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Enhancing effect of corticotropin-releasing neurohormone on the production of interleukin-1 and interleukin-2
Neuroscience Letters, 120 (1990) 151-154
151
Elsevier Scientific Publishers Ireland Ltd. NSL 07340
Enhancing effect of corticotropin-releasing neurohormone on the production of interleukin-1 and interleukin-2 Vijendra K. Singh and Sy-Jye Christine Leu Department of Biology, Biomedical Division, Developmental Center for Handicapped Persons, Utah State University, Logan, UT 84322-6800 (U.S.A.) (Received 7 July 1990; Revised version received 6 August 1990; Accepted l0 August 1990)
Key words: Corticotropin-releasing factor; Interleukin production; Neuroimmunomodulation Based upon an immunomodulatory role for Corticotropin-Releasing Factor (CRF), a low molecular weight neurohormone, we investigated the effect of CRF on the production of interleukin-I (IL-I) and interleukin-2 (IL-2) activities of mononuclear cells isolated from the peripheral blood of healthy subjects. The production of both IL-1 and IL-2 was stimulated by a nanomolar concentration of CRF by itself. In addition, CRF augmented the production of IL- 1 as induced by lipopolysaeeharide and the production of IL-2 as induced by phytohemagglutinin. These results suggest that CRF modulates the function of the cells of the immune system presumably by acting as a blood-borne mediator of the neuroendocrine-immune pathways.
It is now generally accepted that a unique structural and functional relationship exists between the immune system and the central nervous system. Accordingly, the cells of the immune system modify the function of the cells of the CNS, and vice versa, the cells of the CNS can modulate the function of the immune system [8]. In this respect, we recently hypothesized that the corticotropinreleasing factor (CRF) is a modulator of immune functions [9, I1]. CRF is a low molecular weight peptide neurohormone of the hypothalamus [12] and a putative neurotransmitter in the CNS [4]. One of the important biological functions of CRF is to induce the release of adrenocorticotropin hormone (ACTH) and fl-endorphin from the pituitary gland [13], which occurs via specific receptors for CRF [2, 15]. The receptor sites of CRF have also been found on human blood lymphocytes and monocytes [10] and mouse splenic macrophages [14]. Moreover, CRF can induce the release of immunoreactive fl-endorphin from human peripheral blood mononuclear cells [5]. Our current research showed that the CRF stimulates two immune functions in vitro, namely, the proliferation of human lymphocytes and the expression of interleukin-2-reeeptor (IL-2R) antigen on human T cells [9, 11]. The objective of the present study was to investigate the effect of CRF on the production of interleukin-1 (IL-l) and interleukin-2 (IL-2), and as described
Correspondence: V. K. Singh, Utah State University, UMC 6800, Logan, UT 84322, U.S.A. 0304-3940/90/$ 03.50 O 1990 Elsevier Scientific Publishers Ireland Ltd.
