The kinetics of ACTH expression in rat leukocyte subpopulations

Journal of Neuroimmunology ELSEVIER

Journal of Neuroimmunology 63 (1995) 103- 112

The kinetics of ACTH expression in rat leukocyte subpopulations Paul D. Lyons, J. Edwin Blalock

*

Deparhnent of Physiology and Biophysics, University ofAlabama, BHSB, Room 896, 1918 University Blvd., Birmingham, AL 35294-0005, USA

Received 29 June 1995; revised 16 August 1995; accepted 16 August 1995

Abstract The pro-opiomelanocortin (POMC) gene encodes a family of peptides originally identified in the pituitary gland. An important POMC-derived peptide hormone, corticotropin (ACTH), is also produced by leukocytes and modulates in vitro immune functions. The present investigation was undertaken to determine the kinetics and cellular distribution of ACTH immunoreactivity (ACTH-ir) in vitro in rat splenic leukocyte subpopulations. Cells were cultured with Concanavalin A (ConA), lipopolysaccharide (LPS), or media alone. ACTH-ir was identified with a specific antiserum raised against ACTH l-24. Double indirect-immunofluorescence was done at 0, 21, and 48 h for B, T-helper (Th), and T-cytotoxic (CTL) cells. Initial kinetic studies demonstrated peak ACTH-ir in all cell types at 18-21 h for both ConA and LPS treatments. A few leukocytes (l-2%) expressed ACTH-ir at 0 h and these were found to be macrophages (M0). Lymphocyte ACTH-ir is 0% at 0 h and rises to 90 + 5% and 75 + 6% at 21 h with ConA and LPS, respectively. This sharply contrasts with 9 + 4% of each cell type positive in media alone at 21 h. The percent immunoreactivity among the three lymphocyte subpopulations did not significantly differ at any single treatment at a single time point. However, there were significant differences in the intensity levels among the subpopulations. At 48 h of ConA or LPS treatment only 10 + 4% of B, Th and Tc were positive, while none were positive in media alone. Stimulated peritoneal M0 also increase positivity for ACTH-ir (85 f 5%). These results indicate that rat splenic B, CTL, and Th lymphocytes can be immunologically stimulated to express the peptide hormone ACTH and that basal ACTH expression in macrophages is distinct from that in lymphocytes. Thus, lymphocyte-derived ACTH may be a paracrine or autocrine regulator of immune function. Keywords: ACTH; Lymphocyte subsets; Immunocytochemistry; Macrophage; Mitogen

1. Introduction An animal’s response to infectious or inflammatory stressors has physiological consequences beyond the immune system and includes altered neuronal firing patterns [ 11 and hormone levels [2]. Moreover, emotional and physical stressors are capable of altering the function of the immune system [3-71. A molecular understanding of the conduit for the brain-immune axis came, in part, with the discovery that leukocytes produce neuroendocrine peptide hormones and their receptors [S-lo] and the subsequent identification of cytokines and their receptors in neurons and endocrine cells [ 1 I-131. Collectively, these data support a molecular mechanism of bidirectional communication between the neuroendocrine and immune systems which involves shared receptors and ligands. ACTH was the first neuroendocrine peptide discovered

* Corresponding author. Phone (205) 934 3032; Fax (205) 934 1446. 0165-5728/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0165-5728(95)00133-6

in lymphocytes and macrophages [8,14]. Stimulated leukocytes contain POMC mRNA [I 5-201 and produce immunoreactive (ir) ACTH having sequence identity with pituitary ACTH [16]. The expression of ACTH receptors on leukocytes [9] and their secretion of corticotropin [21] suggest that leukocyte-derived ACTH may mediate autocrine or paracrine functions in the immune system [22]. In order to further characterize the expression of ACTH in immune cells, we determined the kinetics and cellular distribution of ACTH immunoreactivity (ACTH-ir) in vitro in rat splenic leukocyte subpopulations.

