Ultraviolet B induces high mobility group box 1 release from mouse peritoneal macrophages in vitro via caspase-1 mediated secretion pathway.

High mobility group box 1 (HMGB1) protein is a unique non histone nuclear protein that acts extracellularly as a mediator of delayed inflammation. Sub lethal dose of UVB triggers the release of cytokines from macrophages (MΦs). Adding to the panoply of UVB induced cytokines; it is reported that UVB induces HMGB1 release from mouse peritoneal MΦs in time and partially dose dependent manner, independent of TNF-α. UVB also enhanced the transcription of HMGB1 gene and expression of cellular protein, which influences its subsequent release. HMGB1 is secreted by an unconventional secretion pathway of unknown mechanism. Caspase-1 has been shown to function as a general regulator of stress induced unconventional secretion for a number of cytokines. In the present study, we have observed that pharmacological inhibitors specific for caspase-1 (ZVAD and YVAD) abrogated UVB induced HMGB1 release from MΦs. This effect was most likely mediated via physical interaction between HMGB1 and active caspase-1 (p10 and p20) as demonstrated by immunoprecipitation. In addition, it was found that HMGB1 and active caspase-1 p20 release depends on UVB mediated enhancement of intracellular Ca(2+). Thus our data suggests that optimal dose of UVB (50 mJ/cm(2)) induces HMGB1 upregulation and active release from mouse peritoneal MΦs which is mediated by caspase-1 in a Ca(2+) dependent manner.

High mobility group box 1 protein synergizes with lipopolysaccharide and peptidoglycan for nitric oxide production in mouse peritoneal macrophages in vitro.

Extracellular high mobility group box 1 (HMGB1) protein and nitric oxide (NO) has been credited with multiple inflammatory functions using in vivo and in vitro systems. Therefore, delineating their regulation may be an important therapeutic strategy for the treatment of sepsis. In the present study, it is demonstrated that recombinant HMGB1 (rHMGB1) synergizes with sub threshold concentration of TLR2 agonist (PGN; 1 µg/ml) as well as with TLR4 agonist (LPS; 1 ng/ml) to induce NO release in mouse peritoneal macrophages. The enhanced iNOS expression was also observed at the transcription and translational level. Co-incubation of macrophages with rHMGB1 with either PGN or LPS showed enhanced expression of TLR2, TLR4 and RAGE. TLR2, TLR4 or RAGE knockdown macrophages effectively inhibited the rHMGB1+PGN or LPS induced NO synergy. It was further observed that the JNK MAPK inhibitor SP600125 attenuated the PGN+rHMGB1 induced iNOS/NO synergy whereas p38 MAPK inhibitor SB908912 inhibited iNOS/NO synergy induced by LPS+rHMGB1. It was also observed that the activation of NF-κB is essential for the synergy as the pharmacological inhibition or siRNA knockdown of NF-κB (cRel) significantly reduced the rHMGB1+PGN or rHMGB1+LPS induced enhanced iNOS/NO expression. Altogether, the data suggests that the co-incubation of macrophages with rHMGB1 with either LPS or PGN induces the synergistic effect on iNOS expression and NO release by the upregulation of surface receptors (TLR2, TLR4 and RAGE) which in turn amplifies the MAPKs (p38 and JNK) and NF-κB activation and results in enhanced iNOS expression and NO production.

Synthesis, cytotoxic evaluation, docking and in silico pharmacokinetic prediction of 4-arylideneamino/cycloalkylidineamino 1, 2-naphthoquinone thiosemicarbazones.

In an attempt to develop potent anticancer agents, a series of 4-arylideneamino/cycloalkylidineamino-1, 2-naphthoquinone thiosemicarbazones were synthesized and characterized using FT-IR, (1)H NMR, (13)C NMR spectroscopy and elemental analysis. The compounds were screened for antiproliferative activity against three human cancer cell lines (Hep-G2, MG-63 and MCF-7) using the MTT assay. Significant anticancer activity was observed for several members of the series. The compounds 4-(3, 4, 5-trimethoxybenzylidene amino) 1, 2-naphthoquinone-2-thiosemicarbazone (TS10) and 4-(4-hydroxy-3-methoxy benzylideneamino) 1, 2-naphthoquinone-2-thiosemicarbazone (TS13) were active cytotoxic agents in all three cancer cell lines, with IC50 values in the range of 3.5-6.4 µM. Further evaluation of some of these potent cytotoxic compounds demonstrated their good safety profile in a normal cell line (MCF-12A). Docking experiments showed a good correlation between the predicted glide scores and the IC50 values of these compounds. In silico ADME studies revealed that these compounds can be used for second generation development.