in this report, the production of both of these soluble mediators of immunity is stimulated by CRF. Venous blood was drawn by venipuncture into a syringe containing approximately 20 units/ml of sodium heparin (Sigma). The blood donors were all healthy adult humans in the age range of 21-40 years. None of them was taking any prescription medication or showed any evidence of a clinical disease. For the separation of mononuclear cells (MNC), heparinized blood was subjected to a density-gradient centrifugation method in the Histopaque solution (Sigma) as described elsewhere [9]. The final MNC pellet was resuspended at a concentration of 10 x 106 cells/ml of complete growth medium which contained RPMI-1640 with L-glutamine (Gibco), streptomycin-penicillin mixture (100 units/ml of each, Gibco) and 10% fetal bovine serum (Hyclone Labs., Logan, UT). Monocytes were purified by adherance of MNC to the plastic surface as described previously [9] and they appeared to be nearly 99% pure as judged by the staining of monoclonal antibody to Mo2 antigen, a specific marker of human monocytes (Coulter Immunology, Hialeah, FL). The cell viability was > 98%. Replicative experiments were performed using up to 9 different blood donors. For the assay of IL-1 activity, monocytes were first stimulated with CRF in the absence or presence of lipopolysaccharide (LPS) to produce IL-1 and then the cellfree supernatant was assayed for its activity in the murine thymocyte proliferation assay. For the production of IL-1, monocytes were preseparated from 5 x l0 6 MNC/
152
ml as mentioned above [9] and cultured in the presence of various concentrations of CRF (Sigma, code C2024) for 24 h in a 37°C incubator humified with 5% CO2. The concentration of LPS (Sigma) was 20/zg/ml whenever used as the standard stimulator of IL-I. The cell cultures were centrifuged at 1500 rpm for 10 min and the supernatants were stored frozen at -20°C usually for 3-4 days before their use in the thymocyte proliferation assay. The source of thymocytes was thymus surgically removed from C57/BL6 mice under CO2-gas anesthesia. In the thymocyte proliferation assay, as adapted from the report of Gillis and Mizel [3], 0.1 ml of a 1:4 dilution
of various culture supernatants was treated with 1.5x 10 6 thymocytes in 0.1 ml of complete growth medium containing a 2% concentration of 2-mercaptoethanol and a 1% concentration of phytohemagglutinin (PHA) as the co-stimulant. This reaction was set up in triplicate wells of a flat-bottomed 96-well microculture plate (Corning). After 72 h of incubation, the thymocytes were labelled for 24 h with 0.4 /tCi of [methyl3H]thymidine (spec. act. 2 Ci/mmol, purchased from NEN) and harvested using a semi-automatic cell harvester (Flow Labs.). Thereafter, the filter paper discs were transferred into counting vials, mixed with 2 ml of 2.0"
[A]
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Concentration o f CRF [nMl 260"
75
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Fig. 1. Effect of CRF on the production of IL-1 and IL-2. For IL- 1 production, various concentrations of CRF were added to the cultures of monocytes preseparated from 5 x 106 MNC/ml and the microculture plate was allowed to incubate for 24 h. The cultures were centrifuged at 1500 rpm/10 min, supernatant diluted 1:4 and tested for IL-1 activity in the murine thymocyte proliferation assay. The IL-I activity is given as the units/ml (mean+S.E.) of supernatant of monocyte cultures derived from 1 x 106 MNC in the absence of any mitogen [A] ( n = 9 blood donors) or in the presence of 20 gg/ml LPS [C] (n = 5 blood donors). For IL-2 production, 2 × 106 MNC/ml were cultured in the presence of various concentrations of CRF for 48 h, centrifuged at 1500 rpm/10 min and the supernatant assayed undiluted for IL-2 activity as measured in the 24-h proliferation assay of HT-2 cells. The IL-2 activity is given as the units/ml (mean + S.E.) of supernatant derived from 1 × 106 MNC in the absence of any mitogen [B] (n = 5 donors) or in the presence of 1% PHA [D] (n = 3 donors).
