Journal of Neuroimmunology, 33 (1991)43-54
43
© 1991 ElsevierSciencePublishers B.V. 0165-5728/91/$03.50 JNI 02012
Expression of immunoreactive growth hormone in leukocytes in vivo Judith B. Baxter, J. Edwin Blalock and Douglas A. Weigent Department of Physiology and Biophysics, Universityof A labama at Birmingham, Birmingham, A L 35294, U.S.A.
(Received23 October1990) (Revised, received8 February1991) (Accepted 8 February1991)
Key words: Growthhormone; Leukocyte;RNA; (In vivo)
Summary In the present study, we investigated the production of growth hormone (GH)-related RNA and protein in vivo by rat leukocytes after intraperitoneal treatment with different inducing agents including bacterial lipopolysaccharide (LPS) and Freund's complete adjuvant (FCA). The data showed that in rats after exposure to LPS or FCA leukocytes obtained from the spleen, thymus, and peritoneum all showed a dose-dependent increase in GH-related RNA content. The peak production of GH-related RNA was observed 48 h after treatment in the spleen and thymus and 96 h after treatment in the peritoneum. We also evaluated the ability of LPS-sensitive (C3HeB/FeJ) and resistant (C3H/HeJ) inbred mice treated with LPS to produce GH-related RNA. The LPS-sensitive mice presented with a typical pathophysiologic response pattern and higher levels of GH-related RNA in the spleen and thymus than the LPS-resistant mice. An increase in the production of immunoreactive G H (irGH) was also observed by direct immunofluorescence with specific antibodies to rat GH. We validated that the GH-related RNA produced in vivo by leukocytes was similar in structure to pituitary G H RNA using reverse transcription and the polymerase chain reaction (PCR). A sample of the PCR reaction, analyzed by gel electrophoresis, showed a single major DNA band corresponding in length (600 base pairs) to the distance between the 5'-ends of the two GH-specific primers that was specifically detected with a GH-specific probe after Southern transfer. In other studies with normal nontreated animals, the G H RNA levels are higher in the evening hours and early on in the first month of life. Taken together, our data are the first demonstration that G H RNA and immunoreactive protein can be detected in leukocytes in vivo both in normal and stimulated animals and support the idea that G H may be active in an immune response.
Address for correspondence:Dr. DouglasA. Weigent,University of Alabama at Birmingham, Department of Physiology and Biophysics, UAB Station, BHSB 894, Birmingham, AL 35294, U.S.A.
Introduction Recent studies indicate that bidirectional communication exists between the immune and neuro-
44
endocrine systems (Blalock, 1989). In this model, it has been suggested that neuroendocrine hormones may serve as signal molecules since cells of the immune system produce neuroendocrine peptide hormones. In particular, corticotropin (ACTH) and endorphins are synthesized after virus infection or interaction with transformed cells or bacterial lipopolysaccharide (Blalock and Smith, 1980; Smith and Blalock, 1981; Harbour-McMenamin et al., 1985). Chorionic gonadotropin (CG) and thyrotropin (TSH) are produced during mixed lymphocyte reactions (Harbour-McMenamin et al., 1986) and after stimulation with a T cell mitogen (Smith et al., 1983), respectively. More recently, luteinizing hormone (LH) (Ebaugh and Smith, 1988) and prolactin (PRL) (Shah et al., 1988) have also been shown to be synthesized by stimulated immune cells. More recent studies in the case of PRL indicate that the molecule found in leukocytes may have been previously internalized from bovine serum (Clevenger et al., 1990; Kenner et al., 1990). Very recently, our work (Weigent et al., 1988) and that of others (Kao et al., 1989; Hattori et al., 1990) have provided strong evidence that mononuclear leukocytes can de novo synthesize and secrete an immunoreactive growth hormone (irGH) in vitro. IrGH produced by leukocytes in vitro is similar to pituitary GH in terms of antigenicity, molecular weight, and bioactivity (Weigent et al., 1988). The factors involved in the in vitro synthesis and secretion of leukocyte irGH are under active investigation. The preliminary findings suggest both similarities and differences to the regulation of irGH in the pituitary gland. Thus, growth hormone releasing hormone (GHRH) and thyrotropin releasing hormone appear to modestly stimulate irGH production (Kao et al., 1989) similar to the regulation of pituitary GH, whereas GH added during the culture of phytohemagglutinin-A stimulated cells augmented irGH secretion unlike the regulation of pituitary GH (Hattori et al., 1990). Our own recent findings show that leukocytes also de novo synthesize an irGHRH and suggest a possible autocrine mechanism of stimulation of irGH synthesis by immune cells (Weigent and Blalock, 1990a). Although no data are available on leukocyte irGH production in vivo, some data are published concerning cytokine effects on
pituitary GH secretion. GH secretion is enhanced by interleukin (IL)-2 therapy in cancer patients (Atkins et al., 1986) and by intracerebroventricular (i.c.v.) injection of IL-1 in rats (Rettori et al., 1987). Intravenous tumor necrosis factor a appears to suppress GH secretion (Elsassar et al., 1989). In another study, i.c.v, or arcuate nucleus injection of IL-lj3 suppressed rat GH levels suggesting an inhibition of hypothalamic GHRH a n d / o r stimulation of hypothalamic somatostatin release (Lumpkin and Hartmann, 1989). The release of GH by a direct action of IL-1 on pituitary cells has also been reported (Bernton et al., 1987). Although many studies have addressed the ability of the immune system to control pituitary GH secretion, no studies have been reported on the effect of immune activation in the control of leukocyte-derived irGH in vivo. Therefore, in the present study, we have attempted to determine if irGH production by leukocytes is detectable in vivo after intraperitoneal injection with substances known to stimulate leukocytes. The data reported here show for the first time the synthesis of GH RNA and protein in vivo from leukocytes of treated animals and support the idea that GH may be active in an immune response.
Materials and methods
Cell preparations Adult (150-200 g, 4-8 weeks old) male Sprague-Dawley rats were obtained from Harlan (Prattville, AL, U.S.A.). LPS-sensitive C3HeB/FeJ and LPS-resistant C 3 H / H e J male mice (6 weeks old) were obtained from The Jackson Laboratories (Bar Harbour, ME, U.S.A.). Animals were injected in the early morning or late afternoon. Following sacrifice, the spleens and thymi were immediately collected, teased with sterile groundglass-edged slides and frozen in microfuge tubes for RNA isolation. Peritoneal exudate cells were collected by standard techniques (Weir, 1978) into RPM| containing heparin, centrifuged, and quickly frozen. Greater than 95% cell viability by trypan blue exclusion was observed in all cell fractions (Kruse and Patterson, 1973). Rat pituitaries were carefully removed and washed in phosphatebuffered saline (PBS), quickly frozen, and the
45 RNA isolated as described below. Rat pituitary GH3 cells used for some of the studies reported here were kindly provided by Dr. J. Mulchahey of the University of Alabama at Birmingham. RNA &olation 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 measurements at 260 and 280 nm. The RNA (10/~g) was blotted on nitrocellulose membranes with the Minifold II slot blotter (Schleicher and Schuell, Keene, NH, U.S.A.) as described by the manufacturers. After hybridization with a GH-specific cDNA probe as described below, the membranes were washed with 0.16 M NaCI, 14 mM sodium citrate and 0.1% sodium dodecyl sulfate (SDS) at 100°C for 30 min to elute the cDNA probe. The membrane was then hybridized to a synthetic oligonucleotide 18 S ribosomal probe to detect differences in the amount of RNA bound to the membrane. Small corrections determined by proportion were applied to the densitometric GH eDNA data according to the amount of RNA bound to the nitrocellulose determined after probing with the 18 S ribosomal probe. Total RNA was prepared by homogenizing leukocytes in 5 M guanidine thiocyanate, 1% sarkosyl, 20 mM EDTA, 1% 2mercaptoethanol, 50 mM Tris-HC1, pH 7.5, with subsequent protease K digestion and extraction with phenol/chloroform (Maniatis et al., 1982). For PCR, this RNA was purified through oligo-dT columns prior to first-strand eDNA synthesis (Maniatis et al., 1982). GH eDNA and hybridization Plasmids containing specific rat (Seeburg et al., 1977) GH eDNA 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). 800 base-pair HindIII inserts (nucleotides - 2 0 to 780) were purified from these plasmids and labeled with [32p]dCTP by nick translation (Bethesda Research Laboratories, Rockville, MD, U.S.A.) to a
specific activity of 1-2 x 108 cpm//~g. Synthetic oligonucleotide probes were prepared and purified in our laboratory by Dr. Robert LeBoeuf. The GH probes used to identify the RNA encoding the 20 kDa and 22 kDa protein, correspond to amino acids 1-7 and 32-46 and the mouse 18 S ribosomal probe corresponds to amino acids 10561067. The synthetic oligonucleotide probes were end-labeled with T4 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 insert (2 x 106 cpm/ml) (Maniatis et al., 1982). 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. The autoradiographs were analyzed using a GS300 densitometer (Hoefer Scientific Instruments). Reuerse transcription and amplification by polymerase chain reaction First-strand eDNA synthesis was performed using a commercially available reagent kit (Amersham) that is based on the procedure of Okyama and Berg (1982). Target sequences from this eDNA (3 /xl from a total reaction volume of 30 #1) were amplified (30 cycles) in a polymerase chain reaction (PCR) using the antisense primer (corresponding to amino acids 180-187) (1 fig) and a sense primer (corresponding to amino acids 4-11) (1/~g) by standard procedures as described (LeBoeuf et al., 1989). The primers were prepared and purified in our laboratory. PCR reactions are performed in a Perkin-Elmer DNA thermal cycler. Generally, reactions are performed in a total volume of 0.1 ml containing 200 /~M of each dNTP, 1 /~g of each primer, 1-10 ng of template DNA, and 2.5 U of Taq polymerase. The final reaction mixture is overlaid with 0.1 ml of mineral oil (Perkin Elmer) to prevent evaporation. A usual cycle consists of 1 min at 94 °C (denaturation), 2 min at 52°C (annealing of primer) and 3 min at 72 °C (extension). 25-40 cycles (7 rain total/cycle) are usually run over a 3-5 h period. Excess primers are separated from amplified DNA on a Sephadex
46 G50 spin column (DuPont). Amplified samples are then run on a 2% agarose gel and stained with ethidium bromide to determine efficiency and size. A control template and primers derived from bacteriophage A which define a 500 bp target is always run to assure reliability of the procedure (Saiki et al., 1985; Scharf et al., 1986). To confirm that the G H cDNA was specifically amplified, a Southern transfer was performed as previously described (Maniatis et al., 1982). The membrane was hybridized to the radiolabeled GH-specific c D N A probe as described above.
