BRAIN,

BEHAVIOR,

AND

IMMUNITY

6,

365-376 (1992)

The Production of Growth Hormone and Insulin-like Growth Factor-l by the Same Subpopulation of Rat Mononuclear Leukocytes’ DOUGLAS

A. WEIGENT,*

Department

of Physiology

JUDITH

B. BAXTER,

AND J. EDWIN

and Biophysics. The University of Alabama Birmingham. Alabama 35294-0005

BLALOCK

at Birmingham.

In the present study. we evaluated the subpopulation of lymphoid cells from normal and hypophysectomized rats producing GH and IGF-I in vitro. The data show that removal of the pituitary results in depression of GH production in spleen, thymus, and bone marrow and an increase in the peripheral blood leukocytes. The changes in the percentage of cells producing GH in hypophysectomized animals are not due to a single cell type but appears to influence the T-helper. T-cytotoxic. and B-cell subsets. Interestingly, no significant changes in the levels of GH RNA were detected between control and hypophysectomized animals after the in vitro culture. We also found that the increase in GH production in spleen cell cultures after mitogen stimulation could be accounted for by an increase in the percentage of T cells producing GH. Lastly, we demonstrated that the cells positive for GH production were also positive for IGF-I production. This later finding coupled with our previous results suggest that an autocrine regulatory circuit may be important for the production of leukocyte-derived irGH and irIGF-I within the immune system. ‘c 1992 Academic Prea\. Inc. INTRODUCTION

Numerous studies over the past 10 years have established the fact that cells of the immune system have the capacity to produce and secrete neuroendocrine hormones (Blalock, 1989; Weigent & Blalock, 1987). The accumulated evidence indicates that lymphocytes synthesize and secrete ACTH and endorphins (Smith & Blalock, 1981), thyrotropin (Smith. Phan, Coppenhaver, Kruger, & Blalock. 1983), follicle-stimulating hormone (Kasson & Tuchel, 1989), luteinizing hormone (Ebaugh & Smith, 1988), prolactin (Montgomery, Zukoski, Shah, Buckley, Pacholczyk, & Russell, 1987), and growth hormone (Weigent, Baxter, Wear, Smith, Bost, & Blalock, 1987). In addition to this, several hypothalamic-releasing hormones, including corticotropin-releasing hormone (Stephanou, Jessop, Knight, & Lightman, 1990) and growth hormone-releasing hormone (Weigent & Blalock, 1990b), have been shown to be secreted by lymphocytes. Our work (Baxter, Blalock, & Weigent, 1991a; Weigent, Baxter, Wear, Smith, Bost, & Blalock. 1988) and that of others (Hattori, Shimatsu, Sugita, Kumagai, & Imura, 1990; Kao, Harbour, Smith, & Meyer, 1989) have provided evidence that rat leukocytes produce GH-related RNA and protein both in vitro and in vivo. Although differences in the molecular weight of GH RNA (Hiestand, Mekler, Nordmann, Grieder, & Permmongkol, 1986) and protein (Baglia, Cruz, & Shaw, 1992) have been suggested, our findings suggest that the RNA molecules and the GH protein ’ Supported by NIH Grants NS24636 to D.A.W. and DK38024 to J.E.B. ’ To whom correspondence should be addressed at Department of Physiology and Biophysics, 1918 University Blvd., BHSB 894. Birmingham, AL 35294-0005. 365 0889-1591192 $5.00 Copyright Q 1992 by Academic Press. Inc. All nghta of reproduction in any form reserved.

