity of IFN (data not shown).
Effect of SOM on IFN-y production by SEA -stimulated PBMC IFN-y production by SEA-stimulated peripheral blood mononuclear cells with or without increasing concentrations of SOM is shown in Figure 1. The inhibitory effect of SOM on IFN-y production occurred at all doses and was maximal at concentrations between 10-9 M and 10-8 M. The subjects examined had different ranges of IFN-y production by SEA-stimulated PBMC in the absence or presence of SOM; the ranges of percentage inhibition of IFN-y production at all concentrations tested are presented in Table 1. The time course of IFN-y production by SEA-stimulated PBMC, cultured in the absence or presence of 10-9 M SOM, and its percentage inhibition are shown in Figure 2.
Effect of VIP on IFN-y production by SEA-stimulated PBMC IFN -y production by SEA-stimulated peripheral blood mononuclear cells in the absence or presence of increasing concentrations of VIP is
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shown in Figure 3. The inhibitory effect of VIP on IFN-y production 'occurred at all doses and was maximal at concentrations between 10-9 M and 10-8 M. The subjects examined had different ranges of IFN-y production by SEA-stimulated PBMC in the absence or presence of VIP; the ranges of percentage inhibition IFN-y production at all concentrations tested are Table 2. Range (expressed in %) of inhibition of IFN-y production by SEA-stimulated peripheral blood mononuclear cells in presence of increasing concentrations of VIP Stimulus
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Figure 4. Time course of IFN-y production by peripheral blood mononuclear cells (PBMC) of 4 healthy donors. PBMC, stimulated with polyclonal T cell activator SEA (100 ng/l06 cells) were cultured in the absence (0--0) or presence (e--e) of 1O-9 M VIP. The effect of VIP on IFN-y production is expressed IU/l0 6 cells and as percentage inhibition (e----e) with respect to controls. The values are the means ± SD of 4 experiments and all samples were tested in quadruplicate and twice.
presented in Table 2. The time course of IFN-y production by SEAstimulated PBMC, cultured in the absence or presence of 10-9 M VIP, and its percentage inhibition are shown in Figure 4.
Discussion N europeptides, known to be released from peripheral nerve varicosities and produced by neuroendocrine cells, have also been localized in cells of the immune system (for review see 12). There is increasing evidence that neuropeptide modulation of the immune response is an important physiological phenomenon which involves the interaction of peptidergic neuromodulators with specific neuropeptide receptors on the plasma membrane of immune effector cells (for review see 6, 26). Specific binding sites for VIP (8, 27, 28) and SOM (29) have been detected on normal human peripheral blood lymphocytes suggesting that they play a role in lympho-
426 .
MICHELA MUSCETTOLA
and
GIOVANNI GRASSO
cyte function. It has been demonstrated that SOM is a potent inhibitor of the proliferation of PHA-stimulated human peripheral T lymphocytes and Molt-4 lymphoblasts (10), and mouse spleen lymphocytes (13). VIP furthermore inhibits the proliferative response of PHA - and Con A -stimulated murine lymphocytes from secondary lymphoid tissues (30, 31). Moreover, both SOM and VIP modulate immunoglobulin synthesis in murine lymphocytes from spleen, mesenteric lymph nodes and Peyer's patches stimulated with Con A. This modulation is neuropeptide-, organ-, and isotypespecific (31). The precise role of such neuropeptides in regulating immune function is still unclear and no information exists about their possible effect on IFN-y production. It is known that IFN-y is a lymphokine produced by T lymphocytes after mitogenic or antigenic stimulation, and that it plays a crucial role in the modulation of a variety of immune functions (for review see 32). IFN-y secretion appears to be a better indicator of specific T cell response than eH] thymidine uptake (21); cell proliferation is not a necessary prerequisite for IFN-y production (33-36). Accordingly, we decided to investigate the effect of SOM and VIP on IFN-y production by normal human peripheral blood mononuclear cells. Our findings provide experimental evidence that both neuropeptides reduce the IFN-y production by SEA-stimulated PBMC in a time- and dose-dependent manner. This effect was absent in resting PBMC. The suppressive effect of VIP and SOM was also observed with Con A and PHA (data not shown), but the great difference between these mitogens and SEA as regards the level of IFN-y produced prompted us to perform our experiments with SEA. That the inhibitory effects of SOM and VIP are not due to a direct cytotoxic effect on lymphocyte function was demonstrated by the similar exclusion of trypan-blue dye under all culture