0013-7227/91/1294-1727$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society

Vol. 129, No. 4 Printed in U.S.A.

Characterization of Immunoreactive Insulin-Like Growth Factor-I from Leukocytes and Its Regulation by Growth Hormone* JUDITH B. BAXTER, J. EDWIN BLALOCK, AND DOUGLAS A. WEIGENT University of Alabama at Birmingham, Department of Physiology and Biophysics, Birmingham, Alabama 35294-0005

ABSTRACT. In the present study, we investigated the production of insulin-like growth factor I (IGF-I) by leukocytes and its production after treatment with GH. Immunoreactive (ir) IGF-I was observed in leukocytes by direct immunofluorescence with fluorescein isothiocynate-conjugated antibodies to IGF-I. Studies using immunoaffinity purification, HPLC and a fibroblast proliferation bioassay suggests that the de novo synthesized leukocyte-derived irIGF-I is similar in mol wt, antigenicity, and bioactivity to serum IGF-I. We also evaluated the effect of GH on the production of leukocyte-derived irIGF-I. Spleen cells cultured for 24 h in the presence of exogenous GH caused a 2fold elevation of irIGF-I as demonstrated by RIA and immuno-

T

HE DEMONSTRATION that neuroendocrine hormones can be synthesized by the immune system has allowed for the development of a model in which the neuroendocrine hormones may serve as signal molecules between the neuroendocrine and immune systems (1). Several leukocyte-derived peptides that are similar to neuroendocrine hormones have been identified (1-3). Very recently, our work (4, 5) and that of others (6, 7) have provided strong evidence that human and rat leukocytes produce GH-related RNA and immunoreactive (ir) protein both in vitro and in vivo. The leukocyte GH protein is similar to pituitary GH in terms of antigenicity, mol wt, and bioactivity. Although the factors important for the synthesis of pituitary GH are well studied, the specific mechanisms governing the regulation of leukocyte-derived irGH remain unknown. The primary hypothalamic regulation of pituitary GH secretion is mediated through the stimulatory actions of GH-releasing hormone (GHRH) (8, 9) and the inhibitory actions of somatostatin (SRIF) (10). In addition to the Received March 18, 1991. Address all correspondence and requests for reprints to: Dr. Douglas Weigent, University of Alabama at Birmingham, Department of Physiology and Biophysics, UAB Station, BHSB 894, Birmingham, Alabama 35294-0005. * This work was supported in part by NINCD Grant RO1-NS-24636 and NIDDK Grant RO2-DK-38024.

fluorescence. In order to determine if leukocyte-derived irGH can stimulate the production of irIGF-I, we cultured spleen cells for 24 h in the presence of antibodies specific for GH. The data showed a decrease in the number of cells positive for irIGF-I, suggesting that leukocyte-derived irGH may stimulate the synthesis of irIGF-I by leukocytes. We also demonstrated that exogenous IGF-I can decrease the levels of leukocyte GH-related RNA and ir protein. Taken together, our data demonstrate the synthesis and secretion of bioactive irIGF-I from leukocytes and suggest a regulatory circuit for leukocyte-derived irGH and irIGF-I within the immune system. {Endocrinology 129: 17271734,1991)

hypothalamic regulation of GH, the peripheral hormone insulin-like growth factor I (IGF-I) can participate in the negative feedback regulation of pituitary GH expression (11, 12). To date, some evidence has accumulated to suggest that the hypothalamic regulators of pituitary GH, somatostatin (6,13), and GHRH (7,14,15), may be involved in the control of irGH synthesis by leukocytes. The role of IGF-I in the regulation of leukocyte irGH to our knowledge has not yet been investigated. Although the major source of IGF-I is the liver, many extra-hepatic sources have been identified (see review Ref. 16). Northern analysis of RNA from the rat spleen and thymus demonstrated similar sized transcripts to those seen in the liver (17). The administration of GH to hypophysectomized rats was observed to cause a 4- to 5-fold increase in the IGF-I messenger (m) RNA transcripts from the tissues examined, suggesting GH dependence (17). IGF-I appears to be produced by and to have numerous effects on immune cells. Human aveolar macrophages, activated by asbestos (18) and transformed human T cell lines, produce an irIGF-I (19). Epstein-Barr virus-transformed human B lymphocytes secrete an irIGF-I, and the addition of GH to the cultured transformed lymphocytes augmented the production of irIGF-I and stimulated the rate of cell multiplication (20). IGF-I is chemotactic to resting and activated T cells,

1727

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 11 April 2016. at 06:24 For personal use only. No other uses without permission. . All rights reserved.

IGF-I REGULATION BY GH

1728

and these cells bear cell surface receptors for IGF-I (21), as do human IM-9 lymphocytes (22). IGF-I also appears to enhance DNA synthesis (23) an effect also attributed to GH (24). In addition, enhancement of human marrow myeloid colony formation by IGF-I and GH was detected in serum-free cultures stimulated with human recombinant granulocyte/macrophage colony stimulating factor (25). All of these reports considered together strongly support a physiological role for GH and IGF-I in immunoregulation. In this paper we show that irIGF-I produced by leukocytes is bioactive and that the levels increase after treatment with GH. Furthermore, the levels of leukocyte-derived irGH are lower after IGF-I treatment. Together with our previous GH and GHRH results, these findings suggest a complete immune regulatory circuit for GH.