366

WEIGENT,

BAXTER,

AND

BLALOCK

are similar to pituitary GH in terms of antigenicity, molecular weight, bioactivity, and nucleic acid sequence (Weigent et al., 1988; Weigent, Blalock, & LeBoeuf. 1991a). Although the GH molecule produced by leukocytes appears to be substantially similar to pituitary GH, the mechanisms regulating the synthesis and secretion of this protein by leukocytes appears to be both similar and dissimilar to the mechanisms operating at the level of the pituitary. Thus, GHRH stimulates the production of leukocyte-derived GH (Guarcello, Weigent, & Blalock, 1991) whereas exogenous levels of GH or somatostatin appear to have little or no effect on leukocyte production of GH (Hattori et al., 1990). More recently, we have shown the synthesis and secretion of bioactive IGF-I from leukocytes which can be inhibited by antibodies specific to GH. Further, the addition of IGF-I to leukocytes can decrease the levels of leukocyte GH RNA and protein (Baxter, Blalock, & Weigent, 1991b). In view of the production of neuroendocrine hormones by cells of the immune system, it is important to establish the cellular origin to better understand the role they may play during an immune response. It is known that when human peripheral blood cells are infected with Newcastle disease virus, all the major immune cell types produce POMC peptides (Blalock & Smith, 1980). However, when peripheral leukocytes are treated with bacterial LPS only B cells produce the POMC peptides (Harbour, Smith. & Blalock, 1987). It has also been shown that corticotropin-releasing factor stimulates monocytes to produce IL-I which in turn stimulates B cells to secrete p-endorphin (Kavelaars, Ballieux, & Heijnen, 1989). The production of thyroid-stimulating hormone seems to be limited to T lymphocytes regardless of the stimulus (Smith et al., 1983). In our previous work with GH, we have observed that after removal of leukocytes from animals, they spontaneously produce and secrete GH (Weigent et al., 1988). In another study, we demonstrated that mononuclear leukocytes from various tissues including spleen, thymus, bone marrow, Peyer’s patches, and peripheral blood all have the ability to produce GH RNA and secrete GH. An analysis of subpopulations suggested there was heterogeneity within lymphocytes regarding their ability to produce GH (Weigent & Blalock, 1991). The purpose of the present research was to extend our prior investigations on the cell types producing growth hormone. The present study evaluated the GHproducer cell types in several different tissues and the effect of hypophysectomy on GH production, The production of IGF-I was similarly investigated to determine whether the same cells were producing GH and IGF-I. The data suggest that numerous cell types may produce GH and that these same cells are also the producers of IGF-I. MATERIALS

AND METHODS

Reagents. Monkey anti-rat GH antiserum was generously donated by Dr. S. Raiti of The National Hormone and Pituitary Program (NIDDK, Bethesda, MD). The antisera is highly specific and shows less than 0.0001% cross-reactivity to other anterior pituitary hormones according to the suppliers. The rat GH we used was from the Pituitary Hormones and Antisera Center (Torrance, CA; Dr. A. F. Parlow) for RIA and judged to be contaminated with less than 0.1% of the anterior pituitary hormones as determined by RIAs. The IGF-I antiserum (UBK487) was generously donated by Drs. L. E. Underwood and J. J. Van Wyk of the University of North Carolina at Chapel Hill through the National Hormones and Pituitary

GROWTH

HORMONE

PRODUCTION

BY LEUKOCYTES

367

Program. The antisera is highly specific and shows a 0.5% cross-reactivity with IGF-II and crossreacts minimally with insulin at 10ph M according to the suppliers. The biotin-labeled mouse monoclonal antibodies to rat T-helper (W-325). T-cytotoxic (0X-8). and B-cell (MR 18.5) lymphocytes and the avidin-PE conjugate were prepared and graciously provided by Dr. Eldridge from the Department of Microbiology at the University of Alabama at Birmingham. The W-325 antibody stains approximately 50% of the rat spleen cells, 95% of thymus cells, and 60% of cells obtained from the peripheral blood. The OX-8 antibody stains approximately 30% of the rat spleen cells, 95% of thymus cells, and 25% of cells obtained from the peripheral blood. The MR 18.5 antibody stains approximately 30% of the rat spleen cells, 0% of thymus cells. and 13% of cells obtained from the peripheral blood. Additional reagents of the highest purity available were purchased from GIBCO (Grand Island, NY) and Sigma (St. Louis, MO) unless otherwise stated. Cell preparations. Adult (150-200 g) male Sprague-Dawley rats were obtained from Harlan Sprague-Dawley, Inc. (Prattville, AL). Ten-week-old male rats were hypophysectomized or sham-operated by the parapharyngeal approach at the Harlan Laboratories and were used after an observation of 2 weeks. Rats were weighed prior to experiment. At the time of sacrifice. autopsies on hypophysectomized rats were performed in order to determine whether any residual pituitary tissue remained. Following sacrifice, lymphoid tissues including the spleen. thymus, peripheral blood, and bone marrow were prepared as single-cell suspensions in RPMI-1640 medium supplemented with penicillin, streptomycin, and mycostatin (100 U/ml). Peripheral blood lymphocytes were obtained after passage over Ficoll-hypaque gradients as previously described (Boyum, 1968). Contaminating red blood cells from the cell preparations were removed by NH&L lysis and cell viability was monitored by trypan blue exclusion. Cells were incubated for 18 h at 37°C in RPMI-1640 containing 10% fetal calf serum in multiwell plates (Falcon). After incubation cells were either immediately frozen in microfuge tubes for RNA isolation or washed and spotted onto glass slides for immunofluorescent studies or stained with the appropriate antibodies and analyzed by FACS after fixation in 1% paraformaldehyde (see below). Rat pituitaries were carefully removed, washed in PBS, and quickly frozen, and the mRNA was isolated as described below. Mitogerz stirnulution. The mitogenic response of enriched lymphoid cell populations was determined by adding the T-cell mitogen concanavalin A (ConA) to cell suspensions (1 X IOh/ml) as previously described (Langford, Weigent, Georgides, Johnson, & Stanton, 1981). Replicate cultures containing I x 10’ cells/O.2 ml in RPMI-1640 medium supplemented with Hepes, antibiotics, 2 mM glutamine, 5 X IO-” M 2-mercaptoethanol, and 10% heat-inactivated pooled FBS were set up for controls. At 24 h after initiation, I FCi of tritiated thymidine Q3H]TdR; New England Nuclear Corp., Boston, MA) was added to the mitogenstimulated and nonstimulated cultures. The cultures were harvested 24 h after the addition of [3H]TdR and the incorporated radioactivity was determined by liquid scintillation spectrometry. RNA isolation and blottirzg. Total cytoplasmic RNA was isolated by the proteinase K method (Maniatis, Fritsch, & Sambrook, 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 absorbance measurements at 260 and 280 ti. The RNA (10 pg) was blotted on nitrocellulose mem-

