ARTICLE IN PRESS

Immunobiology 214 (2009) 430–440

0171-2985/$ - se

doi:10.1016/j.im

Abbreviations

protein kinases

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PD98059, ERK

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Growth hormone-induced production of cytokines in murine peritoneal

macrophages in vitro: Role of JAK/STAT, PI3K, PKC and MAP kinases

Anurag Tripathi, Ajit Sodhi�

School of Biotechnology, Faculty of Science, Banaras Hindu University, Varanasi 221005, India

Received 10 November 2008; accepted 10 November 2008

Abstract

The immunomodulatory properties of growth hormone (GH) are well recognized. Enhanced production of NO andcytokines by macrophages on treatment with GH was reported by us recently. The present investigation elucidates thesignaling mechanism(s) by which GH activates macrophages in vitro. It is observed that GH induces thephosphorylation (activation) of JAK2, PI3K, PKC and MAP kinases. Studies with pharmacological inhibitors ofvarious signaling molecules also indicated that GH-induced proinflammatory responses in macrophages are mediatedby JAK2/PI3K/PKC/ERK1/2, JAK2/JNK and JAK/STAT signaling cascades. It was further observed that GHinduced the enhanced expression/phosphorylation of transcription factors c-fos, c-jun, Elk-1 and Stat1.

It is also demonstrated that GH-induced ERK1/2 cascade regulates the production of TNF-a and IL-1b inmacrophages, whereas JNK cascade mediated the production of TNF-a, IFN-g and IL-12. These results suggest thatJAK2 plays a central role in mediating proinflammatory responses of macrophages on GH treatment.r 2008 Elsevier GmbH. All rights reserved.

Keywords: Growth hormone; Macrophage; Cytokines; Tyrosine kinases; ERK1/2; JNK MAP kinase

Introduction

Macrophages are phagocytic cells that recognize andkill microbial and tumor targets by cell-to-cell contact or

e front matter r 2008 Elsevier GmbH. All rights reserved.

bio.2008.11.013

: GH, growth hormone; MAPK, mitogen-activated

; JAK, Janus kinase; PI3K, phosphoinositide 3-kinase;

inase C; JNK, c-Jun N-terminal kinases; ERK,

nal-regulated kinases; STAT, signal transducers and

anscription; TNF-a, tumor necrosis factor-a; IL-1b,IFN, interferon; IL-12, interleukin-12; NO, nitric

tress-activated protein kinases; H7, PKC inhibitor;

1/2 inhibitor; SP600125, JNK inhibitor; AG490, JAK-

Wortmannin (PI3K inhibitor); DMSO, dimethyl

, o-Phenylenediamine dihydrochloride; EDTA, ethy-

traacetic acid; PMSF, phenyl methyl sulfonyl fluoride.

ing author. Tel.: +91 542 2307314/2368331;

68693.

ess: ajit.sodhi@lycos.com (A. Sodhi).

through secretion of a wide array of products includingreactive oxygen species, reactive nitrogen intermediates,cytokines, chemokines, etc. Immune modulationthrough the production of cytokines such as IL-1b,TNF-a, IFN-g, IL-12 is central to their regulatoryrole. They are also important antigen-presentingcells and play a role in the regulation of T celldifferentiation and activation (Nathan, 1987; Germainand Margulies, 1993; Adams and Hamilton, 1984;Sodhi and Chauhan, 2007; Stuehr and Nathan, 1989;Klostergaard et al., 1990). Exposure to macrophage-activating factors, like IFN-g, primes or activates thecells to mount a vigorous response against the tumorcells or microbial challenge (Nathan et al., 1984;Murray, 1988). Growth hormone (GH) has long beenknown to possess macrophage-activating properties(Warwick-Davies et al., 1995). There are numerous

ARTICLE IN PRESSA. Tripathi, A. Sodhi / Immunobiology 214 (2009) 430–440 431

reports that suggest a role for GH in the regulation ofthe immune system, its role as a cytokine andreconstitution of the immune system (Gala, 1991;Hooghe-Peters and Hooghe, 1995; Chappel, 1999).GH has been postulated to be an important mediatorin thymic development and increases the thymiccellularity (Kelley et al., 1986; Murphy et al., 1993).Apart from the pituitary, production of immunoreactiveGH from the cells of the immune system has beendocumented, indicating a paracrine/autocrine role ofGH (Weigent et al., 1988; Kao et al., 1992; Lytras et al.,1993). The treatment of cells of the immune system withGH modulates antibody production by B cells, NK cellactivity, phagocytosis and the killing capacity ofneutrophils and macrophages (Kooijman et al., 1996;Weigent,1996).