Direct imrnunofluorescence Cells were obtained from the spleen, thymus, pituitary, and peritoneal exudate from normal rats or from rats sacrificed 48 h or 96 h after injection with 5 ml FCA by standard procedures. The cells were washed 3 times in 0.01 M PBS, p H 7.2, resuspended in PBS (1 x 10 6 cells/ml) and airdried onto glass slides. After fixation with ice-cold 95% ethanol, the cells were rehydrated in PBS and incubated with monkey serum (1 : 200) for 1 h at 37 ° C. The cells were washed 3 times with PBS and then covered in a 1 : 50 dilution of fluorescein isothiocyanate (FITC)-conjugated monkey anti-rat G H prepared in our laboratory by standard techniques (Johnstone and Thorpe, 1987). The slides were allowed to incubate 1 h at 37 ° C followed by washing 3 times in PBS and rinsing in distilled water. To test the specificity of the FITC-conjugated antibody, the labeled antibody and excess rat G H were mixed and incubated at 4 ° C for 2 h before addition to cells. The cells were observed using an Olympus vertical fluorescence illuminator model A-RFL (Marietta, GA, U.S.A.). Materials Monkey anti-rat G H antiserum was generously donated by Dr. Raiti of the National Hormone and Pituitary Program (National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases, NIH, Bethesda, MD, U.S.A.). The antiserum is highly specific and shows < 0.0001% crossreactivity to other anterior pituitary hormones according to the suppliers. The rat G H we used was from the Pituitary Hormones and Antisera Center of Torrance, CA, U.S.A. (Dr. A.F. Parlow) for radioimmunoassay (RIA) and judged to be
contaminated with < 0.1% of the anterior pituitary hormones as determined by RIAs. The other chemicals used in these experiments were obtained from the Sigma Chemical Company.
Data analysis Each data point from the experiments represents replicate samples from 3-5 rats. Each experiment was replicated at least 3 times. Data are presented as mean + SEM. Rat pituitary (5 /~g/slot) was used as a standard in each probing experiment to normalize the values in experimentto-experiment variation. Differences between groups were determined by the Student t-test and Mann-Whitney test where differences are indicated as being significant, p is less than 0.05.
Results
Leukocyte GH production in rats We have previously described the in vitro production of bioactive irGH from human and rat leukocyte populations (Weigent et al., 1988). In the present studies, we wanted to determine whether the leukocyte-derived irGH could be produced in vivo in response to chemical agents known to stimulate immune cells. Our approach was to inject rats intraperitoneally with the agents listed in Table 1, sacrifice animals after 48 h and immediately freeze the lymphoid tissues. The cytoplasmic R N A was isolated, quantitated, and slotted onto nitrocellulose paper and subsequently probed with a rat pituitary G H specific cDNA. The results shown in Table 1 are presented as the percent of control, where the control animals were nontreated. The data show that animals treated with sheep red blood cells (SRBC), Escherichia coli (HB 101), FCA and purified lipopolysaccharide (LPS) all showed elevated levels (1.7- to 2.0-fold) of GH-related R N A in the spleen 48 h after treatment. The thymus also showed elevated levels (1.5- to 1.7-fold) after treatment compared to the control nontreated animals. The percentage of G H R N A obtained from leukocytes from noninjected animals showed no difference to animals injected with 5 ml PBS. Fig. 1 shows the effect of increasing amounts of intraperitoneal injection of FCA on GH-related R N A levels. Groups of
47 TABLE 1 LEUKOCYTE-DERIVED GH-RELATED RNA PRODUCTION IN RATS Treatment a
Noninjected PBS SRBC E. coli
Freund's complete adjuvant Lipopolysaccharide a
GH RNA b Percent of control Spleen
Thymus
100-4-_17 111+14 184+_ 9 220_+26 196 5:22 271 _+52
100 5:12 127-+10 153_+ 7 219_+29 247 -+31 223 5:41
Rats were injected i.p. with PBS (5 ml); SRBC (5 × 10l°); E. coli (1>(109); FCA (5 ml); LPS (1 mg/kg), or were non-
treated. Animals were sacrificed 48 h after injection and spleens and thymi immediately frozen. b Cytoplasmic RNA from spleen and thymus cells was isolated, slotted onto nitrocellulose, and probed as described in Materials and Methods. After a 48 h exposure to X-ray film, the autoradiograph was scanned by a densitometer and is presented as the percent of control as described in Materials and Methods. The data shown represents the results from four experiments + standard error of the mean. All values are significant at p < 0.05 except for the values obtained from leukocytes of rats injected with PBS.
a n i m a l s were injected with different a m o u n t s of F C A a n d the spleen a n d t h y m u s h a r v e s t e d f r o m o n e group of a n i m a l s 48 h after injection. Perit o n e a l e x u d a t e cells were o b t a i n e d f r o m a n o t h e r g r o u p of a n i m a l s 96 h after injection. The d a t a show that the p r o d u c t i o n of G H - r e l a t e d R N A in the spleen, thymus, a n d p e r i t o n e u m was dose-dependent. Fig. 2 shows the levels of G H R N A in spleen a n d p e r i t o n e a l e x u d a t e cells in rats at specific time p o i n t s after injection of F C A . C y t o p l a s m i c R N A was isolated from all samples at the e n d of the experiment, p r o b e d with the specific G H c D N A insert, a n d the results p r e s e n t e d as the p e r c e n t of control. The d a t a show that significant G H - r e l a t e d R N A p r o d u c t i o n f r o m the spleen of rats was o b s e r v e d at the 48 h time p o i n t after injection of F C A . Similar data, with a p e a k at 48 h, were o b t a i n e d for G H - r e l a t e d R N A p r o d u c t i o n b y the t h y m u s ( d a t a n o t shown). O n the o t h e r hand, p e a k p r o d u c t i o n of G H - r e l a t e d R N A was n o t o b s e r v e d in p o p u l a t i o n s of p e r i t o n e a l e x u d a t e cells until 96 h after the injection of F C A . T h e d a t a show that l e u k o c y t e - d e r i v e d G H - r e l a t e d R N A p r o d u c t i o n was d e t e c t a b l e earlier in b o t h the
spleen a n d t h y m u s t h a n in the p e r i t o n e u m after i n t r a p e r i t o n e a l injection o f F C A . Since o u r d a t a suggested that the leukocytes f r o m a n i m a l s t r e a t e d with F C A p r o d u c e G H - r e l a t e d R N A in vivo, we d e t e r m i n e d if the message was expressed b y m e a s u r i n g the presence of cytop l a s m i c i r G H b y direct i m m u n o f l u o r e s c e n c e . Table 2 shows the results of direct fluorescent meas u r e m e n t s on spleen, t h y m u s , p e r i t o n e a l exudate, a n d p i t u i t a r y cells f r o m b o t h n o n t r e a t e d rats a n d f r o m rats injected with F C A . T h e spleen, thymus, a n d p e r i t o n e a l e x u d a t e cells were l a b e l e d with a F I T C - c o n j u g a t e d a n t i b o d y to rat G H . The results show an elevated level of fluorescence in cells
350
300
_
250
200
o
~5o 100 50 [
0
1
I
I
I
I
2
3
4
5
FCA (mL)
Fig. 1. Induction of GH-related RNA in rats injected with FCA. Male (150 g) Sprague-Dawley rats were each injected intraperitoneally with 1, 2, 3, 4 or 5 ml of FCA or sterile PBS. Groups of animals (five/group) were' sacrificed and cytoplasmic RNA isolated from the spleen and thymus at 48 h and peritoneal exudate cells from another group at 96 h. The RNA was slotted onto nitrocellulose, and probed as described in Materials and Methods. Data points represent the percent of control of average densitometric scans+ SEM obtained from five individual animals slotted in 'triplicate. The average densitometric control values were as follows: spleen, 3.8_+0.5; thymus, 7.3_+1.5; peritoneal exudate, 22.8-+4.2. The data shown above are typical of a single experiment performed three times. Key: (@) spleen; (O) thymus; (A) peritoneal exudate cells; * significant at p < 0.05 except for the value for thymus cells in rats injected with 3 ml of FCA which is significant at p < 0.1.
48
250
200 0 I,. tO
rj
~6 O
150
100
0.
Leukocyte production of GH-related RNA during endotoxic shock
_Z I
50
0
0
I
I
24
48
I
72 Time (h)
L
I
96
120
Fig. 2. Kinetics of induction of GH-related R N A in rats injected with FCA. Groups of animals (five/group) were injected intraperitoneally with F C A (4 ml) or PBS (4 ml) at 0 h. Animals were sacrificed at the indicated time points and the spleen and peritoneal exudate cells harvested and frozen as pellets at - 2 0 o C. After 120 h, the R N A was isolated from the replicate samples and slotted onto nitrocellulose and probed as described in Materials and Methods. Data points represent the percent of control of average densitometric scans + SEM. The average densitometric value for the spleen control was 25.7 +_5.5 and the peritoneal exudate control value was 20.7+4.8. The data shown are from a typical experiment performed 3 times. Key: (O) spleen; (A) peritoneal exudate cells; * significantly different from control at p < 0.05.
obtained from FCA treated animals as compared to cells from nontreated controls. Approximately 20% of the dispersed pituitary cells were labeled positive for GH as expected. An example of a microscopic field containing fluorescent peritoneal exudate cells labeled with the FITC-conjugated antibody specific to rat G H is shown in Fig. 3A. Similar fields were observed with cells obtained from the spleen and thymus (data not shown). Pituitary cells labelled with the same antisera are shown in Fig. 3B. The labeling of pituitary and immune cells could be blocked by prior incubation of the FITC-conjugated antibodies with excess rat GH (data not shown). Taken together, the data show that rat leukocytes stimulated by FCA produce an irGH in vivo.