2. Materials and methods 2.1. Anti-ACTH

antibody preparation

Synthetic peptides, ACTH l-24 (Sigma) and ACTH 18-39 (Sigma), were coupled to bovine serum albumin (BSA). Peptide l-24 was coupled to carrier molecules

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using the carbodiimide procedure in which carboxyl groups of the peptide are linked to amino groups of BSA [23]. Peptide 18-39 was coupled to BSA using glutaraldehyde. Each conjugate was dialyzed against phosphate-buffered saline (PBS), pH 7.4, before use. Emulsions were made with peptide conjugates and Freund adjuvant (FA) mixed 1:l. Preimmune serum samples were drawn from male, New Zealand white rabbits (6 months old) prior to immunization. Complete FA emulsions were injected intradermally (i.d.) with 50 pg antigen per site in 100 ~1. 1.0 mg of each antigen was used in primary immunizations. Subsequent (i.d.) boosts with 0.4 mg antigen were similarly given every 4 weeks in Incomplete FA. A total of four injections were made. Blood samples were drawn in lo14day intervals after the first boost. Serum was isolated from clotted blood. Antisera titers were monitored by ELISA with the appropriate solid-phase bound antigen. Once high titers developed, the animals were deeply anesthetized with ketamine and xylazine (40 and 10 mg/kg, respectively) and were exsanguinated by cardiac puncture. Filtered sterilized serum was stored at -70°C. 2.2. ACTH ELlSA Antisera titers were determined with enzyme-linked immunosorbent assays (ELISA). ACTH l-24 or 18-39 were diluted to 1 pg/ml in 0.1 M bicarbonate/carbonate buffer, pH 9.6. Modified-polystyrene 96-well microtiter plates (Nunc) were coated over night at 4°C with 100 ~1 of the diluted peptide per well. The plates were washed three times with PBS, pH 7.4. Nonspecific binding sites were blocked by a 1 h incubation at room temperature with 1.0% w/v BSA solution in PBS/3 mM NaN,, The plates were washed with PBS, pH 7.4 containing 0.05% Tween20. All further dilutions and washes were carried out with 1% BSA/PBS, pH7.4 unless otherwise stated. Prediluted antisera were added to wells coated with the peptide immunogen used to elicit the antiserum. Control wells used to normalize absorbance readings consisted of substitution of dilution buffer for prediluted antiserum with all subsequent steps being the same. Samples were incubated at room temperature for 2 h in replicates of 5 and washed 3 X . Goat anti-rabbit IgG conjugated to alkaline phosphatase CAP) (Southern Biotechnology, Birmingham, AL) was used as a secondary antibody at a 1:2000 dilution and incubated for 1.5 h at room temperature. Plates were washed 3 X . AP substrate, p-nitrophenyl phosphate (Sigma), was dissolved in fresh carbonate/bicarbonate buffer to 1 mg/ml and was added to all wells and incubated at room temperature. The absorbance of the resulting product was measured at 405 nm at 15, 30 and 60 min time points (Bio-Rad Model 2550 EIA Reader). The titration of antiserum against its immunogen was monitored over time and compared to preimmune. Competitive inhibition ELISA was also performed. This procedure was the same as the above ELISA except for the

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preincubation of prediluted primary antisera with peptide. Antiserum prediluted to 1: 10 000 was added to an equal volume of peptide at 2-times its final concentration. The final working concentration of antiserum in this assay was 1:20 000. Fluid-phase peptide and antiserum were incubated at room temperature for 3 h, after which each sample was added to wells coated with solid-phase peptide as previously described. 2.3. Monoclonal

antibodies

T lymphocyte subsets were defined using two mouse monoclonal antibodies: MRC OX-8 and W3/25. Hybridomas producing these antibodies were used with the kind permission of Don Mason, Medical Research Council, Oxford, England. OX-8 is directed against the rat homolog of the human CD8 antigen and mouse Ly-2, and was used as a marker for cytotoxic T cells (CTL) [24]. W3/25 is directed against the rat homolog of the human CD4 antigen and was used as a marker for helper T cells (Th) [25]. Hybridomas were cultured in a protein-free hybridoma medium (PFHM-II, GIBCO, Grand Island, NY, 430-3600 EA) at 37°C under 7% CO,. Culture supematants were collected. Precipitated immunoglobulin was reconstituted in saline and dialyzed against PBS, pH 7.4. Immunoglobulin was isolated by passage over a protein G-Sep-

80

60

Peptide concentration

(M)

Fig. 1. Competitive inhibition ELISA. (A) Emincubation of anti-l-24 serum with ACTH l-24 peptide inhibited this antiserum’s interactions with solid-phase bound l-24 in a dose-dependent fashion, while ACTH 18-39 failed to significantly affect such interactions. (Bl Preincubation of anti-18-39 serum with ACTH 18-39 peptide inhibited its interactions with solid-phase bound 18-39, while ACTH l-24 failed to significantly affect such interactions (n = 5 = f S.E.M.).