153 Ready Safe scintillation fluid (Beckman) and samples counted for radioactivity using a liquid scintillation counter (Packard Model Tri-Carb 1500). The blank activity (without any IL-1 containing sample) was subtracted from the test activity (samples containing IL-1) and the difference between the two was taken as the indicator of IL-1 activity. In parallel, a standard of recombinant IL-1 (Cistron Biotechnology, Pine Brook, N J) was used to calculate the units of IL-1 activity. The assay for IL-2 activity involved first the stimulation of MNC by C R F to produce IL-2 followed by the determination of activity in the IL-2 dependent proliferation of HT-2 cells. For IL-2 production, 2 x 10 6 MNC/ ml were cultured with various concentrations of CRF for 48 h in a 37°C incubator humified with 5% CO2. The concentration of PHA was 1% whenever used as the standard stimulator of IL-2. The tubes were centrifuged at 1500 rpm for 10 min to harvest the cell-free supernatants which were stored frozen at - 2 0 ° C usually for 3 to 4 days before the assay of IL-2 activity. The IL-2 activity was assayed by a modification of the method of Noble and Warren [6]. For this purpose, HT2 cells (4,000 cells) in 0.1 ml were cultured with 0.1 ml of undiluted culture supernatants. This reaction was set up in triplicate wells of a fiat-bottomed microculture plate containing 96 wells (Coming). After 24 h of incubation inside the humidified chamber of a 37°C/5% CO zincubator, the cells were labelled for 4 h with 0.4/tCi of [methyl-3H]thymidine (spec. act. 2 Ci/mmol, purchased from NEN). The cells were harvested and processed for the counting of radioactivity in the same fashion as described above for the assay of IL-1 activity. The blank activity of HT-2 cells (without any IL-2 containing sample) was subtracted from the test activity (samples containing IL-2) and the difference between the two was taken as the indicator of IL-2 activity. Simultaneously, a standard of recombinant IL-2 (Genzyme Corp., Boston, MA) was used as the positive control and the units of IL-2 activity were calculated in reference to this standard. The effect of C R F on the production of IL-1 (24 h) and IL-2 (48 h) is shown in Fig. 1A-D. The production of both of these interleukins was also studied at different time intervals: 1, 3, 6, 12, 24 and 48 h of culture incubation. From this kinetic study, the greatest production of IL-1 and IL-2 was found at 24 h and 48 h, respectively and, thus, these two intervals were chosen in the experiments reported herein. The C R F (0.1 nM), by itself caused about an 8-fold increase in the production of ILl activity of isolated human monocytes (Fig. 1A). In the presence of LPS as the standard stimulator of IL-1, CRF caused a further augmentation of the production of this interleukin (Fig. 1C). The concentration of CRF
required to yield a maximum stimulation was approximately 0.1 nanomolar. The C R F (1 nM), by itself caused about 2-fold increase in the production of IL-2 activity (Fig. 1B). In the presence of PHA as the standard stimulator of IL-2 production, the CRF was able to manifest an additional increase in the production of IL-2 activity (Fig. 1D). On average, the maximum effect of C R F on IL-2 production was seen at a 1 nM concentration of CRF. As tested in separate experiments (Fig. 2), C R F did not have any effect on the proliferation of mouse thymocytes or HT-2 cells used as inducer cells in the bioassay of IL-1 and IL-2, respectively. Both the IL-1 and IL-2 data varied from one donor to another but this variability is frequently observed in the bioassays used, e.g., all of the 9 donors responded to CRF-induced stimulation of IL-1 activity but each to a different degree. Moreover, the effect of C R F on IL-1 production was a much greater effect than the effect on IL-2 production. Also, it should be pointed out that the enhancing effect of C R F was more pronounced in the absence of mitogens (Fig. 1A, B) than in the presence of mitogens, e.g., LPS for IL-1 (Fig. 1C) and PHA for IL-2 (Fig. 1D): note that the greatest stimulation was 797% ( - L P S ) and 123% ( + L P S ) for IL-I and 190% ( - P H A ) and 127% ( + P H A ) for IL-2. Thus, the CRF-induced baseline production of IL-1 and IL-2 is potentiated more than the mitogenic production, suggesting that CRF by itself is an inducer of IL-1 and IL-2 activity, and this property of CRF may be biologically important in the modulation of immune responses.
100000' Mouse thymocytes
I00(~ '
1000 "~ HT-2 cells
100 0.0
!
I
u
0.001
0.1
10
Concentrationof CRF [nM] Fig. 2. Effect of CRF on the proliferationof mouse thymocytesand HT-2-cells. Mouse thymocytesand HT-2 cells were incubated in the presence of various concentrationsof CRF, but in the absenceof IL-1and IL-2-containingculture supernatants, as describedin the Methods section.