We next studied the production of leukocyte irGH in mice to test whether there might be differential synthesis of irGH in response to LPS in LPS-sensitive C3HeB/FeJ and LPS-resistant C 3 H / H e J mice. In previous studies, we have shown that leukocytes from rats and mice are comparable in that the GH-related RNA levels increase in leukocytes after removal from animals. It has been shown by others that C 3 H / H e J mice do not exhibit the pathophysiologic changes seen in endotoxic shock (Haas et al., 1978; Morrison and Ryan, 1979). We hypothesize that the ability to produce irGH may influence the response characterized by LPS-sensitive mice. Therefore, we injected groups of LPS-sensitive and -resistant mice with LPS and monitored the pathophysiologic changes and GH-related RNA content 6 h after injection. The LPS-injected C3H e/ FeJ had reduced respiration rates, diarrhea, a white discharge from the eyes, and lethargy while the LPSresistant C 3 H / H e J did not manifest any pathophysiologic abnormalities (data not shown). Fig. 4 shows the results of GH-related RNA levels in
TABLE 2 D E T E C T I O N OF irGH P R O D U C T I O N IN L E U K O C Y T E S BY D I R E C T I M M U N O F L U O R E S C E N C E Source of cells a
Time of harvest
Percent positive by immunofluorescence
Spleen
0 48 0 48 0 96 0 48 96
3.9+_ 1.2 10.4_+ 1.5 6.9 + 1.3 13.5_+1.6 16.3 _+1.5 30.1 _+4.3 21.3 +_2.4 26.8 _+2.1 25.3_+2.3
Thymus Peritoneal exudate Pituitary
h h h h h h h h h
a Rats were injected i.p. with 5 ml F C A and sacrificed either 48 h or 96 h later. The cells were washed 3 times in PBS and 10 ALl of 1 × 106 cells/ml were fixed onto glass slides and labeled with FITC-conjugated monkey anti-rat G H antibodies as previously described in Materials and Methods. The slides were washed 3 times in PBS before determining the percentage of cells positive for G H production. 500 cells from each group were counted from randomly chosen fields. Data is presented as the percent of positive cells + standard error of the mean.
49
A
@
Analysis of GH RNA by reverse transcription and polymerase chain reaction Our previous work has shown the presence of GH-related mRNA molecules spontaneously expressed in rat spleen and thymus cells in vitro (Weigent et al., 1988). To confirm that the leukocyte GH-related RNA produced after in vivo stimulation is similar to the leukocyte GH-related RNA produced in vitro, we selectively enriched
B 300
250 o
0
200
"E a) Fig. 3. Fluorescent photomicrographs of cells stained with FITC-conjugated antibodies to rat GH. Cells from both nontreated rats and rats injected i.p. with FCA (5 ml) were washed 3 times in PBS and resuspended at a concentration of 1 x 106 cells/ml. The cells were air-dried to glass slides and fixed in 95% ethanol. The cells were then labeled with FITC-conjugated antibody to rat GH. A: Peritoneal exudate cells from rats injected with FCA and sacrificed 96 h after injection. B: Pituitary cells from rats injected with FCA. Magnification 400 X.
o
150 ~
III
I"~
I:L
100
so L 0
I
I
0
5
I
I
10
20
LPS (mg/kg)
LPS-sensitive and resistant groups of mice 6 h after injection with various doses of LPS. The results show that at doses between 5 and 20 mg/kg mouse, the LPS-sensitive C3HeB/FeJ animals produced higher levels (2-fold) of GH-related RNA than LPS-resistant animals in the spleen and thymus. The thymus in both groups of animals produced less GH-related RNA than the spleen tissues and in the LPS-resistant C3H/HeJ mice the levels of thymus GH-related RNA were significantly lower than control animals. The lower level of GH-related RNA in the thymus found in LPS-resistant animals is unexplained.
Fig. 4. Induction of GH-related RNA in mice injected with LPS. C 3 H e B / F e J (LPS-sensitive) and C 3 H / H e J (LPS-resistant) mice were injected intraperitoneally with various doses of LPS as indicated or sterile PBS. Groups of animals (five/group) were sacrificed after 6 h and the spleen and thymus cells immediately frozen. RNA was isolated, slotted, and probed with a rat GH cDNA as described in Materials and Methods. Data points represent the percent of control of average densitometric scans + SEM obtained from five individual animals slotted in triplicate. The controls are the spleen and thymus cells from animals of the same species sacrificed 6 h after injection with sterile PBS. The average densitometric s c a n + S E M for each control is listed in the key. The data shown above are typical of a single experiment performed 3 times. Key: (e) C 3 H e B / F e J (sensitive) spleen, control value, 8.2+1.90; ( O ) C 3 H e B / F e J (sensitive) thymus, control value, 16.0+ 1.3; (I), C 3 H / H e J (resistant) spleen, control value, 4.0 + 0.4; (13), C 3 H / H e J (resistant) thymus, control value, 43.8 + 8.6; * significantly different from control at p < 0.05.
50 the c o n c e n t r a t i o n of G H - s p e c i f i c n u c l e o t i d e sequences b y reverse t r a n s c r i p t i o n a n d P C R so that they c o u l d be o b s e r v e d b y c o n v e n t i o n a l gel electrophoresis. F o r this e x p e r i m e n t , total R N A was e x t r a c t e d f r o m the t h y m u s of c o n t r o l a n d LPStreated rats, a n d c o n t r o l G H , p i t u i t a r y cells. T h e m R N A was reverse t r a n s c r i b e d a n d then a m p l i fied b y P C R using two o l i g o n u c l e o t i d e p r i m e r s specific for rat p i t u i t a r y G H . R N A was used as a t e m p l a t e for selective first-strand c D N A synthesis with an anti-sense o l i g o n u c l e o t i d e p r i m e r c o m p l e m e n t a r y to the 3' end of exon 5 of G H m R N A . W h e n this purified c D N A is i n t r o d u c e d as a temp l a t e into a P C R with a second-sense oligonucleotide p r i m e r r e p r e s e n t i n g the G H sequence, ten bases 5' to the G H c o n t a i n i n g region of exon 1, there is a m p l i f i c a t i o n of the a p p r o p r i a t e - s i z e d (600 bp) c D N A fragment. T h e p o t e n t i a l p r o b l e m of a m p l i f y i n g g e n o m i c D N A is c o n s i d e r e d unlikely since we have used o l i g o - d T p u r i f i e d R N A a n d p r i m e r s for two different exons for the first-strand synthesis a n d P C R , respectively. T h e selective a m p l i f i c a t i o n of a G H - r e l a t e d target c D N A sequence was c o n f i r m e d b y S o u t h e r n analysis (Fig. 5). Since the P C R reactions were not set up to b e
LANE
q u a n t i t a t i v e , n o c o n c l u s i o n s can be d r a w n a b o u t the relative a m o u n t of G H - r e l a t e d R N A in the original sample. I n a d d i t i o n to the a b o v e study, we have asked the q u e s t i o n w h e t h e r l e u k o c y t e s e n c o d e an R N A for a p r e v i o u s l y d e s c r i b e d smaller m o l e c u l a r weight v a r i a n t f o r m of G H . A v a r i a n t f o r m of G H with a m a s s of 20 k D a , l a c k i n g residues 3 2 - 4 6 of 22 k D a G H has b e e n i d e n t i f i e d as b e i n g g e n e r a t e d b y an a l t e r n a t i v e splicing m e c h a n i s m ( M a s u d a et al., 1988). By using o l i g o n u c l e o t i d e p r o b e s c o m p l e m e n t a r y to the c o d i n g regions of a m i n o acids 1 - 7 a n d a m i n o acids 3 2 - 4 6 of 22 k D a G H ( 1 - 2 x 10 s c p m / t ~ g ) , we tested w h e t h e r the G H - r e l a t e d R N A f r o m l e u k o c y t e s c o n t a i n e d the sequence i n f o r m a tion d e l e t e d f r o m the 20 k D a variant. T h e results ( T a b l e 3) show that there was no significant difference in the h y b r i d i z a t i o n to the R N A utilizing the same n u m b e r of c p m for b o t h o l i g o n u c l e o t i d e probes. T h e d a t a suggest t h a t the m a j o r a m o u n t of l e u k o c y t e G H - r e l a t e d R N A a p p e a r s to c o n t a i n the sequence i n f o r m a t i o n e n c o d i n g a m i n o acids 3 2 - 4 6 which c o r r e s p o n d s to the 22 k D a G H .
Endogenous regulation of leukocyte irGH Several r e p o r t s i n d i c a t e that rat serum G H levels fluctuate m a r k e d l y d u r i n g a 24 h p e r i o d
I 2 3
TABLE 3 ANALYSIS OF CYTOPLASMIC GH-RELATED RNA EXPRESSION IN VIVO Source of leukocytes
-~600 bp
Fig. 5. Confirmation of selective amplification of rat GH-related RNA by Southern analysis. The Southern analysis and hybridization with a radiolabeled GH-specific probe were carried out as described in Materials and Methods. Lane 1, thymus control; lane 2, thymus from rat treated with LPS (30 mg/kg) 6 h prior to sacrifice; lane 3, GH 3, pituitary control.
a
Spleen Thymus Peritoneal exudate cells
Time of harvest
GH RNA b Densitometric units
(h)
Probe 1-7 amino acids
Probe 32-46 amino acids
48 48
64 _+4 72 _+5
59 _+4 70 + 4
96
75 _+6
71 + 5
Rats were injected i.p. with FCA (5 ml) and one set of animals sacrificed 48 h later and another set at 96 h after treatment. Animals were sacrificed as described and tissues frozen. Total cytoplasmic RNA from spleen cells was isolated, slotted onto nitrocellulose, and probed as described in Materials and Methods. After a 48 h exposure to X-ray film, the autoradiograph was scanned by a densitometer as described in Materials and Methods. The data shown represents the results from four experiments.
51 detectable changes in leukocyte GH-related RNA production between 4 and 36 weeks after birth and between the sexes (data not shown).