Materials and Methods Materials Monkey antirat 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 Hormone and Pituitary Program. The antisera is highly specific and shows a 0.5% crossreactivity with IGF-II and cross-reacts minimally with insulin at 10~6 M according to the suppliers. The recombinant human IGF-I used in these studies was obtained from Chemicon (El Segundo, CA). 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 female Sprague-Dawley rats (150-200 g) used for the experiments were purchased from Harlan (Prattville, AL). Spleens and thymi were aseptically removed and teased apart in RPMI-1640 medium supplemented with penicillin, streptomycin, and mycostatin (100 U/ml). Pieces of liver were cut into small pieces and dispersed using trypsin (0.25%). The dispersed liver cells were washed three times in Dulbecco's modified Eagle's medium (DMEM), and a dilute suspension of cells was passed over a Ficoll-hypaque gradient (26). Cell viability was monitored by trypan blue exclusion. Only cell preparations with a 90% viability or greater were used. Before use in any assay, the cells were washed in serum-free RPMI-1640 and antibiotics. After isolation, cells were either immediately frozen in microfuge tubes for RNA isolation or washed and spotted onto glass slides for immunofluorescent studies (see below). BALB/c 3T3 fibroblast cells used for the bioassay described

Endo • 1991 Voll29«No4

below were kindly provided by Ms. Cindy Bull and Dr. Richard Marchase of the University of Alabama at Birmingham. RNA isolation and blotting Total cytoplasmic RNA was isolated by the proteinase K method (27). 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 nM. The RNA (10 Mg) was blotted on nitrocellulose membranes with the Minifold II slot blotter (Schleicher and Schuell, Inc., Keene, NH) as described by the manufacturers. After hybridization with either a GH-specific complementary (c) DNA probe or an IGF-I specific cDNA probe as described below, the membranes were 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 (27). Densitometric values of IGF-I and GH RNA were determined as a percentage with 5 ng control liver RNA or pituitary RNA as the 100% reference point, respectively. Membranes were then reprobed with a synthetic oligodeoxynucleotide 188 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 18$ ribosomal probe. IGF-I and GH cDNAs and hybridization Plasmids containing the rat (28) 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 (27). Eight hundred-base pair HindlU inserts (nucleotides -20 to 780) were purified from the plasmid (27). Plasmids containing the rat IGF-I cDNA were kindly provided by Dr. Liam Murphy (Department of Physiology, University of Manitoba, Manitoba, Canada) (17). A 0.5-kilobase IGF-I cDNA insert (containing amino acid —3 to amino acid 105) was released from the plasmid (pGEM-3) using the primers flanking the pGEM-3 cloning site in a polymerase chain reaction using standard techniques (29). The primers were to the SP6 and T7 RNA polymerase transcription initiation sites. The cDNAs were labeled with [32P] deoxycytidine triphosphate by nick translation using a commercially available kit (Bethesda Research Laboratories, Gaithersburg, MD) to a specific activity of 1-2 x 103 cpm/^g. The mouse 18$ 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 (27). 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, NaCl, NaH2PO4, H2O, EDTA, and standard buffer containing 32P-labeled insert (27). After hybridization, the membranes were extensively washed until the radioactivity in the final wash was close to background using 0.4 M NaCl, 0.04 M sodium citrate, and 0.1% SDS for 10 min, and 0.04 M NaCl, 0.004 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 NaCl, 0.003 M sodium citrate, and 0.1% SDS for 30 min at 55 C with gentle mixing. The

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 11 April 2016. at 06:24 For personal use only. No other uses without permission. . All rights reserved.

IGF-I REGULATION BY GH 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 immunofluorescence The cells used for immunofluorescence were washed three times in 0.01 M PBS, pH 7.2, resuspended in PBS (1 x 106 cells/ml) and air-dried onto glass slides. After fixation with icecold 95% ethanol, the cells were rehydrated in PBS and incubated with appropriate serum (1:200) for 1 h at 37 C. The cells were washed three times with PBS and then covered in a 1:50 dilution of either fluorescein isothiocyanate (FITC)-conjugated monkey-antirat GH or FITC-conjugated rabbit antihuman IGF-I prepared in our laboratory by standard techniques (30). The slides were allowed to incubate 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 observed using an Olympus (Marietta, GA) vertical fluorescence illuminator model A-RFL. Immunoaffinity chromatography The immunoaffinity columns were prepared by conjugating highly specific rabbit antihuman IGF-I antibodies to Affigel 10 (Bio-Rad, Richmond, CA) according to the manufacturer's instructions. The column was washed in PBS with 0.1 M sodium azide (pH 7.4) before use. In a typical experiment, cells (50 X 106 cells/ml) were cultured for 24 h in RPMI-1640 without serum containing 2 /uCi/ml of a uniformly tritium-labeled mixture of amino acids (Amersham, Arlington Heights, IL). Spent supernatant fluids were centrifuged at 3000 x g for 15 min and applied to the column. The column was washed with 20 void vol PBS-azide. Material bound to the column was then eluted with 0.2 M glycine-HCl buffer containing 0.5 M NaCl (pH 2.0) as described (31). The column was regenerated by washing with PBS-azide, and then the initial effluent was reapplied and eluted as before. Pooled effluents were dialyzed against distilled water and then dried by lyophilization and reconstituted in buffer for further analysis. HPLC Peak fractions of leukocyte-derived irIGF-I from immunoaffinity columns were pooled, dialyzed, and reduced in vol by lyophilization. Samples were reconstituted in the mobile phase buffer (0.1 M Na2SO4, 0.02 M NaH2PO4, pH 6.91) and applied to a SOTA GF-200 analytical column (Rainin Instrument Co., Emeryville, CA). The column was run at 0.6 ml/min over 30 min. The HPLC column was calibrated with the Bio-Rad standards of thyroglobulin, 7-globulin, ovalbumin, myoglobulin, and vitamin B-12. RIA Freshly dispersed spleen cells (50 x 106 cells/ml) were cultured 24 h in 3 ml RPMI-1640 without serum. The supernatant