Peptidoglycan induced expression of peroxisome proliferator-activated receptor γ in mouse peritoneal macrophages: role of ERK and JNK MAP kinases.

The peroxisome proliferator-activated receptor (PPAR) γ plays an important role in macrophage inflammatory homeostasis. Here we investigate the cross talk between PPARγ and TLR2 signaling pathway in mouse peritoneal macrophages. Real time RT-PCR and immunoblot analysis revealed that peptidoglycan (PGN) treatment of macrophages leads to biphasic effect on PPARγ expression i.e. an early upregulation and a late suppression. Inhibition of ERK MAP kinase by PD98059 abolished the early and rapid induction of PPARγ, while the inhibition of JNK MAP kinase by SP600125 nullifies the late inhibitory effect on the PPARγ expression in a dose-dependent manner. Furthermore, PPARγ knockdown macrophages showed enhanced NF-κB activity after PGN treatment. PGN treatment also enhances PPARγ interaction with p65 as observed by immunoprecipitation. This interaction may inhibit NF-κB (p65) activity as increased nuclear localization of p65 was observed in PPARγ knockdown macrophages after PGN treatment. PPARγ knockdown also increased the PGN-induced inflammatory cytokines (TNF-α, IL-1ß, IL-12p40) production. Thus, our observations suggest that PGN induces PPARγ expression which is regulated by MAPKs activation and this enhanced PPARγ in turn attenuate NF-κB activity probably via enhancing p65 nuclear export. These results provide insight into how these pathways could be modulated in inflammatory diseases.

Mycobacterium indicus pranii mediates macrophage activation through TLR2 and NOD2 in a MyD88 dependent manner.

Mycobacterium indicus pranii (MIP) is a non-pathogenic strain of mycobacterium and has been used as a vaccine against tuberculosis and leprosy. Here, we investigated the role of different pattern recognition receptors in the recognition of heat-killed MIP by macrophages. Treatment of macrophages with MIP caused upregulation of pro-inflammatory cytokines (like TNFα and IL-1ß) which was mediated through both TLR2 and NOD2, as revealed by our knockdown and/or knockout studies. Mechanistically, MIP-induced macrophage activation was shown to result in NF-κB activation and drastically abrogated by MyD88 deficiency, suggesting its regulation via an MyD88-dependent, NF-κB pathway. Interestingly, the IFN-inducible cytokine, CXCL10, which is known target of the TRIF-dependent TLR pathway was found to be upregulated in response to MIP but, in an MyD88-dependent manner. Collectively, these results demonstrate macrophages to recognize and respond to MIP through a TLR2, NOD2 and an MyD88-dependent pathway. However, further studies should clarify whether additional TLR-dependent or -independent pathways also exist in regulating the full spectrum of MIP action on macrophage activation.

Mycobacterium indicus pranii downregulates MMP-9 and iNOS through COX-2 dependent and TNF-α independent pathway in mouse peritoneal macrophages in vitro.

Despite the popular belief that granulomas are innate immune mechanism to restrict mycobacterial growth, evidences suggest that granulomas facilitate growth of Mycobacterium by recruiting large numbers of uninfected macrophages to the site of infection. Matrix metalloproteinase-9 (MMP-9) has been shown to be directly involved in recruitment of macrophages at the site of infection, contributing to nascent granuloma maturation and bacterial growth. In this manuscript it is reported that heat-killed Mycobacterium indicus pranii (MIP) leads to a significant downregulation of MMP-9 in murine peritoneal macrophages in vitro. The downregulation of MMP-9 is mediated through cyclooxygenase-2 (COX-2), but independent of tumor necrosis factor-α (TNF-α). By limiting nuclear to cytoplasmic export of COX-2 and iNOS transcripts, MIP inhibits excessively-high levels of nitric oxide which can be damaging to the host during acute phases of infection. MIP has been shown to provide clinical improvement in all phases of leprosy and used for treatment of leprosy and tuberculosis.