An understanding of the intracellular signal transduc-tion pathways initiated by GH is necessary to knowhow GH may act as a growth factor or immunostimu-lant. The JAK/STAT pathway is the most studiedcascade in GH signaling (Kopchick and Andry, 2000).JAK2, a non-receptor tyrosine kinase, was identifiedas growth hormone receptor (GHR)-associatedtyrosine kinase. When GH binds to its receptor, JAK2rapidly associates with the GHR, which leads to itsactivation and phosphorylation of tyrosines on JAK2(Argetsinger et al., 1993). Apart from JAK/STAT, theMAP kinase pathway is also considered important forthe regulation of GH-induced gene transcription (Smitet al., 1999). Recently, we have demonstrated theinvolvement of p-38 MAP kinase in the GH-inducedproduction of cytokines by murine macrophages (Sodhiand Tripathi, 2008).

The signaling pathways involved in GH signaling inmacrophages remain largely unknown. We have pre-viously reported that GH induces production of NO andcytokines in macrophages; it was observed that tyrosinekinases, Ca2+ signaling and MAP kinases were involvedin GH-induced NO production (Sodhi and Tripathi,2008; Tripathi and Sodhi, 2007). We have also shownthe involvement of tyrosine kinases and MAP kinases inPRL-induced macrophage activation (Tripathi andSodhi, 2008). The present manuscript investigates theinvolvement of the JAK/STAT pathway, ERK1/2 andJNK MAP kinase signaling cascades in the GH-inducedproduction of cytokines.

Materials and methods

Mice

Inbred strains of Balb/c mice of either sex at 8–10weeks of age were used for obtaining peritonealmacrophages.

Cell cultures and reagents

Macrophage monolayers were cultured in RPMI 1640medium supplemented with heat-inactivated fetal calfserum (10%), penicillin (100U/ml), streptomycin(100U/ml) and gentamycin (20 mg/ml) at 37 1C inhumidified air containing 5% CO2. Medium RPMI1640, TRI reagent, growth hormone (from porcinepituitaries), Wortmannin and most of the other reagentswere obtained from Sigma-Aldrich Chemicals, USA.Fetal calf serum was purchased from BiologicalIndustries, Israel. H7, SP600125, PD98059 and Tyr-phostin (AG490) were purchased from Calbiochem, LaJolla, CA, USA. Polyclonal antibodies against phospho-JAK2, phospho-PI3K, phospho-PKC, phospho-ERK1/2, phospho-JNK, phospho-STAT1, phospho-c-jun,phospho-Elk-1, c-Fos, actin and HRP-conjugated anti-rabbit, anti-goat and anti-mouse IgGs were obtainedfrom Santa Cruz Biotechnology Inc, California, USA.All the reagents were endotoxin-free as determined bythe Limulus amoebocyte lysate assay (sensitivity limit,0.1 ng/ml).

Isolation and activation of macrophages

Macrophage monolayers were prepared as describedpreviously (Sodhi et al., 1992). Peritoneal exudate cellswere harvested from peritoneal cavity using chilledserum-free RPMI 1640 medium and added to wells of24-well tissue culture plates (Nunc, Denmark). After 2 hincubation at 37 1C in an atmosphere of 5% CO2 in airin a CO2 incubator, the non-adherent cells wereremoved by vigorous washing (3 times) with warmserum-free medium and the adherent cells were incu-bated in complete medium overnight to form macro-phage monolayers. More than 95% of the adherent cellpopulation was macrophages as determined by mor-phology and non-specific esterase staining.