1729

fluids were lyophilized and then resuspended in 300 n\ sterile water. Concentrated fluids were measured for their IGF-I content by a standard RIA procedure using a commercial kit available from Incstar Corporation (Stillwater, MN). The RIA kit includes an octadecasilyl (ODS) -silica extraction procedure to separate IGF-I from binding proteins. This involves incubating the controls and unknown samples with 0.5 N HC1 and then applying the samples to ODS-silica/Cl8 columns. The columns are then washed two times with 4% acetic acid, and the samples are eluted with methanol, evaporated to dryness, and reconstituted in a BSA-borate buffer provided in the kit. According to the manufacturer of the RIA kit, the rabbit antisomatomedin C antibody used in this assay has less than 1% cross-reactivity with IGF-II, human GH, fibroblast growth factor, transforming growth factor, and platelet-derived growth factor. The assay has a sensitivity of not less than 15 ng/ml. pHJThymidine incorporation into BALB/c 3T3 fibroblast cells The bioassay based on the proliferative response of BALB/ c 3T3 fibroblast cells to somatomedins was used to study the bioactivity of leukocyte-derived irIGF-I (31). The 3T3 fibroblast cells were cultured (37 C, 5% CO2) in DMEM containing 10% calf serum (Hyclone, Logan, UT) in 96-well plates. After 48 h, the cells were washed twice with DMEM. The media was changed to DMEM containing 0.5% calf serum, and the cells were cultured for 24 h. The cells were then washed three times with DMEM. After the addition of either commercial IGF-I or affinity purified irIGF-I, the cells were cultured for 8 h and pulsed for 16 h with [3H]thymidine (0.2 ^Ci/well). The media was aspirated off, and the cells were dispersed from the plate by the addition of 0.025% trypsin in PBS and harvested on a glass filter by a multiple cell harvester (Cambridge Technology, Inc., Watertown, MA). The incorporated [3H]thymidine was counted by a scintillation counter (TM Analytic, Oak Grove Village, IL). The affinity purified irIGF-I was prepared as described above. The lyophilized irIGF-I was resuspended in 3 ml water and was dialyzed 72 h against PBS. Fifty microliters were used in each well. 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 mean ± SEM. Rat pituitary or liver RNA (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 Student's t test and the Mann-Whitney test where differences are indicated as being significant, P is less than 0.05.

Results Detection of IGF-I-related RNA and immunoreactiue protein in rat leukocytes Previous investigators have shown that IGF-I-related mRNA can be identified in rat spleen and thymus cells by Northern analysis (17). To confirm these findings and in addition show that irIGF-I molecules were bioactive,

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 11 April 2016. at 06:24 For personal use only. No other uses without permission. . All rights reserved.

IGF-I REGULATION BY GH

1730

we determined whether the message is translated and the product secreted. Analysis of spent culture supernatant fluids from leukocytes by RIA for IGF-I suggest that leukocytes secrete approximately 1.38 ng/liter x 106 leukocytes in a 24-h culture period. This value is approximately 60 times less the amount of IGF-I produced on a per cell basis by cultured hepatocytes over a similar time period (32). In addition, the supernatant fluids from the cultured cells were collected and passed twice over an Affigel 10 affinity column coupled with antibodies to IGF-I. This affinity-purified leukocyte-derived irIGF-I was chromatographed by HPLC. The data are shown in Fig. 1. The mol wt of serum IGF-I is 7.6K (16). The majority of the protein appeared in fractions 22 and 23 which corresponds to a mol wt of 4-8K and suggests a similar size irIGF-I is produced by leukocytes. The peak at fraction 16 corresponds to the mol wt of the 150K binding protein, suggesting a very small portion of the irIGF-I might be bound to binding protein, although this may be considered unlikely since irIGF-I from human leukocytes is not associated with a binding protein (20, 25, 33). The peak at fraction 20 is approximately 17K, perhaps corresponding to a larger mol wt IGF-I-like peptide (25, 34, 35). However, the majority of the protein is in fractions 22 and 23, and the incorporation of the