50
4O
i.~
3O
~
2o
Discussion &
10
0
112 1
2:3
4 5 6 7 8 9 1011112 1 2 3 4 5 6 7 8 9 10111 PM AM Time(h)
Fig. 6. Analysis of the leukocyteGH-related RNA rhythm in the light-dark condition. Two animals were sacrificed every hour over a 24 h time period. At each time point, the spleen was quickly frozen at -20 ° C. After 24 h, the RNA was isolated from the replicate animals and slotted in triplicate and probed as described in Materials and Methods. The data shown are from a typical experimentperformedtwice. suggesting an ultradian rhythm with a periodicity of approximately 3.3 h most likely synchronized to a light-dark cycle (Takahashi et al., 1971; Scheving and Turpen, 1974; Tannenbaum and Martin, 1976). To investigate the possibility of a rhythm controlling leukocyte-derived GH, we sacrificed nontreated animals every hour for a period of 24 h and determined their content of cytoplasmic GHrelated RNA. The results (Fig. 6) show that the levels of spleen GH-related RNA were 2- to 4-fold higher during the dark phase of a 12 h light-dark cycle. Similar findings were observed in the regulation of thymus GH-related RNA (data not shown). In both man and laboratory animals, it is also known that episodic G H secretion is maximal during adolescence and early adulthood and diminishes as individuals age (Finklestein et al., 1972; Sonntag et al., 1980; Takahashi et al., 1987). We investigated whether the production of leukocyte GH-related RNA is reduced in aged rats or undergoes any alteration with age. A group of animals were sacrificed every week for 4 weeks and every fourth week thereafter for GH-related RNA determinations in the spleen and thymus. The results showed that significantly higher levels of GH-related RNA were found within the first month of life in the spleen and thymus where a 4-fold increase in GH-related RNA was observed in the third and fourth week of life. There were no
In the present studies, we have shown that spleen and thymic leukocytes and peritoneal exudate cells produce higher levels of GH-related RNA and protein in vivo when stimulated with LPS or FCA. The increase in GH-related RNA was dose-dependent with peak production in the spleen and thymus observed 48 h after treatment. The peak increase in GH-related RNA was not observed in peritoneal exudate cells until 96 h after treatment. Our present data do not permit us to define the mechanism for the delayed peritoneal response. One can speculate on several possible mechanisms. For example, leukocytes from the spleen a n d / o r thymus which are already producing irGH, may redistribute to the peritoneum. A second possible mechanism may be that different G H inducer substances may stimulate irGH production in leukocytes at different sites. The scenario is obviously complex and more studies are required to identify the mechanism for the delayed peritoneal response. In other results reported here, we have obtained data from reverse transcription and PCR that support the idea that GH-related RNA produced in vivo from leukocytes is structurally similar to the pituitary G H RNA. RNA from the thymus and the GH, pituitary cell line were both used to synthesize a 600 bp c D N A that reacted with a specific rat G H c D N A after Southern transfer. The idea that genomic D N A was amplified rather than R N A is considered to be highly unlikely. This is due to the fact that oligo-dT purified RNA was used in the synthesis of first strand and the primers for PCR were designed from two different exons. The same RNA could be hybridized to a specific oligonucleotide complementary to the sequence of R N A specifying the deletion in 20 kDa GH. This result suggests that the major product of translation of leukocyte GH-related RNA may be the 22 kDa species. The data do not rule out the presence of the 20 kDa species of GH. The data we have obtained strongly support the existence of
52 GH-related RNA induced in leukocytes. Additional studies by direct immunofluorescence using an FITC-conjugated antibody to rat G H have verified the translation of the GH-related RNA in vivo. Previous studies have shown that immune stimulation with LPS or cytokine administration in vivo is associated with changes in pituitary hormone secretion. In general, there is a stimulation of ACTH, inhibition of TSH, species-dependent effects on G H and modest effects on prolactin (review, see Scarborough, 1990). The G H response to endotoxin in both rat and humans is the same as the G H response to stress, that is, an increase in humans and a decrease in the rat (Kasting and Martin, 1982). It was, therefore, somewhat surprising in the rat that the leukocyte response to LPS was an increase in GH-related RNA. It has been reported that mediators such as IL-1 are produced in large quantities by LPStreated macrophages and play an important role in endotoxic shock (Beutler et al., 1986). Recently, it has also been reported that IL-1 stimulates the release of pituitary G H (Bernton et al., 1987; Rettori et al., 1987). We investigated the effect of LPS on the leukocytes from the LPS-sensitive and -resistant mice and showed that the mice also increase the production of GH-related RNA after LPS treatment in vivo. However, the C 3 H / H e J resistant mice injected with LPS did not manifest the pathophysiologic abnormalities associated with endotoxic shock and produced significantly lower levels of GH-related RNA after treatment compared with the C3HeB/FeJ-sensitive mice. It is tempting to speculate that the defect in LPS-resistant mice which results in lower levels of IL-1 also prevents the induction of GH-related RNA. However, more studies are needed to define the mechanism of reduced GH-related RNA production in LPS-resistant mice. Finally, we have presented data that suggest the regulation of leukocyte irGH may be under an endogenous 24 h rhythm as well as decrease with the onset of adulthood. These results are similar to published data measuring serum levels of G H (Tannenbaum, 1976; Millard et al., 1990) over a 24 h time period .and in aged rats. The present results do not explain the mechanism of induction of leukocyte-derived irGH or the cell types in-
volved in vivo. Our previous in vitro findings on the spontaneous production of irGH from cells separated by adherence, nylon wool columns, and positive and negative sorting with monoclonal antibodies that define B, monocyte, T helper and T cytotoxic cells show that several different cell types have the ability to produce GH-related m R N A (Weigent and Blalock, 1990b). The results suggest that B cells and macrophages produce more GHrelated m R N A than T cells. Taken together, the in vitro data suggest there is heterogeneity within leukocytes regarding their ability to produce irGH and more studies are required to identify G H producer cell types in vivo after stimulation. The significance of irGH release by leukocytes during intraperitoneal injection of LPS is not clear. Since G H is involved in substrate metabolism, the results may suggest that G H facilitates the increase in energy utilization during fever. On the other hand, G H may promote a host defense mechanism. It has been demonstrated that high levels of G H injected in vivo double both basal and lectin-induced proliferative responses from spleens of aged rats (Davila et al., 1987; Kelley, 1989). 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 G H in vitro. G H affects the functional activity of cytolytic cells (Snow et al., 1981) and N K cells (Saxena et al., 1982) and has been shown to be as potent as "/-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. We anticipate that future studies on leukocyte-derived irGH will help explain its effects on the immune response. Finally, the postulated sensory role that the immune system plays in responding to noncognitive stimuli (LPS) coupled with the role of G H in the immune system provides a molecular mechanism whereby a physiological response is associated with infectious disease.
Acknowledgements We thank Dr. Robert LeBoeuf for providing the synthetic oligonucleotides and Dr. J. Mulcha-
53 h e y for h e l p o n t h e i m m u n o f l u o r e s c e n t studies. 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 for p r e p a r i n g t h e m a n u s c r i p t . T h i s w o r k was s u p p o r t e d in p a r t b y g r a n t s f r o m The National Institute of Neurology and Comm u n i c a t i v e D i s o r d e r s (R01 N S 2 4 6 3 6 ) a n d T h e N a t i o n a l I n s t i t u t e of A r t h r i t i s , D i a b e t e s , D i g e s t i v e a n d K i d n e y D i s e a s e s (R01 D K 3 8 0 2 4 ) .
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Finklestein, J.W., Roffwarg, H.P., Boyar, R.M., Kream, J. and Hellman, L. (1972) Age-related change in the twenty-fourhour spontaneous secretion of growth hormone. J. Clin. Endocrinol. Metab. 35, 665-670. Haas, G.P., Johnson, A.G. and Nowotny, A. (1978) Suppression of the immune response in C3H/HeJ mice by protein-free lipopolysaccharides. J. Exp. Med. 148, 10811086. Harbour-McMenamin, D.V., Smith, E.M. and Blalock, J.E. (1985) Bacterial lipopolysaccharide induction of leukocytederived ACTH and endorphins. Infect. Immun. 48, 813817. Harbour-McMenamin, D.V., Smith, E.M. and Blalock, J.E. (1986) Production of chorionic gonadotropin in a mixed leukocyte reaction: a possible selective mechanism for genetic diversity. Proc. Natl. Acad. Sci. U.S.A. 83, 68346838. Hattori, N., Shimatsu, A., Sugita, M., Kumagai, S. and Imura, H. (1990) Immunoreactive growth hormone (GH) secretion by human lymphocytes: augmented release by exogenous GH. Biochem. Biophys. Res. Commun. 168, 396-401. Johnstone, A. and Thorpe, R. (1987) Immunochemistry in Practice, 2nd edn., Blackwell Scientific Publishing, London. Kao, T.-L., Harbour, D.V., Smith, E.M. and Meyer, W.J. (1989) Immunoreactive growth hormone production by cultured lymphocytes. 71st Annual Meeting of the Endocrine Society, p. 108 (Abstract). Kasting, N.W. and Martin, J.B. (1982) Altered release of growth hormone and thyrotropin induced by endotoxin in the rat. Am. J. Physiol. 243, E332-E337. Kelley, K.W. (1989) Growth hormone, lymphocytes, and macrophages. Biochem. Pharmacol. 35, 705-713. Kenner, J.R., Holaday, J.W., Bernton, E.W. and Smith, P.F. (1990) Prolactin-like protein in murine splenocytes: morphological and biochemical evidence. Prog. Neuro-Endocr. Immunol. 3, 188-195. Kiess, W., Holtmann, H., Buteriandt, O. and Eife, R. (1983) Modulation of lymphoproliferation by human growth hormone. Eur. J. Pediatr. 140, 47-50. Kruse, Jr., P.F. and Patterson, M.K. (Eds.) (1973) Tissue Culture: Methods and Applications, Academic Press, New York. LeBoeuf, R.D., Galin, F.S., Hollinger, S.K., Peiper, S.C. and Blalock, J.E. (1989) Cloning and sequencing of immunoglobulin variable region genes using degenerate oligonucleotides and the polymerase chain reaction. Gene 82, 371-377. Lumpkin, M.D. and Hartmann, D.P. (1989) Recombinant human interleukin-lfl acts within the hypothalamic arcuate nucleus to inhibit pulsatile growth hormone secretion. 71st Annual Meeting of the Endocrine Society, Abstract 789. Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Masuda, N., Watahiki, M., Tanaka, M., Yamakawa, M., Shimizu, K., Nagai, J. and Nakashima, K. (1988) Molecular cloning of cDNA encoding 20 kDa variant human GH and the alternative splicing mechanism. Biochim. Biophys. Acta 949, 125-131.