After overnight culture, the medium was removedfrom the wells and macrophage mono-layers (1� 106 cells per well) were treated with GH(300 ng/ml) in complete medium for 24 h. We havepreviously reported that GH induces macrophageactivation at an optimal dose of 300 ng/ml (Sodhi andTripathi, 2008; Tripathi and Sodhi, 2007). Untreatedmacrophages were used as control. In another set, themacrophage cultures were pretreated for 1 h with PI3Kinhibitor, Wortmannin (200 nM) or ERK1/2 inhibitor,PD98059, phospho-JNK inhibitor SP600125 (10 mM) orJAK2 inhibitor, Tyrphostin (AG490) (25 mM) or PKCinhibitor, H7. Thereafter, macrophages were washedand treated with GH (300 ng/ml) in fresh complete orincomplete medium for indicated time periods. Thestrength of DMSO in these stock solutions of the aboveinhibitor were adjusted so that its effective concentra-

ARTICLE IN PRESSA. Tripathi, A. Sodhi / Immunobiology 214 (2009) 430–440432

tion is less than 0.1% when used at the recommendeddoses.

Protein tyrosine kinase (PTK) assay

PTK activity was measured using a colorimetric kit.Briefly, cell lysate was prepared using lysis buffercontaining activated sodium vanadate solution (accord-ing to kit instruction). Assay was performed in a 96-wellmicrotiter plate. One hundred and twenty-five micro-liters of PTK substrate solution was added to each welland the plate was incubated overnight at 37 1C. Thecoating solution was removed and each well was washedwith 200 ml of washing buffer. The buffer was removedand wells were dried for 2 h at 37 1C. Ninety microlitersof 1� tyrosine kinase buffer containing ATP was addedto each well. Twenty microliters of cell lysates was addedin each well. The plate was covered and incubated atroom temperature for 30min. The reaction mixture wasremoved and each well was washed with 200 ml ofwashing buffer five times. In each well 100 ml conjugatedantibody was added. The plate was covered andincubated at room temperature for 30min. The anti-body solution was removed and each well was washedwith 200 ml of washing buffer five times. One hundredmicroliters of freshly prepared OPD substrate solutionwas added to each well and incubated for 7min in darkat room temperature. One hundred microliters of 2.5NH2SO4 was added to each well to stop the reaction. Theplate was read in a microplate ELISA reader (Emax,Molecular Devices, USA) at 492 nm within 30min ofaddition of the stop solution.

Preparation of cell lysates and immunoblotting

The macrophage monolayers with or without treat-ment were washed with ice-cold phosphate-bufferedsaline containing 1mM Na3VO4, lysed in 50 ml of lysisbuffer [20mM Tris-HCl, pH 8, 137mM NaCl, 10%glycerol (v/v), 1% Triton X-100 (v/v), 1mM Na3VO4,2mM EDTA, 1mM PMSF, 20 mM leupeptin and0.15 units/ml aprotonin] for 20min at 4 1C. The lysateswere centrifuged at 15 000g for 15min and the super-natants (containing Triton X-100 soluble proteins) wereseparated on 10% SDS-polyacrylamide gels. Theseparated proteins (40 mg/lane) were transferred to anitrocellulose membrane (1 h at 350mA) using a Bio-Rad Mini Transblotter and immunoblotted with pri-mary antibody, incubated with secondary antibodyconjugated with horseradish peroxidase and visualizedby the Chemiluminescence Western Blotting Kit (SantaCruz Biotechnology, California, USA). To monitorequal loading of protein, Western blot analysis usingantibody directed against actin was performed for each

treated or untreated cell extract as shown in the lowerpanels.

The densitometric analysis is shown below the blots.Densitometric analysis was carried out using GeneTools software from Syngene, a Division of SynopticsLtd., UK. The densities of each band are represented asRaw volume.

Assay for IL-1b, IL-12p40, IFN-c and TNF-a

Murine peritoneal macrophages were treated withGH (300 ng/ml) for different time periods. IL-1b orIL-12p40 or IFN-g or TNF-a was measured in super-natant by commercial ELISA kits (BD Pharmingen,California, USA).

Percentage viability by MTT assay

Percentage viability of macrophages was determinedby the MTT [3(4,5) dimethyl thiazol-2, 5-diphenyltetrazolium bromide] assay as described earlier (Mizel,1982). The relative cell viability was calculated accord-ing to the formula

Relative cell viability ¼Absorbance experimental

Absorbance control� 100

where ‘Absorbance control’ represents macrophagesincubated in medium alone and ‘Absorbance experi-mental’ represents macrophages treated with the GH orGH plus AG490 or Wortmannin or H7 or PD98059 orSP600125 or their vehicles. In each of the experiments,the viability of the peritoneal macrophages was notaffected by the doses of the PRL or AG490 orWortmannin or H7 or PD98059 or SP600125 or theirvehicles used up to 48 h.

