Journal of Neuroimmunology, 29 (1990) 1-13

1

Elsevier JNI 00967

Immunoreactive growth hormone-releasing hormone in rat leukocytes Douglas A. Weigent and J. Edwin Blalock Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, AL 35294, U.S.A.

(Received8 January 1990) (Revisedreceived20 February 1990) (Accepted21 February 1990)

Key words: Growth hormone-releasinghormone; Leukocyte;High-performanceliquid chromatography; RNA; Bioactivity

Summary In the present study, we evaluated whether mononuclear leukocytes could synthesize and secrete growth hormone-releasing hormone (GHRH) in vitro. By using RNA slot-blot analysis, we detected maximum basal levels of specific G H R H mRNA in the cytoplasm of rat leukocytes after an 8 h in vitro incubation. Northern gel analysis demonstrated that the specific G H R H RNA was polyadenylated and had a molecular mass of approximately 0.8 kDa. Further studies using antibody affinity chromatography followed by size separation on high-performance liquid chromatography (HPLC) columns showed two peaks of immunoreactive (ir) material, a large molecular weight species, and a smaller molecular weight species at approximately 5 kDa. The smaller molecular weight irGHRH appeared to be de novo synthesized since it could be radiolabeled with tritiated amino acids. Both molecular species were detectable in enzyme-linked immunosorbent assay (ELISA) with specific antibodies made to the first 23 amino acids as well as specific antibody obtained commercially made to the entire molecule (1-43). Although the larger molecular weight form appeared to be the more predominant, only the lower molecular weight form could block the binding of 12SI-hGHRH to pituitary cells. Most importantly, the lower molecular weight leukocyte-derived G H R H stimulated an increase in the level of GH RNA in the pituitary. We conclude that lymphocytes produce an irGHRH that is similar to hypothalamic G H R H in terms of bioactivity, antigenicity, and molecular weight. The findings demonstrate a potential regulatory loop between the immune and neuroendocrine tissues.

Introduction The primary hypothalamic regulation of pituitary growth hormone (GH) secretion is mediated Address for correspondence: Dr. DouglasA. Weigent, Department of Physiology and Biophysics, UAB Station, Birmingham, AL 35294, U.S.A.

through the stimulatory actions of growth hormone-releasing hormone (GHRH) (Guillemin et al., 1982; Frohman and Jansson, 1986) and the inhibitory actions of somatostatin (Brazeau et al.; 1973). More recently, GH has also been shown to be produced by leukocytes, but nothing is known about the mechanisms that regulate this response (Weigent et al., 1988). Since the initial isolation

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and characterization of G H R H from two separate pancreatic tumors (Guillemin et al., 1982; Rivier et al., 1982), the peptide has been sequenced from the hypothalami of a number of animal species (Spiess et al., 1983; Ling et al., 1984). The cDNA coding sequences for both human and rat G H R H have been deduced from cloning studies (Gubler et al., 1983; Mayo et al., 1983, 1985). G H R H s belong structurally to the glucagon-secretion family where considerable homology exists between human, ovine, porcine and bovine whereas the rat molecule shares approximately 70% homology with human (Spiess et al., 1983). Immunoreactive GHRH-like molecules have been detected in plasma from normal subjects, acromegalics and from tumor tissue (Christofides et al., 1984; Penny et al., 1984; Saito et al., 1984; Thorner et al., 1984; Asa et al., 1985; Donnadieu et al., 1985; Sopwith et al., 1985). The source(s) of G H R H in plasma is uncertain and immunoreactive (ir) G H R H is present in extrahypothalamic tissues such as brain, lung, liver, pancreas, and the gastrointestinal tract (Shibasaki et al., 1984). It has been reported that the human placenta contains several kinds of hypothalamic releasing hormones such as corticotropin-releasing factor (CRF) (Shibasaki et al., 1982; Sasaki et al., 1987), thyrotropin-releasing hormone (TRH) (Gibbons et al., 1975), and luteinizing hormone-releasing hormone (LHRH) (Gibbons et al., 1975; Khodr and Siler-Khodr, 1980). The presence of immunoreactive and biologically active G H R H in rat placenta has also been described (Sasaki et al., 1989). It is interesting that in pregnant rats the serum levels of G H R H are not altered and a higher molecular weight form was detected (Meigan et al., 1988). The data suggested that post-translational processing of G H R H may be different between the placenta and the median eminence and that placental G H R H may have a paracrine function. Most recently, the precursor structure and expression of mouse G H R H in brain and placenta have also been reported (Suhr et al., 1989). The possibility that leukocytes synthesize and secrete hypothalamic releasing hormones is just beginning to be investigated; however, the effect of releasing hormones and the presence .of releasing hormone receptors on leukocytes have now