54 Mercola, K.E., Cline, M.J. and Golde, D.W. (1981) Growth hormone stimulation of normal and leukemic human Tlymphocyte proliferation in vitro. Blood 58, 337-340. Millard, W.J., Ramano, T.M. and Simpkins, J.W. (1990) Growth hormone and thyrotropin secretory profiles and provocative testing in aged rats. Neurobiol. Aging 11,229235. Morrison, C. and Ryan, J.L. (1979) Bacterial endotoxins and the immune response. Adv. Immunol. 28, 293-450. Okayama, H. and Berg, P. (1982) High efficiency cloning of full-length cDNA. Mol. Cell. Biol. 2, 161-170. Rettori, V., Jurcovicova, J. and McCann, S.M. (1987) Central action on interleukin-1 in altering the release of TSH, growth hormone, and prolactin in the male rat. J. Neurosci. Res. 18, 179-183. Saiki, R.K., Scharf, S., Faloona, F., Mullis, K.B., Horn, G.T., Erlich, H.A. and Arnheim, N. (1985) Enzymatic amplification of B-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230, 1350-1354. Saxena, Q.B., Saxena, R.K. and Adler, W.H. (1982) Regulation of natural killer activity in vivo. IlL Effect of hypophysectomy and growth hormone treatment on the natural killer activity of the mouse spleen cell population. Int. Arch. Allergy Appl. Immunol. 67, 169-174. Scarborough, D.E. (1990) Cytokine modulation of pituitary hormone secretion. In: Neuropeptides and Immunopeptides: Messengers in a Neuroimmune Axis. Ann. N.Y. Acad. Sci. 495, 169-187. Scharf, S.J., Horn, G.T. and Erlich, H.A. (1986) Direct cloning and sequence analysis of enzymatically amplified genomic sequences. Science 233, 1076-1078. Scheving, L.E. and Turpen, C. (1974) Daily variation in rat GH concentration and the effect of stress on periodicity. Neuroendocrinology 13, 69-78. Seeburg, P.H., Shine, J., Martial, J., Baxter, J.D. and Goodman, H.M. (1977) Nucleotide sequence and amplification in bacteria of a structural gene for rat growth hormone. Nature 270, 486-494. Shah, G.N., Montgomery, D.W., Sredzinski, J.A. and Russell, D.H. (1988) Characterization of a novel prolactin synthe-
sized by immunologically stimulated lymphocytes. Fed. Am. Soc. Exp. Biol. Abstract 1354, A528. Smith, E.M. and Blalock, J.E. (1981) Human lymphocyte production of ACTH and endorphin-like substances: association with leukocyte interferon. Proc. Natl. Acad. Sci. U.S.A. 78, 7530-7534. Smith, E.M., Phan, M., Coppenhaver, D., Kruger, T.E. and Blalock, J.E. (1983) Human lymphocyte production of immunoreactive thyrotropin. Proc. Natl. Acad. Sci. U.S.A. 80, 6010-6013. Snow, E.C., Feldbush, T.L. and Oaks, J.A. (1981) The effect of growth hormone and insulin upon MLC responses and the generation of cytotoxic lymphocytes. J. Immunol. 126, 161-164. Sonntag, W.E., Steger, R.W., Forman, L.J. and Meites, J. (1980) Decreased pulsatile release of growth hormone in old male rats. Endocrinology 107, 1875-1879. Takahashi, K., Daughaday, W.H. and Kipnis, D.M. (1971) Regulation of immunoreactive GH secretion in male rats. Endocrinology 88, 909-917. Takahashi, S., Gottschall, P.E., Quigley, K.L., Gya, R.G. and Meites, J. (1987) Growth hormone secretory patterns in young, middle-aged and old female rats. Neuroendocrinology 46, 137-142. Tannenbaum, G.S. and Martin, J.B. (1976) Evidence for an endogenous ultraradian rhythm governing growth hormone secretion in the rat. Endocrinology 98, 562-570. Weigent, D.A. and Blalock, J.E. (1990a) Growth hormone releasing hormone production by rat leukocytes. J. Neuroimmunol. 29, 1-13. Weigent, D.A. and Blalock, J.E. (1990b) The production of growth hormone by subpopulations of rat leukocytes. Cell. Immunol. (in press). Weigent, D.A., Baxter, J.B., Wear, W.E., Smith, L.R., Bost, K.L and Blalock, J.E. (1988) Production of irnmunoreactive growth hormone by mononuclear leukocytes. FASEB J. 2, 2812-2818. Weir, D.M. (Ed.) (1978) Handbook of Experimental Immunology: Cellular Immunology, Vol. 2, 3rd edn., Blackwell Scientific Publishing, London.
43
© 1991 ElsevierSciencePublishers B.V. 0165-5728/91/$03.50 JNI 02012
Expression of immunoreactive growth hormone in leukocytes in vivo Judith B. Baxter, J. Edwin Blalock and Douglas A. Weigent Department of Physiology and Biophysics, Universityof A labama at Birmingham, Birmingham, A L 35294, U.S.A.
(Received23 October1990) (Revised, received8 February1991) (Accepted 8 February1991)
Key words: Growthhormone; Leukocyte;RNA; (In vivo)
Summary In the present study, we investigated the production of growth hormone (GH)-related RNA and protein in vivo by rat leukocytes after intraperitoneal treatment with different inducing agents including bacterial lipopolysaccharide (LPS) and Freund's complete adjuvant (FCA). The data showed that in rats after exposure to LPS or FCA leukocytes obtained from the spleen, thymus, and peritoneum all showed a dose-dependent increase in GH-related RNA content. The peak production of GH-related RNA was observed 48 h after treatment in the spleen and thymus and 96 h after treatment in the peritoneum. We also evaluated the ability of LPS-sensitive (C3HeB/FeJ) and resistant (C3H/HeJ) inbred mice treated with LPS to produce GH-related RNA. The LPS-sensitive mice presented with a typical pathophysiologic response pattern and higher levels of GH-related RNA in the spleen and thymus than the LPS-resistant mice. An increase in the production of immunoreactive G H (irGH) was also observed by direct immunofluorescence with specific antibodies to rat GH. We validated that the GH-related RNA produced in vivo by leukocytes was similar in structure to pituitary G H RNA using reverse transcription and the polymerase chain reaction (PCR). A sample of the PCR reaction, analyzed by gel electrophoresis, showed a single major DNA band corresponding in length (600 base pairs) to the distance between the 5'-ends of the two GH-specific primers that was specifically detected with a GH-specific probe after Southern transfer. In other studies with normal nontreated animals, the G H RNA levels are higher in the evening hours and early on in the first month of life. Taken together, our data are the first demonstration that G H RNA and immunoreactive protein can be detected in leukocytes in vivo both in normal and stimulated animals and support the idea that G H may be active in an immune response.
Address for correspondence:Dr. DouglasA. Weigent,University of Alabama at Birmingham, Department of Physiology and Biophysics, UAB Station, BHSB 894, Birmingham, AL 35294, U.S.A.
Introduction Recent studies indicate that bidirectional communication exists between the immune and neuro-
44
endocrine systems (Blalock, 1989). In this model, it has been suggested that neuroendocrine hormones may serve as signal molecules since cells of the immune system produce neuroendocrine peptide hormones. In particular, corticotropin (ACTH) and endorphins are synthesized after virus infection or interaction with transformed cells or bacterial lipopolysaccharide (Blalock and Smith, 1980; Smith and Blalock, 1981; Harbour-McMenamin et al., 1985). Chorionic gonadotropin (CG) and thyrotropin (TSH) are produced during mixed lymphocyte reactions (Harbour-McMenamin et al., 1986) and after stimulation with a T cell mitogen (Smith et al., 1983), respectively. More recently, luteinizing hormone (LH) (Ebaugh and Smith, 1988) and prolactin (PRL) (Shah et al., 1988) have also been shown to be synthesized by stimulated immune cells. More recent studies in the case of PRL indicate that the molecule found in leukocytes may have been previously internalized from bovine serum (Clevenger et al., 1990; Kenner et al., 1990). Very recently, our work (Weigent et al., 1988) and that of others (Kao et al., 1989; Hattori et al., 1990) have provided strong evidence that mononuclear leukocytes can de novo synthesize and secrete an immunoreactive growth hormone (irGH) in vitro. IrGH produced by leukocytes in vitro is similar to pituitary GH in terms of antigenicity, molecular weight, and bioactivity (Weigent et al., 1988). The factors involved in the in vitro synthesis and secretion of leukocyte irGH are under active investigation. The preliminary findings suggest both similarities and differences to the regulation of irGH in the pituitary gland. Thus, growth hormone releasing hormone (GHRH) and thyrotropin releasing hormone appear to modestly stimulate irGH production (Kao et al., 1989) similar to the regulation of pituitary GH, whereas GH added during the culture of phytohemagglutinin-A stimulated cells augmented irGH secretion unlike the regulation of pituitary GH (Hattori et al., 1990). Our own recent findings show that leukocytes also de novo synthesize an irGHRH and suggest a possible autocrine mechanism of stimulation of irGH synthesis by immune cells (Weigent and Blalock, 1990a). Although no data are available on leukocyte irGH production in vivo, some data are published concerning cytokine effects on
pituitary GH secretion. GH secretion is enhanced by interleukin (IL)-2 therapy in cancer patients (Atkins et al., 1986) and by intracerebroventricular (i.c.v.) injection of IL-1 in rats (Rettori et al., 1987). Intravenous tumor necrosis factor a appears to suppress GH secretion (Elsassar et al., 1989). In another study, i.c.v, or arcuate nucleus injection of IL-lj3 suppressed rat GH levels suggesting an inhibition of hypothalamic GHRH a n d / o r stimulation of hypothalamic somatostatin release (Lumpkin and Hartmann, 1989). The release of GH by a direct action of IL-1 on pituitary cells has also been reported (Bernton et al., 1987). Although many studies have addressed the ability of the immune system to control pituitary GH secretion, no studies have been reported on the effect of immune activation in the control of leukocyte-derived irGH in vivo. Therefore, in the present study, we have attempted to determine if irGH production by leukocytes is detectable in vivo after intraperitoneal injection with substances known to stimulate leukocytes. The data reported here show for the first time the synthesis of GH RNA and protein in vivo from leukocytes of treated animals and support the idea that GH may be active in an immune response.