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harose (Pharmacia) column and acid elution. Samples were immediately neutralized and dialyzed. Protein content was determined by Bradford Assay (Bio-Rad). 2.4. Cell isolation and culture Splenic leukocytes were isolated from adult 200-300 g male Sprague-Dawley rats (Harlan Sprague-Dawley, Indianapolis, IN). Spleens were excised aseptically and single cell preparations were made by pressing organs through sterile wire sieve (60-mesh). Leukocytes were further purified by density gradient centrifugation using Ficoll-hypaque ( p = 1.089). Cells were washed and their viability was determined by trypan blue exclusion (> 95%). Cells were resuspended at 6 X lo6 cells/ml in serum-free RPMI-1640 supplemented with 1% Nutridoma SP (Boehringer Mannheim Biochemicals, Indianapolis, IN), 0.5% BSA, 200 mM/ml L-glutamine, 25 mM HEPES, 100 pg/ml streptomycin, 100 U/ml penicillin and 0.22%

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NaHCO,. Lipopolysaccharide (LPS, 0127:B8) (Sigma) and concanavalin A (ConA) (Sigma) were added to separate cultures at 20 and 0.78 pg/ml, respectively. In some experiments, leukocytes were separated into adherent and nonadherent subpopulations prior to culture. In this case, leukocytes were resuspended in RPMI-1640 supplemented with 5% fetal calf serum (FCS) at 1 X 10’ cells/ml, and introduced into FCS pretreated plastic petri dishes (Coming) and incubated for 2 h at 37°C and 5% CO, to allow macrophage adhesion [26]. Nonadherent cells were removed after four washes with medium. Adherent cells were removed with ice-cold Ca’+-, Mg2+-free PBS. The purity of the resulting enriched plastic adherent macrophages was about 95% as evaluated by nonspecific esterase staining [27]. Peritoneal macrophages were obtained by intraperitoneal lavage 4d after injection of 3 ml of sterile thioglycollate broth (4%) (DIFCO, Detroit, MI) in the peritoneal cavity. Plastic-adherent cells were isolated and treated as above.

ConA

Fig. 2 Double immunofluorescence for ACTH I-24-ir and T-helper cell marker (CD4). Cells were ConA stimulated (ConA) or untreated (Null). Time points are 0 h (A), 21 h (B), and 48 h (C). ACTH-ir is presented in the large frames, while cell-surface staining for T, lymphocytes in the same fiel d is presenlted in the inserts. The insert images were reduced Cfold. The scale bar is equivalent to 10 pm. Equivalent images in Fig. 3 and Fig. 4 are of the same Iscale.

106 2.5. Immunocytochemistry

P.D. Lyons,J.E. Blalock/Journal of Neuroimmunology63 (1995) 103-l 12 and microscopy

In initial experiments, leukocyte cultures were processed for ACTH-ir at the initiation of culture, and at 3 h intervals up to 27 h and again at 36, 48, and 72 h. Subsequent experiments had time points of 0,21, and 48 h. Cultured cells were collected with ice-cold 5% FCS/RPMI 1640 with 0.1% NaN, (RPMI/A& Cells were washed, centrifuged at 4°C at 250 X g, and counted. All subsequent steps were done on ice. Double indirect immunofluorescence employed 1 million cells in 100 ~1 RPMI/Az, cell surface stained with either OX-8 IgG, W3/25 IgG or Goat IgG anti-rat Ig (Cappel, Durham, NC) for 30 min. Washed cells were air-dried, rehydrated, and fixed in fresh 1% paraformaldehyde solution in PBS, pH 7.4 at 4°C for 75 min. Cells washed with 0.1 M glycine/PBS, pH 8.5 were permeabilized with 0.1% Triton X-lOO/PBS, pH 7.4. Cells were blocked with 10% normal porcine serum in PBS at RT for 45 min. Cells were incubated overnight at 4°C with specific anti-ACTH serum or pre-immune at 1:200 dilutions in humid chambers and washed in PBS. A porcine anti-rabbit secondary antibody (Dako, Carpinteria, CA) conjugated to tetramethyl-rhodamine isothiocyanate