been well documented. CRF receptors (Rivier and Plotsky, 1986) were suggested on immune cells as a stimulus for lymphocytes to synthesize proopiomelanocortin (POMC) m R N A (Smith et al., 1986) and subsequently secrete corticotropin (ACTH) which has been shown to serve as a proliferative signal for B cells (Bost et al., 1987; Wear et al., 1987). Thyrotropin (TSH) receptors were first suggested on leukocytes when it was observed that this hormone enhanced the in vitro antibody production against T-dependent and Tindependent antigens (Blalock et al., 1985; Kruger and Blalock, 1986; Kruger et al., 1987). Additional studies have demonstrated that TRH has a similar ability to augment in vitro antibody responses (Harbour et al., 1988; Kruger et al., 1989). The enhancing ability of TRH is apparently mediated through the synthesis of TSH by leukocytes since antibody directed against the TSH fl-subunit was able to block the ability of TRH to enhance antibody production. In previous work we have reported that leukocytes produce and secrete an immunoreactive growth hormone similar to that described for the pituitary (Weigent et al., 1988). A major part of this work demonstrated the presence of GH RNA in cells and the secretion of a 22 kDa immunoreactive protein. In other studies, we have demonstrated that G H R H treatment of leukocytes resulted in an increase in Ca uptake, [3H]thymidine and [3H]uridine incorporation, as well as a 2-fold increase in the synthesis of GH RNA (Guarcello et al., 1989). The binding of G H R H was specific and saturable and suggested a single class of receptor with a nanomolar K a. We have initiated studies to characterize the possible production of G H R H in the periphery by leukocytes in an effort to answer the question whether this molecule can be produced by leukocytes and function at the G H R H receptor as a signal for the synthesis of leukocyte-derived GH. In this study, we have characterized immunoreactive GHRH-Iike material from leukocytes and compared it with rat hypothalamic G H R H . The data suggest that rat leukocytes secrete a bioactive immunoreactive G H R H similar to hypothalamic-derived G H R H . Overall, the data support the idea that G H and G H R H may be produced by leukocytes and be active in a local immune response.

Materials and methods

Cell preparations Adult (150-200 g) male Sprague-Dawley rats were obtained from Harlan (Prattville, AL, U.S.A.). Following sacrifice, lymphoid tissues including spleens, thymus, peripheral blood lymphocytes, and bone marrow were prepared as single-cell suspensions by standard techniques and frozen in microfuge tubes for RNA isolation. Peritoneal exudate cells were collected from rats injected i.p. with Freund's adjuvant by standard techniques and washed in RPMI (Roswell Park Memorial Institute) containing heparin, centrifuged, and quickly frozen. Greater than 95% cell viability by trypan blue exclusion was observed in all cell fractions (Julius et al., 1973). Rat pituitaries and rat hypothalamic tissues were carefully removed and washed in phosphate-buffered saline (PBS), quickly frozen, and the RNA isolated as described below. RNA isolation and blotting Total cytoplasmic RNA was isolated by the proteinase K method (Maniatis et al., 1982). After ethanol precipitation, the RNA pellet was dried under vacuum and dissolved in sterile water. An aliquot was removed to determine the yield and purity by optical density (OD) measurements at 260 and 280 nm. Total RNA (10 #g) was blotted on nitrocellulose membranes with the Minifold II slot blotter (Schleicher and Schueil, Keene, NH, U.S.A.) as described by the manufacturer. Total RNA was prepared by homogenizing lymphocytes in 5 M guanidine thiocyanate, 1% sarkosyl, 20 mM EDTA, 1% 2-mercaptoethanol, 50 mM TrisHCI, pH 7.5, with subsequent protease K digestion and extraction with phenol/chloroform. Polyadenylated mRNA was isolated on oligo(dT)cellulose as previously described (Maniatis et al., 1982). Northern gel analysis was performed on 1% agarose gels and blotted to nytran paper overnight by capillary action. Nytran paper was hybridized with GH cDNA probes as described later. GH and GHRH cDNA and hybridization Plasmid containing specific rat (Seeburg et al., 1977) GH cDNA were kindly provided by Dr.