368

WEIGENT,

BAXTER,

AND

BLALOCK

branes with the Minifold II slot blotter (Schleicher and Schuell, Inc., Keene, NH) as described by the manufacturers. After hybridization with a GH-specific complementary DNA probe as described below, the membranes were densitometritally scanned and then washed with 0.16 M NaCl, 14 mM sodium citrate, and 0.1% sodium dodecyl sulfate (SDS) at 100°C for 30 min to elute the cDNA probe (Maniatis et al., 1982). Membranes were then reprobed with a synthetic oligodeoxynucleotide 18s ribosomal probe to detect differences in the amount of RNA bound to the membrane. Small corrections determined by proportion were applied to the densitometric cDNA data according to the amount of RNA bound to the nitrocellulose determined after probing with the 18s ribosomal probe. Densitometric values of GH RNA (10 p,g) were corrected as a percentage with 2.5 kg control pituitary RNA as the 100% reference point. GH cDNA and hybridization. Plasmids containing the rat (Seeburg et al., 1977) GH cDNA were kindly provided by Dr. John Baxter and Dr. Fran Denoto (Neurochemistry Laboratories, VA Medical Center, Sepulveda, CA). Plasmid DNA was prepared essentially as described (Maniatis et al., 1982). Eight hundred bp Hind11 inserts (nucleotides -20 to 780) were purified from the plasmid. The cDNAs were labeled with [3’P]deoxycytidine triphosphate by nick translation using a commercially available kit (Bethesda Research Laboratories, Gaithersburg, MD) to a specific activity of 1-2 x IO* cpm/pg. The mouse 18s synthetic oligodeoxynucleotide ribosomal probe was prepared and purified by our laboratory. The probe corresponds to nucleotides 1056-1067 and was 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 SDS, denatured salmon sperm DNA, Denhardt’s solution, formamide, NaCI, NaH,PO,, H20, EDTA, and standard buffer containing 3’P-labeled insert (Maniatis et al., 1982). After hybridization, the membranes were extensively washed until the radioactivity in the final wash was close to background using 0.4 M NaCI, 0.04 M sodium citrate, and 0.1% SDS for another 10 min. Both washings were done at 25°C with gentle mixing. Two washes were done in 0.03 M NaCI, 0.003 M sodium citrate, and 0.1% SDS for 30 min at 44°C with gentle mixing. The papers were then rinsed in 0.04 M NaCl and 0.004 M sodium citrate. The nitrocellulose papers were exposed to X-ray film at -70°C with DuPont (Wilmington, DE) Cronex Lightning-Plus intensifying screens for 2-3 days. The autoradiographs were analyzed using a GS 300 densitometer (Hoefer Scientific Instruments, San Francisco, CA). Direct immunufluorescence. The cells used for immunofluorescence were washed three times in 0.01 M PBS, pH 7.2, resuspended in PBS (1 X IO6 cells/ml) and air-dried onto glass slides. After fixation with ice-cold 95% ethanol, the cells were rehydrated in PBS and incubated with rat serum (I :200) for 1 h at 37°C. The cells were washed three times with PBS and then covered in a 1: 10 dilution of tetramethylrhodamine isothiocyanate (TRITC)-conjugated monkey-anti-rat GH and a 1:50 dilution of fluorescein isothiocyanate (FITC)-conjugated rabbit antihuman IGF-I prepared in our laboratory by standard techniques (Johnstone & Thorpe, 1987). The slides were allowed to incubate for 1 h at 37°C followed by washing three times in PBS and rinsing in distilled water. To confirm the specificity of the conjugated antibodies, the labeled antibody and excess hormone were mixed and incubated at 4°C for 2 h before addition to cells. The cells were ob-

GROWTH

served using an Olympus A-RFL.