154 property o f C R F m a y be biologically important in the modulation o f immune response. The aforementioned data clearly demonstrated that the C R F , by itself, causes an increase in the production o f both IL-1 and IL-2 activities. Moreover, it can potentiate the response o f certain standard stimulators, e.g., LPS for IL-1 and P H A for IL-2. These data also correlated with the l z s I - C R F binding data o f h u m a n M N C . As reported elsewhere [10], the binding o f ~25I-CRF to m o n o c y t e s (IL-1 producing cells) was about 3 times greater than the binding to T cells (IL-2 producing cells). Based u p o n these circumstantial results, we suggest that C R F receptors are functionally coupled to immunological activities o f m o n o c y t e s and lymphocytes. C R F is a well-known peptide h o r m o n e o f the neuroendocrine system [12]. It is p r o d u c e d by hypothalamus, the site o f homeostasis, and it acts on the pituitary gland regulating the synthesis and secretion o f A C T H and flendorphins [12, 13]. There is also some evidence that C R F acts as a neurotransmitter in the C N S [4]. Moreover, C R F can cause the secretion o f fl-endorphin from h u m a n blood m o n o n u c l e a r cells and this action apparently involves IL-1 since IL-1 can be substituted for C R F and antibodies to IL-1 can block this action [5], however, the actual production o f IL-1 was not measured in this system. As reported herein, our experiments clearly demonstrated that one o f the immune effects o f C R F is to cause stimulation o f IL- 1 production. Using h u m a n blood m o n o n u c l e a r cells, we recently reported that the C R F stimulates two immune functions: (i) proliferation o f lymphocytes both in the absence and presence o f T cell mitogens, and (ii) the expression of IL2R antigen on T cells [9, 11]. We have also shown the presence o f binding sites for azsI-labelled C R F on h u m a n blood monocytes, T and B lymphocytes and thymus [10]. As reported in this paper, we demonstrated that the C R F is also a stimulator o f production of two interleukins, namely, IL-I and IL-2. It should also be noted that the concentration o f C R F required to manifest immunostimulation in vitro was in the n a n o m o l a r range (0.1 to 1), which is a very low concentration expected to exist in the tissues. IL-I has recently been shown to stimulate the secretion o f C R F by hypothalamic ceils [1, 7] and C R F stimulates the production o f IL- 1 and IL-2 (present data), suggesting the existence o f a complete circuitry between the neuroendocrine system and the immune system. Based u p o n the i m m u n o s t i m u l a t o r y properties o f C R F , we suggest that C R F plays an i m p o r t a n t role in
the m o d u l a t i o n o f the neuroendocrine-immune axis, presumably by acting as a soluble mediator o f C N S control o f the immune system. This research was supported by a U S U faculty G r a n t SB1196. 1 Berkenbosch, F., van Oers, J., del Rey, A., Tilders, F. and Besedovsky, H., Corticotropin-Releasing Factor-producing neurons in the rat activated by interleukin-1, Science, 238 (1987) 524-526. 2 De Souza, E.B., Perrin, M.H., Insel, T.R., Rivier, J., Vale, W.W. and Kuhar, M., Corticotropin-releasing factor in rat forebrain: autoradiographic identification, Science, 224 (1984) 1449-1451. 3 Gillis, S. and Mizel, S.B, T-cell lymphoma model for the analysis of interleukin l-mediated T-cell activation, Proc. Natl. Acad. Sci. U.S.A., 78 (1981) 1133 1137. 4 Joseph, S.A., Pilcher, W.H. and Knigge, K.M., Anatomy of the corticotropin-releasing factor and opiomelanocortin systems in the brain, Fed. Proc., 44 (1985) 100-107. 5 Kavelaars, A., Ballieux, R.E. and Heijnen, C.J., The role of IL-I in the corticotropin-releasing factor and arginine-vasopressininduced secretion of immunoreactive fl-endorphin by human peripheral blood mononuclear cells, J. Immunol., 142 (1989) 23382342. 6 Noble, R.L. and Warren, R.P., Altered T cell subsets and defective T-cell function in young children with Down syndrome (trisomy21), Immunol. Invest., 16 (1987) 371 382. 7 Sapolsky, R., Rivier, C., Yamamoto, G., Plotsky, P. and Vale, W., Interleukin-I stimulates the secretion of hypothalamic corticotropin-releasing factor, Science, 238 (1987) 522-524. 