Materials and methods
Cell preparations Adult (150-200 g, 4-8 weeks old) male Sprague-Dawley rats were obtained from Harlan (Prattville, AL, U.S.A.). LPS-sensitive C3HeB/FeJ and LPS-resistant C 3 H / H e J male mice (6 weeks old) were obtained from The Jackson Laboratories (Bar Harbour, ME, U.S.A.). Animals were injected in the early morning or late afternoon. Following sacrifice, the spleens and thymi were immediately collected, teased with sterile groundglass-edged slides and frozen in microfuge tubes for RNA isolation. Peritoneal exudate cells were collected by standard techniques (Weir, 1978) into RPM| containing heparin, centrifuged, and quickly frozen. Greater than 95% cell viability by trypan blue exclusion was observed in all cell fractions (Kruse and Patterson, 1973). Rat pituitaries were carefully removed and washed in phosphatebuffered saline (PBS), quickly frozen, and the
45 RNA isolated as described below. Rat pituitary GH3 cells used for some of the studies reported here were kindly provided by Dr. J. Mulchahey of the University of Alabama at Birmingham. RNA &olation 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 measurements at 260 and 280 nm. The RNA (10/~g) was blotted on nitrocellulose membranes with the Minifold II slot blotter (Schleicher and Schuell, Keene, NH, U.S.A.) as described by the manufacturers. After hybridization with a GH-specific cDNA probe as described below, the membranes were washed with 0.16 M NaCI, 14 mM sodium citrate and 0.1% sodium dodecyl sulfate (SDS) at 100°C for 30 min to elute the cDNA probe. The membrane was then hybridized to a synthetic oligonucleotide 18 S ribosomal probe to detect differences in the amount of RNA bound to the membrane. Small corrections determined by proportion were applied to the densitometric GH eDNA data according to the amount of RNA bound to the nitrocellulose determined after probing with the 18 S ribosomal probe. Total RNA was prepared by homogenizing leukocytes in 5 M guanidine thiocyanate, 1% sarkosyl, 20 mM EDTA, 1% 2mercaptoethanol, 50 mM Tris-HC1, pH 7.5, with subsequent protease K digestion and extraction with phenol/chloroform (Maniatis et al., 1982). For PCR, this RNA was purified through oligo-dT columns prior to first-strand eDNA synthesis (Maniatis et al., 1982). GH eDNA and hybridization Plasmids containing specific rat (Seeburg et al., 1977) GH eDNA 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). 800 base-pair HindIII inserts (nucleotides - 2 0 to 780) were purified from these plasmids and labeled with [32p]dCTP by nick translation (Bethesda Research Laboratories, Rockville, MD, U.S.A.) to a
specific activity of 1-2 x 108 cpm//~g. Synthetic oligonucleotide probes were prepared and purified in our laboratory by Dr. Robert LeBoeuf. The GH probes used to identify the RNA encoding the 20 kDa and 22 kDa protein, correspond to amino acids 1-7 and 32-46 and the mouse 18 S ribosomal probe corresponds to amino acids 10561067. The synthetic oligonucleotide probes were end-labeled with T4 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 insert (2 x 106 cpm/ml) (Maniatis et al., 1982). 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. The autoradiographs were analyzed using a GS300 densitometer (Hoefer Scientific Instruments). Reuerse transcription and amplification by polymerase chain reaction First-strand eDNA synthesis was performed using a commercially available reagent kit (Amersham) that is based on the procedure of Okyama and Berg (1982). Target sequences from this eDNA (3 /xl from a total reaction volume of 30 #1) were amplified (30 cycles) in a polymerase chain reaction (PCR) using the antisense primer (corresponding to amino acids 180-187) (1 fig) and a sense primer (corresponding to amino acids 4-11) (1/~g) by standard procedures as described (LeBoeuf et al., 1989). The primers were prepared and purified in our laboratory. PCR reactions are performed in a Perkin-Elmer DNA thermal cycler. Generally, reactions are performed in a total volume of 0.1 ml containing 200 /~M of each dNTP, 1 /~g of each primer, 1-10 ng of template DNA, and 2.5 U of Taq polymerase. The final reaction mixture is overlaid with 0.1 ml of mineral oil (Perkin Elmer) to prevent evaporation. A usual cycle consists of 1 min at 94 °C (denaturation), 2 min at 52°C (annealing of primer) and 3 min at 72 °C (extension). 25-40 cycles (7 rain total/cycle) are usually run over a 3-5 h period. Excess primers are separated from amplified DNA on a Sephadex
46 G50 spin column (DuPont). Amplified samples are then run on a 2% agarose gel and stained with ethidium bromide to determine efficiency and size. A control template and primers derived from bacteriophage A which define a 500 bp target is always run to assure reliability of the procedure (Saiki et al., 1985; Scharf et al., 1986). To confirm that the G H cDNA was specifically amplified, a Southern transfer was performed as previously described (Maniatis et al., 1982). The membrane was hybridized to the radiolabeled GH-specific c D N A probe as described above.
Direct imrnunofluorescence Cells were obtained from the spleen, thymus, pituitary, and peritoneal exudate from normal rats or from rats sacrificed 48 h or 96 h after injection with 5 ml FCA by standard procedures. The cells were washed 3 times in 0.01 M PBS, p H 7.2, resuspended in PBS (1 x 10 6 cells/ml) and airdried onto glass slides. After fixation with ice-cold 95% ethanol, the cells were rehydrated in PBS and incubated with monkey serum (1 : 200) for 1 h at 37 ° C. The cells were washed 3 times with PBS and then covered in a 1 : 50 dilution of fluorescein isothiocyanate (FITC)-conjugated monkey anti-rat G H prepared in our laboratory by standard techniques (Johnstone and Thorpe, 1987). The slides were allowed to incubate 1 h at 37 ° C followed by washing 3 times in PBS and rinsing in distilled water. To test the specificity of the FITC-conjugated antibody, the labeled antibody and excess rat G H were mixed and incubated at 4 ° C for 2 h before addition to cells. The cells were observed using an Olympus vertical fluorescence illuminator model A-RFL (Marietta, GA, U.S.A.). Materials Monkey anti-rat G H antiserum was generously donated by Dr. Raiti of the National Hormone and Pituitary Program (National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases, NIH, Bethesda, MD, U.S.A.). The antiserum is highly specific and shows < 0.0001% crossreactivity to other anterior pituitary hormones according to the suppliers. The rat G H we used was from the Pituitary Hormones and Antisera Center of Torrance, CA, U.S.A. (Dr. A.F. Parlow) for radioimmunoassay (RIA) and judged to be
contaminated with < 0.1% of the anterior pituitary hormones as determined by RIAs. The other chemicals used in these experiments were obtained from the Sigma Chemical Company.
Data analysis Each data point from the experiments represents replicate samples from 3-5 rats. Each experiment was replicated at least 3 times. Data are presented as mean + SEM. Rat pituitary (5 /~g/slot) was used as a standard in each probing experiment to normalize the values in experimentto-experiment variation. Differences between groups were determined by the Student t-test and Mann-Whitney test where differences are indicated as being significant, p is less than 0.05.
Results
Leukocyte GH production in rats We have previously described the in vitro production of bioactive irGH from human and rat leukocyte populations (Weigent et al., 1988). In the present studies, we wanted to determine whether the leukocyte-derived irGH could be produced in vivo in response to chemical agents known to stimulate immune cells. Our approach was to inject rats intraperitoneally with the agents listed in Table 1, sacrifice animals after 48 h and immediately freeze the lymphoid tissues. The cytoplasmic R N A was isolated, quantitated, and slotted onto nitrocellulose paper and subsequently probed with a rat pituitary G H specific cDNA. The results shown in Table 1 are presented as the percent of control, where the control animals were nontreated. The data show that animals treated with sheep red blood cells (SRBC), Escherichia coli (HB 101), FCA and purified lipopolysaccharide (LPS) all showed elevated levels (1.7- to 2.0-fold) of GH-related R N A in the spleen 48 h after treatment. The thymus also showed elevated levels (1.5- to 1.7-fold) after treatment compared to the control nontreated animals. The percentage of G H R N A obtained from leukocytes from noninjected animals showed no difference to animals injected with 5 ml PBS. Fig. 1 shows the effect of increasing amounts of intraperitoneal injection of FCA on GH-related R N A levels. Groups of
47 TABLE 1 LEUKOCYTE-DERIVED GH-RELATED RNA PRODUCTION IN RATS Treatment a
Noninjected PBS SRBC E. coli
Freund's complete adjuvant Lipopolysaccharide a
GH RNA b Percent of control Spleen
Thymus
100-4-_17 111+14 184+_ 9 220_+26 196 5:22 271 _+52
100 5:12 127-+10 153_+ 7 219_+29 247 -+31 223 5:41
Rats were injected i.p. with PBS (5 ml); SRBC (5 × 10l°); E. coli (1>(109); FCA (5 ml); LPS (1 mg/kg), or were non-
treated. Animals were sacrificed 48 h after injection and spleens and thymi immediately frozen. b Cytoplasmic RNA from spleen and thymus cells was isolated, slotted onto nitrocellulose, and probed as described in Materials and Methods. After a 48 h exposure to X-ray film, the autoradiograph was scanned by a densitometer and is presented as the percent of control as described in Materials and Methods. The data shown represents the results from four experiments + standard error of the mean. All values are significant at p < 0.05 except for the values obtained from leukocytes of rats injected with PBS.
a n i m a l s were injected with different a m o u n t s of F C A a n d the spleen a n d t h y m u s h a r v e s t e d f r o m o n e group of a n i m a l s 48 h after injection. Perit o n e a l e x u d a t e cells were o b t a i n e d f r o m a n o t h e r g r o u p of a n i m a l s 96 h after injection. The d a t a show that the p r o d u c t i o n of G H - r e l a t e d R N A in the spleen, thymus, a n d p e r i t o n e u m was dose-dependent. Fig. 2 shows the levels of G H R N A in spleen a n d p e r i t o n e a l e x u d a t e cells in rats at specific time p o i n t s after injection of F C A . C y t o p l a s m i c R N A was isolated from all samples at the e n d of the experiment, p r o b e d with the specific G H c D N A insert, a n d the results p r e s e n t e d as the p e r c e n t of control. The d a t a show that significant G H - r e l a t e d R N A p r o d u c t i o n f r o m the spleen of rats was o b s e r v e d at the 48 h time p o i n t after injection of F C A . Similar data, with a p e a k at 48 h, were o b t a i n e d for G H - r e l a t e d R N A p r o d u c t i o n b y the t h y m u s ( d a t a n o t shown). O n the o t h e r hand, p e a k p r o d u c t i o n of G H - r e l a t e d R N A was n o t o b s e r v e d in p o p u l a t i o n s of p e r i t o n e a l e x u d a t e cells until 96 h after the injection of F C A . T h e d a t a show that l e u k o c y t e - d e r i v e d G H - r e l a t e d R N A p r o d u c t i o n was d e t e c t a b l e earlier in b o t h the
spleen a n d t h y m u s t h a n in the p e r i t o n e u m after i n t r a p e r i t o n e a l injection o f F C A . Since o u r d a t a suggested that the leukocytes f r o m a n i m a l s t r e a t e d with F C A p r o d u c e G H - r e l a t e d R N A in vivo, we d e t e r m i n e d if the message was expressed b y m e a s u r i n g the presence of cytop l a s m i c i r G H b y direct i m m u n o f l u o r e s c e n c e . Table 2 shows the results of direct fluorescent meas u r e m e n t s on spleen, t h y m u s , p e r i t o n e a l exudate, a n d p i t u i t a r y cells f r o m b o t h n o n t r e a t e d rats a n d f r o m rats injected with F C A . T h e spleen, thymus, a n d p e r i t o n e a l e x u d a t e cells were l a b e l e d with a F I T C - c o n j u g a t e d a n t i b o d y to rat G H . The results show an elevated level of fluorescence in cells
350
300
_
250
200
o
~5o 100 50 [
0
1
I
I
I
I
2
3
4
5
FCA (mL)
Fig. 1. Induction of GH-related RNA in rats injected with FCA. Male (150 g) Sprague-Dawley rats were each injected intraperitoneally with 1, 2, 3, 4 or 5 ml of FCA or sterile PBS. Groups of animals (five/group) were' sacrificed and cytoplasmic RNA isolated from the spleen and thymus at 48 h and peritoneal exudate cells from another group at 96 h. The RNA was slotted onto nitrocellulose, and probed as described in Materials and Methods. Data points represent the percent of control of average densitometric scans+ SEM obtained from five individual animals slotted in 'triplicate. The average densitometric control values were as follows: spleen, 3.8_+0.5; thymus, 7.3_+1.5; peritoneal exudate, 22.8-+4.2. The data shown above are typical of a single experiment performed three times. Key: (@) spleen; (O) thymus; (A) peritoneal exudate cells; * significant at p < 0.05 except for the value for thymus cells in rats injected with 3 ml of FCA which is significant at p < 0.1.