HORMONE

(Marietta,

PRODUCTION

369

BY LEUKOCYTES

GA) vertical fluorescence

illuminator

Model

Cells (1 x 10’) were first incubated with 5% normal rat serum for 15 min prior to staining to block Fc receptors. For double staining, cells were stained first with primary monoclonal antibodies followed by treatment with phycoerythrin (PE)-conjugated avidin and finally by a FITC-conjugated monkey anti-rat GH. Cells stained with only one fluorochrome reagent (PE or FITC) were used to determine the proper window settings. Cells were washed extensively after each incubation, suspended in PBS containing 1% paraformaldehyde, and analyzed using an EPICS cell sorter (Coulter Electronics, Hialeah, FL) for unlabeled and for single and double positive populations. Data were collected on 5000 cells and shown as a contour plot of fluorescence intensity in the logarithmic scale with red fluorescence (PE) on the y-axis and green fluorescence (FITC) on the x-axis. Data analysis. Each data point from the experiments represent replicate samples from three to five rats. Each experiment was replicated at least three times. Data are presented as means ? SEM. Rat pituitary (2.5 pg/slot) was used as a standard in each probing experiment to normalize the values in experiment-toexperiment variation. Differences between groups were determined by Student’s t test where differences are indicated as being significant, p is less than .05. Phenotypic

analysis.

RESULTS Spontaneous production of GH mRNA and protein by different lymphoid tissues from normul and hypophysectomized rats. In a previous work, we estab-

lished that cells obtained from numerous lymphoid tissues after removal from animals immediately begin to synthesize GH mRNA and secrete GH into supernatant fluids (Weigent & Blalock, 1991). The mechanism resulting in this negative control in viva has not yet been definitely established. In the course of this investigation, we asked whether this same negative pattern of regulation might be observed in animals without a pituitary gland to get an idea whether this endocrine gland was involved in the negative control of lymphocyte GH. Thus GH RNA and protein synthesis was studied in lymphoid cell preparations from normal male rats that underwent hypophysectomy or sham operation. The body weight (335.0 g versus 112.5 g), spleen weight (0.6 g versus 0.25 g), and serum GH levels (97 rig/ml versus 8 rig/ml) in control versus hypophysectomized animals, respectively, were significantly reduced as a result of the loss of the pituitary gland. In three experiments, GH synthesis in immune cells from hypophysectomized rats was significantly lower than that in sham-operated rats in the spleen, thymus. bone marrow. whereas slightly elevated levels were observed in cells obtained from the peripheral blood (Table I). Interestingly, no significant differences were observed in the levels of GH RNA observed from immune cells after a 24 h in vitro incubation. Lymphoid cells frozen immediately after sacrifice of animals and probed for GH RNA and stained for the GH protein were negative, suggesting the lack of involvement of pituitary-derived hormones in maintaining the endogenous low in viva levels of GH RNA and protein produced by cells of the immune system. Phenotypic analysis of GH-producing cells in the peripheral blood, spleen, and thymus of normal and hypophysectomized rats. In a previous report, separating

cells by common fractionation protocols (i.e., plastic and nylon wool) and separating lymphocyte subsets by magnetic particles and monoclonal antibodies indi-

370

WEIGENT,

Determination

BAXTER,

AND

BLALOCK

TABLE 1 of GH-Producing Cells in Different Tissues from Normal and Hypophysectomized Rats GH mRNA densitometric scan (5%of control)”

Cell source Peripheral blood Spleen Thymus Bone marrow

Normal 12 I4 I5 13

k ? 2 2

2 1 2 I

Hypophysectomized IO-+ I 13 zk 2 14 k 2 IO t I

Percentage positive for GH productior? Fold change -

1.2 I.1 I.2 1.3

Normal 5.0 16.7 9.6 9.0

t k k +

Hypophysectomized

I.2 1.7 0.9 3.0

8.7 7.4 5.9 4.4

2 2 -t 2

1.4 1.3 0.3 1.0

Fold change + I .7’ - 2.3* - 1.6* - 2.0*

” Mononuclear cells were collected and the RNA was extracted after 24 h of incubation. Cytoplasmic RNA (IO (*g/slot) was isolated and slotted onto nitrocellulose and probed as described under Materials and Methods. The autoradiograph was exposed for 4X h and scanned with a densitometer. The value obtained with 2.5 p,g of pituitary RNA was used as the GH mRNA standard and arbitrarily set at 100% relative intensity on the scanning densitometer. Each value represents the mean k SEM of duplicate samples assayed in three different experiments. ’ Mononuclear cells were collected at 24 h of incubation and treated with FITC-labeled antibodies to GH and subjected to FACS analysis as described under Materials and Methods. Five thousand cells were counted and the data are presented as the percentage of positive cells !I SEM. *I) < .05 (two-way analysis of variance) compared with controls.