John Baxter and Dr. Fran Denoto (Neurochemistry Laboratories, V.A. Medical Center, Sepulveda, CA, U.S.A.). Plasmid DNA was prepared essentially as described (Maniatis et al., 1982). Eight hundred base pair HindIII inserts were purified from these plasmids (Maniatis et al., 1982) and labeled with [32p]dCTP by nick translation (Bethesda Research Laboratories, Rockville, MD, U.S.A.) to a specific activity of 1-2 × 105 cpm//~g. The synthetic oligonucleotide G H R H probe was prepared and purified in our laboratory by Dr. Robert LeBoeuf. This probe corresponded to amino acids 13-27 (48 bases; CCTGTTCATGATTTCGTGCAGCAGTTTGCGGGCATATAATTGGCCCAG) and was end-labeled with polynucleotide kinase by standard procedures (Maniatis et al., 1982). Prehybridization was done for 4 h at 42°C and hybridization was done for 18 h at 42°C in standard buffer containing 32p-labeled probes (2 x 106 cpm/ml). After hybridization, the membranes were extensively washed by standard techniques until the radioactivity in the final wash was close to background. The nitrocellulose papers were exposed to X-ray film at - 7 0 ° C with Dupont Cronex Lightning-Plus intensifying screens for 2-3 days.

Preparation of pituitary cells Rat pituitaries were removed from male Sprague-Dawley rats (180-200 g) that had been killed by rapid decapitation. Anterior lobes were first separated from posterior lobes, and then dissociated in PBS containing 1.5% trypsin for 60 rain at 37°C and cultured in F-10 medium supplemented with 15% horse serum (HS), '2.5 fetal calf serum (FCS), and 100 units each of penicillin and streptomycin per ml. Binding of [~25I]GHRH Binding assays were carried out in the presence of [125I]hGHRH (IM.180, Amersham; 1812 Ci/mmol) in a total volume of 0.2 ml. Nonspecific binding was calculated using 100-fold unlabeled h G H R H in the control tube. The incubations were carried out in microfuge tubes treated with Sigmacote in RPMI-1640 containing 25 mM Hepes (pH 7.3) and 0.1% bovine serum albumin (BSA) at 4°C for 60 min. At the end of the incubations, the

cells were centrifuged for 3 min in a Beckman 12 microfuge, triplicate 100 /~1 aliquots of supernate were removed for the measurement of free. The pellets were washed 3 times in ice-cold buffer, and the tip of the tube was cut off and counted in a gamma counter. Specific binding fraction (SB) is defined as total binding fraction (TB) minus nonspecific binding fraction (NSB). Radioactivity removed in the presence of an excess (100-fold) of unlabeled G H R H was considered specific. Nonspecific binding was 10-15% of total cell binding.

Immunoaffinity chromatography The immunoaffinity columns were prepared by conjugating the immunoglobulin-enriched fraction from 50 ml of r G H R H antiserum (1-23aa, 10,000 units/ml) or 2 ml of r G H R H (1-43aa, 1000 assays/ml, Accurate Chemical Co.) to Affigel 10 (Bio-Rad) according to the manufacturer's instructions. The column was washed in PBS (pH 7.4) prior to use. In a typical experiment, cells (10-20 × 10 6 per ml) were cultured for 24 h in RPMI without serum containing 2 # C i / m l of a uniformly tritium-labeled mixture of amino acids (Amersham). Spent supernatant fluids were centrifuged at 3000 × g for 15 min and applied to the column at 30 ml/h. The effluent containing the unbound material was saved for further study and the column washed with 20 void volumes of PBS. Material bound to the column was then eluted with 2 M potassium bromide, 15 mM NaC1 and 1 mM Hepes at a pH of 5.5. The column was regenerated by washing with PBS and the initial effluent reapplied and eluted as before. Pooled effluents were dialyzed against PBS and then dried by lyophilization and reconstituted in buffer for further analysis.

white rabbits by intradermal/subcutaneous injections of the rat peptide 1-23 corijugated to bovine serum albumin with glutaraldehyde. The rabbit serum we prepared was sequentially purified by protein A chromatography and affinity chromatography on columns of Affi-gel-10 coupled with synthetic rat G H R H 1-23 peptide. The ELISAs were performed in microtiter plates in a total volume of 0.2 rnl with carbonate buffer with the pH adjusted to 9.0. Rat G H R H antiserum (1-23) and Accurate 1-43 were used at final dilutions of 1:100 and 1:1000, respectively. Assays were incubated at 4°C for 2 h with antiserum and then a further 2 h after the addition of goat anti-rabbit alkaline phosphatase-conjugated secondary antibody before incubation with substrate. Both antisera were highly specific and showed no crossreactivity with CRF, ACTH, or interleukin-1. The least detectable doses of G H R H were 10 ng/tube.