conditions. Because of well known great interindividual variation in IFN-y production by SEA-stimulated PBMC, the subjects examined and the time of blood collection were the same for both neuropeptides. The inhibitory effect of both neuropeptides on IFN -y production can be detected just after 12 h of culture and reaches a maximum at 72 h, which is the time with maximal IFN -y levels induced by SEA, and then decreases. The suppressive effect of VIP and SOM can be detected even at concentrations as low as 10-12_10-11 M with maximal reduction at concentrations between 10-9-10-8 M, but at higher neuropeptide concentrations (10-7-10-6 M) there is a reversal of inhibition of IFN -y production. This loss of response to neuropeptide pharmacological concentrations could be related to the down regulation of the cell surface receptors. In fact it has been demonstrated that 10-7 M VIP treatment of T cells resulted in a dosedependent loss of the ability of T cells to specifically bind radio iodinated VIP (37). This altered binding of the treated cells was observed in different biological and physiological models and was due to destruction of part of the receptors into the prelysosomal and lysosomal compartments (25, 38).
Neuropeptides and IFN-y production· 427
Because peripheral blood concentrations of VIP and SOM are 10-12 M whereas at nerve endings in the intestine they are 10-7M (39), the effect of neuropeptides on lymphokine production by activated lymphocytes obtained from spleen and lymphoid tissues is of interest. Since organspecific effects of neuropeptide have been reported (31), we are currently investigating the effect of SOM and VIP on IFN-y production by murine lymphocytes from Peyer's patches. Whether or not the inhibitory effects of SOM and VIP on IFN~y production are direct effects or the result of the inhibition of interleukin-2 (IL 2) production is presently under investigation. However, recent studies on murine lymphocytes (40) and our preliminary results on human lymphocytes (data not shown) suggest that the inhibitory effect of VIP on IFNy production is due to a decreased production of IL2 by CD4+ Tcells. However, the experimental system used by us contained a multitude of immunocompetent cell populations because the binding of SEA to the accessory cells (monocytes and B cells) is an absolute requirement for T cell activation and subsequent IFN-y production (41). Since high-affinity specific receptors for VIP have been identified on CD3+ T cells, CD8+ T cells, CD4+ T cells, LGL and B cells (42), the inhibitory effect of VIP on IFN-y production could be explicable also by its direct effect on suppressor and LGL cells. The results of the present Study raise the question of the meaning of the inhibitory effects of VIP and SOM on IFN-y production and of its role in immune response in vivo. It is known that human intestinal mucosal mononuclear cells produce IFN-y (43) and surface receptors for this lymphokine are also found on epithelial cells (44). HLA-DR antigens are also found on enterocytes of the human small intestine (45, 46). Since IFNy enhances HLA-DR antigen expression in a human colonic adenocarcinoma cell line (47) and affects the barrier function of cultured intestinal epithelial monolayers (48), SOM and VIP may modulate intestinal immunophysiology by regulating IFN-y production. Finally, it has been clearly established that the traffic of lymphocytes is of considerable importance to the initiation and propagation of the immune response. The lymphoid tissues associated with mucosal membranes are major sites of lymphoid cell migration. Moreover, the adherence of lymphocytes to the endothelial cells is an essential step for their emigration from the blood into lymphoid tissue. Exposure of T cells to VIP, at concentrations that downregulate the VIP receptors, resulted in a decrease in their ability to migrate in Peyer's patches and mesenteric lymph nodes of recipient animals (37). Furthermore, the presence of VIB-ergic nerve fibers in very close proximity to the specialized postcapillary venules of these lymphoid tissues (49) suggests that VIP alters the capacity of lymphocytes to bind to the high endothelial cells. The demonstration that IFN -y increases the binding of T lymphocytes to endothelial cells (50) and the results presented here suggest
428 . MICHELA MUSCETTOLA and GIOVANNI GRASSO
that VIP can indirectly affect the traffic of lymphocytes via the reduction of IFN -y production. In conclusion, the present findings further emphasize the interdependence of the immune and nervous systems. Acknowledgements This study was financed by a grant (60 %) from the Ministry of Education, Rome. The authors are grateful to Professor JAN VILCEK (New York) for the generous gift of monoclonal antibodies to human IFN-y and to Mrs. ANNA ROMEI for skilful secretarial assistance.