Statistical analysis

The statistical significance of difference between thetest groups was analyzed by Student’s t-test (two-tailed)using SPSS 7.5 software. Statistical significance wasassumed at po0.05. The error bars of the valuesrepresent a 95% confidence interval.

Results

Protein tyrosine kinase activity in

GH-treated macrophages

PTK activity was measured in macrophages treatedwith GH (300 ng/ml) for various time intervals. Asillustrated in Fig. 1a, maximum tyrosine kinase activitywas observed at 5–15min of GH stimulation.

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Fig. 1. (a) Time kinetics of PTK activity in macrophages

treated with GH (300 ng/ml). Each bar represents the standard

error of three independent experiments. All values are

mean7SD and representative of three independent experi-

ments done in triplicate. (b) Effect of inhibitors on PTK

activity in macrophages after 15min of GH (300 ng/ml)

treatment. Each bar represents the standard error of three

independent experiments. All values are mean7SD and

representative of three independent experiments done in

triplicate. *, values significantly different from control,

po0.05 versus control. ], values significantly different from

GH-treated macrophages, po0.05 versus macrophages treated

with GH alone.

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Fig. 2. Expression of phospho-JAK2 (a), phospho-PI3K (b),

phospho-PKC (c) in macrophages treated with GH (300 ng/

A. Tripathi, A. Sodhi / Immunobiology 214 (2009) 430–440 433

To examine the contribution of JAK2, PI3K andPKC in the tyrosine kinase activity induced by GH,their respective pharmacological inhibitors AG490,Wortmannin and H7 were used. Pretreatment ofmacrophages with AG490 or Wortmannin or H7significantly inhibited the GH-induced PTK activity inmacrophages (Fig. 1b).

ml) for different time periods as checked by immunoblotting.

Lane 1: untreated; lane 2: GH (5min); lane 3: GH (15min);

lane 4: GH (30min); lane 5: GH (60min). The figures are

representative of three independent experiments having similar

results.

GH induces phosphorylation of

JAK2 in macrophages

Western blot analysis clearly demonstrated the GH-induced phosphorylation of JAK2, which was found topeak at 5min of GH treatment and decrease thereafter(Fig. 2a).

Pretreatment of macrophages with AG490, a specificinhibitor of JAK2, significantly inhibited GH-inducedphosphorylation of JAK2. Wortmannin, an inhibitor of

PI3K, or H7, an inhibitor of PKC, or PD98059, aninhibitor of ERK1/2, or SP600125, an inhibitor of JNK,did not significantly affect the GH-induced phosphor-ylation of JAK2 (Fig. 3a).

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Fig. 3. Effect of Wortmannin (W), H7, AG490, PD98059 and

SP600125 on the expression of phospho-JAK2 (a), phospho-

PI3K (b), in GH-treated macrophages, as checked by

immunoblotting. Macrophages were pretreated with various

inhibitors for 1 h. Thereafter, macrophages were washed,

treated with GH (300 ng/ml) in fresh medium for 5min and

expression of phospho-JAK2 or phospho-PI3K checked by

immunoblotting. Lane 1: untreated; lane 2: GH (300 ng/ml);

lane 3: GH+W; lane 4: GH+H7; lane 5: GH+AG490; lane 6:

GH+PD98059; lane 7: GH+SP600125. (c) Effect of Wort-

mannin (W), H7, AG490, PD98059 and SP600125 on the

expression of p-PKC in GH-treated macrophages, as checked

by immunoblotting. Macrophages were pretreated with

various inhibitors for 1 h. Thereafter, macrophages were

washed, treated with GH (300 ng/ml) in fresh medium for

60min and expression of phospho-PKC checked by immuno-

blotting. Lane 1: untreated; lane 2: GH (300 ng/ml); lane 3:

GH+W; lane 4: GH+H7; lane 5: GH+AG490; lane 6:

GH+PD98059; lane 7: GH+SP600125. The figures are

representative of three independent experiments having similar

results.