360

B-12

r

r

0.05

Endo • 1991 Vol 129 • No 4

3

H-amino acids suggests the protein was de novo synthesized. IrIGF-I molecules also can be detected in leukocytes by immunofluorescence. Table 1 shows the results of direct immunofluorescence measurements on spleen, thymus, and liver cells. Spleen and thymus cells incubated with a FITC-conjugated antibody to IGF-I show an elevated level of fluorescence when cultured 24 h compared to noncultured cells. The labeling of spleen cells was blocked by prior incubation of the FITC-conjugated antibodies to IGF-I with excess IGF-I (0.8%). Trypsinized liver cells labeled positive for IGF-I as expected. Figure 2, A and B shows a photomicrograph of cultured spleen and thymus cells, respectively, labeled with the FITC-conjugated antibody specific to IGF-I. The prior incubation of antibodies to IGF-I with excess IGF-I also completely blocked the labeling of thymus cells (data not shown). Overall, the data confirm that rat leukocytes cultured for 24 h produce an irIGF-I that is similar to hepatic IGF-I in terms of size and antigenicity. Growth stimulatory activity of leukocyte-derived irIGF-I The replication of BALB/c 3T3 cells in microtiter culture is specifically stimulated by IGF-I (31). We have used this assay to study the biological activity of leukocyte-derived irIGF-I. The effect of incubating cell cultures for 24 h with IGF-I from commercial sources as well as from leukocytes is shown in Fig. 3. The data show that 50 n\, which was equivalent to 11.5 ng/ml of leukocyte-derived irIGF-I, was able to stimulate the incorporation of thymidine in the BALB/c 3T3 cell line similar

240 - 0.03

L

0

5

10

15

20

™.

0.01

TABLE 1. Detection of irIGF-I production in leukocytes by direct immunofluorescence Source of cells Spleen"

0

25

Fraction Number

FIG. 1. HPLC of leukocyte-derived irIGF-I from rat spleen. Supernatant fluids (500 ml) were collected from 24-h-old cultured cells (50 x 106/ml) cultured in the presence of 2 /iCi/ml uniformly tritium-labeled mixture of amino acids and purified over an anti-IGF-I affinity column as described in Materials and Methods. The lyophilized material was resuspended in 1.5 ml mobile phase buffer. Three hundred fifty microliters of the irIGF-I were then chromatographed on a HPLC mol wt sizing column (Sota GF 200). Fractions of 0.6 ml were collected for 30 min. The absorbance at 280 nM is shown by the solid line. One hundred microliters of the fractions were counted in a TM Analytic 0-counter, and counts per minute are shown by the dashed line. One microCurie of commercial [125I]IGF-I (Amersham) was also chromatographed over the same column. The arrow represents the fraction where the IGF-I eluted from the column. The standards used to calibrate the column were thyroglobulin (TG) (670,000), immunoglobulin (GG) (158,000), ovalbumin (ON) (44,000), myoglobin (MY) (17,000), and vitamin B-12 (B-12) (1350) (Bio-Rad).

Thymus6 Liverd

Time of harvest 0 24 24 0 24 0

Percent positive by immunofluorescence0 4.4 ± 1.1 9.5 ± 1.0 0.8 ± 0.4c 4.3 ± 1.0 8.1 ± 2.6 32.4 ± 3.1

" Five hundred cells from each group were counted from randomly chosen fields. Data is presented as the percent of positive cells ± SEM. 6 Spleen and thymus cells were removed from rats and washed three times in PBS. Cells were resuspended to a concentration of 1 x 106 cells/ml, and 10 nl were fixed onto glass slides and labeled as described in Materials and Methods. The remaining cells were incubated 24 h at 37 C. The slides were washed three times in PBS before determining the percentage of fluorescent cells. c FITC-conjugated antibodies to IGF-I were mixed with excess IGFI (10 Mg/ml) and incubated at 4 C for 2 h. This mixture was then added to glass slides with fixed spleen cells which had been previously treated with normal rabbit serum. d Liver cells from rat were dispersed using trypsin (0.25%). The cells were then washed three times in RPMI-1640 with 10% fetal calf serum and antibiotics and then washed three times in PBS. The cells were labeled with FITC-conjugated antibody to IGF-I as described in Materials and Methods.

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 11 April 2016. at 06:24 For personal use only. No other uses without permission. . All rights reserved.

IGF-I REGULATION BY GH E

1731

5

°

40

2

30

*

20

10

..llll I

None 0.8

1.5

3.0 12.5

IGF-I (ng/mL)

25

irIGF-I

FIG. 3. Effect of affinity-purified leukocyte-derived irIGF-I on [3H] thymidine incorporation by BALB/c 3T3 fibroblast cells. 3T3 fibroblast cells were cultured as described in Materials and Methods. The cells were cultured in the presence of diluent (control), commercial IGF-I (0.85-25 ng/ml), and affinity-purified irIGF-I (50 /tl) and [3H]thymidine (0.2 ^d/well) and mash harvested to determine the extent of thymidine incorporation. Each value represents the mean ± SEM of 12 replicate samples. The results are representative of three different experiments. Asterisks denote statistical significance from control at P < 0.05.