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430 . MICHELA MUSCETIOLA and GIOVANNI GRASSO 37. OTIAWAY, C. A. 1984. In vitro alteration of receptors for vasoactive intestinal peptide changes the in vivo localization of mouse T cells. J. Exp. Med. 160: 1054. 38. BOISSARD, c.,J. C. MARIE, G. HEJBLUM, C. GESPACH, and G. ROSSELIN. 1986. Vasoactive intestinal peptide receptor regulation and reversible desensitization in human colonic carcinoma cells in culture. Cancer Res. 46: 4406. 39. LONG, R. G., and M. G. BRYANT. 1982. Vasoactive intestinal polypeptide. In: S. R. BLOOM, and R. G. LONG (Eds.), Radioimmunoassay of Gut Regulatory Peptides, Saunder, London, p. 120. 40. OTIAWAY, C. A. 1987. Selective effects of vasoactive intestinal peptide on the mitogenic response of murine T cells. Immunology 62: 291. 41. CARLSSON, R., H. FISCHER, and H. O. SJOGREN. 1988. Binding of staphylococcal enterotoxin A to accessory cells is a requirement for its ability to activate human T cells. J. Immunol. 140: 2484. 42. OTIAWAY, C. A. 1988. Vasoactive intestinal peptide as a modulator of lymphocyte and immune function. Ann. N.Y. Acad. Sci. 527: 486. 43. LIEBERMAN, B. Y., C. FIOCCHI, K. R. YOUNGMAN, W. K. SAPATNEKAR, and M. R. PROFFITI. 1988. Interferon y production by human intestinal mucosal mononuclear cells. Decreased levels in inflammatory bowel disease. Dig. Dis. Sci. 33: 1297. 44. UCER, U., H. BARTSCH, P. SCHEURICH, and K. PFIZENMAIER. 1985. Biological effects of gamma interferon on human tumor cells. Int. J. Cancer 36: 103. 45. SCOTI, H., B. G. SOLHEIM, P. BRANDTZAEG, and E. THORSBY. 1980. HLA-DR-like antigens in the epithelium of the human small intestine. Scand. J. Immunol. 12: 77. 46. SPENCER, J., T. FINN, and P. G. ISAACSON. 1986. Expression of HLA-DR antigens on epithelium associated with lymphoid tissue in the human gastrointestinal tract. Gut 27: 153. 47. SOLLID, L. M., G. GAUDERNACK, G. MARKUSSEN, D. KVALE, P. BRANDTZAEG, and E. THORSBY. 1987. Induction of various HLA class II molecules in a human colonic adenocarcinoma cell line. Scand. J. Immunol. 25: 175. 48. MADARA, J. L., and J. STAFFORD. 1989. Interferon-y directly affects barrier function of cultured intestinal epithelial monolayers. J. Clin. Invest. 83: 724. 49. OTIAWAY, C. A., D. L. LEWIS, and S. L. ASA. 1987. Vasoactive intestinal peptidecontaining nerves in Peyer's patches. Brain Behav. Immunol. 1: 148. 50. Yu, c.-L., D. O. HASKARD, D. CAVENDER, A. R. JOHNSON, and M. ZIFF. 1985. Human gamma interferon increases the binding of T lymphocytes to endothelial cells. Clin. expo Immunol. 62: 554. Dr. MICHELA MUSCETIOLA, Institute of General Physiology, University of Siena, Via del Laterino 8, 53100 Siena, Italy
Somatostatin and Vasoactive Intestinal Peptide Reduce Interferon Gamma Production by Human Peripheral Blood Mononuclear Cells MICHELA MUSCE1TOLA 1 and GIOVANNI GRASS02 Received May 30, 1989 . Accepted in Revised Form February 9, 1990
Abstract There is increasing evidence that neuropeptide modulation of the immune response is an important physiological phenomenon which involves the interaction of peptidergic neuromodulators with specific neuropeptide receptors on the plasma membrane of immune effector cells. Many studies have examined the effect of neuropeptides on mitogen-induced lymphocyte proliferation and immunoglobulin synthesis but very little is known about specific lymphokine production. In this study, we describe the effect of somatostatin (SOM) and vasoactive intestinal peptide (VIP) on interferon gamma (IFN-y) production by normal human peripheral blood mononuclear cells (PBMC) stimulated in vitro with polyclonal T cell activator staphylococcal enterotoxin A (SEA). Our findings provide experimental evidence that both SOM and VIP reduce the IFN-y production by SEA-stimulated PBMe. This reduction was time- (with maximal effect at 72h) and dose-dependent (at doses as low as 10-11 M with maximal effect at concentrations between 10-9 and 10-8M of neuropeptides). This effect was absent in resting PBMe. The meaning of inhibitory effect of VIP and SOM on IFNy production and its role in immune response in vivo are discussed.