A. Tripathi, A. Sodhi / Immunobiology 214 (2009) 430–440434

GH induces phosphorylation of PI3K

in macrophages

GH induced phosphorylation of PI3K, which peakedbetween 5 and 15min of GH treatment and decreasethereafter (Fig. 2b).

To investigate the upstream signaling events leadingto phosphorylation of PI3K, macrophages were pre-treated with pharmacological inhibitors of JAK2, PKC,ERK1/2 and JNK MAP kinases. It was observed thatpretreatment of macrophages with AG490, an inhibitorof JAK2, and Wortmannin, an inhibitor of PI3K,blocked GH-induced phosphorylation of PI3K. H7 orPD98059 or SP600125 did not affect the GH-inducedphosphorylation of PI3K (Fig. 3b).

GH induces phosphorylation of PKC in macrophages

GH treatment of macrophages resulted in thephosphorylation of PKC. A gradual increase in theexpression of phosphorylated PKC was observed from 5to 60min of GH treatment (Fig. 2c).

Pretreatment of macrophages with AG490, an in-hibitor of JAK2, or Wortmannin, an inhibitor of PI3K,or H7, an inhibitor of PKC, significantly inhibited theGH-induced phosphorylation of PKC. As evident fromimmunoblotting, PD98059 or SP600125 did not cause asignificant change in the expression of phosphorylatedPKC (Fig. 3c).

GH induces phosphorylation of ERK1/2 MAP

kinase in macrophages

To examine the kinetics of phosphorylation ofERK1/2, macrophages were treated with GH forindicated time periods. GH induced phosphorylationof ERK1/2, which was found to peak between 15 and30min of GH treatment (Fig. 4a).

Pretreatment of macrophages with AG490, an in-hibitor of JAK2, or Wortmannin, an inhibitor of PI3K,or H7, an inhibitor of PKC, or PD98059, an inhibitor ofERK1/2, moderately inhibited GH-induced phosphor-ylation of ERK1/2, although JNK inhibitor SP600125did not cause a significant change in the GH-inducedphosphorylation of ERK1/2 MAP kinase (Fig. 4b).

GH induces phosphorylation of JNK MAP kinase

in macrophages

GH treatment induced phosphorylation of JNKMAPkinase in macrophages. The maximum phosphorylationof JNK MAP kinase was observed at 15min of GHtreatment (Fig. 5a).

Pretreatment of macrophages with AG490, aninhibitor of JAK2, or JNK inhibitor SP600125significantly inhibited the GH-induced phosphorylation

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Fig. 5. (a) Expression of phospho-JNK MAP kinase in

macrophages treated with GH (300 ng/ml) for different time

periods as checked by immunoblotting. Lane 1: untreated; lane

2: GH (5min); lane 3: GH (15min); lane 4: GH (30min); lane

5: GH (60min). The figures are representative of three

independent experiments having similar results done in

triplicate. (b) Effect of Wortmannin (W), H7, AG490,

PD98059 and SP600125 on the expression of phospho-JNK

MAP kinase protein in GH (300 ng/ml)-treated macrophages,

as checked by immunoblotting. Macrophages were pretreated

with various inhibitors for 1 h. Thereafter, macrophages were

washed, treated with GH (300 ng/ml) in fresh medium for

15min and expression of phospho-JNK checked by immuno-

blotting. Lane 1: untreated; lane 2: GH (300 ng/ml); lane 3:

GH+W; lane 4: GH+H7; lane 5: GH+AG490; lane 6:

GH+PD98059; lane 7: GH+SP600125. The figures are

representative of three independent experiments having similar

results.

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Fig. 4. (a) Expression of phospho-ERK1/2 MAP kinase in

macrophages treated with GH (300 ng/ml) for different time

periods as checked by immunoblotting. Lane 1: untreated; lane

2: GH (5min); lane 3: GH (15min); lane 4: GH (30min); lane

5: GH (60min). The figures are representative of three

independent experiments having similar results done in

triplicate. (b) Effect of Wortmannin (W), H7, AG490,

PD98059 and SP600125 on the expression of phospho-

ERK1/2 MAP kinase protein in GH-(300 ng/ml) treated

macrophages, as checked by immunoblotting. Macrophages

were pretreated with various inhibitors for 1 h. Thereafter,

macrophages were washed, treated with GH (300 ng/ml) in

fresh medium for 30min and expression of phospho-ERK1/2

checked by immunoblotting. Lane 1: untreated; lane 2: GH

(300 ng/ml); lane 3: GH+W; lane 4: GH+H7; lane 5:

GH+AG490; lane 6: GH+PD98059; lane 7: GH+SP600125.