B

TABLE 2. An increase in irIGF-I production from leukocytes after treatment with exogenous GH irIGF-I (ng/liter x 106 cells)6 4 0.86 ± 0.03 No addition 1.38 ± 0.06 24. 0.74 ± 0.03 4 500 ng/ml GH 2.74 ± 0.15 24 0 Spleens were removed from animals (3 rats per group) and immediately dispersed. The cells were washed in RPMI-1640 without serum. The cells were then incubated in 3 ml RPMI-1640 (50 X 106 cells/ml). Half of the cells were incubated in the presence of 500 ng/ml rat GH. The supernatant fluids were collected at 4 h and 24 h after the start of incubation. 6 The supernatant fluid was lyophilized and then resuspended in 300 fil distilled water. The fluid was then analyzed (50 ^I/tube) by RIA as described in Materials and Methods. Data is presented as the average amount of irIGF-I (ng/liter X 106 cells) ± SEM. Treatment0

FIG. 2. Fluorescent photomicrographs of cells labeled with FITC-conjugated antibodies to IGF-I. Cultured cells were washed three 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 IGF-I. A, Spleen cell. B, Thymus cells. Magnification, 400x.

to the control commercial IGF-I. Although the stimulation is 40% lower than predicted, this value was not unexpected based on the instability of IGF-I during purification. Affinity-purified irIGF-I from leukocytes stimulated [3H]thymidine incorporation in a dose-dependent manner (data not shown). Taken together, the findings are consistent with the de novo synthesis of a bioactive leukocyte-derived irIGF-I. Increased levels of irIGF-I in GH-treated cultures of leukocytes In the neuroendocrine system, the level of IGF-I gene expression and secretion is thought to be regulated in

Time (h)

part by GH (12). In view of this, we investigated the effect of GH on the production irIGF-I by leukocytes. Spleens were removed from killed rats and the cells dispersed and washed in RPMI-1640 with antibiotics and without serum. The cells (50 x 106 cells/ml) were then cultured in 3 ml RPMI-1640 and treated with 500 ng/ml rat GH for either 4 h or 24 h. The supernatant fluids were collected for analysis of irIGF-I content by RIA as described in Materials and Methods. The data in Table 2 show an increased amount (2.74 ± 0.15 ng/liter X 106 cells) of irIGF-I in the supernatant fluids from leukocytes cultured for 24 h in the presence of 500 ng/ mlGH. In order to confirm our results from the RIA, we determined if there was also an increase in the presence of cytoplasmic irIGF-I by direct immunofluorescence.

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 11 April 2016. at 06:24 For personal use only. No other uses without permission. . All rights reserved.

1732

IGF-I REGULATION BY GH

Endo • 1991 Vol 129 • No 4

Spleen cells cultured for 24 h in the presence of GH were labeled with FITC-conjugated antibodies to IGF-I. A 2fold increase in the number of cells positive for IGF-I was seen in cells treated with GH (data not shown). The cultured spleen cells were also examined for IGF-I RNA by slot blot analysis and no significant difference was seen in the amount of cytoplasmic IGF-I-related RNA in GH-treated cells compared to nontreated cells (data not shown). Taken together, it appears that the stimulatory effect of GH on irIGF-I production may be at the level of translation. Since we have shown that the addition of exogenous GH can increase the production of irIGF-I, we next determined if irGH can influence irIGF-I production in leukocytes by determining the effect of adding antibody

pared for immunofluorescence, and the remaining cells were used to isolate cytoplasmic RNA. The cytoplasmic RNA was slotted onto nitrocellulose and probed with a rat GH-specific cDNA as described in Materials and Methods. After 24 h of in vitro culture, a significant decrease in GH-related RNA was seen in cells treated with 50 ng/ml IGF-I (Fig. 4). Figure 5 shows a decrease in the number of cells labeled positive for irGH production after IGF-I treatment. Taken together, these data demonstrate that IGF-I can decrease leukocyte-derived irGH which is similar to its effect on pituitary GH.

to GH to leukocyte cultures. Freshly dispersed spleen

the ability of rat leukocytes to produce a bioactive irlGF-

cells were cultured in RPMI-1640 with antibiotics and the dilution of GH antibodies listed in Table 3. After 24 h, the cells were washed three times with PBS and air dried onto glass slides and fixed in 95% ethanol. The spleen cells were then labeled with FITC-conjugated antibodies to IGF-I. The data show that at the highest concentration of antibodies to GH the number of cells positive for irIGF-I decreased 2-fold. Taken together, the data suggests that the production of irIGF-I by leukocytes can be mediated by GH. Decreased levels of irGH in IGF-I-treated cultures of leukocytes

Discussion The results of the studies reported here demonstrate I molecule. Secreted irIGF-I could be demonstrated using immunoaffinity purification, HPLC, fibroblast proliferation assay, and direct immunofluorescence. IrIGF-I was also measured by RIA using a commercial kit which includes an ODS-silica extraction procedure. The amount of irIGF-I from both untreated and acid-extracted spleen cell supernatant fluids were found to be equal by RIA, which is consistent with the idea that 1600 - , 1400

-

Since we demonstrated that GH can increase the production of irIGF-I from leukocytes, the next question we addressed was whether IGF-I could influence the production of leukocyte-derived GH. In the neuroendocrine system, IGF-I has been shown to participate in a negative feedback regulation of pituitary GH secretion and gene expression (11). Spleen cells were dispersed and cultured in RPMI-1640 with antibiotics and 0-50 ng/ml IGF-I. After 24 h, the cells were harvested and washed three times in PBS. A small portion of these cells were preTABLE 3. Detection of a decrease in irIGF-I production from leukocytes treated with antibodies to GH by direct immunofluorescence0 Dilution of antibody

Percent positive by immunofluorescence6

No antibody 1:50 1:500 1:5000

6.6 ± 1.0 3.0 ± 0.7 3.9 ± 0.9 5.6 ± 1.2

0 Freshly dispersed spleen cells were cultured in RPMI-1640 without serum at 37 C, 5% CO2 with the above final dilution of antibodies to rat GH (NIDDK anti-rGH-S-5, 0.4 ml). After 24 h, the cells were washed three times in PBS, fixed onto glass slides, and stained with FITC-conjugated antibodies to IGF-I as described in Materials and Methods. 6 At least 500 cells from each group were counted from randomly chosen fields. Data is presented as the percent of positive cells ± SEM.