Introduction
The involvement of the nervous system in the direct and indirect regulation and modulation of immune response is now well recognized (1-4). Neuroendocrine and immune systems constitute a totally integrated circuit and share a common set of hormones, neurotransmitters and polypeptides, and their receptors (5, 6). Many newly identified neurotransmitters and neuromodulators are neuropeptides which produce receptor-mediated Abbreviations: SOM = somatostatin; VIP = vasoactive intestinal peptide; IFN-y = interferon gamma; PBMC = peripheral blood mononuclear cells; SEA = staphylococcal enterotoxin A; WISH = human amnionic cells; VSV = vesicular stomatitis virus; IL2 = interleukin-2;PFU = plaque-forming units; LGL = large granular lymphocytes; PBS = phosphate-buffered saline; EMEM = Eagle's Minimum Essential Medium. Part of this work was presented at the Annual Meeting of the International Society for Interferon Research, Kyoto, Japan, November 14-18, 1988.
420 . MICHELA MUSCETTOLA and GIOVANNI GRASSO
behavioral effects (7). In recent years, in vitro and in vivo studies have shown that neuropeptides, including vasoactive intestinal polypeptide (VIP), substance P and somatostatin (SOM), can modulate lymphocyte function (8-16). These studies have examined the effect of neuropeptides on mitogen-induced lymphocyte proliferation and immunoglobulin synthesis but very little is known about specific lymphokine production (17, 18). Because lymphokines (nonimmunoglobulin glycoproteins) are central in the regulation and modulation of immune and inflammatory responses, the possible effect of neuropeptides on lymphokine production is of interest. In this study we describe the inhibitory role of somatostatin (SOM) and vasoactive intestinal peptide (VIP) on interferon gamma (IFN-y) production by normal human peripheral blood mononuclear cells (PBMC) stimulated in vitro with polyclonal T cell activator staphylococcal enterotoxin A
(SEA).
Materials and Methods Chemicals Pure somatostatinl_14 and VIP I _28 (purchased from Boehringer and Sigma respectively) were initially dissolved in phosphate-buffered saline (PBS, pH 7.4) to 10-4 M and kept at -80 DC for up to two weeks. They were freshly diluted in RPMI-1640 medium before each experiment. Lymphocyte isolation PBMC were isolated from heparinized buffy coats of ten healthy donors (age range 28-45 years) by Ficoll-Paque (Pharmacia, Fine Chemicals) gradient centrifugation (19). The same donors were used for both neuropeptides. Blood samples were taken at 8 AM. The PBMC in the interface were washed twice with PBS and once with RPMI-1640 (Gibco) containing 20 mM L-glutamine (Gibco), 10 mM Hepes (Gibco) and 1 % penicillin-streptomycin solution (Labtek, Eurobio). Finally the cells were suspended in RPMI-1640 supplemented with 8 % heat-inactivated fetal-calf serum (Flow Laboratories). Cell numbers were determined by lightmicroscope count and viability was assayed by the trypan-blue dye exclusion technique. The technique routinely resulted in cell populations consisting of approximately 90 % lymphocytes and 10 % monocytes. Culture conditions and cell responsiveness The incubation was carried out in quadruplicate in 48-well plates (Costar) at 37 DC in a humidified atmosphere of 5 % CO 2 : 95 % air; each well contained 5 x 105 cells/0.5 m!. Cell responsiveness was checked by means of the potent mitogen staphylococcal enterotoxin A (Toxin Technology, Inc.) (20, 21) added at a dose of 0.1 [!g/106 cells at the start of the culture. Each plate contained unstimulated and stimulated cells with or without increasing concentrations of neuropeptides (10-12 M to 10-6 M) added at the beginning of the culture. At the end of incubation, the cultures were collected and centrifuged at 2,000 x g for 15 min at 4 DC. The supernatants were stored at -20 DC until IFN-y assay. Interferon assay The microplaque reduction assay (22) was used throughout for the titration of IFN activity using human amnionic cells (WISH) as indicator cells and vesicular stomatitis virus (VSV, Indiana strain) as challenge virus.