The figures are representative of three independent experi-

ments having similar results.

A. Tripathi, A. Sodhi / Immunobiology 214 (2009) 430–440 435

of JNK. Wortmannin, an inhibitor of PI3K, or H7, aninhibitor of PKC, or PD98059, an inhibitor of ERK1/2,did not cause any significant change in the GH-inducedphosphorylation of JNK (Fig. 5b).

Expression of phosphorylated Elk-1, STAT1, c-jun

and c-fos in GH-treated macrophages and effect of

Wortmannin, H7, AG490, PD98059 and SP600125

on their expression

GH (300 ng/ml) treatment induced the enhancedexpression of phosphorylated Elk-1, c-jun, STAT1 and

expression of c-fos in macrophages. The maximumexpression of phosphorylated Elk-1, c-fos and phos-phorylated c-jun was observed at 60min of GHtreatment (Fig. 6a–c). The phosphorylation of STAT1was found to peak between 5 and 15min of GHtreatment (Fig. 6d).

Pretreatment of macrophages with Wortmannin, aninhibitor of PI3K, or H7, an inhibitor of PKC, orAG490, an inhibitor of JAK2, or PD98059, an inhibitor

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Fig. 6. Expression of phospho-Elk-1 (a), c-fos (b), phospho-c-

jun (c) and phospho-STAT1 (d) in macrophages treated with

GH (300 ng/ml) for different time periods as checked by

immunoblotting. Lane 1: untreated; lane 2: GH (5min); lane 3:

GH (15min); lane 4: GH (30min); lane 5: GH (60min). The

figures are representative of three independent experiments

having similar results.

A. Tripathi, A. Sodhi / Immunobiology 214 (2009) 430–440436

of p42/44 MAP kinase, or SP600125, an inhibitor ofJNK, significantly inhibited GH-induced phosphoryla-tion of Elk-1 and expression of c-fos. Wortmannin, H7,AG490 and SP600125 also inhibited phosphorylation ofc-jun although PD98059 only partially suppressed itsactivation (Fig. 7a–c). The phosphorylation of STAT1was unaffected by all the pharmacological inhibitorsused except for AG490 (Fig. 7d).

Role of JAK2, PI3K, PKC, ERK1/2 and JNK MAP

kinase in the production of TNF-a, IL-1b, IFN-cand IL-12

Macrophages on treatment with GH (300 ng/ml)produced significantly enhanced amounts of TNF-a,IL-1b, IFN-g and IL-12 as assayed by ELISA.Pretreatment of macrophages with Wortmannin, aninhibitor of PI3K, or H7, an inhibitor of PKC, orAG490, a inhibitor of JAK2, or PD98059, an inhibitorof p42/44 MAP kinase, or SP600125, an inhibitor ofJNK MAP kinase, significantly inhibited GH-inducedproduction of TNF-a (Fig. 8a). Wortmannin, H7,AG490 and PD98059 were also effective in down-regulating GH-induced IL-1b production althoughSP600125 did not cause any significant change in theproduction of IL-1b (Fig. 8b).

It was further observed that the GH-induced produc-tion of IFN-g by macrophages was significantlyinhibited only by AG490 or SP600125. Wortmanninonly partially suppressed while H7 and PD98059 did notcause any significant change in IFN-g production bymacrophages (Fig. 8c). Macrophages pretreated withAG490 or SP600125 produced significantly reducedIL-12 on treatment with GH; Wortmannin or H7partially suppressed the production of IL-12. PD98059did not cause any noticeable change in the production ofIL-12 (Fig. 8d).

Macrophages treated with Wortmannin or H7 orAG490 or PD98059 or SP600125 alone exhibitedminimal production of cytokines as in the untreatedmacrophages.