48
250
200 0 I,. tO
rj
~6 O
150
100
0.
Leukocyte production of GH-related RNA during endotoxic shock
_Z I
50
0
0
I
I
24
48
I
72 Time (h)
L
I
96
120
Fig. 2. Kinetics of induction of GH-related R N A in rats injected with FCA. Groups of animals (five/group) were injected intraperitoneally with F C A (4 ml) or PBS (4 ml) at 0 h. Animals were sacrificed at the indicated time points and the spleen and peritoneal exudate cells harvested and frozen as pellets at - 2 0 o C. After 120 h, the R N A was isolated from the replicate samples and slotted onto nitrocellulose and probed as described in Materials and Methods. Data points represent the percent of control of average densitometric scans + SEM. The average densitometric value for the spleen control was 25.7 +_5.5 and the peritoneal exudate control value was 20.7+4.8. The data shown are from a typical experiment performed 3 times. Key: (O) spleen; (A) peritoneal exudate cells; * significantly different from control at p < 0.05.
obtained from FCA treated animals as compared to cells from nontreated controls. Approximately 20% of the dispersed pituitary cells were labeled positive for GH as expected. An example of a microscopic field containing fluorescent peritoneal exudate cells labeled with the FITC-conjugated antibody specific to rat G H is shown in Fig. 3A. Similar fields were observed with cells obtained from the spleen and thymus (data not shown). Pituitary cells labelled with the same antisera are shown in Fig. 3B. The labeling of pituitary and immune cells could be blocked by prior incubation of the FITC-conjugated antibodies with excess rat GH (data not shown). Taken together, the data show that rat leukocytes stimulated by FCA produce an irGH in vivo.
We next studied the production of leukocyte irGH in mice to test whether there might be differential synthesis of irGH in response to LPS in LPS-sensitive C3HeB/FeJ and LPS-resistant C 3 H / H e J mice. In previous studies, we have shown that leukocytes from rats and mice are comparable in that the GH-related RNA levels increase in leukocytes after removal from animals. It has been shown by others that C 3 H / H e J mice do not exhibit the pathophysiologic changes seen in endotoxic shock (Haas et al., 1978; Morrison and Ryan, 1979). We hypothesize that the ability to produce irGH may influence the response characterized by LPS-sensitive mice. Therefore, we injected groups of LPS-sensitive and -resistant mice with LPS and monitored the pathophysiologic changes and GH-related RNA content 6 h after injection. The LPS-injected C3H e/ FeJ had reduced respiration rates, diarrhea, a white discharge from the eyes, and lethargy while the LPSresistant C 3 H / H e J did not manifest any pathophysiologic abnormalities (data not shown). Fig. 4 shows the results of GH-related RNA levels in
TABLE 2 D E T E C T I O N OF irGH P R O D U C T I O N IN L E U K O C Y T E S BY D I R E C T I M M U N O F L U O R E S C E N C E Source of cells a
Time of harvest
Percent positive by immunofluorescence
Spleen
0 48 0 48 0 96 0 48 96
3.9+_ 1.2 10.4_+ 1.5 6.9 + 1.3 13.5_+1.6 16.3 _+1.5 30.1 _+4.3 21.3 +_2.4 26.8 _+2.1 25.3_+2.3
Thymus Peritoneal exudate Pituitary
h h h h h h h h h
a Rats were injected i.p. with 5 ml F C A and sacrificed either 48 h or 96 h later. The cells were washed 3 times in PBS and 10 ALl of 1 × 106 cells/ml were fixed onto glass slides and labeled with FITC-conjugated monkey anti-rat G H antibodies as previously described in Materials and Methods. The slides were washed 3 times in PBS before determining the percentage of cells positive for G H production. 500 cells from each group were counted from randomly chosen fields. Data is presented as the percent of positive cells + standard error of the mean.
49
A
@
Analysis of GH RNA by reverse transcription and polymerase chain reaction Our previous work has shown the presence of GH-related mRNA molecules spontaneously expressed in rat spleen and thymus cells in vitro (Weigent et al., 1988). To confirm that the leukocyte GH-related RNA produced after in vivo stimulation is similar to the leukocyte GH-related RNA produced in vitro, we selectively enriched
B 300
250 o
0
200
"E a) Fig. 3. Fluorescent photomicrographs of cells stained with FITC-conjugated antibodies to rat GH. Cells from both nontreated rats and rats injected i.p. with FCA (5 ml) were washed 3 times in PBS and resuspended at a concentration of 1 x 106 cells/ml. The cells were air-dried to glass slides and fixed in 95% ethanol. The cells were then labeled with FITC-conjugated antibody to rat GH. A: Peritoneal exudate cells from rats injected with FCA and sacrificed 96 h after injection. B: Pituitary cells from rats injected with FCA. Magnification 400 X.
o
150 ~
III
I"~
I:L
100
so L 0
I
I
0
5
I
I
10
20
LPS (mg/kg)
LPS-sensitive and resistant groups of mice 6 h after injection with various doses of LPS. The results show that at doses between 5 and 20 mg/kg mouse, the LPS-sensitive C3HeB/FeJ animals produced higher levels (2-fold) of GH-related RNA than LPS-resistant animals in the spleen and thymus. The thymus in both groups of animals produced less GH-related RNA than the spleen tissues and in the LPS-resistant C3H/HeJ mice the levels of thymus GH-related RNA were significantly lower than control animals. The lower level of GH-related RNA in the thymus found in LPS-resistant animals is unexplained.
Fig. 4. Induction of GH-related RNA in mice injected with LPS. C 3 H e B / F e J (LPS-sensitive) and C 3 H / H e J (LPS-resistant) mice were injected intraperitoneally with various doses of LPS as indicated or sterile PBS. Groups of animals (five/group) were sacrificed after 6 h and the spleen and thymus cells immediately frozen. RNA was isolated, slotted, and probed with a rat GH cDNA as described in Materials and Methods. Data points represent the percent of control of average densitometric scans + SEM obtained from five individual animals slotted in triplicate. The controls are the spleen and thymus cells from animals of the same species sacrificed 6 h after injection with sterile PBS. The average densitometric s c a n + S E M for each control is listed in the key. The data shown above are typical of a single experiment performed 3 times. Key: (e) C 3 H e B / F e J (sensitive) spleen, control value, 8.2+1.90; ( O ) C 3 H e B / F e J (sensitive) thymus, control value, 16.0+ 1.3; (I), C 3 H / H e J (resistant) spleen, control value, 4.0 + 0.4; (13), C 3 H / H e J (resistant) thymus, control value, 43.8 + 8.6; * significantly different from control at p < 0.05.
50 the c o n c e n t r a t i o n of G H - s p e c i f i c n u c l e o t i d e sequences b y reverse t r a n s c r i p t i o n a n d P C R so that they c o u l d be o b s e r v e d b y c o n v e n t i o n a l gel electrophoresis. F o r this e x p e r i m e n t , total R N A was e x t r a c t e d f r o m the t h y m u s of c o n t r o l a n d LPStreated rats, a n d c o n t r o l G H , p i t u i t a r y cells. T h e m R N A was reverse t r a n s c r i b e d a n d then a m p l i fied b y P C R using two o l i g o n u c l e o t i d e p r i m e r s specific for rat p i t u i t a r y G H . R N A was used as a t e m p l a t e for selective first-strand c D N A synthesis with an anti-sense o l i g o n u c l e o t i d e p r i m e r c o m p l e m e n t a r y to the 3' end of exon 5 of G H m R N A . W h e n this purified c D N A is i n t r o d u c e d as a temp l a t e into a P C R with a second-sense oligonucleotide p r i m e r r e p r e s e n t i n g the G H sequence, ten bases 5' to the G H c o n t a i n i n g region of exon 1, there is a m p l i f i c a t i o n of the a p p r o p r i a t e - s i z e d (600 bp) c D N A fragment. T h e p o t e n t i a l p r o b l e m of a m p l i f y i n g g e n o m i c D N A is c o n s i d e r e d unlikely since we have used o l i g o - d T p u r i f i e d R N A a n d p r i m e r s for two different exons for the first-strand synthesis a n d P C R , respectively. T h e selective a m p l i f i c a t i o n of a G H - r e l a t e d target c D N A sequence was c o n f i r m e d b y S o u t h e r n analysis (Fig. 5). Since the P C R reactions were not set up to b e
LANE
q u a n t i t a t i v e , n o c o n c l u s i o n s can be d r a w n a b o u t the relative a m o u n t of G H - r e l a t e d R N A in the original sample. I n a d d i t i o n to the a b o v e study, we have asked the q u e s t i o n w h e t h e r l e u k o c y t e s e n c o d e an R N A for a p r e v i o u s l y d e s c r i b e d smaller m o l e c u l a r weight v a r i a n t f o r m of G H . A v a r i a n t f o r m of G H with a m a s s of 20 k D a , l a c k i n g residues 3 2 - 4 6 of 22 k D a G H has b e e n i d e n t i f i e d as b e i n g g e n e r a t e d b y an a l t e r n a t i v e splicing m e c h a n i s m ( M a s u d a et al., 1988). By using o l i g o n u c l e o t i d e p r o b e s c o m p l e m e n t a r y to the c o d i n g regions of a m i n o acids 1 - 7 a n d a m i n o acids 3 2 - 4 6 of 22 k D a G H ( 1 - 2 x 10 s c p m / t ~ g ) , we tested w h e t h e r the G H - r e l a t e d R N A f r o m l e u k o c y t e s c o n t a i n e d the sequence i n f o r m a tion d e l e t e d f r o m the 20 k D a variant. T h e results ( T a b l e 3) show that there was no significant difference in the h y b r i d i z a t i o n to the R N A utilizing the same n u m b e r of c p m for b o t h o l i g o n u c l e o t i d e probes. T h e d a t a suggest t h a t the m a j o r a m o u n t of l e u k o c y t e G H - r e l a t e d R N A a p p e a r s to c o n t a i n the sequence i n f o r m a t i o n e n c o d i n g a m i n o acids 3 2 - 4 6 which c o r r e s p o n d s to the 22 k D a G H .