(TRITC) was used for all ACTH immunofluorescence. A goat anti-mouse secondary antibody (Dako) conjugated to fluorescein isothiocyanate (FITC) was used to detect cell surface staining of CD4 and CDS. A porcine anti-goat IgG (Tago, Burlingame, CA) was used to indirectly detect B cells. After incubation with primary antisera, washed cells were incubated with secondary antibodies at I:50 dilutions at 37°C for 30 min. Slides were washed, mounted in glycerol and analyzed. Fluorescent microscopic analysis was performed on a Zeiss IM-35 epifluorescent microscope, equipped for Normaski imaging. Images were obtained with a series 200 cooled charge-coupled device camera system (Photometrics, Tucson, AZ) configured for a Macintosh IIci computer (Apple, Cupertino, CA). A 40 X planachromat objective was used for all images and analysis. Illumination for epifluorescence was achieved with a 100 W mercury arc lamp. Standard FITC (485 f 20 nm excitation filter; 520560 nm emission filter) and TRITC (546 f 10 nm excitation filter; > 590 nm emission filter) filters were used for visualizing FITC and TRITC fluorescence, respectively [28]. Digitized images of ACTH-ir were normalized to the same intensity scale with Photometrics software. Digitized

ConA

Null

Fig. 3. Double immunfluorescence for ACTH l-24-ir and T-cytotoxic cell marker (CDS). Ceils were ConA stimulated (ConA) or untreated (Null). Time points are 0 h (A), 21 h (B), and 48 h (0.

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images of staining for each cell surface marker were also normalized to the same intensity scale. To establish specificity, antisera were preincubated with 10 PM ACTH l-24, 18-39, or l-39 for 3 h at RT prior to use. Quantitation of double indirect-immunofluorescence of lymphocyte subpopulations involved counting 200 cells per slide for each cell subtype for each treatment at each time point of 0, 21, and 48 h. All conditions were replicated a total of 5 times with 5 animals. Quantitation was reported as the mean of the 5 replicates f standard error of the mean (S.E.M.). The intensity of the immunofluroescence was quantitated visually in a blinded fashion on a scale with 3 intensity levels: (-1 negative; (+> strong ACTH-ir; (+ + ) very strong ACTH-ir. 2.6. Statistics All reported values are given as the mean f S.E.M. from replicate samples. Analysis of variance was performed on values of percentages of cells expressing ACTH-ir using GraphPAD Instat Software (GraphPad Software, San Diego, CA).

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3. Results 3.1. Specificity of polyclonal antisera against ACTH peptides I-24 and 18-39 The specificities of the two polyclonal antisera were tested with the competitive inhibition ELISA. This ELISA is based on the dose-dependent inhibitory effect of fluidphase ACTH on binding of a specific antiserum to ACTH immobilized on microtiter plate wells. Preincubation of anti l-24 serum with ACTH l-24 peptide inhibited its solid phase interaction in a dose-dependent fashion (Fig. IA). 7 X 10e8 M ACTH l-24 produced a 50% inhibition while preincubation with an equimolar concentration of ACTH 18-39 peptide resulted in 1% inhibition. Also, preincubation with 10 PM ACTH 18-39 only inhibited 15 f 5% of anti-ACTH l-24 binding to l-24 peptide while 10 PM ACTH l-24 inhibited 83%. Likewise, preincubation of anti-18-39 serum with ACTH 18-39 peptide dose-dependently inhibited solid phase interactions (Fig. IB). 4 X 10d8 M ACTH 18-39 produced a 50% inhibition while preincubation with an

LPS

for ACTH l-243 are 0 h (A), 21 h (B), and 48 h CC).

Fig. 4. Double immunofluorescence

and B cell marker (surface Ig). Cells were LPS-stimulated (LPS) or untreated (Null). Time points

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equimolar concentration of ACTH l-24 resulted in a 2% inhibition. Preincubation with 10 PM ACTH l-24 inhibited 10 f 5% of anti-ACTH 18-39 binding to 18-39 peptide while 10 PM ACTH 18-39 inhibited 100%. Preimmune serum failed to interact with solid-phase bound peptides. 3.2. The kinetics and cellular distribution of ACTH-ir in rat splenic leukocytes Rat splenic leukocytes were cultured with or without ConA (0.75 pg/rnl) or LPS (20 pg/ml). In initial experiments, leukocyte cultures were processed for indirect immunofluorescence for ACTH-ir at the initiation of culture, and at 3 h intervals up to 27 h. Thereafter, cells were processed at 36, 48, and 72 h. The percent of cells immunoreactive for ACTH with

A

Fig. 6. Specificity of ACTH-ir with anti-ACTH l-24 serum. Diluted anti-ACTH l-24 serum was pre-incubated with ACTH peptides l-24, 18-39, or l-39 for 2 h at RT prior to use in immunofluorescence. Pm-incubation with ACM-I l-24 or l-39 abolished positive ACTH-ir, while ACTH 18-39 failed to alter it.