Discussion

Auto-immune pathologies like rheumatoid arthritisare characterized by a dysfunctional cellular andhumoral response, enhanced migration and attachmentof peripheral macrophages to the joints and an elevatedlevel of proinflammatory cytokines. GH is suspected toplay an important role in such auto-immune disorders(Malemud, 2007). At the intracellular level, the mole-cular mechanisms underlying these actions have notbeen fully elucidated. We have previously reported theproduction of NO, TNF-a, IL-1b, IFN-g and IL-12 on

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A. Tripathi, A. Sodhi / Immunobiology 214 (2009) 430–440 437

treatment of macrophages with GH (Sodhi and Tri-pathi, 2008; Tripathi and Sodhi, 2007). The presentstudy was undertaken to investigate the involvementof JAK2, PI3K, PKC, p42/44 and JNK MAP kinase inthe activation of macrophages on treatment with GHin vitro.

GH-induced tyrosine phosphorylation of a number ofintracellular proteins in its target cells has been reportedpreviously (Anderson, 1992; Campbell et al., 1993). It isalso known that many of the actions of GH aretransduced through the JAK2 pathway (Xu et al.,1996; Han et al., 1996). We report that GH treatmentinduced a significantly enhanced tyrosine kinase activityin macrophages, which was observed to peak between 5and 15min of GH stimulation. AG490, an inhibitor ofJAK2, Wortmannin, an inhibitor of PI3K, and H7, aninhibitor of PKC, significantly inhibited the GH-induced tyrosine kinase activity in macrophages. It isalso demonstrated that GH induces rapid increase in theprotein tyrosine phosphorylation of JAK2, PI3K andPKC in macrophages. Pretreatment of macrophageswith Wortmannin or H7 or PD98059, an inhibitor ofERK1/2, or SP600125, an inhibitor of JNK, did notinhibit the phosphorylation of JAK2. GH-inducedphosphorylation of PI3K was significantly inhibited bypretreatment of macrophages with JAK2 inhibitorAG490, although there was no effect of H7 orPD98059 or SP600125. These observations suggest thatJAK2 is an upstream regulator of PI3K. Furthermore,inhibition of tyrosine phosphorylation of PKC by JAK2and PI3K pharmacological inhibitors implies that GHinduces JAK2/ PI3K/ PKC signaling cascade in macro-phages.

MAP kinases are a general feature observed in mostinflammatory reactions and have gained importance as

Fig. 7. Effect of Wortmannin (W), H7, AG490, PD98059 and

SP600125 on the expression of phospho-Elk-1 (a), c-fos (b),

phospho-c-jun (c) in GH-treated macrophages, as checked by

immunoblotting. Macrophages were pretreated with various

inhibitors for 1 h. Thereafter, macrophages were washed,

treated with GH (300 ng/ml) in fresh medium for 60min and

expression of phospho-Elk-1 or c-fos or phospho-c-jun

checked by immunoblotting. Lane 1: untreated; lane 2: GH

(300 ng/ml); lane 3: GH+W; lane 4: GH+H7; lane 5:

GH+AG490; lane 6: GH+PD98059; lane 7: GH+SP600125.

(d) Effect of Wortmannin (W), H7, AG490, PD98059 and

SP600125 on the expression of phospho-STAT1 in GH-treated

macrophages, as checked by immunoblotting. Macrophages

were pretreated with various inhibitors for 1 h. Thereafter,

macrophages were washed, treated with GH (300 ng/ml) in

fresh medium for 15min and expression of phospho-STAT1

checked by immunoblotting. Lane 1: untreated; lane 2: GH

(300 ng/ml); lane 3: GH+W; lane 4: GH+H7; lane 5:

GH+AG490; lane 6: GH+PD98059; lane 7: GH+SP600125.

The figures are representative of three independent experi-

ments having similar results.

ARTICLE IN PRESS

Medium

0

100

200

300

400

500

600

700

800

W H7

AG490

PD9805

9SP

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AG490

PD9805

9 SP

Medium W H7

AG490

PD9805

9 SP

Medium W H7

AG490

PD9805

9 SP

TNF-

α (p

g/m

l/106

cells

) MediumGH

MediumGH

0

50

100

150

200

250

IL-1

β (p

g/m

l/106

cells

)

IL-1

2 (p

g/m

l/106

cells

)

*

# #

#

##

∗ ∗

##

#

#

0

200

400

600

800

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1400

IFN

-γ (p

g/m

l/106

cells

)

0

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1200

∗

#

#

*

#

#

MediumGH

MediumGH

Fig. 8. Effect of Wortmannin (W), H7, AG490, PD98059 and SP600125 on GH-induced production of TNF-a (a), IL-1b (b), IFN-g(c) and IL-12 (d). Macrophages were pretreated with or without Wortmannin (W) or H7 or AG490 or PD98059 or SP600125.