Neuropeptides and IFN-y production· 421 Briefly, two-fold dilutions of samples in Eagle's Minimum Essential Medium (EMEM, Gibco) supplemented with 2 % heat~inactivated fetal calf ,syr~m . (Flow Laboratories) were added to cell monolayers in flat-boti:om tnicrotiter wells (Costar): After 16-18 h incubation, the medium was removed by inversion and the monolayers carefully washed three times with EMEM without serum to remove interfering substances such as lipoproteins and immunoglobulins. The monolayers were then infected with about 60 plaque-forming units (PFU) of VSV in 1 % fetal-calf serum-EMEM. The samples were tested at least in quadruplicate and twice. The highest dilution of a sample causing a 50 % reduction in virus microplaque was determined to be the endpoint, and the reciprocal of this dilution was recorded as the IFN titer. Each assay included a human IFN-y WHO International standard (obtained from NIH, Bethesda, MD, USA) to check the sensitivity and reproducibility of our test system according to the Expert Committee on Biological Standardization of Interferons (23). The IFN activity was expressed in International Units (IU) and the results are presented as IFN-y production per 106 cells (IU/l0 6 cells) and percentage change in IFN-y production calculated as follows: IFN -y produced in the presence of neuropeptide IFN-y produced in the absence of neuropeptide IFN -y produced in the absence of neuropeptide
x 100
Statistical analysis The data were processed by one-way analysis of variance (ANOVA) for repeated samples.
Results
The cell viability with all the concentrations of SOM and VIP tested was not significantly different from that of control cultures as quantified by similar uptake of about 4 % of the trypan blue after 72 h culture. The geometric mean titer of the human IFN-y NIH reference preparation (Gg23-901-530) with a defined potency of 3.61 Log lO IV/vial when reconstituted in 1 ml of sterile distilled water, in our laboratory conditions, was 3.4834 Log lO IV/ml (with a standard deviation of 0.2128 Log lO corresponding to about a 1.6322-fold variation; total number of titrations = 25). The antiviral activity induced by SEA was characterized as IFN-y (20, 24). This IFN was labile at pH 2, neutralized by anti-IFN-y antibodies (monoclonal antibody B3, specific for human IFN-y, kindly provided by Professor Jan Vilcek) and was not affected by antibodies to human IFN-a or B(Boehringer Biochemia Robin). SEA-unstimulated cells produced only negligible amounts of IFN-y (16 ± 4.7 IV/106 cells). On the other hand, when neuropeptides alone were added to PBMC cultures, no effect on IFN-y production was observed. Maximal IFN-y levels were found 72h after stimulation with SEA (Figs. 2, 4) and therefore, in our experiments on dose response, the effect of neuropeptides on IFN-y production was assessed after 3 days of exposure. It has been demonstrated that the levels of SOM in the T-Iymphocyte cultures diminished as a function of time and after 24 hover 95 % of SOM was degraded (10). Furthermore, in the medium of human transformed
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colonic epithelial cells, after 20 min there were no more intact VIP molecules (25). Nevertheless, we determined what effect if any, neuropeptides had on the IFN assay. We found that over a wide range of concentrations the neuropeptides. 1) were not toxic to WISH cells, 2) did not protect Table 1. Range (expressed in %) of inhibition of IFN-y production by SEA-stimulated peripheral blood mononuclear cells in presence of increasing concentrations of SOM Stimulus
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Neuropeptides and IFN-y production . 423
cells from viral plaque formation and 3) did not inhibit the antiviral activity of IFN (data not shown).