C

oh

Zlh

CD4

4%

Oh

all

4%

MI

Cell Type/

2,h

48h

AZ?-

B

Time

(hr)

Fig. 5. ACTS-ir quantitation in subpopulations of lymphocytes. ConA (A), LPS (B). or untreated (C) leukocytes were processed by double-indirect immunofluorescence for ACTH-ir and T,. T, and B lymphocyte surface markers at three time points (0. 21 and 48 h). Each value is the mean of 5 replicate experiments. 200 cells were counted for each replicate per treatment per time point for each cell type (+ = strong ACTH-ir, + + = very strong ACTH-ir).

anti l-24 serum at time 0 h was l-2% of total cells. Separation of these cells into adherent and nonadherent cells identified this subpopulation as adherent cells, of which 18 f 4% were positive (data not shown). Ninety-five percent of adherent cells stained positive for nonspecific esterase. All T,, T, and B lymphocytes at 0 h were negative with anti l-24 serum (frame A of Figs. 2-4). ACTH l-24 immunofhtorescent intensity peaked at 18-21 h for all treatments (frame B of Figs. 2-4). However, 90 f 5% Cot&treated and 75 f 6% of LPS-treated cells were positive, while only 9 f 4% of unstimulated cells were positive at 21 h, as shown in Fig. 5A, B and C, respectively. By 48 h of culture, the percent of cells expressing ACTH-ir in all three lymphocyte subpopulations was very low (frame C of Figs. 2-4). ACTH-ir in leukocytes was localized to the cytoplasm in a diffuse, nonptmctate pattern. The specificity of the ACTH-ir with anti-ACTH l-24 was established with substitution of rabbit pre-immune serum for anti-ACTH 1-24 serum, as well as preincubation with peptide. Prediluted anti-ACTH l-24 was preincubated for 3 h at room temperature with ACTH peptides

P.D. Lyons, J.E. Blalock/Journd

Fig. 7. Indiit immunofluorescence with pm-immune serum. Cells were ConA-stimulated. Time points are 0 h (A), 21 h (B), and 48 h (Cl.

l-24, 18-39, and l-39 at 10 PM. Preincubation with both 1-24 and l-39, but not 18-39, inhibited ACTH-ir in leukocytes stimulated with ConA for 21 h (Fig. 6). AntiACTH l-24 serum was used for all indirect immunofluorescence. Anti-ACTH 18-39 serum failed to specifically identify ACTH-ir in leukocytes, though both anti-18-39 and -1-24 sera specifically stained the corticotroph cell line AtT-20/D16V-F2 (ATCC, Rockville, MD), for ACTH-ir (data not shown). The inability of anti-18-39 to detect ACTH-ir in leukocytes may be a consequence of alternative POMC processing in stimulated leukocytes as proposed by Harbour-McMenamin et al. [29] in which mature ACTH is further processed to truncated ACTH l-25. Alternatively, epitopes recognized by anti-18-39 serum may only be suitably presented in an ACTH biosynthetic precursor which is not found in leukocytes. Substitution of pre-immune for specific antiserum resulted in negative ACTH-ir (Fig. 7). Double indirect immunofluorescence was done on rat splenic leukocytes at three time points: 0, 21, and 48 h. The quantitative results for all three treatments are given in Fig. 5A, B and C. T-helper (Th) and T-cytotoxic (CTL) cells were identified by their respective phenotypes, namely CD4+CD8- (Th) and CD4_CDS+ (CTL). B cells were identified by surface immunoglobulin. The total percent of

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cells with ACTH-ir in the three lymphocyte subpopulations did not significantly differ at any single time point and treatment (Fig. 5A-C). However, there were quantitative differences in the intensity of ACTH immunofluorescence between cell subpopulations at 21 h with ConA or LPS treatment. As shown in Fig. 5A, ConA treatment of leukocytes for 21 h stimulated 76 f 4% (S.E.M.) of Th to express very strong ACTH-ir ( + + ) and 14 f 2% of Th to express strong ACTH-ir ( + >. This significantly differed from comparable values for CTL cells of 43 f 3% ( + + ) (P = 0.0002> and 45 f 3% (+> (P
Fig. 8. ACTH-ir in activated macrophages. Peritoneal macrophages were stimulated with thioglycollate for 4 days and isolated as described in Section 2. Cells were stained with either anti-ACTH l-24 semm (l-24) or pre-immune serum (p.i.).