Endogenous regulation of leukocyte irGH Several r e p o r t s i n d i c a t e that rat serum G H levels fluctuate m a r k e d l y d u r i n g a 24 h p e r i o d
I 2 3
TABLE 3 ANALYSIS OF CYTOPLASMIC GH-RELATED RNA EXPRESSION IN VIVO Source of leukocytes
-~600 bp
Fig. 5. Confirmation of selective amplification of rat GH-related RNA by Southern analysis. The Southern analysis and hybridization with a radiolabeled GH-specific probe were carried out as described in Materials and Methods. Lane 1, thymus control; lane 2, thymus from rat treated with LPS (30 mg/kg) 6 h prior to sacrifice; lane 3, GH 3, pituitary control.
a
Spleen Thymus Peritoneal exudate cells
Time of harvest
GH RNA b Densitometric units
(h)
Probe 1-7 amino acids
Probe 32-46 amino acids
48 48
64 _+4 72 _+5
59 _+4 70 + 4
96
75 _+6
71 + 5
Rats were injected i.p. with FCA (5 ml) and one set of animals sacrificed 48 h later and another set at 96 h after treatment. Animals were sacrificed as described and tissues frozen. Total cytoplasmic RNA from spleen cells was isolated, slotted onto nitrocellulose, and probed as described in Materials and Methods. After a 48 h exposure to X-ray film, the autoradiograph was scanned by a densitometer as described in Materials and Methods. The data shown represents the results from four experiments.
51 detectable changes in leukocyte GH-related RNA production between 4 and 36 weeks after birth and between the sexes (data not shown).
50
4O
i.~
3O
~
2o
Discussion &
10
0
112 1
2:3
4 5 6 7 8 9 1011112 1 2 3 4 5 6 7 8 9 10111 PM AM Time(h)
Fig. 6. Analysis of the leukocyteGH-related RNA rhythm in the light-dark condition. Two animals were sacrificed every hour over a 24 h time period. At each time point, the spleen was quickly frozen at -20 ° C. After 24 h, the RNA was isolated from the replicate animals and slotted in triplicate and probed as described in Materials and Methods. The data shown are from a typical experimentperformedtwice. suggesting an ultradian rhythm with a periodicity of approximately 3.3 h most likely synchronized to a light-dark cycle (Takahashi et al., 1971; Scheving and Turpen, 1974; Tannenbaum and Martin, 1976). To investigate the possibility of a rhythm controlling leukocyte-derived GH, we sacrificed nontreated animals every hour for a period of 24 h and determined their content of cytoplasmic GHrelated RNA. The results (Fig. 6) show that the levels of spleen GH-related RNA were 2- to 4-fold higher during the dark phase of a 12 h light-dark cycle. Similar findings were observed in the regulation of thymus GH-related RNA (data not shown). In both man and laboratory animals, it is also known that episodic G H secretion is maximal during adolescence and early adulthood and diminishes as individuals age (Finklestein et al., 1972; Sonntag et al., 1980; Takahashi et al., 1987). We investigated whether the production of leukocyte GH-related RNA is reduced in aged rats or undergoes any alteration with age. A group of animals were sacrificed every week for 4 weeks and every fourth week thereafter for GH-related RNA determinations in the spleen and thymus. The results showed that significantly higher levels of GH-related RNA were found within the first month of life in the spleen and thymus where a 4-fold increase in GH-related RNA was observed in the third and fourth week of life. There were no
In the present studies, we have shown that spleen and thymic leukocytes and peritoneal exudate cells produce higher levels of GH-related RNA and protein in vivo when stimulated with LPS or FCA. The increase in GH-related RNA was dose-dependent with peak production in the spleen and thymus observed 48 h after treatment. The peak increase in GH-related RNA was not observed in peritoneal exudate cells until 96 h after treatment. Our present data do not permit us to define the mechanism for the delayed peritoneal response. One can speculate on several possible mechanisms. For example, leukocytes from the spleen a n d / o r thymus which are already producing irGH, may redistribute to the peritoneum. A second possible mechanism may be that different G H inducer substances may stimulate irGH production in leukocytes at different sites. The scenario is obviously complex and more studies are required to identify the mechanism for the delayed peritoneal response. In other results reported here, we have obtained data from reverse transcription and PCR that support the idea that GH-related RNA produced in vivo from leukocytes is structurally similar to the pituitary G H RNA. RNA from the thymus and the GH, pituitary cell line were both used to synthesize a 600 bp c D N A that reacted with a specific rat G H c D N A after Southern transfer. The idea that genomic D N A was amplified rather than R N A is considered to be highly unlikely. This is due to the fact that oligo-dT purified RNA was used in the synthesis of first strand and the primers for PCR were designed from two different exons. The same RNA could be hybridized to a specific oligonucleotide complementary to the sequence of R N A specifying the deletion in 20 kDa GH. This result suggests that the major product of translation of leukocyte GH-related RNA may be the 22 kDa species. The data do not rule out the presence of the 20 kDa species of GH. The data we have obtained strongly support the existence of
52 GH-related RNA induced in leukocytes. Additional studies by direct immunofluorescence using an FITC-conjugated antibody to rat G H have verified the translation of the GH-related RNA in vivo. Previous studies have shown that immune stimulation with LPS or cytokine administration in vivo is associated with changes in pituitary hormone secretion. In general, there is a stimulation of ACTH, inhibition of TSH, species-dependent effects on G H and modest effects on prolactin (review, see Scarborough, 1990). The G H response to endotoxin in both rat and humans is the same as the G H response to stress, that is, an increase in humans and a decrease in the rat (Kasting and Martin, 1982). It was, therefore, somewhat surprising in the rat that the leukocyte response to LPS was an increase in GH-related RNA. It has been reported that mediators such as IL-1 are produced in large quantities by LPStreated macrophages and play an important role in endotoxic shock (Beutler et al., 1986). Recently, it has also been reported that IL-1 stimulates the release of pituitary G H (Bernton et al., 1987; Rettori et al., 1987). We investigated the effect of LPS on the leukocytes from the LPS-sensitive and -resistant mice and showed that the mice also increase the production of GH-related RNA after LPS treatment in vivo. However, the C 3 H / H e J resistant mice injected with LPS did not manifest the pathophysiologic abnormalities associated with endotoxic shock and produced significantly lower levels of GH-related RNA after treatment compared with the C3HeB/FeJ-sensitive mice. It is tempting to speculate that the defect in LPS-resistant mice which results in lower levels of IL-1 also prevents the induction of GH-related RNA. However, more studies are needed to define the mechanism of reduced GH-related RNA production in LPS-resistant mice. Finally, we have presented data that suggest the regulation of leukocyte irGH may be under an endogenous 24 h rhythm as well as decrease with the onset of adulthood. These results are similar to published data measuring serum levels of G H (Tannenbaum, 1976; Millard et al., 1990) over a 24 h time period .and in aged rats. The present results do not explain the mechanism of induction of leukocyte-derived irGH or the cell types in-
volved in vivo. Our previous in vitro findings on the spontaneous production of irGH from cells separated by adherence, nylon wool columns, and positive and negative sorting with monoclonal antibodies that define B, monocyte, T helper and T cytotoxic cells show that several different cell types have the ability to produce GH-related m R N A (Weigent and Blalock, 1990b). The results suggest that B cells and macrophages produce more GHrelated m R N A than T cells. Taken together, the in vitro data suggest there is heterogeneity within leukocytes regarding their ability to produce irGH and more studies are required to identify G H producer cell types in vivo after stimulation. The significance of irGH release by leukocytes during intraperitoneal injection of LPS is not clear. Since G H is involved in substrate metabolism, the results may suggest that G H facilitates the increase in energy utilization during fever. On the other hand, G H may promote a host defense mechanism. It has been demonstrated that high levels of G H injected in vivo double both basal and lectin-induced proliferative responses from spleens of aged rats (Davila et al., 1987; Kelley, 1989). 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 G H in vitro. G H affects the functional activity of cytolytic cells (Snow et al., 1981) and N K cells (Saxena et al., 1982) and has been shown to be as potent as "/-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. We anticipate that future studies on leukocyte-derived irGH will help explain its effects on the immune response. Finally, the postulated sensory role that the immune system plays in responding to noncognitive stimuli (LPS) coupled with the role of G H in the immune system provides a molecular mechanism whereby a physiological response is associated with infectious disease.
Acknowledgements We thank Dr. Robert LeBoeuf for providing the synthetic oligonucleotides and Dr. J. Mulcha-
53 h e y for h e l p o n t h e i m m u n o f l u o r e s c e n t studies. 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 for p r e p a r i n g t h e m a n u s c r i p t . T h i s w o r k was s u p p o r t e d in p a r t b y g r a n t s f r o m The National Institute of Neurology and Comm u n i c a t i v e D i s o r d e r s (R01 N S 2 4 6 3 6 ) a n d T h e N a t i o n a l I n s t i t u t e of A r t h r i t i s , D i a b e t e s , D i g e s t i v e a n d K i d n e y D i s e a s e s (R01 D K 3 8 0 2 4 ) .
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