HPLC Peak fractions of leukocyte-derived G H R H from immunoaffinity columns were pooled, dialyzed, and reduced in volume by lyophilization. Samples were reconstituted in the mobile phase buffer (0.1 M Na2SO 4, 0.02 M NaH2PO 4, pH 6.91) and applied to a SOTA GF-200 analytical column (Rainin Instrument Co.). The column was run at 1 m l / m i n over 30 min. 1 ml fractions were collected and an aliquot assayed by ELISA for GHRH. The high-performance liquid chromatography (HPLC) column was calibrated with the Bio-Rad standards of thyroglobulin, gamma globulin, ovalbumin, myoglobin, and vitamin B-12.

Results

ELISA

Production of leukocyte-derived GHRH RNA

A standard enzyme-linked immunosorbent assay (ELISA) protocol was used. Lymphocyte peptides were prepared as described before while rat G H R H (1-43) was obtained from Peninsula Laboratories, h G H R H (1-29) from Sigma and rat G H R H (1-23) prepared by Ken Bost in our laboratory. Antisera for the assay was obtained from Accurate Chemical and Scientific Corporation (i413/001) and also raised in New Zealand

The production and release of most pituitary hormones are regulated by peptides released from the hypothalamus as a result of central nervous stimulation. Since we know that cells of the immune system produce GH, we asked the question whether G H synthesis in these cells might be subject to regulation by leukocyte-derived GHRH. To test the idea, we isolated RNA from lymphoid tissues cultured for 24 h in vitro and probed for

TABLE 1 DETECTION OF irOHRI-I RNA IN RAT IMMUNE CELLS BY SLOT BLOT ANALYSIS RNA was isolated from the tissues by the NP-40 method and the amount slotted onto nitrocellulose is shown in parentheses. Spleen and thymus leukocytes were cultured in vitro for 24 h before harvest, while the peritoneal exudate cells were obtained from rats injected 4 days previously with 2 ml of Frennd's complete adjuvant. Nitrocellulose papers were probed as described in Materials and Methods with an end-labelled ([5'-32p]ATP) GHRH-specific probe, stringently washed (0.1 x SSC, 70°C, 0.1% SDS, 30 min), exposed to film and the autoradiograph densitometricallyscanned. Values are listed as a percentage with 5/~g hypothalamic RNA as the 100% reference point.

Source of cells

Duplicate slots

Percent densitometric scan

Spleen (I0 ~g)

60+3

Thymus (I0 ~g)

75+2

Peritoneal exudate cells (I0 ug)

65+4

Pituitary (I0 ~g)

5+1

Muscle (I0 ~g)

7+1

P388 (I0 ~g)

106 + 6

Rat hypothalamus (5 .g)

I00 + 5

the presence of specific G H R H R N A with a ~2p. end-labeled oligonucleotide specific for rat G H R H (amino acids 13-27, 48 bases). The results shown in Table 1 demonstrate the presence of G H R H R N A in the cytoplasm of immune cells. The data show that we were able to detect G H R H message in spleen, thymus, and peritoneal exudate cells. In addition, we could detect G H R H R N A in the mouse P-388 maerophage cell line, whereas rat pituitary and muscle cells were essentially negative. The use of freshly obtained rat hypothalamus was used as a reference point for G H R H RNA. To determine the kinetics of G H R H R N A synthesis, we cultured rat mononuclear spleen and thymus cells after removal from animals for various periods of time and isolated, slotted, and probed for R N A as described in Materials and Methods. The data (Table 2) from both spleen and thymus show that maximal levels were obtained

after 8 h and 16 h of in vitro culture for thymus and spleen cells, respectively. The expression of G H R H R N A was also detectable after 128 h but at reduced levels. We next determined whether mononuclear leukocytes from rat thymus showed an increase in GHRH-specific R N A by Northern blot analysis (Fig. 1). The data show that the R N A from the thymus a n d hypothalamus was detectable as a broad band with a molecular weight in the range of 500-900 bp. The same pattern was observed by Northern analysis with R N A obtained from the thymus and spleen in the presence of the ribonuclease inhibitor vanadyl ribonucleoside complex (data not shown). The data show that the labeling pattern from leukocytes after hybridization was similar to that observed with hypothalamus R N A and is consistent with published results by others on the size of hypothalamic