Effect of SOM on IFN-y production by SEA -stimulated PBMC IFN-y production by SEA-stimulated peripheral blood mononuclear cells with or without increasing concentrations of SOM is shown in Figure 1. The inhibitory effect of SOM on IFN-y production occurred at all doses and was maximal at concentrations between 10-9 M and 10-8 M. The subjects examined had different ranges of IFN-y production by SEA-stimulated PBMC in the absence or presence of SOM; the ranges of percentage inhibition of IFN-y production at all concentrations tested are presented in Table 1. The time course of IFN-y production by SEA-stimulated PBMC, cultured in the absence or presence of 10-9 M SOM, and its percentage inhibition are shown in Figure 2.
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shown in Figure 3. The inhibitory effect of VIP on IFN-y production 'occurred at all doses and was maximal at concentrations between 10-9 M and 10-8 M. The subjects examined had different ranges of IFN-y production by SEA-stimulated PBMC in the absence or presence of VIP; the ranges of percentage inhibition IFN-y production at all concentrations tested are Table 2. Range (expressed in %) of inhibition of IFN-y production by SEA-stimulated peripheral blood mononuclear cells in presence of increasing concentrations of VIP Stimulus
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presented in Table 2. The time course of IFN-y production by SEAstimulated PBMC, cultured in the absence or presence of 10-9 M VIP, and its percentage inhibition are shown in Figure 4.
Discussion N europeptides, known to be released from peripheral nerve varicosities and produced by neuroendocrine cells, have also been localized in cells of the immune system (for review see 12). There is increasing evidence that neuropeptide modulation of the immune response is an important physiological phenomenon which involves the interaction of peptidergic neuromodulators with specific neuropeptide receptors on the plasma membrane of immune effector cells (for review see 6, 26). Specific binding sites for VIP (8, 27, 28) and SOM (29) have been detected on normal human peripheral blood lymphocytes suggesting that they play a role in lympho-
426 .
MICHELA MUSCETTOLA
and
GIOVANNI GRASSO
cyte function. It has been demonstrated that SOM is a potent inhibitor of the proliferation of PHA-stimulated human peripheral T lymphocytes and Molt-4 lymphoblasts (10), and mouse spleen lymphocytes (13). VIP furthermore inhibits the proliferative response of PHA - and Con A -stimulated murine lymphocytes from secondary lymphoid tissues (30, 31). Moreover, both SOM and VIP modulate immunoglobulin synthesis in murine lymphocytes from spleen, mesenteric lymph nodes and Peyer's patches stimulated with Con A. This modulation is neuropeptide-, organ-, and isotypespecific (31). The precise role of such neuropeptides in regulating immune function is still unclear and no information exists about their possible effect on IFN-y production. It is known that IFN-y is a lymphokine produced by T lymphocytes after mitogenic or antigenic stimulation, and that it plays a crucial role in the modulation of a variety of immune functions (for review see 32). IFN-y secretion appears to be a better indicator of specific T cell response than eH] thymidine uptake (21); cell proliferation is not a necessary prerequisite for IFN-y production (33-36). Accordingly, we decided to investigate the effect of SOM and VIP on IFN-y production by normal human peripheral blood mononuclear cells. Our findings provide experimental evidence that both neuropeptides reduce the IFN-y production by SEA-stimulated PBMC in a time- and dose-dependent manner. This effect was absent in resting PBMC. The suppressive effect of VIP and SOM was also observed with Con A and PHA (data not shown), but the great difference between these mitogens and SEA as regards the level of IFN-y produced prompted us to perform our experiments with SEA. That the inhibitory effects of SOM and VIP are not due to a direct cytotoxic effect on lymphocyte function was demonstrated by the similar exclusion of trypan-blue dye under all culture conditions. Because of well known great interindividual variation in IFN-y production by SEA-stimulated PBMC, the subjects examined and the time of blood collection were the same for both neuropeptides. The inhibitory effect of both neuropeptides on IFN -y production can be detected just after 12 h of culture and reaches a maximum at 72 h, which is the time with maximal IFN -y levels induced by SEA, and then decreases. The suppressive effect of VIP and SOM can be detected even at concentrations as low as 10-12_10-11 M with maximal reduction at concentrations between 10-9-10-8 M, but at higher neuropeptide concentrations (10-7-10-6 M) there is a reversal of inhibition of IFN -y production. This loss of response to neuropeptide pharmacological concentrations could be related to the down regulation of the cell surface receptors. In fact it has been demonstrated that 10-7 M VIP treatment of T cells resulted in a dosedependent loss of the ability of T cells to specifically bind radio iodinated VIP (37). This altered binding of the treated cells was observed in different biological and physiological models and was due to destruction of part of the receptors into the prelysosomal and lysosomal compartments (25, 38).