cated that T cells, B cells, and macrophages all produce GH FsNA and protein during a 24-h in vitro culture period (Weigent & Blalock, 1991). Since all the major cell types are believed to produce GH, attempts were made to determine whether the decline in GH activity in hypophysectomized animals was accompanied by a parallel decline in a particular subset or whether all cell types were similarly affected. The data shown in Table 2 suggest that the increase seen in peripheral blood cells and the decrease in the thymus in the levels of GH produced was not specific to one cell type but effects T-helper, T-cytotoxic, and B cells approximately equally. Similar findings were observed with cells from the spleen and bone marrow (data not shown). Effect of T-cell mitogen on GH production by subpopulations of rcrt spleen cells. Several different groups, including ours, have reported the increase in GH

production after stimulation of cultures with the T-cell mitogen concanavalin A (ConA) (Weigent & Blalock, 1989). We have investigated here whether this increase can be measured to occur among the major cell types of the immune system or whether T cells are principally affected. In these experiments, spleen cells from normal rats were cultured in vitro for 48 h with and without ConA. After the culture period, cells were collected and analyzed for the percentage of T-helper, T-cytotoxic, and B cells producing GH. The results (Table 3) show that cultures treated with ConA contain IS- to 1.6-fold more cells positive for GH production. In addition, the percentage of both T-helper and T-cytotoxic cells producing GH was observed to increase 2.2- and 1.5-fold, respectively. These results indicate that T cells appear to be the major cell type that increases in their ability to produce GH when treated with ConA. The production of GH and insulin-like growth fuctor-I

cell type. In previous work in our laboratory,

by the same lymphoid

we demonstrated

that rat leukocytes

source

-.

type

T helper T cytotoxic B T helper T cytotoxic B

Mab

Analysis

9.9 4.1 91.9

49.3 67.6 88.7

CDGHnormal-hypox

of GH-Producing

4.2 3.3 90.2

67.6 66.6 71.4

-

Cells

0.1 45.9 27.3 9.2 0.1 78.4 87.0 0.2

CD+ GHnormal-hypox 0.2 43.5 24.5 10.1 0.1 89.8 91.4 0.4

.-

Percentage

5.0 3.2 2.4 3.9 9.6 I .4 0.6 7.9

8.7 5.1 4.3 5.3 5.9 0.5 0.4 5.1

0.0 1.6 2.7 1.2 0.0 10.3 8.3 1.8

CD+ GH+ normal-hypox

and Hypophysectomized

by FACS

-CDGH+ normal-hypox

positive

of Normal

0.1 3.8 4.6 3.1 0.2 5.5 5.2 1.0

Rats”

Fold

f1.7 f2.4 + 1.7 + 2.6 - 1.6 - 1.9 - 1.6 - 1.8

change

0 Rats were sacrificed and peripheral blood lymphocytes and thymus removed, teased, and cultured for 24 h as described under Materials and Methods. Cells were washed and labeled with the specific antisera as described previously under Materials and Methods and subjected to FACS analysis. Five thousand ceils were counted and the data are presented as the percentage of positive and negative cells for a selected CD phenotype and GH. The fold change is calculated by comparing the values of double-positive normal and hypophysectomized animals except in the case of the control cells treated with labeled GH alone.

Thymus

Peripheral blood

Cell

Phenotypic

TABLE 2 in the Peripheral Blood and Thymus

3 m

!

G c

z

Ft

5

Q

g

is

E

g

2 3:

%

372

WEIGENT,

Effect Cell SOWCC

Unlabeled T helper cytotoxic B cell

of T-Cell

Mitogen treatment (ConA) + + t +

Mitogen

CD-

.-

GH-

63.9 61.2 73.4 64.7 53.3 65.1

BAXTER,

on GH

CD+

AND

TABLE Production

-

GH-

25.1 21.0 15.1 16.6 34.8 37.5

CD-

BLALOCK

3 by Subpopulations

GH+

8.4 12.1 6.9 II.8 7.8 12.2

CD+

of Rat Spleen

GH+

2.6 5.6 4.6 6.9 4. I 5.1

Total fold increase

+2.2* + 1.s* + 1.2

Cells”