Synthesis, Molecular docking and Biological evaluation of 4-Cycloalkylidineamino 1, 2-Naphthoquinone Semicarbazones as Anticancer agents

Objective: In an effort to etablish new candidates with improved antineoplastic activity, 4-cycloalkylidineamino 1,2-naphthoquinone semicarbazones were synthesized, characterized and evaluated for anticancer activity. Method: The desired compounds were synthesized by condensation of 4- amino1, 2-naphthoquinone with cyclic ketones and further subsequent reaction of this product with semicarbazide hydrochloride. Compounds were characterized by FT-IR, 1H NMR, 13C NMR, elemental analysis and screened for antiproliferative activity against three human cancer cell lines (HepG2, MG-63 and MCF-7) by MTT assay using doxorubicin as standard. Docking was performed by using the Glide 5.7 integrated with Maestro 9.2 (Schr?dinger, LLC, 2011) to understand the binding preference of synthesized compounds with target enzyme topoisomerase-II. Results: 4-(cyclohexylideneamino) [1, 2] naphthoquinone 2-semicarbazone was found to be most active cytotoxic agent against all cancer cell lines with IC50 values in the range of 5.58–6.31 μM. Results of molecular docking were also well correlated in vitro cytotoxicity assay. Insilico ADME studies revealed the drug likeliness of compounds. Conclusions: The synthesized compounds were found to have significant anticancer activity and able to enter in higher phases of the drug development process due to favorable pharmacokinetic properties.

Nod2 downregulates TLR2/1 mediated IL1ß gene expression in mouse peritoneal macrophages.

Nod2 is a cytosolic pattern recognition receptor. It has been implicated in many inflammatory conditions. Its signaling has been suggested to modulate TLR responses in a variety of ways, yet little is known about the mechanistic details of the process. We show in this study that Nod2 knockdown mouse peritoneal macrophages secrete more IL1ß than normal macrophages when stimulated with peptidoglycan (PGN). Muramyl dipeptide (MDP, a Nod2 ligand) + PGN co-stimulated macrophages have lower expression of IL1ß than PGN (TLR2/1 ligand) stimulated macrophages. MDP co-stimulation have similar effects on Pam3CSK4 (synthetic TLR2/1 ligand) mediated IL1ß expression suggesting that MDP mediated down regulating effects are receptor dependent and ligand independent. MDP mediated down regulation was specific for TLR2/1 signaling as MDP does not affect LPS (TLR4 ligand) or zymosan A (TLR2/6 ligand) mediated IL1ß expression. Mechanistically, MDP exerts its down regulating effects by lowering PGN/Pam3CSK4 mediated nuclear cRel levels. Lower nuclear cRel level were observed to be because of enhanced transporting back rather than reduced nuclear translocation of cRel in MDP + PGN stimulated macrophages. These results demonstrate that Nod2 and TLR2/1 signaling pathways are independent and do not interact at the level of MAPK or NF-κB activation.

Role of mitogen-activated protein kinases in peptidoglycan-induced expression of inducible nitric oxide synthase and nitric oxide in mouse peritoneal macrophages: extracellular signal-related kinase, a negative regulator.

The expression of inducible nitric oxide synthase (iNOS) and the production of nitric oxide (NO) are important host defense mechanisms against pathogens in mononuclear phagocytes. The objectives of this study were to examine the roles of mitogen-activated protein kinases (MAPKs) and transcription factors (nuclear factor-κB [NF-κB] and activating protein 1 [AP-1]) in peptidoglycan (PGN)-induced iNOS expression and NO production in macrophages. PGN is a cell wall component of Gram-positive bacteria that stimulates inflammatory responses both ex vivo and in vivo. PGN stimulates the activation of all three classes of MAPKs, extracellular signal-related kinase (ERK), c-Jun N-terminal kinase (JNK), and p38(mapk) in macrophages, albeit with differential activation kinetics. Using a selective inhibitor of JNK (SP600125) and JNK1/2 small interfering RNA (siRNA) knocked-down macrophages, it was observed that PGN-induced iNOS and NO expression is significantly inhibited. This suggested that JNK MAPK plays an essential role in PGN-induced iNOS expression and NO production. In contrast, inhibition of the ERK pathway using PD98059 dose dependently enhanced PGN-induced iNOS expression and NO production. PGN-induced ERK activation was attenuated in ERK1/2 siRNA knocked-down macrophages; however, NO and iNOS expression were significantly enhanced. An electrophoretic mobility shift assay showed that SP600125 inhibited PGN-induced NF-κB and AP-1 activation, whereas inhibition of the ERK pathway enhanced NF-κB activation, but with no effect on AP-1. These results indicate that the JNK MAPK positively regulate PGN-induced iNOS and NO expression by activating NF-κB and AP-1 transcription factors, whereas the ERK pathway plays a negative regulatory role via affecting NF-κB activity.