8 Singh, V.K., Neuroimmune axis as a basis of therapy in Alzheimer's disease, Prog. Drug Res., 34 (1990) 383-393. 9 Singh, V.K., Stimulatory effect of corticotropin-releasing neurohormone on human lymphocyte proliferation and interleukin-2 receptor expression, J. Neuroimmunol., 23 (1989) 257-262. 10 Singh, V.K. and Fudenberg, H.H., Binding of [~251]corticotropinreleasing factor to blood immunocytes and its reduction in Alzheimer's disease, Immunol. Lett., 18 (1988) 5-8. 11 Singh, V.K., Warren, R.P., White, E.D. and Leu, S.J.C., Corticotropin-releasing factor-induced stimulation of immune functions. Ann. N.Y. Acad. Sci., 594 (1990) 416--419. 12 Vale, W.W., Spies, J., Rivier, C. and Rivier, J., Characterization of a 41-residue ovine hypothalamic peptide that stimulates secretion of corticotropin anbd beta-endorphin, Science, 213 (1981) 1394-1397. 13 Vale, W.W., Rivier, C., Brown, M.R., Spies, J., Koob, J., Swanson, L., Blizikijan, E., Bloom, F. and Rivier, E., Chemical and biological characterization of corticotropin releasing factor, Rec. Prog. Horm. Res., 39 (1983) 245-269. 14 Webster, E.L. and DeSouza, E.B., Corticotropin-releasing factor receptors in mouse spleen: identification, autoradiographic localization and regulation by divalent cations and guanine nucleotides, Endocrinology, 122 (1983) 609~617. 15 Wynn, P.C., Aguilera, C., Morell, J. and Can, K.J., Properties and regulation of high-affinity pituitary receptors for corticotropinreleasing factor, Biochem. Biophys. Res. Commun., 110 (1983) 602 -608.
151
Elsevier Scientific Publishers Ireland Ltd. NSL 07340
Enhancing effect of corticotropin-releasing neurohormone on the production of interleukin-1 and interleukin-2 Vijendra K. Singh and Sy-Jye Christine Leu Department of Biology, Biomedical Division, Developmental Center for Handicapped Persons, Utah State University, Logan, UT 84322-6800 (U.S.A.) (Received 7 July 1990; Revised version received 6 August 1990; Accepted l0 August 1990)
Key words: Corticotropin-releasing factor; Interleukin production; Neuroimmunomodulation Based upon an immunomodulatory role for Corticotropin-Releasing Factor (CRF), a low molecular weight neurohormone, we investigated the effect of CRF on the production of interleukin-I (IL-I) and interleukin-2 (IL-2) activities of mononuclear cells isolated from the peripheral blood of healthy subjects. The production of both IL-1 and IL-2 was stimulated by a nanomolar concentration of CRF by itself. In addition, CRF augmented the production of IL- 1 as induced by lipopolysaeeharide and the production of IL-2 as induced by phytohemagglutinin. These results suggest that CRF modulates the function of the cells of the immune system presumably by acting as a blood-borne mediator of the neuroendocrine-immune pathways.
It is now generally accepted that a unique structural and functional relationship exists between the immune system and the central nervous system. Accordingly, the cells of the immune system modify the function of the cells of the CNS, and vice versa, the cells of the CNS can modulate the function of the immune system [8]. In this respect, we recently hypothesized that the corticotropinreleasing factor (CRF) is a modulator of immune functions [9, I1]. CRF is a low molecular weight peptide neurohormone of the hypothalamus [12] and a putative neurotransmitter in the CNS [4]. One of the important biological functions of CRF is to induce the release of adrenocorticotropin hormone (ACTH) and fl-endorphin from the pituitary gland [13], which occurs via specific receptors for CRF [2, 15]. The receptor sites of CRF have also been found on human blood lymphocytes and monocytes [10] and mouse splenic macrophages [14]. Moreover, CRF can induce the release of immunoreactive fl-endorphin from human peripheral blood mononuclear cells [5]. Our current research showed that the CRF stimulates two immune functions in vitro, namely, the proliferation of human lymphocytes and the expression of interleukin-2-reeeptor (IL-2R) antigen on human T cells [9, 11]. The objective of the present study was to investigate the effect of CRF on the production of interleukin-1 (IL-l) and interleukin-2 (IL-2), and as described
Correspondence: V. K. Singh, Utah State University, UMC 6800, Logan, UT 84322, U.S.A. 0304-3940/90/$ 03.50 O 1990 Elsevier Scientific Publishers Ireland Ltd.