Thereafter, macrophages were washed, treated with GH in fresh medium for 24 h and cytokine production was assayed by ELISA.

Each error bar represents the standard error of three independent experiments. *, values significantly different from control, po.05

versus control. ], values significantly different from GH-treated macrophages, po.05 versus macrophages treated with GH alone.

A. Tripathi, A. Sodhi / Immunobiology 214 (2009) 430–440438

novel anti-inflammatory targets (Hobbie et al., 1997;Hommes et al., 2003). It is observed that GH inducedthe activation (phosphorylation) of JNK and ERK1/2MAP kinase in macrophages. AG490 or Wortmannin orH7 brought about a significant reduction in thephosphorylation of ERK1/2, though only AG490significantly inhibited the phosphorylation/activationof JNK. The present observations on the signaltransduction pathway(s), using the pharmacologicalinhibitors, suggest that JAK2/ PI3K/ PKC/ ERK1/2and JAK2/ JNK are important cascades governingproinflammatory responses induced by GH in macro-phages.

Next we looked into the expression of transcriptionfactors involved in the regulation of gene expression byGH. The data suggest the involvement of transcriptionfactors Elk-1, c-fos, c-jun and STAT1 in GH-inducedresponses in macrophages. Enhanced expression ofphospho-Elk-1, phospho-c-jun, phospho-STAT1 andc-fos is observed in GH-stimulated macrophages. Theenhancement in the expression of phospho-Elk-1 and

c-fos was significantly reduced by inhibitors of JAK2,PI3K, PKC, ERK1/2 and JNK MAP kinase. Theinhibitors of JAK2, PI3K, PKC and JNK alsoabrogated the activation (phosphorylation) of c-junalthough the ERK1/2 inhibitor, PD98059, only partiallysuppressed its activation. Interestingly, among all thepharmacological inhibitors used, only the JAK2inhibitor, AG490, blocked the phosphorylation ofSTAT1, which identifies JAK2 as an upstream regulatorof STAT1.

Proinflammatory cytokines, such as TNF-a, IL-1b,IL-12 and IFN-g, play a key role in sepsis and septicshock during bacterial infections (Martin et al., 2003;Cook et al., 2004; Rothe et al., 1993). Efficient reductionin the production of proinflammatory cytokines duringimmune responses is essential to prevent potentiallydetrimental pathogenic states such as septic shock andautoimmunity. Pretreatment of macrophages withWortmannin, H7, AG490, PD98059 brought about asignificant downregulation in the GH-induced produc-tion of TNF-a and IL-1b, implicating JAK2, PI3K,

ARTICLE IN PRESSA. Tripathi, A. Sodhi / Immunobiology 214 (2009) 430–440 439

PKC, ERK1/2 in the regulation of TNF-a and IL-1bproduction. SP600125, an inhibitor of JNK, inhibitedTNF-a production but had no effect on the expressionof IL-1b. Furthermore, it was observed that AG490 andSP600125 significantly inhibited, Wortmannin partiallysuppressed and H7 or PD98059 did not have anysignificant affect on GH-induced IFN-g production bymurine macrophages. Pretreatment of macrophageswith Wortmannin or H7 partially suppressed, AG490or SP600125 significantly reduced and PD98059 did nothave any affect on GH-induced IL-12 production.

The present findings add to our understanding of thesignaling mechanisms adopted by GH to mount aproinflammatory response by macrophages. It clearlyestablishes that JAK2 lies at the hub and initiatesmost of the signaling cascades involved in the produc-tion of GH-induced cytokines in macrophages. Thepresent study demonstrates that PI3K, PKC, ERK1/2and JNK are also components of prominent pathwaysother than JAK2, which are involved in GH-inducedmacrophage activation. The current investigation isinteresting in view of formulating new anti-inflamma-tory targets and devising new strategies to combatGH-induced disorders.

Acknowledgment

Anurag Tripathi is a UGC-JRF fellow.

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