24 Time (h)

FIG. 4. Inhibition of the induction of GH-related RNA by IGF-I. Groups of animals (three per group) were killed and the spleens removed. Zero-hour samples of dispersed spleen cells were immediately frozen for RNA isolation. The remaining cells were cultured in RPMI1640 with antibiotics at 37 C, 5% CO2. Half of the cells were also cultured with 50 ng/ml IGF-I. At 2,4, and 24 h the cells were harvested and frozen as pellets at -20 C. Cytoplasmic RNA was isolated from each sample, and 10 ng was slotted onto nitrocellulose and probed for GH RNA as described in Materials and Methods. Data points represent the percent of control of average densitometric scans ± SEM. The average densitometric value for the 0-h control was 3.0 ± 0.3. The data shown are from a typical experiment performed three times. O, No addition; • , cultured in the presence of 50 ng/ml IGF-I.

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 11 April 2016. at 06:24 For personal use only. No other uses without permission. . All rights reserved.

IGF-I REGULATION BY GH 12 -i

o

FIG. 5. Detection of a decrease in irGH production from leukocytes treated with exogenous IGF-I by direct immunofluorescence (IF). Spleens were removed from animals and were cultured in RPMI-1640 without serum for the times listed in the figure along with 50 ng/ml IGF-I. At each harvest time, the cells were washed three times in PBS. Ten microliters were fixed onto glass slides and labeled as described in the methods with FITC-conjugated antibody to rat GH. At least 500 cells from each group were counted from randomly chosen fields. Data is presented as the percent of positive cells ± SEM. O, No addition; • , cultured in the presence of 50 ng/ml IGF-I.

leukocytes may not produce IGF-I binding proteins (data not shown). The data suggests that the de novo synthesized leukocyte-derived irIGF-I is similar in size, antigenicity, and bioactivity to serum IGF-I. GH has been established as the primary regulator of IGF-I gene expression for the liver as well as many extrahepatic tissues, including the heart, lung, and pancreas (12,36). GH regulation of IGF-I gene expression in these tissues appears to be rapid and occurs at the level of transcription (37). In our studies, it appears that in leukocytes GH acts principally at the level of translation to enhance irIGF-I levels. In addition to GH, other tropic hormones such as ACTH, TSH, LH, and FSH can stimulate the paracrine synthesis of IGF-I from their target organs (38). Thus, it was not surprising that GH was able to enhance the levels of leukocyte irIGF-I produced. It is also important to note that antibodies to GH caused a decrease in irIGF-I levels. One can speculate that the GH antibodies have interfered with the action of leukocyte-derived GH, and thus the signal for irIGF-I production has been removed. This is important in that it suggests a regulatory mechanism for the production of a leukocyte-derived peptide that is similar to the neuroendocrine mechanism. The idea that IGF-I might feedback inhibit the production of leukocyte irGH also was not unexpected. The data taken together suggest that endogenously produced GH and IGF-I can regulate the production of the other hormone and are consistent with the possibility that they may function in intrasystem

1733

communication. The overall levels of hormones produced by leukocytes, however, are much smaller and therefore most likely function by an autocrine or paracrine mechanism. The effect of IGF-I on the immune system is varied and includes the stimulation of chemotaxis (21) and DNA synthesis (22). GH has been shown to have an effect on most immune cell types (39). Recently, we have shown that leukocytes can be induced to produce GHrelated RNA in vivo in rats injected with lipopolysaccharide (5). In another study, we have examined the role of GH in lymphocyte proliferation by examining the effect of an antisense oligodeoxynucleotide complementary to GH mRNA. The results of these studies showed that antisense GH oligodeoxynucleotide treatment inhibits lymphocyte production of irGH and that antisense GH oligodeoxynucleotide-mediated inhibition of irGH production resulted in a decrease in lymphocyte proliferation. The data indicate that irGH produced by lymphocytes can stimulate proliferation, suggesting that irGH may play an autocrine/paracrine role in lymphocyte replication (40). All of these reports considered together strongly support a physiological role for GH in immunoregulation. Several reports indicate that GHRH may also be involved in immunomodulation. Thus, GHRH has been shown to stimulate lymphocyte proliferation (41), inhibit NK activity (42), and inhibit the chemotactic response (43). Our own studies have identified a specific GHRH receptor on immune cells as well as measured an increase in Ca2+ uptake, thymidine incorporation, and the levels of GH RNA (14, 44). Our data (15), along with others (45-47) show the extra-hypothalamic production of irGHRH. Since we know that leukocytes can function as a source of irGH (4), this suggests the possibility that irGHRH synthesis by leukocytes may function as a signal for the synthesis of leukocyte-derived irGH. The new data we have obtained and report here demonstrate the ability of leukocyte-derived irGH to stimulate irIGF-I as shown by using antibodies to GH as well as the ability of exogenous IGF-I to inhibit the levels of irGH. Further studies including identifying the cell types which produce these peptides are needed to fully understand and unravel the communication networks that appear to exist among lymphoid cells.