Mycobacterium indicus pranii supernatant induces apoptotic cell death in mouse peritoneal macrophages in vitro.

Mycobacterium indicus pranii (MIP), also known as Mw, is a saprophytic, non-pathogenic strain of Mycobacterium and is commercially available as a heat-killed vaccine for leprosy and recently tuberculosis (TB) as part of MDT. In this study we provide evidence that cell-free supernatant collected from original MIP suspension induces rapid and enhanced apoptosis in mouse peritoneal macrophages in vitro. It is demonstrated that the MIP cell-free supernatant induced apoptosis is mitochondria-mediated and caspase independent and involves mitochondrial translocation of Bax and subsequent release of AIF and cytochrome c from the mitochondria. Experiments with pharmacological inhibitors suggest a possible role of PKC in mitochondria-mediated apoptosis of macrophages.

In vitro activation of murine peritoneal macrophages by recombinant YopJ: production of nitric oxide, proinflammatory cytokines and chemokines.

Recently it was reported that 3 µg/ml of recombinant YopJ induced apoptosis in murine peritoneal macrophages in vitro. However, in this study, we report the activation of murine peritoneal macrophages in vitro on treatment with sub-apoptotic dose of recombinant YopJ protein (1 µg/ml). The activation involves enhanced production of nitric oxide (NO), tumor necrosis factor-α (TNF-α), IL-12, and IL-6. Production of NO and IL-6 was found to peak at 24 h of rYopJ treatment, whereas IL-12 and IFN-γ production peaked at 18 h of rYopJ treatment. Increased mRNAs expression of nitric oxide, IL-12, IL-6 and IFN-γ molecules, was also observed in rYopJ-treated macrophages by RT-PCR. rYopJ induced the enhanced activity of protein tyrosine kinases which was inhibited by pharmacological inhibitor genestein, wortmanin and H-7 suggesting the role of tyrosine kinases, PI3K and PKC in the above process. rYopJ also induced increased enhanced production chemokines MIP-1α, MCP-1, and RANTES in macrophages. Significantly, increased expression of TLR-2, TLR-6, MyD 88 and IRAK-1 was also observed by immunoblotting in rYopJ-treated macrophages. rYopJ induced production of NO, TNF-α and IL-6 was significantly inhibited in macrophages pretreated with pharmacological inhibitor wortmanin, genestein and H-7 demonstrating the probable involvement of protein tyrosine kinases in the above process.

Recombinant YopJ induces apoptotic cell death in macrophages through TLR2.

Bacterial species evolved evasive maneuvers to bypass their recognition by the receptors primarily TLRs of the innate immune cells. We have reported that 3µg/ml of recombinant YopJ when provided extracellularly induced apoptosis in murine peritoneal macrophages in vitro. The present investigations demonstrate the role of TLR2 in apoptotic signals induced by rYopJ protein in murine peritoneal macrophages. The role of TLR2 in rYopJ induced macrophage apoptosis was shown by neutralization experiments and its co-immunoprecipitation with downstream molecule MyD88. The observed functional consequence of TLR2 neutralization were the inhibition of caspase-8 and caspase-3 activation, change in mitochondrial membrane potential (Δψm) and DNA fragmentation induced by rYopJ in macrophages. Further, rYopJ induced enhanced expression of IRAK-4, FADD, phosphorylation of IκB and p38 MAP kinase in macrophages. Pharmacological inhibitor of p38 MAP kinase and neutralization of TLR2 with neutralizing antibodies significantly inhibited the rYopJ induced caspases activation and DNA fragmentation, suggesting the possible involvement of TLR2 and p38 MAP kinase in rYopJ induced macrophages apoptosis.

Role of prostaglandin E2 in peptidoglycan mediated iNOS expression in mouse peritoneal macrophages in vitro.

Many extracellular stimuli, e.g. microbial products, cytokines etc., result in the expression of inducible nitric oxide synthase (iNOS) in macrophages. However, it is not known whether expression of the iNOS gene in response to microbial products is a primary response of macrophages, or is the result of paracrine/autocrine signalling induced by endogenous biomolecules that are synthesised as a result of host cell-microbe interaction. In this paper we demonstrate that iNOS expression in mouse peritoneal macrophages in response to bacterial peptidoglycan (PGN) is a secondary effect requiring autocrine signalling of endogenously produced prostaglandin E2, and that PGN stimulation is mandatory, but not sufficient in itself, for induction of iNOS expression.