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ACTH-ir at 21 h (Fig. 5C). All three lymphocyte subtypes treated with LPS or ConA for 48 h had approximately 10% of their population expressing (+> ACTH-ir, while ACTH-ir at 48 h in unstimulated lymphocytes was negative. In order to further characterize ACTH-ir expression in macrophages, peritoneal macrophages were elicited with the irritant, thioglycollate. Such macrophages are termed elicited or inflammatory, and are high in secretory activity, but do not possess enhanced microbial activity as do activated macrophages [30]. 80 -I: 8% of such cells had very strong immunofluorescence for ACTH with antiACTH l-24 serum (Fig. 8). Thus, unlike rat splenic lymphocytes at 0 h, tissues macrophage basally express ACTH-ir. Yet, both lymphocytes and macrophages respond to immunological stimuli with production of ACTHir.

4. Discussion Our results demonstrate that the expression of ACTH-ir in rat splenic lymphocytes is dependent on cell stimulation. Quiescent T-helper, T cytotoxic and B cells fail to express ACTH-ir, while quiescent tissue macrophages can express ACTH-ir. These results are in agreement with previous studies [ 14,20,31-331. This may also explain the failure to detect POMC products in large numbers of splenic lymphocytes by other studies [34-371. Intracellular ACTH-ir was maximal at 18-21 h of culture in Th, CTL, and B lymphocytes, being first detectable at approximately 12 h of culture. Both T(ConA)- and B(LPS)-cell polyclonal activators greatly increased ACTH-ir in the three lymphocyte populations, as opposed to media alone which resulted in a small increase in ACTH-ir by 18-21 h. The total percent of positive cells and the intensity of ACTH-ir at 21 h was higher with ConA stimulation as compared to LPS. Moreover, comparable percentages of ConA stimulated T, and B cells expressing very strong (+ + ) ACTH-ir were significantly higher than that for CTL cells (P = 0.0002 Th vs. CTL, P = 0.011 B vs. CTL). In comparison, only LPS-treated B cells, and not Th, were significantly higher in percent expressing ( + + > ACTH-ir relative to CTL cells (P = 0.0384). ConA and LPS were used at concentrations which resulted in near maximal proliferation in treated leukocytes at 72 h. The ability of ConA stimulation to result in a higher percentage of cells expressing ACTH-ir and with greater intensity relative to LPS may be partly due to the second messengers each elicits. LPS has been shown to fail to enhance the concentration of intracellular Ca++ [38], while ConA increases cytosolic free Ca” [39]. Interestingly, CRH and AVP both increase cytosolic free Ca2+, as well as other second messengers, within corticotrophs in stimulating ACTH secretion from the anterior pituitary [al. We unexpectedly observed that ConA is able to