Acknowledgments We thank Dr. Robert LeBoeuf for providing the synthetic oligonucleotide probes and Ms. Cindy Bull for assistance with the BALB/c 3T3 fibroblast cell line. We also thank John Riley and Vincent Law for excellent technical assistance and Diane Weigent for preparing the manuscript.

References 1. Blalock JE 1989 A molecular basis for bidirectional communication between the immune and neuroendocrine systems. Physiol Rev

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 11 April 2016. at 06:24 For personal use only. No other uses without permission. . All rights reserved.

1734

IGF-I REGULATION BY GH

69:1-27 2. Ebaugh MJ, Smith EM 1988 Human lymphocyte production of immunoreactive luteinizing hormone. FASEB J 2:7811 (Abstract A1642) 3. Shah GN, Montgomery DW, Sredzinski JA, Russell DH 1988 Characterization of a novel prolactin synthesized by immunologically stimulated lymphocytes. FASEB J 2:1354 (Abstract A528) 4. Weigent DA, Baxter JB, Wear WE, Smith LR, Bost KL, Blalock JE 1988 Production of immunoreactive growth hormone by mononuclear leukocytes. FASEB J 2:2812-2818 5. Baxter JB, Blalock JE, Weigent DA 1991 Expression of immunoreactive growth hormone in leukocytes in vivo. J Neuroimmunol 33:43-54 6. Hattori N, Shimatsu A, Sugita M, Kumagai S, Imura H 1990 Immunoreactive growth hormone (GH) secretion by human lymphocytes: augmented release by exogenous GH. Biochem Biophys Res Commun 168:396-401 7. Kao T-L, Harbour DV, Smith EM, Meyer WJ Immunoreactive growth hormone production by cultured lymphocytes. Program of the 71st Annual Meeting of the Endocrine Society, Seattle, 1989 p 108 (Abstract) 8. Guillemin R, Brazeau P, Bohlin P, Esch F, Sing N, Wehrenberg WB 1982 Growth hormone releasing factor from a human pancreatic tumor that caused acromegaly. Science 218:585-587 9. Frohman LA, Jansson J 1986 Growth hormone releasing hormone. Endocr Rev 7:223-253 10. Brazeau P, Vale W, Burges R, Sing N, Butcher M, Rivier J, Guillemin R 1973 Hypothalamic polypeptide that inhibits the secretion of immunoreactive pituitary growth hormone. Science 79:77-79 11. Berelowitz M, Szato M, Frohman LH, Firistone S, Chu L, Hintz RL 1981 Somatomedin-C mediates growth hormone negative feedback by effects on both the hypothalamus and the pituitary. Science 212:1279-1281 12. Mathews LS, Norstedt G, Palmiter RD 1986 Regulation of insulinlike growth factor I gene expression by growth hormone. Proc Natl Acad Sci USA 83:9343-9347 13. Fuller PJ, Verity K 1989 Somatostatin gene expression in the thymus gland. J Immunol 143:1015-1017 14. Weigent DA, Baxter JB, Guarcello V, Blalock JE 1990 Growth hormone production and growth hormone releasing hormone receptors in the rat immune system. Ann NY Acad Sci 594:432-434 15. Weigent DA, Blalock JE 1990 Immunoreactive growth hormonereleasing hormone in rat leukocytes. J Neuroimmunol 29:1-13 16. Daughaday WH, Rotwein P 1989 Insulin-like growth factors I and II. Peptide, messenger ribonucleic acid and gene structures, serum, and tissue concentrations. Endocr Rev 10:68-91 17. Murphy LJ, Bell GI, Duckworth ML, Friesen HG 1987 Identification, characterization, and regulation of a rat complementary deoxyribonucleic acid which encodes insulin-like growth factor I. Endocrinology 121:684-691 18. Rom WN, Baoset P, Fells GA, Nukiwa T, Trapnell BC, Crystal RG 1988 Aveolar macrophages release our insulin-like growth factor I-type molecule. J Clin Invest 82:1685-1693 19. Geffner ME, Bersch N, Lippe BM, Rosenfeld RG, Hintz RL, Golde DW 1990 Growth hormone mediates the growth of T lymphoblast cell lines via locally generated insulin-like growth factor-I. J Clin Endocrinol Metab 71:464-469 20. Merimee TJ, Grant MB, Broder CM, Cavalli-Sforza LL 1989 Insulin-like growth factor secretion by human B-lymphocytes: a comparison of cells from normal and pygmy subjects. J Clin Endocrinol Metab 69:978-984 21. Tapson VF, Boni-Schnetzler M, Pilch PF, Center DM, Berman JS 1988 Structural and functional characterization of the human T lymphocyte receptor for insulin-like growth factor I in vitro. J Clin Invest 82:950-957 22. Rosenfeld RG, Hintz RL 1980 Characterization of a specific receptor for somatomedin C (SM-C) on cultured human lymphocytes: evidence that SM-C modulates homologous receptor concentrations. Endocrinology 107:1841-1848 23. Schimpff RM, Repellin AM, Salvatoni A, Thieriot-Prevost G, Chotelain P 1983 Effect of purified somatomedins on thymidine incorporation into lectin-activated human lymphocytes. Acta Endocrinol (Copenh) 102:21-26 24. Pandian MR, Talwar CP 1971 Effect of growth hormone on the