Neuropeptides and IFN-y production· 427
Because peripheral blood concentrations of VIP and SOM are 10-12 M whereas at nerve endings in the intestine they are 10-7M (39), the effect of neuropeptides on lymphokine production by activated lymphocytes obtained from spleen and lymphoid tissues is of interest. Since organspecific effects of neuropeptide have been reported (31), we are currently investigating the effect of SOM and VIP on IFN-y production by murine lymphocytes from Peyer's patches. Whether or not the inhibitory effects of SOM and VIP on IFN~y production are direct effects or the result of the inhibition of interleukin-2 (IL 2) production is presently under investigation. However, recent studies on murine lymphocytes (40) and our preliminary results on human lymphocytes (data not shown) suggest that the inhibitory effect of VIP on IFNy production is due to a decreased production of IL2 by CD4+ Tcells. However, the experimental system used by us contained a multitude of immunocompetent cell populations because the binding of SEA to the accessory cells (monocytes and B cells) is an absolute requirement for T cell activation and subsequent IFN-y production (41). Since high-affinity specific receptors for VIP have been identified on CD3+ T cells, CD8+ T cells, CD4+ T cells, LGL and B cells (42), the inhibitory effect of VIP on IFN-y production could be explicable also by its direct effect on suppressor and LGL cells. The results of the present Study raise the question of the meaning of the inhibitory effects of VIP and SOM on IFN-y production and of its role in immune response in vivo. It is known that human intestinal mucosal mononuclear cells produce IFN-y (43) and surface receptors for this lymphokine are also found on epithelial cells (44). HLA-DR antigens are also found on enterocytes of the human small intestine (45, 46). Since IFNy enhances HLA-DR antigen expression in a human colonic adenocarcinoma cell line (47) and affects the barrier function of cultured intestinal epithelial monolayers (48), SOM and VIP may modulate intestinal immunophysiology by regulating IFN-y production. Finally, it has been clearly established that the traffic of lymphocytes is of considerable importance to the initiation and propagation of the immune response. The lymphoid tissues associated with mucosal membranes are major sites of lymphoid cell migration. Moreover, the adherence of lymphocytes to the endothelial cells is an essential step for their emigration from the blood into lymphoid tissue. Exposure of T cells to VIP, at concentrations that downregulate the VIP receptors, resulted in a decrease in their ability to migrate in Peyer's patches and mesenteric lymph nodes of recipient animals (37). Furthermore, the presence of VIB-ergic nerve fibers in very close proximity to the specialized postcapillary venules of these lymphoid tissues (49) suggests that VIP alters the capacity of lymphocytes to bind to the high endothelial cells. The demonstration that IFN -y increases the binding of T lymphocytes to endothelial cells (50) and the results presented here suggest
428 . MICHELA MUSCETTOLA and GIOVANNI GRASSO
that VIP can indirectly affect the traffic of lymphocytes via the reduction of IFN -y production. In conclusion, the present findings further emphasize the interdependence of the immune and nervous systems. Acknowledgements This study was financed by a grant (60 %) from the Ministry of Education, Rome. The authors are grateful to Professor JAN VILCEK (New York) for the generous gift of monoclonal antibodies to human IFN-y and to Mrs. ANNA ROMEI for skilful secretarial assistance.
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