Protein kinase Cdelta and protein tyrosine kinase regulate peptidoglycan-induced nuclear factor-kappaB activation and inducible nitric oxide synthase expression in mouse peritoneal macrophages in vitro.

Bacteria and their ubiquitous cell wall component peptidoglycan (PGN) activate the innate immune system of the host and induce the release of inflammatory molecules. Nitric oxide (NO) is a potent molecule involved in the cytotoxic effects mediated by macrophages (MPhi) against microorganisms. This study investigates the signaling pathway involved in inducible nitric oxide synthase (iNOS) expression and nitric oxide release caused by peptidoglycan from Staphylococcus aureus in mouse peritoneal macrophages. Protein tyrosine kinase inhibitor, genestein and PKCdelta inhibitor, rottlerin attenuated the PGN-induced expression of iNOS and NO. H-7, a PKC inhibitor did not significantly affected the PGN-induced iNOS expression and NO release. NF-kappaB inhibitor, curcumin also inhibited PGN-induced NO release. Treatment of MPhi with PGN caused an increase in protein tyrosine kinase activity, expression and activation of PKCdelta, IkappaB phosphorylation and p65 (NF-kappaB) nuclear translocation. The PGN-induced IkappaB phosphorylation and p65 nuclear translocation was inhibited in macrophages pretreated with rottlerin and genestein. No paracrine or autocrine effect of TNF-alpha on PGN-induced iNOS expression and NO release was observed. These observations suggest that PGN induces enhanced expression of iNOS and NO production through activation of protein tyrosine kinases and PKCdelta, which in turn initiates NF-kappaB activation and translocation to nucleus.

Recombinant YopJ induces apoptosis in murine peritoneal macrophages in vitro: involvement of mitochondrial death pathway.

Yersinia species during infection adhere to host immune cells primarily to macrophages and employ its secretary proteins known as Yersinia outer proteins to trigger death in infected cells. In the present study, it is shown that recombinant Yersinia outer protein J (rYopJ) could induce apoptosis in murine peritoneal macrophages in vitro as assessed by morphological features, internucleosomal DNA fragmentation, change in mitochondrial membrane potential (MMP) (Deltapsim), activation of caspases and Annexin V binding. rYopJ-induced cell death was dose and time dependent. Pre-treatment with broad-spectrum caspase inhibitor Z-VAD-FMK, caspase-3 inhibitor Ac-DEVD-CHO and caspase-8 inhibitor Z-IETD-FMK prevented the change in MMP and DNA fragmentation, suggesting caspase-dependent apoptosis of rYopJ-treated macrophages. Blocking the endocytosis by pre-treatment of cells with cytochalasin B did not prevent the rYopJ-induced macrophages apoptosis. The data further suggest that rYopJ-induced apoptosis is mediated by molecules upstream of caspase-8 and relay through mitochondrial pathway involving Bax, Bcl-2, activation of caspase-8 and caspase-3, Bid and polyadenosine diphosphate-ribose polymerase cleavage, cytochrome c release and DNA fragmentation.

Growth hormone-induced production of cytokines in murine peritoneal macrophages in vitro: role of JAK/STAT, PI3K, PKC and MAP kinases.

The immunomodulatory properties of growth hormone (GH) are well recognized. Enhanced production of NO and cytokines by macrophages on treatment with GH was reported by us recently. The present investigation elucidates the signaling mechanism(s) by which GH activates macrophages in vitro. It is observed that GH induces the phosphorylation (activation) of JAK2, PI3K, PKC and MAP kinases. Studies with pharmacological inhibitors of various signaling molecules also indicated that GH-induced proinflammatory responses in macrophages are mediated by JAK2/PI3K/PKC/ERK1/2, JAK2/JNK and JAK/STAT signaling cascades. It was further observed that GH induced 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-alpha and IL-1beta in macrophages, whereas JNK cascade mediated the production of TNF-alpha, IFN-gamma and IL-12. These results suggest that JAK2 plays a central role in mediating proinflammatory responses of macrophages on GH treatment.

Cisplatin primes murine peritoneal macrophages for enhanced expression of nitric oxide, proinflammatory cytokines, TLRs, transcription factors and activation of MAP kinases upon co-incubation with L929 cells.