GH FITC 11.8 18.1 Il.0 17.8 11.5 18.7 I I.9 17.3

Fold increase I.5 1.6 I .6 1.5

“ Rats were sacrificed and spleen cells were cultured for 48 h in vI’~rv with and without Conconavalin A (10 &ml) as described under Materials and Methods. Cells were washed and labeled with the specific antisera as described previously and subjected to FACS analysis. Five thousand cells were counted and the data are presented as the percentage of positive cells as described under Materials and Methods and in the legend to Table 2. The fold change is calculated by comparing the double-positive cells in control cultures to those treated with mitogen. *p i .OS (two-way analysis of variance) compared with controls.

produce a bioactive IGF-I molecule. In the same series of studies, we showed that exogenous IGF-I can inhibit the levels of leukocyte-derived GH and that leukocyte-derived GH is in part involved in the induction of leukocyte-derived IGF-I. As a result of these findings, we investigated whether leukocyte-derived GH and IGF-I molecules can be produced by the same lymphoid cell type. After removal of spleen cells from animals and in rifro culture, we stained the cells with a mixture of specific antibodies labeled with IGF-I (Fig. IB) (FITC) and GH (Fig. 1C) (TRITC). The photographs shown together in Fig. I strongly support the notion that GH and IGF-I are produced by the same cell type. In this study, approximately 10% of the cells were positive for GH and 10% of the cells were positive for IGF-I. Few single (fluorescein or rhodamine) labeled cells were observed, suggesting that a large percentage of the cells are producing both GH and IGF-I at the same time. Taken together, the data support the existence of an autocrine regulatory circuit for leukocyte-derived GH and IGF-I within the immune system. DISCUSSION

In the present study, we have confirmed our earlier findings showing GH production by many different cell types of the immune system. In addition, we have shown that animals without a pituitary produce less lymphocyte GH in the spleen, thymus, and bone marrow and somewhat higher levels (1.6-fold) in the peripheral blood. Significant changes in the levels of GH RNA were not observed in the different tissues in control or hypophysectomized animals. In other studies, data obtained from reverse transcription and PCR support the idea that the GH RNA detected on our slot blots is similar to rat pituitary GH RNA. RNA from spleen or thymus and pituitary has been used to synthesize a 600-bp cDNA that reacts with the specific rat GH cDNA after Southern transfer (Weigent et al., 1991). Further, the leukocyte message is translated and results in the secretion of a bioactive molecule (Weigent et al., 1987; Weigent & Blalock, 1991). The results show that the levels of GH production in rats by most tissues was lower except in the periphery. The mechanism(s) is not yet understood but may suggest that cells containing the ability to produce GH may have undergone a redistribution and/or that the concentration of certain factors may be different in

GROWTH

G. 1. Fluorescent

HORMONE

PRODUCTION

BY LEUKOCYTES

373

photomicrographs of spleen cells labeled with FITC-conjugated ant ibodies to I and TRITC-conjugated antibodies to GH. Cultured ceils were washed three times it I PBS and spended at a concentration of I X 10’ cells/ml. The cells were air-dried to glass slides and fixed in 95i% ethanol. The cells were then labeled as described under Materials and Methods. A. phase rast; B, FITC-labeled antibodies to IGF-I: C, TRITC-labeled antibodies to GH. Mag nification.

374

WEIGENT,

BAXTER,

AND

BLALOCK

the periphery in an animal without a pituitary. We originally thought that substances in the serum such as GH from the pituitary might be mediating a negativefeedback effect on GH synthesis by leukocytes. However, the data presented here show that GH levels in cells of the immune system are not elevated in hypophysectomized animals but are significantly lower. In other studies consistent with these findings, we have found that adding back rat serum or GH to cells already cultured for 24 h has no significant effect on the synthesis of leukocyte-derived GH (data not shown). At the present time, several investigators have reported positive effects by GH, TRH, and GHRH (Guarcello et al., 1991; Hattori et al., 1990; Kao et al., 1989) and a negative effect by IGF-I (Baxter et al., 1991b) on the expression of leukocytederived GH. The cytokine regulation of pituitary GH has been documented (Weigent & Blalock, 1990b), and it is suspected that leukocyte GH may be under a similar regulatory control but no data to establish this have been reported. Although some controversy has existed, it appears that leukocytes have receptors for GH (Kiess & Butenandt. 1985) and IGF-I (Tapson et al., 1988). Thus, the expression of GH and its receptor and IGF-I and its receptor on leukocytes may allow the cells to autoregulate their growth. Consistent with this hypothesis, we have other data using a GH-antisense oligodeoxynucleotide showing that leukocyte-derived GH may stimulate proliferation (Weigent et al., 1991). Our finding that the same cells that produce GH also produce IGF-I supports an autocrine mechanism for the synthesis of this lymphocyte neuroendocrine hormone. Although other scenarios may be identified, these results support the synthesis of GH and IGF-I by the same cells, at least in the spleen under the experimental conditions we have examined. It should also be pointed out that the level of lymphocyte GH secretion overall is low and that differences exist between the percentage positive by indirect immunofluorescence and the number of cells showing signs of secretion by plaque assay (Kao, Harbour, & Meyer, 1992). Although the effect of GH on the immune system is being extensively investigated (Kelley, 1989; Weigent & Blalock, 1990b), the biological significance of GH synthesis by leukocytes is still poorly defined. GH alone and/or through the production of IGF-I appears to enhance the functional activities of T and B lymphocytes and macrophages and NK cells (Kelley, 1989). The ability of leukocytes to produce GH, GHRH, and IGF-I in regional lymphatic tissues or at sites of inflammation (Weigent, Riley, Galin, LeBoeuf, & Blalock, 199lb) suggests the importance of these hormones in acting locally in an autocrine and/or paracrine fashion. ACKNOWLEDGMENTS We thank Dr. Robert LeBoeuf for providing the synthetic oligonucleotide probes and Mr. Keith Berry for performing the FACS analysis. We also thank Vincent Law for excellent technical assistance and Diane Weigent for preparing the manuscript.