TABLE 2 KINETICS OF irGHRH RNA INDUCTION IN RAT MONONUCLEAR LEUKOCYTES Rat spleen and thymus cell cultures (107/ml, 5 ml) were isolated for GHRH RNA analysis after time intervals as indicated in the figure. The RNA (10 /~g) was slotted onto nitrocellulose and probed as described in Materials and Methods. The blot was autoradiographed for 48 h with intensifying screens. The data shown are from quadruplicate cultures from a single experiment performed 4 times.

Source of cells a

Time (hr) 0

2

4

8

16

32

64

128

Thymus

Spleen

G H R H m R N A (Gubler et al., 1983; Mayo et al., 1983, 1985).

Structure of leukocyte-derived irGHRH The structures of G H R H from several species have been determined and except for the rat they exhibit nearly complete homology with h G H R H . To further study the molecular details of leukocyte-derived rat i r G H R H , we attempted to intrinsically radiolabel the molecule by incubating the cells with a tritium-labeled mixture of amino acids. In a typical experiment, 50 rat thymi or approximately 1.3 × 1011 cells were cultured in 2.5 liters of radioactive R P M I medium without serum for 24 h at 37°C. The culture fluids were passed twice over an affinity column (1-43 antibody) and eluted material dialyzed and concentrated as described in Materials and Methods. This procedure gave a

yield of approximately 300 #g of total protein from which approximately 6.3/xg of i r G H R H was detectable with specific antibodies to the G H R H molecule (1-43). This latter value suggests that leukocytes secrete approximately 1.4 n g / m g protein of i r G H R H in a 24 h in vitro culture period. Thus it appears that leukocytes m a y be a substantial source of G H R H . An H P L C profile of immunoaffinity-purified leukocyte-derived G H R H material is shown in Fig. 2A. Interestingly, two major peaks of immunoreactivity were detected, one in the void volume of the column while the other peak was at a position corresponding to the size of hypothalamic-derived G H R H . The data show that the majority (85%) of the immunoreactive material is a high molecular weight form whereas the remainder ( - 15%) is approximately 5 kDa. Bona fide rat G H R H 1-43 when analyzed

KB

Sepharose affinity column. This column was extensively washed and subsequently eluted with 2 M KBr, pH 7. The eluate was pooled, dialyzed, and lyophilized prior to trichloroacetic acid precipitation and counting. The results showed that the column bound 4800 cpm and suggests that leukocyte-derived irGHRH was de novo synthesized by leukocytes. The amount of irGHRH produced in this particular experiment based on ELISA was approximately 1.2 ng/mg protein. Most importantly, when HPLC column fractions were precipitated and counted, approximately 60% of the input counts were recovered in the lower

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by this same HPLC protocol migrates with exactly the same pattern as the lower molecular weight form (arrow). Taken together, these data suggest that approximately 210 pg of the lower molecular weight irGHRH ( - 5 kDa) is synthesized per mg thymus protein over a 24 h in vitro culture period by leukocytes. In another experiment, we have examined the profile of de novo synthesized irGHRH by HPLC chromatography (Fig. 2B). In a representative experiment, 3H-labeled amino acids (2.0 ~tCi/ml) were added to a rat thymus cell culture (25 thymi, 360 ml RPMI) for overnight incubation at 37°C). After 24 h, the supernatant fluids were removed and passed over an anti-GHRH (1-43) antibody

~

Q .4 0

3

Fig. 1. Northern blot analysis of R N A isolated from rat pituitary and hypotlialamic tissues and mononuclear leukocytes. RNA was loaded onto a 1% agarose-formaldehyde gel. The blot was stringently washed (0.1 ×saline sodium citrate (SSC), 7 0 ° C , 0.1% sodium dodecyl sulfate (SDS), 30 rain) and autoradiographed for 24 h with intensifying screens. Lane 1: rat hypothalamus (5 #g); lane 2: rat pituitary RNA (30/~g); lane 3: rat thymus (30 #g).