in this report, the production of both of these soluble mediators of immunity is stimulated by CRF. Venous blood was drawn by venipuncture into a syringe containing approximately 20 units/ml of sodium heparin (Sigma). The blood donors were all healthy adult humans in the age range of 21-40 years. None of them was taking any prescription medication or showed any evidence of a clinical disease. For the separation of mononuclear cells (MNC), heparinized blood was subjected to a density-gradient centrifugation method in the Histopaque solution (Sigma) as described elsewhere [9]. The final MNC pellet was resuspended at a concentration of 10 x 106 cells/ml of complete growth medium which contained RPMI-1640 with L-glutamine (Gibco), streptomycin-penicillin mixture (100 units/ml of each, Gibco) and 10% fetal bovine serum (Hyclone Labs., Logan, UT). Monocytes were purified by adherance of MNC to the plastic surface as described previously [9] and they appeared to be nearly 99% pure as judged by the staining of monoclonal antibody to Mo2 antigen, a specific marker of human monocytes (Coulter Immunology, Hialeah, FL). The cell viability was > 98%. Replicative experiments were performed using up to 9 different blood donors. For the assay of IL-1 activity, monocytes were first stimulated with CRF in the absence or presence of lipopolysaccharide (LPS) to produce IL-1 and then the cellfree supernatant was assayed for its activity in the murine thymocyte proliferation assay. For the production of IL-1, monocytes were preseparated from 5 x l0 6 MNC/
152
ml as mentioned above [9] and cultured in the presence of various concentrations of CRF (Sigma, code C2024) for 24 h in a 37°C incubator humified with 5% CO2. The concentration of LPS (Sigma) was 20/zg/ml whenever used as the standard stimulator of IL-I. The cell cultures were centrifuged at 1500 rpm for 10 min and the supernatants were stored frozen at -20°C usually for 3-4 days before their use in the thymocyte proliferation assay. The source of thymocytes was thymus surgically removed from C57/BL6 mice under CO2-gas anesthesia. In the thymocyte proliferation assay, as adapted from the report of Gillis and Mizel [3], 0.1 ml of a 1:4 dilution
of various culture supernatants was treated with 1.5x 10 6 thymocytes in 0.1 ml of complete growth medium containing a 2% concentration of 2-mercaptoethanol and a 1% concentration of phytohemagglutinin (PHA) as the co-stimulant. This reaction was set up in triplicate wells of a flat-bottomed 96-well microculture plate (Corning). After 72 h of incubation, the thymocytes were labelled for 24 h with 0.4 /tCi of [methyl3H]thymidine (spec. act. 2 Ci/mmol, purchased from NEN) and harvested using a semi-automatic cell harvester (Flow Labs.). Thereafter, the filter paper discs were transferred into counting vials, mixed with 2 ml of 2.0"
[A]
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Concentration of CRF {nMl
Concentration o f CRF [nMl 260"
75
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Fig. 1. Effect of CRF on the production of IL-1 and IL-2. For IL- 1 production, various concentrations of CRF were added to the cultures of monocytes preseparated from 5 x 106 MNC/ml and the microculture plate was allowed to incubate for 24 h. The cultures were centrifuged at 1500 rpm/10 min, supernatant diluted 1:4 and tested for IL-1 activity in the murine thymocyte proliferation assay. The IL-I activity is given as the units/ml (mean+S.E.) of supernatant of monocyte cultures derived from 1 x 106 MNC in the absence of any mitogen [A] ( n = 9 blood donors) or in the presence of 20 gg/ml LPS [C] (n = 5 blood donors). For IL-2 production, 2 × 106 MNC/ml were cultured in the presence of various concentrations of CRF for 48 h, centrifuged at 1500 rpm/10 min and the supernatant assayed undiluted for IL-2 activity as measured in the 24-h proliferation assay of HT-2 cells. The IL-2 activity is given as the units/ml (mean + S.E.) of supernatant derived from 1 × 106 MNC in the absence of any mitogen [B] (n = 5 donors) or in the presence of 1% PHA [D] (n = 3 donors).