Endo • 1991 Vol 129 • No 4

metabolism of thymus and on the immune response against sheep erythrocytes. J Exp Med 134:1095-1112 25. Merchav S, Tatarsky I, Hochberg Z 1988 Enhancement of human granulopoiesis in vitro by biosynthetic insulin-like growth factor I/somatomedin C and human growth hormone. J Clin Invest 81:791-797 26. Boyum A 1968 Isolation of mononuclear cells and granulocytes from human blood. Scand J Clin Lab Invest [Suppl 97] 21:77-82 27. Maniatis T, Fritsch EF, Sambrook J 1982 In: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 28. Seeburg PH, Shine J, Martial J, Baxter JD, Goodman HM 1977 Nucleotide sequence and amplification in bacteria of a structural gene to rat GH. Nature 270:486-494 29. LeBoeuf RD, Galin FS, Hollinger SK, Peiper SC, Blalock JE 1989 Cloning and sequencing of immunoglobulin variable region genes using degenerate oligonucleotides and the polymerase chain reaction. Gene 82:371-377 30. Johnstone A, Thorpe R 1987 Immunochemistry in practice, ed 2. Blackwell Scientific Publishing, London 31. Tamura K, Kobayashi M, Ishii Y, Tamura T, Hashimoto K, Nakamura S, Niwa M, Zapf J 1989 Primary structure of rat insulinlike growth factor I and its biological activities. J Biol Chem 264:5616-5621 32. Scott CD, Martin JL, Baxter RC 1985 Production of insulin-like growth factor I and its binding protein by adult rat hepatocytes in primary culture. Endocrinology 116:1094-1101 33. Rappolee DA, Mark D, Banda MJ, Web Z 1988 Wound macrophages express TGF-a and other growth factors in vivo: analysis by mRNA phenotyping. Science 241:708-712 34. Vassilopoulou-Sellin R, Phillips LS 1982 Extractions of somatomedin activity from rat liver. Endocrinology 110:582-588 35. Clemmons DR, Shaw DS 1986 Purification and biologic properties of fibroblast somatomedin. J Biol Chem 261:10293-10298 36. Roberts Jr CT, Lasky SR, Lowe Jr WL, Seaman WT, Leroith D 1987 Molecular cloning of rat insulin-like growth factor I complementary deoxyribonucleic acids: differential messenger ribonucleic acid processing and regulation by growth hormone in extraheptic tissue. Mol Endocrinol 1:243-248 37. Doglio A, Dani C, Fredrikson G, Grimaldi P, Ailhaud G 1987 Acute regulation of insulin-like growth factor-I gene expression by growth hormone during adipose cell differentiation. EMBO J 6:4011-4016 38. Sara VR, Hall K 1990 Insulin-like growth factors and their binding proteins. Physiol Rev 70:591-614 39. Kelley KW 1989 Growth hormone, lymphocytes, and macrophages. Biochem Pharmacol 35:705-713 40. Weigent DA, Blalock JE, LeBoeuf RD 1991 An antisense oligodeoxynucleotide to growth hormone mRNA inhibits lymphocyte proliferation. Endocrinology 128:2053-2057 41. Bozzola M, Moretta A, Maccario R, Bohen M, Burgio GR Modulating effect of growth hormone (GH) on in vitro lymphoproliferation. 1st European Congress on Endocrinology, Copenhagen, 1987, Abstract 148 (Abstract) Pawlikowski M, Zelazowski P, Dohler KD, Stepien H 1988 Effects 42. of two neuropeptides: somatoliberin (GRF) and corticolierin (CRF) on human lymphocyte natural killer activity. Brain Behav Immun 2:50-56 43. Zelazowski P, Dohler KD, Stepien H, Pawlikowski M 1989 Effect of growth hormone releasing hormone on human peripheral blood leukocyte chemotaxis and migration in normal subjects. Neuroendocrinology 50:236-239 44. Weigent DA, Baxter JB, Wear WE, Smith LR, Bost KL, Blalock JE 1987 Production of immunoreactive growth hormone by mononuclear leukocytes. Fed Proc 46:926 (Abstract) 45. Shibasaki T, Kiyosawa Y, Masuda A, Nakahara M, Imaki T, Wakabayashi I, Demura H, Shizume K, Ling N 1984 Distribution of growth hormone-releasing hormone-like immunoreactivity in human tissue extracts. J Clin Endocrinol Metab 59:263-268 46. Meigan G, Sasaki A, Yoshinaga K 1988 Immunoreactive growth hormone-releasing hormone in rat placenta. Endocrinology 123:1098-1102 47. Sasaki A, Shuichi S, Shigeru Y, Hanew K, Miru Y, Yoshinaga K 1989 Multiple forms of immunoreactive growth hormone-releasing hormone in human plasma, hypothalamus, and tumor tissues. J Clin Endocrinol Metab 68:180-185

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 11 April 2016. at 06:24 For personal use only. No other uses without permission. . All rights reserved.

Characterization of immunoreactive insulin-like growth factor-I from leukocytes and its regulation by growth hormone.

In the present study, we investigated the production of insulin-like growth factor I (IGF-I) by leukocytes and its production after treatment with GH...
1MB Sizes 0 Downloads 0 Views