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Fig. 2. HPLC of leukocyte-derived irGHRH from rat thymus. Supernatant fluids were collected from 24-h-old cultured cells and purified over an anti-GHRH (1-43) affinity column as described in the text. Approximately 5 pg of irGHRH was applied to the column and 1.0 ml fractions collected as described in Materials and Methods. In (A), aliquots were removed for ELISA (© . . . . . . ©) against specific antibodies as well as binding and biological studies described later. In (B), aliquots (0.5 ml) of 1.0 ml fractions were trichloroacetic acid (TCA) precipitated and collected on Whatman filters, dried, and counted in a TM Analytic Beta Counter ( o . . . . . . o). The standards used to calibrate the column were thyroglobulin (670,000) immunoglobulin (158,000), ovalbumin (44,000), myoglobulin (17,000), and vitamin B-12 (1350) (Bio-Rad).

TABLE 3 INHIBITION OF [125I]GHRH BINDING TO PITUITARY CELLS BY PURIFIED LEUKOCYTE-DERIVED GHRH Rat GHRH (1-43) was obtained from Peninsula Laboratories and the irGHRH preparations were prepared from spent supernatant culture fluids, immunoaffinity- and HPLC-purified, dialyzed, and lyophilized as previously described. Pituitary cells were dispersed with trypsin and after overnight culture, counted and used in the binding assay. Binding studies were performed in triplicate Sigmacote-treated microfuge tubes in a volume of 200 #1. Pituitary cells (1 × 105) in RPMI-1640 (pH 7.3) with 0.1% bovine serum albumin, 25 mM Hepes (binding buffer) were incubated (60 min at 4°C) in the presence of 70,000 cpm/tube of [12SI]GHRH (1860 Ci/mmol) with or without excess (100-fold) unlabeled GHRH. Incubated, labeled cells were washed with binding buffer (3 times) and free and bound ligand were separated by centrifugation. Cell-associated radioactivity was determined by a TM Analytical Gamma Counter. Values in parentheses in picomoles were calculated from the specific activity. The percent inhibition is derived from the displaced cpm divided by the total cpm. The above data are representative of a single experiment performed 3 times with similar results in each experiment. Source of sample

Concentration (/~g/ml)

cpm bound

Specific cpm displaced (pmol)

% Inhibition

None rGHRH Spleen affinity purified Thymus affinity purified Thymus (HPLC, high MW) Thymus (HPLC, low MW)

0.1 0.5 0.5 0.5 0.5

13,260 1,636 3,172 2,529 12,573 6,447

11,624 (6.4) 11,801 (6.5) 10,751 (5.9) 687 (0.4) 6,813 (3.8)

88 79 81 5 51

TABLE 4 BIOLOGICAL ACTIVITY OF LEUKOCYTE-DERIVED irGHRH Rat GHRH (1-43) was obtained from Peninsula Laboratories and the irGHRH preparations were prepared from spent supernatant culture fluids, immunoaffinity- and HPLC-purified, dialyzed, and lyophilized as previously described. Pituitary cells were dispersed with trypsin and allowed to grow for 4 h. After 24 h treatment with GHRH, cytoplasmic RNA was extracted and the RNA was slotted (5 /ig) and probed with GH-specific insert and scanned as described in Materials and Methods. Values are listed as a percentage with 5/xg nonstimulated control pituitary RNA from each experiment as the 100% reference point.

Source of sample

Concentration

Pituitary GH RNA Exp. 1 Exp. 2 Slots Percent Slots Percent Densitometric Densitometric Scan Scan

None

I00

I00

rGHRH

2 ~g/ml

309

203

Spleen affinity purified irGHRH

0.5 ~g/ml

300

206

Thymus affinity purified irGHRH

0.5 ~g/ml

305

210

Thymus (HPLC, high mol wt)

0.5 ~g/ml

114

94

Thymus (HP$C, low mol wt)

0.5 ~g/ml

245

180

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9

molecular weight peak in the same position that irGHRH was detected by ELISA. The remaining 40% of the counts were observed in the void volume of the column. In another experiment, testing the effect of cycloheximide on the synthesis of irGHRH, we found that no radioactivity could be eluted from the GHRH 1-43 immunoaffinity column. The data taken together strongly support the de novo synthesis of an irGHRH by leukocytes.