153 Ready Safe scintillation fluid (Beckman) and samples counted for radioactivity using a liquid scintillation counter (Packard Model Tri-Carb 1500). The blank activity (without any IL-1 containing sample) was subtracted from the test activity (samples containing IL-1) and the difference between the two was taken as the indicator of IL-1 activity. In parallel, a standard of recombinant IL-1 (Cistron Biotechnology, Pine Brook, N J) was used to calculate the units of IL-1 activity. The assay for IL-2 activity involved first the stimulation of MNC by C R F to produce IL-2 followed by the determination of activity in the IL-2 dependent proliferation of HT-2 cells. For IL-2 production, 2 x 10 6 MNC/ ml were cultured with various concentrations of CRF for 48 h in a 37°C incubator humified with 5% CO2. The concentration of PHA was 1% whenever used as the standard stimulator of IL-2. The tubes were centrifuged at 1500 rpm for 10 min to harvest the cell-free supernatants which were stored frozen at - 2 0 ° C usually for 3 to 4 days before the assay of IL-2 activity. The IL-2 activity was assayed by a modification of the method of Noble and Warren [6]. For this purpose, HT2 cells (4,000 cells) in 0.1 ml were cultured with 0.1 ml of undiluted culture supernatants. This reaction was set up in triplicate wells of a fiat-bottomed microculture plate containing 96 wells (Coming). After 24 h of incubation inside the humidified chamber of a 37°C/5% CO zincubator, the cells were labelled for 4 h with 0.4/tCi of [methyl-3H]thymidine (spec. act. 2 Ci/mmol, purchased from NEN). The cells were harvested and processed for the counting of radioactivity in the same fashion as described above for the assay of IL-1 activity. The blank activity of HT-2 cells (without any IL-2 containing sample) was subtracted from the test activity (samples containing IL-2) and the difference between the two was taken as the indicator of IL-2 activity. Simultaneously, a standard of recombinant IL-2 (Genzyme Corp., Boston, MA) was used as the positive control and the units of IL-2 activity were calculated in reference to this standard. The effect of C R F on the production of IL-1 (24 h) and IL-2 (48 h) is shown in Fig. 1A-D. The production of both of these interleukins was also studied at different time intervals: 1, 3, 6, 12, 24 and 48 h of culture incubation. From this kinetic study, the greatest production of IL-1 and IL-2 was found at 24 h and 48 h, respectively and, thus, these two intervals were chosen in the experiments reported herein. The C R F (0.1 nM), by itself caused about an 8-fold increase in the production of ILl activity of isolated human monocytes (Fig. 1A). In the presence of LPS as the standard stimulator of IL-1, CRF caused a further augmentation of the production of this interleukin (Fig. 1C). The concentration of CRF
required to yield a maximum stimulation was approximately 0.1 nanomolar. The C R F (1 nM), by itself caused about 2-fold increase in the production of IL-2 activity (Fig. 1B). In the presence of PHA as the standard stimulator of IL-2 production, the CRF was able to manifest an additional increase in the production of IL-2 activity (Fig. 1D). On average, the maximum effect of C R F on IL-2 production was seen at a 1 nM concentration of CRF. As tested in separate experiments (Fig. 2), C R F did not have any effect on the proliferation of mouse thymocytes or HT-2 cells used as inducer cells in the bioassay of IL-1 and IL-2, respectively. Both the IL-1 and IL-2 data varied from one donor to another but this variability is frequently observed in the bioassays used, e.g., all of the 9 donors responded to CRF-induced stimulation of IL-1 activity but each to a different degree. Moreover, the effect of C R F on IL-1 production was a much greater effect than the effect on IL-2 production. Also, it should be pointed out that the enhancing effect of C R F was more pronounced in the absence of mitogens (Fig. 1A, B) than in the presence of mitogens, e.g., LPS for IL-1 (Fig. 1C) and PHA for IL-2 (Fig. 1D): note that the greatest stimulation was 797% ( - L P S ) and 123% ( + L P S ) for IL-I and 190% ( - P H A ) and 127% ( + P H A ) for IL-2. Thus, the CRF-induced baseline production of IL-1 and IL-2 is potentiated more than the mitogenic production, suggesting that CRF by itself is an inducer of IL-1 and IL-2 activity, and this property of CRF may be biologically important in the modulation of immune responses.