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enhance ACTH-ir in B cells and LPS is able to enhance ACTH-ir in T cells. Kavelaars et al. [41] demonstrated that rIL-lp can directly enhance POMC-derived peptide expression in murine B cells, but not in murine T cells or monocytes. Since ConA stimulation of splenic leukocytes results in IL-l production by macrophages, this could account for the ability of ConA to enhance ACTH-ir in B cells. Also, in vitro proliferative responses of human T cells to LPS have been reported [42]. Both CD4+ and CDS+ T cells respond to LPS, but require the help of macrophages. It may be that this proliferative effect of LPS on T cells, or some facet of it, may account for its effect on T cell ACTH-ir in vitro. The observation that the kinetics of ACTH-ir expression in the splenic lymphocyte subpopulations is the same suggests that they are all producing ACTH-ir, rather than uptake through receptor-mediation or endocytosis. Moreover, both stimulated and unstimulated cells were cultured in serum-free media, thereby suggesting that ACTH production by stimulated lymphocytes was not due to passive uptake from exogenous sources. We also observed significantly more cells expressing very strong ACTH-ir in both ConA and LPS-stimulated B cells, and also in ConAstimulated Th cells. Both activated and resting B cells express 2-3-times more functional, high affinity ACTH receptors than comparative T cells, and exogenous ACTH can biphasically modulate B cell functions 143-461. Intracellular ACTH-ir was quite low in lymphocyte subpopulations by 48 h, which is in accordance with a study by Clarke et al. [21], which demonstrated that in vitro secretion of biologically active corticotropin from mitogen-activated lymphocytes was maximal by 48 h. Moreover, ConA and LPS stimulation substantially increases the number of high affinity ACTH receptors by 72 h on T and B cells, respectively [9]. Collectively, these data define a temporally regulated cascade of events in which ACTH production precedes secretion, which occurs at a time when functional ACTH receptor numbers are being increased. Bost et al. demonstrated that ACTH induces increased IgM secretion from an LPS-activated B-cell line [47]. Also, IgM titers following a primary immune response first increase by the third day post antigen challenge. It is at this same time that ACTH receptors are maximally expressed on mitogen- or antigen-stimulated lymphocytes [9]. This temporal correlation between lymphocyte induction of ACTH receptors and the primary immune response suggest a role for lymphocyte-derived ACTH in modulation of the primary immune response. Thus, the immune system may be using a classical regulatory mechanism of cytokine effects, namely receptor regulation, to modulate its exposure to ACTH in infectious or inflammatory states. Such paracrine or autocrine functions of leukocyte-derived ACTH are supported by the manifold effects of ACTH on immune functions [44,48,49]. It is interesting to note that experimental endotoxemia results in a rapid plasma surge of pituitary-derived ACTH

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which returns to levels close to baseline by glucocorticoid negative feedback at 24 h [50]. It is at this time that ACTH-ir is maximally expressed in lymphocytes. The primary target tissue for ACTH in the endocrine system is the adrenal cortex, where ACTH stimulates glucocorticoid synthesis and secretion. Glucocorticoids down regulate ACTH in both anterior pituitary corticotrophs and in lymphocytes, but by different mechanisms. Glucocorticoids directly down-regulates ACTH in corticotrophs by decreasing POMC mRNA [5 11, whereas glucocorticoids indirectly inhibit B cell production of ACTH by blocking macrophage production of IL-l p [41]. Upon inhibition of IL-1 p, the bacterial endotoxin, LPS, can still directly stimulate production of POMC-derived peptide hormones from B cells [52]. Another point of distinction between the regulation of pituitary and lymphocyte ACTH is the fact that IL-l p directly stimulates B cell production of ACTH, while it appears that IL-l p indirectly stimulates pituitary secretion of ACTH through hypothalamic CRH [53]. Proposed paracrine functions of leukocyte-derived ACTH may also extend beyond immune tissues. For example, macrophage-derived POMC peptides are thought to play a role in the local control of Leydig cell function [54]. Also, opioid receptors on peripheral sensory nerves in inflamed rat paws can be activated by another POMC-derived peptide, P-endorphin, released from local immune cells, thereby mediating antinociception [55,56]. Further studies are obviously needed to determine additional roles for leukocyte-derived ACTH. Undoubtedly, however, the state of activation of both lymphocytes and macrophages will be an important determinant because of the profound influence on their production of ACTH-ir.

Acknowledgements The authors would like to thank Diane Weigent for help in preparation of the manuscript and acknowledge the critical input of Drs. K.L. Kirk and T. Jilling. This research was supported in part by NIH grant DK38024.

References [I] Besedovsky, H., So&in, E., Keller, M. and Miller, J. (1977) Hypothalamic changes during the immune response. Eur. J. Immunol. 7, 323-325. [2] Derijk, R., VanRooijen, N., Tilders, F.J.H., Besedovsky, H.G., DelRey, A. and Berkenbosch, F. (1991) Selective depletion of macrophages prevents pituitary-adrenal activation in response to subpyrogenic, but not to pyrogenic, doses of bacterial endotoxin in rats. Endocrinology 129, 330-338. [3] Berkenbosch, F., Wolvers, D.A.W. and Derijk, R. (1991) Neuroendocrine and immunological mechanisms in stress-induced immunomodulation. J. Steroid B&hem. Mol. Biol. 40, 639-647. [4] Pezzone, M.A., Dohanics, J. and Rabin, B.S. (1994) Effects of footshock stress upon spleen and peripheral blood lymphocyte mito-

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