Inhibition of [1251]hGHRH binding to pituitary cells by leukocyte-derived GHRH To investigate further the identity of leukocytederived GHRH, we have examined the ability of leukocyte-derived GHRH to inhibit the binding of iodinated hypothalamic-derived GHRH to rat pituitary cells. The table of the data shown in Table 3 from pituitary cells was obtained using GHRH from different sources and purity including the HPLC-purified fraction 8 (high molecular weight) and 14 (low molecular weight). The results show that affinity-purified spleen and thymus irGHRH as well as only the low molecular weight material from HPLC were able to compete the binding of [12~I]GHRH to pituitary cells. The high molecular weight HPLC fraction was ineffective in blocking the binding of [12~I]GHRH.

Biological activity of leukocyte-derived irGHRH Since leukocytes appear to be an extrapituitary source of irGHRH production, we conducted experiments to determine if leukocyte-derived irGHRH was biologically active. Therefore, we have tested the ability of leukocyte-derived irGHRH to stimulate the expression of GH RNA in primary pituitary cell cultures in vitro. The results of these experiments summarized in Table 4 support the notion that a substance produced by leukocytes can stimulate rat primary pituitary cell cultures to increase transcription of the gene for GH. On the average, the amount of GH RNA in the pituitary was increased approximately 3-fold. In addition to this study, we have also examined the fractions from the HPLC column for their bioactivity (Table 4). The results show that one peak of bioactivity was present and that was only in the lower molecular weight fraction. The data shown support the production of a bioactive GHRH-like substance from leukocytes that is sim-

ilar in activity to bona fide hypothalamic-derived GHRH.

Discussion

In the present study with a GHRH antisense DNA probe, we have obtained hybridization on slots and Northern blots to a species of mRNA in the thymus and spleen of similar size to that seen in the hypothalamus (Colonna et al., 1988). In addition, our data show the presence of immunoreactive and biologically active de novo synthesized GHRH secreted from rat leukocytes. A highly specific commercial antiserum directed toward the whole hypothalamic molecule (1-43) as well as a highly specific antiserum directed toward the amino terminal region of rat GHRH (1-23) was produced and used to immunoaffinity purify GHRH as well as to develop a specific and sensitive ELISA for GHRH. The data obtained with either antibody were similar. Immunoaffinity-purified culture supernates when analyzed by HPLC contained two peaks. One peak corresponded to the void elution volume of the column whereas the second peak eluted at the approximate position of rat GHRH 1-43 (molecular weight -5000). Approximately 75% of the immunoreactive material migrated in the higher molecular weight peak; however, no bioactivity was contained in these fractions. The lower molecular weight peak although containing less material had a substantial amount of bioactivity. A similar size separation profile was recently reported from cultured hypothalamic cells by two different groups (Leidy and Robbins, 1988; Fernandez et al., 1989). The presence of a high molecular weight material in hypothalamic a n d / o r leukocyte tissues may be related to a GHRH precursor whose post-translational processing may vary in different tissues. In contrast, the HPLC characterization of leukocyte supernatant fluids showed another main component whose retention time (14 min) corresponds to that of synthetic rat GHRH. The rat GHRH was de novo synthesized as evidenced by our ability to radiolabel this molecule. The rat leukocyte GHRH concentration (210 pg/mg protein) under our culture conditions was