100000' Mouse thymocytes
I00(~ '
1000 "~ HT-2 cells
100 0.0
!
I
u
0.001
0.1
10
Concentrationof CRF [nM] Fig. 2. Effect of CRF on the proliferationof mouse thymocytesand HT-2-cells. Mouse thymocytesand HT-2 cells were incubated in the presence of various concentrationsof CRF, but in the absenceof IL-1and IL-2-containingculture supernatants, as describedin the Methods section.
154 property o f C R F m a y be biologically important in the modulation o f immune response. The aforementioned data clearly demonstrated that the C R F , by itself, causes an increase in the production o f both IL-1 and IL-2 activities. Moreover, it can potentiate the response o f certain standard stimulators, e.g., LPS for IL-1 and P H A for IL-2. These data also correlated with the l z s I - C R F binding data o f h u m a n M N C . As reported elsewhere [10], the binding o f ~25I-CRF to m o n o c y t e s (IL-1 producing cells) was about 3 times greater than the binding to T cells (IL-2 producing cells). Based u p o n these circumstantial results, we suggest that C R F receptors are functionally coupled to immunological activities o f m o n o c y t e s and lymphocytes. C R F is a well-known peptide h o r m o n e o f the neuroendocrine system [12]. It is p r o d u c e d by hypothalamus, the site o f homeostasis, and it acts on the pituitary gland regulating the synthesis and secretion o f A C T H and flendorphins [12, 13]. There is also some evidence that C R F acts as a neurotransmitter in the C N S [4]. Moreover, C R F can cause the secretion o f fl-endorphin from h u m a n blood m o n o n u c l e a r cells and this action apparently involves IL-1 since IL-1 can be substituted for C R F and antibodies to IL-1 can block this action [5], however, the actual production o f IL-1 was not measured in this system. As reported herein, our experiments clearly demonstrated that one o f the immune effects o f C R F is to cause stimulation o f IL- 1 production. Using h u m a n blood m o n o n u c l e a r cells, we recently reported that the C R F stimulates two immune functions: (i) proliferation o f lymphocytes both in the absence and presence o f T cell mitogens, and (ii) the expression of IL2R antigen on T cells [9, 11]. We have also shown the presence o f binding sites for azsI-labelled C R F on h u m a n blood monocytes, T and B lymphocytes and thymus [10]. As reported in this paper, we demonstrated that the C R F is also a stimulator o f production of two interleukins, namely, IL-I and IL-2. It should also be noted that the concentration o f C R F required to manifest immunostimulation in vitro was in the n a n o m o l a r range (0.1 to 1), which is a very low concentration expected to exist in the tissues. IL-I has recently been shown to stimulate the secretion o f C R F by hypothalamic ceils [1, 7] and C R F stimulates the production o f IL- 1 and IL-2 (present data), suggesting the existence o f a complete circuitry between the neuroendocrine system and the immune system. Based u p o n the i m m u n o s t i m u l a t o r y properties o f C R F , we suggest that C R F plays an i m p o r t a n t role in
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