REFERENCES Baglia, L. A., Cruz, D., & Shaw. J. E. (1992). Production of immunoreactive forms of growth hormones by the Burkitt tumor serum-free cell line sfRamos. Endocrinology 130, 2446-2452. Baxter, J. B., Blalock. J. E., & Weigent, D. A. (1991a). Expression of immunoreactive growth hormone in leukocytes in vivo. J. Neuroimmunol. 33, 43-54. Baxter, J. B., Blalock, J. E.. & Weigent, D. A. (1991b). Characterization of immunoreactive insulin-

GROWTH

HORMONE

PRODUCTION

BY LEUKOCYTES

375

like growth factor-I from leukocytes and its regulation by growth hormone. Endocrinology 129, Il27-1134. Blalock, J. E. (1989). A molecular basis for bidirectional communication between the immune and neuroendocrine systems. Physiol. Rev. 69, 79-108. Blalock, J. E., & Smith, E. M. (1980). Human leukocyte interferon: Structural and biological relatedness to adrenocorticotropic hormone and endorphins. Proc. Nntl. Acud. Sci. USA 77, 59725914. Boyum. A. (1968). Isolation of mononuclear cells and granulocytes from human blood. Stand. J. C/in. Lab. fnvesf. 21 (Suppl. 97), 77-82. Ebaugh. M. J., & Smith, E. M. (1988). Human lymphocyte production of immunoreactive luteinizing hormone. FASEB J. 2, 7811. [Abstract] Guarcello, V.. Weigent. D. A., & Blalock, J. E. (1991). Growth hormone releasing factor receptors on lymphocytes. Cell. Imm~nol. 136, 291-301. Harbour, D. V.. Smith, E. M.. & Blalock. J. E. (1987). A novel processing pathway for proopiomelanocortin in lymphocytes: Endotoxin induction of a new pro-hormone cleaving enzyme. J. Neurosci. Res. 18, 95-101. Hattori, N.. Shimatsu. A., Sugita, M.. Kumagai. S., & Imura. H. (1990). lmmunoreactive growth hormone (GH) secretion by human lymphocytes: Augmented release by exogenous GH. Biothem. Biophys. Res. Commun. 168, 396401. Hiestand, P. C.. Mekler, P., Nordmann, R., Grieder, A., & Permmongkol, C. (1986). Prolactin as a modulator of lymphocyte responsiveness provides a possible mechanism of action for cyclosporin. Proc. N&l. Acud. Sci. USA 83, 2599-2603. Johnstone, A., & Thorpe. R. (1987). Immunochemi.rtry in practice, ed. 2. Blackwell Scientific Publishing: London. Kao, T.-L., Harbour. D. V.. Smith. E. M.. & Meyer, W. J. (1989). Immunoreactive growth hormone production by cultured lymphocytes. In Program of the 71st Annual Meeting qf the Endocrine Society. p. 108. Seattle. [Abstract] Kao, T.-L., Harbour. D. V.. & Meyer, W. J. (1992). Immunoreactive growth hormone production by cultured lymphocytes. Ann. N. Y. Acud. Sci. 650, 179-181, Kasson, B. G., & Tuchel, T. L. (1989). Identification and characterization of a lymphocyte-produced substance with gonadotropin-like activity. In 7/.vt Annrcul Meeting oflhe Endocrine Society, p, 75. [Abstract] Kavelaars. A., Ballieux, R. E., & Heijnen. C. .I. (1989). The role of interleukin-l in the CRF- and AVP-induced secretion of P-endorphin by human peripheral blood mononuclear cells. J. Neurosci. Res. 18, 95-101. Kelley. K. W. (1989). Growth hormone. lymphocytes, and macrophages. Biochem. Pharmucol. 38, 705-713. Kiess, W., & Butenandt. 0. (1985). Specific growth hormone receptors on human peripheral mononuclear cells: Reexpression. identification, and characterization. J. C/in. Endocrinol. Merub. 60, 740-146. Kruger, T., & Blalock. J. E. (1986). Cellular requirements for thyrotropin enhancement of in vitro antibody production. J. fmmunol. 137, 197-200. Langford, M. P.. Weigent. D. A., Georgiades, J. A., Johnson, H. M., & Stanton. G. J. (1981). Antibody to staphylococcal enterotoxin .4-induced human immune interferon. J. Immuno[. 126, 162&1623. Maniatis, T., Fritsch, E. F., & Sambrook. J. (1982). Molecular cloning: A laboratory munuul. Cold Spring Harbor Laboratory: Cold Spring Harbor, NY. Montgomery, D. W., Zukoski, C. F., Shah, N. G.. Buckley, A. R., Pacholczyk, T., & Russell, D. H. (1987). Concanavalin A-stimulated murine leukocytes produce a factor with prolactin-like bioactivity and immunoreactivity. B&hem. Biophys. Res. Commun. 145, 692-698. Seeburg, P. H.. Shine. J.. Martial, J.. Baxter, J. D., & Goodman. H. M. (1977). Nucleotide sequence and amplification in bacteria of a structural gene to rat GH. Nature 270, 486-494. Smith, E. M.. & Blalock, J. E. (1981). Human lymphocyte production of ACTH and endorphin-like substances: Association with leukocyte interferon. Proc. Nat/. Acud. Scj. USA 78, 753&7534. Smith, E. M.. Phan, M., Coppenhaver, D.. Kruger, T. E., & Blalock, J. E. (1983). Human lymphocyte production of immunoreactive thyrotropin. Proc. Nut/. Acad. Sci. USA 80, 601o-fjOl3. Stephanou, A., Jessop, D. S.. Knight, R. A., & Lightman, S. L. (1990). Corticotropin-releasing fattor-like immunoreactivity and mRNA in human leukocytes. Bruin Behav. Immune/. 4, 67-73.