Cisplatin, a chemotherapeutic drug, may also act as a biological response modifier. Cisplatin (10mug/ml) treatment of macrophages for 24h activates them to produce enhanced amounts of nitric oxide (NO), ROI, proinflammatory cytokines and exhibit increased tumoricidal activity, which may or may not be contact mediated. In the present investigation, we report that the treatment of macrophages with cisplatin for a short period of 2h is sufficient to make them more receptive to interaction with tumor cells. Macrophages pretreated with cisplatin for 2h, and co-incubated with L929 cells, produced enhanced NO, TNF-alpha, IL-1beta, IL-12 and IFN-gamma. Production of NO, TNF-alpha, IL-1beta, IL-12 and IFN-gamma was maximum at 24h of co-incubation. Enhanced transcription of iNOS, TNF-alpha, IL-1beta, IL-12 and IFN-gamma genes in cisplatin-pretreated macrophages were observed between 12 and 24h of co-incubation with L929 cells. Cisplatin-treated macrophages on co-incubation with L929 cells also expressed enhanced transcription of Toll-like receptor (TLR)-2 and TLR-4 genes and their proteins. It is observed that cisplatin-pretreated macrophages on co-incubation with L929 cells showed activation of mitogen-activated protein (MAP) kinases and NF-kappaB. Pharmacological inhibitors like PD98059, SB202190 and wortmannin strongly inhibited the production of NO and proinflammatory cytokines suggesting the probable role of p42/44, p38 MAPK and PI3K in the above process. The c-Jun amino terminal kinase (JNK) inhibitor SP600125 was less effective in inhibiting the production of NO and proinflammatory cytokines. The data thus suggests that pretreatment of macrophages with cisplatin makes them biologically more responsive to interaction with L929 cells and become activated.

Prolactin induced production of cytokines in macrophages involves Ca++ and p42/44 MAP kinase signaling pathway.

The immunomodulatory properties of prolactin (PRL) are well recognized. Recently, we have reported the activation and enhanced production of nitric oxide by macrophages on treatment with PRL. The involvement of protein tyrosine kinases, MAP kinases and Ca++ signaling in the enhanced nitric oxide production by macrophages on PRL treatment was also established. In the present study, it has been observed that PRL induces the intracellular release of Ca++; activates protein kinase C (PKC)-8 and p42/44 MAP kinase. The activation of PKC-delta was found to be inhibited by Pertussis toxin (PTX) (Galpha1-protein inhibitor) and H7 (PKC inhibitor). Pretreatment of macrophages with PTX, H7, TMB8 (intracellular Ca++ immobilizer) significantly down regulated the PRL induced intracellular Ca++ release and the activation of p42/44 MAP kinases. The involvement of Ca++ signaling and p42/44 MAP kinase in regulation of PRL induced IL-1beta and TNF-alpha production by macrophages has also been investigated. PRL is observed to induce the expression of transcription factors phospho-Elk-1, c-fos and phospho-c-myc. These observations clearly suggest the involvement of PKC-delta/Ca++/p42-44 MAP kinase cascade in PRL induced activation of murine peritoneal macrophages.

WITHDRAWN:Involvement of JAK/STAT, PI3K, PKC and MAP kinases in the growth hormone induced production of cytokines in murine peritoneal macrophages in vitro.

The Ahead of Print article entitled "Involvement of JAK/STAT, PI3K, PKC and MAP kinases in the growth hormone induced production of cytokines in murine peritoneal macrophages in vitro", which was published online on 30 May 2008, was withdrawn at the author's request on 8 October 2008.

Prolactin and growth hormone induce differential cytokine and chemokine profile in murine peritoneal macrophages in vitro: involvement of p-38 MAP kinase, STAT3 and NF-kappaB.

The role of immune-neuroendocrine interactions in the autoimmune diseases is well recognized. Autoimmune rheumatoid diseases in their active phase have been characterized by high levels of prolactin (PRL) as well as proinflammatory cytokines which suggest a co-relationship between them. In the present study, we have investigated the profile of cytokines secreted by macrophages on treatment with PRL and growth hormone (GH) in vitro. Significantly enhanced production of cytokines IL-1beta, IL-12p40 and IFN-gamma was observed on treatment of macrophages with PRL or GH. However, higher doses of PRL (1000 ng/ml) induced the production of anti-inflammatory cytokine IL-10, with significant abrogation in production of proinflammatory cytokines. It is further observed that PRL and GH induced the production of chemokines MIP-1alpha and RANTES. PRL but not GH selectively induced significantly enhanced production of MCP-1 and IP-10. It is further shown that p38 MAP kinase, STAT3 and NF-kappaB could play a differential regulatory role in PRL or GH induced production of cytokines by macrophages.