10 5-fold lower than that normally observed for hypothalanfic cells (1000 pg/mg). We have also examined the ability of purified leukocyte-derived G H R H from rats to interact at the receptor for G H R H on pituitary cells and address whether the molecule from leukocytes is biologically active. The data (Tables 3 and 4) clearly show the ability of spleen and thymus i r G H R H affinity and HPLC-purified molecules to block the binding of [12SI]GHRH to rat pituitary cells. The displacement curves generated by serial dilutions of the leukocyte i r G H R H were parallel to the standard curve suggesting the same antigenic determinant is present in the material from leukocytes or hypothalamic origin (data not shown). The ability of i r G H R H to stimulate the transcription of the gene for G H in pituitary cells is consistent with those of a biologically active receptor (Table 4). Taken together, the data are consistent with the idea that irGHRH from leukocytes bears a strong resemblance to hypothalamic G H R H in terms of immunogenicity, molecular mass, and bioactivity. The observation that neuroendocrine peptide hormones are produced by leukocytes and that cells of the immune system have specific receptors for neuroendocrine hormones provides a molecular basis for the interactions between the nervous, endocrine, and immune systems (Blalock and Smith, 1980; Blalock et al., 1985). The first described pituitary peptide hormones in the immune system were ACTH and endorphins (Smith and Blalock, 1981). The leukocyte-derived ACTH and endorphins were observed to be identical to their pituitary gland equivalents in terms of bioactivity, antigenicity, and molecular weight (Smith and Blalock, 1981). Hormone production by the immune system also includes vasoactive intestinal peptide (Giachetti et al., 1978; Lygren et al., 1984), somatostatin (Lygren et al., 1984), thyrotropin (Smith et al., 1983), chorionic gonadotropin (Harbour-McMenamin et al., 1986), folliclestimulating hormone, luteinizing hormone (Ebaugh and Smith, 1987), and G H (Weigent et al., 1987). Recently, thymic peptides have been implicated in the regulation of pituitary hormone release (Spangelo et al., 1989). A factor (thymic neuroendocrine releasing factor or TNRF) contained in thymic epithelial cells was reported to enhance

both prolactin and GH release from rat pituitary cells and had an apparent size of 10-15 kDa. The structural relationship of T N R F to G H R H , if any, to our knowledge has not been investigated and, therefore, the similarity of this molecule to hypothalamic G H R H and the i r G H R H from leukocytes we have described here has not been examined. The molecular weight of i r G H R H is substantially smaller than most lymphokines, including interleukin-1 (IL-1) (17,000), which has been shown to induce several neuroendocrine hormones in vivo (Rettori et al., 1987). The effect of GH on the immune system is being extensively investigated (Kelley, 1989). It has been demonstrated that high levels of GH injected in vivo doubles both basal and lectin-induced proliferative responses from spleens of aged rats (Davila et al., 1987). Proliferative responses of both transformed (Mercola et al., 1981) and normal (Astaldi et al., 1973; Kiess et al., 1983) lymphoid cells are greater when treated with GH in vitro. GH affects the functional activity of cytolytic cells (Snow et al., 1981) and natural killer (NK) cells (Saxena et al, 1982) and has been shown to be as potent as y-interferon in priming macrophages for the production of superoxide anion (Edwards et al., 1988). All of these reports considered together strongly support a physiological role for G H in immunoregulation. In view of the low concentrations of G H R H found in the hypothalamus, it is highly debatable whether G H R H secreted from the hypothalamus is functional in peripheral plasma. Therefore, for G H R H to function in the periphery at leukocyte G H R H receptors ( K d - 2 nM; 43), it must be produced at alternate sites. Our data, along with others (Shibasaki et al., 1984; Leidy and Robbins, 1988; Meigan et al., 1988; Fernandez et al., 1989; Sasaki et al., 1989; Shut et al., 1989) show the extra hypothalamic production of G H R H . Since we know that leukocytes can function as a source of GH (Weigent et al., 1988), this suggests the possibility that G H R H synthesis by leukocytes may function as a signal for the synthesis of leukocyte-derived GH. The finding that leukocytederived G H R H is active on pituitary cells and that hypothalamic G H R H (Guarcello et al., 1989) is active on leukocytes strongly supports a functional basis for bidirectional communication.

11

Acknowledgements We thank Dr. Robert LeBoeuf for providing s y n t h e t i c o l i g o n u c l e o t i d e p r o b e s for G H R H a n d D r . K e n n e t h B o s t for p r o v i d i n g s y n t h e t i c G H R H ( 1 - 2 3 ) . W e also t h a n k J o h n E. Riley, A n d r e a H o l m e s , a n d V i n c e n t L a w for e x c e l l e n t t e c h n i c a l assistance, a n d D i a n e W e i g e n t f o r p r e p a r i n g the m a n u s c r i p t . T h i s w o r k w a s s u p p o r t e d in p a r t b y a grant from The National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases (RO1 AM38024), The National Institute of Neurology and Communicative Disorders (RO1 NS24636), a n d the C a n c e r C e n t e r C o r e g r a n t ( C A 1 3 1 4 8 ) .

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Immunoreactive growth hormone-releasing hormone in rat leukocytes.

In the present study, we evaluated whether mononuclear leukocytes could synthesize and secrete growth hormone-releasing hormone (GHRH) in vitro. By us...
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