376

WEIGENT,

BAXTER,

AND BLALOCK

Tapson, V. F., Born-Schnetzier. M.. Pilch. P. F., Center, D. M.. & Berman, J. S. (1988). Structural and functional characterization of the human T-lymphocyte receptor for insulin-like growth factor I in vitro. J. C/in. Invest. 82, 95&957. Weigent, D. A., Baxter. J. B., Wear, W. E., Smith, L. R.. Best, K. L.. & Blalock. .I. E. (1987). Production of immunoreactive growth hormone by mononuclear leukocytes. Fed, Proc. 46, 926. [Abstract] Weigent, D. A., Baxter, J. B., Wear, W. E., Smith, L. R., Bost, K. L.. & Blalock. J. E. (1988). Production of immunoreactive growth hormone by mononuclear leukocytes. FASEB J. 2, 28122818. Weigent, D. A., & Blalock. J. E. (1987). Interactions between the neuroendocrine and immune systems: Common hormones and receptors. Immnal. Rev. 100, 79-108. Weigent, D. A., & Blalock, J. E. (1989). Expression of growth hormone in leukocytes. Int. Rev. Immuna/. 4, 195-213. Weigent, D. A.. & Blalock. J. E. (1990a). Immunoreactive growth hormone releasing hormone in rat leukocytes. 1. Nerrroimmunol. 29, l-13. Weigent, D. A., & Blalock, J. E. (1990b). Growth hormone and the immune system. Prog. Neuroendocrin. Immunol. 3, 231-241. Weigent, D. A.. & Blalock, J. E. (1991). The production of growth hormone by subpopulations of rat mononuclear leukocytes. Cell. Immunol. 135, 5545. Weigent, D. A., Blalock, J. E.. & LeBoeuf, R. D. (1991a). An antisense oligonucleotide to growth hormone mRNA inhibits lymphocyte proliferation. Endocrinology 128, 2053-2057. Weigent, D. A., Riley. J. E.. Galin, F. S.. LeBoeuf, R. D.. & Blalock, J. E. (1991b). Detection of GH and GHRH-related messenger RNA in rat lymphocytes by the polymerase chain reaction. Proc. Sot.

Exp.

Biol.

Med.

Received July 30. 1992

198, 643448.