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The β-Human Chorionic Gonadotropin-Related Peptide LQGV Exerts Anti-Inflammatory Effects through Activation of the Adrenal Gland and This information is current as Glucocorticoid Receptor in C57BL/6 Mice of September 25, 2021. Marten van der Zee, Jan Willem van den Berg, Conny van Holten-Neelen and Willem A. Dik J Immunol 2010; 185:5066-5073; Prepublished online 6 October 2010; Downloaded from doi: 10.4049/jimmunol.1001414 http://www.jimmunol.org/content/185/9/5066 http://www.jimmunol.org/ References This article cites 54 articles, 7 of which you can access for free at: http://www.jimmunol.org/content/185/9/5066.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

The b-Human Chorionic Gonadotropin-Related Peptide LQGV Exerts Anti-Inﬂammatory Effects through Activation of the Adrenal Gland and Glucocorticoid Receptor in C57BL/6 Mice

Marten van der Zee,1 Jan Willem van den Berg,1 Conny van Holten-Neelen, and Willem A. Dik

The systemic inﬂammatory response syndrome is a complex host response to a variety of clinical insults, generally leading to severe pathology. The human chorionic gonadotropin b-chain–related tetrapeptide leucine-glutamine-glycine-valine (LQGV) reduces

hemorrhagic and LPS-induced systemic inflammatory response syndrome, but its mechanisms of action are not yet fully un- Downloaded from derstood. Through the combination of in vivo, in vitro, and ex vivo approaches, we demonstrate that LQGV actively stimulates corticosterone production in mice and thereby suppresses in vivo TLR4-directed inflammation upon LPS administration. Blocking in vivo glucocorticosteroid receptor signaling reduced the prosurvival effect of LQGV. Also, upon multiple TLR activation by heat-killed Listeria monocytogenes, splenocytes from LQGV-treated mice produced significantly less TNF-a and IL-6, which was absent after in vitro blockage of the glucocorticosteroid receptor. Using adrenal gland and adrenal cell line cultures, we show that

LQGV stimulates corticosterone production. Moreover, by using speciﬁc pharmacological inhibitors of the adrenocorticotropic http://www.jimmunol.org/ hormone (ACTH) and luteinizing hormone receptors as well as of cAMP signaling, we demonstrate that LQGV stimulates the ACTH receptor. These data show that the b-human chorionic gonadotropin–related tetrapeptide LQGV stimulates adrenal glucocorticosteroid production through activation of the ACTH receptor with consequent glucocorticoid receptor activation and immunosuppression in C57BL/6 mice. The Journal of Immunology, 2010, 185: 5066–5073.

regnancy is characterized by specific control of the mater- and urine especially in the late second trimester and the third nal immune system, which is necessary to prevent rejection trimester of pregnancy (10). Benner and Khan (9, 13, 14) postu- P of the fetal allograft (1). Consequently during pregnancy, lated that, besides the generation of hCG b-core and nicked by guest on September 25, 2021 symptoms of autoimmune diseases, such as rheumatoid arthritis b-hCG, small breakdown products of three to seven amino acids and multiple sclerosis, decline, whereas susceptibility to intracel- in length are generated from loop 2 of b-hCG and that such small lular pathogens, such as Listeria monocytogenes, Toxoplasma peptides can exert immunomodulatory effects. gondii, Leishmania major, and Plasmodium spp., increases (1–4). The systemic inflammatory response syndrome (SIRS) is a com- The exact mechanisms that modulate the immune system during plex host response that may be inflicted by a variety of insults, pregnancy are largely unknown, but most likely specific hormonal such as severe trauma-hemorrhage, ischemia-reperfusion injury, changes are involved (5). pancreatitis, sepsis, and septic shock (15). SIRS is characterized by Human chorionic gonadotropin (hCG) is a human pregnancy excessive production of proinflammatory mediators, such as IL-1, hormone that, besides its endocrine functions, also influences the IL-6, TNF-a, CCL2, CXCL1, and CXCL2 (16, 17). High levels of functionality of the immune system (6–9). In addition to intact these proinflammatory mediators contribute to severe organ damage hCG, several other isoforms of hCG exist during pregnancy (10). and multiple organ dysfunction syndrome (18). Recently, we dem- It is well recognized that loop 2 of b-hCG is nicked by leukocyte onstrated that the synthetic b-hCG–related tetrapeptide leucine- elastase-like proteases, generating hCG b-core and several nicked glutamine-glycine-valine (LQGV) reduces hemorrhagic shock- b-hCG forms (11, 12). These molecules can be detected in serum associated inflammation and liver damage, prevents LPS-induced mortality in mice, and ameliorates the early chemokine response after L. monocytogenes infection (19–21). However, the regulatory Department of Immunology, Erasmus Medical Center, Rotterdam, The Netherlands mechanism by which LQGV exerts anti-inflammatory effects re- 1M.v.d.Z. and J.W.v.d.B. contributed equally to this work. mains elusive. Received for publication April 30, 2010. Accepted for publication August 31, 2010. Endogenous glucocorticosteroid production and function are Address correspondence and reprint requests to Willem A. Dik, Department of Im- munology, Erasmus Medical Center, P.O. Box 2040, 3000 CA Rotterdam, The Neth- crucial in the control and resolution of inflammation (22–24). In erlands. E-mail address: [email protected] line with this, administration of synthetic glucocorticosteroids is Abbreviations used in this paper: ACTH, adrenocorticotropic hormone; ADX, adrenal- commonly used in clinical settings to control inflammation (22). ectomy; BW, body weight; CBG, corticosteroid-binding globulin; CIP, corticotropin- Also, specific activation of endogenous glucocorticosteroid pro- inhibiting peptide; EPPE, glutamic acid-proline-proline-glutamic acid; GR, glucocorticosteroid receptor; hCG, human chorionic gonadotropin; HKLM, heat-killed Listeria duction by the adrenal glands has been postulated as a way to monocytogenes; LH, luteinizing hormone; LHR, luteinizing hormone antagonist recep- control an inflammatory event (25). An increase in circulating cor- tor; LHR-Y1, LH receptor-transfected Y1-adrenal; LQGV, leucine-glutamine-glycine- tisol (of which the rodent analogue is corticosterone) is observed valine; SIRS, systemic inflammatory response syndrome; US, unstimulated. during the late second trimester and the third trimester of pregnancy, Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 the phase wherein the highest degree of proteolytic cleavage www.jimmunol.org/cgi/doi/10.4049/jimmunol.1001414 The Journal of Immunology 5067 of b-hCG occurs. Therefore, we examined whether the b-hCG– Evaluation of mRNA expression levels by real-time quantitative related tetrapeptide LQGV exerts anti-inflammatory effects through PCR the stimulation of glucocorticosteroid receptor (GR) signaling and RNA was isolated using the GenElute RNA kit (Sigma-Aldrich). CYP11B1 glucocorticosteroid production. (encoding 11-b-hydroxylase) gene expression levels were determined by By using combined in vivo, in vitro, and ex vivo approaches, we real-time quantitative PCR using an Applied Biosystems 7900 PCR ma- demonstrate that LQGV exerts anti-inflammatory effects through chine (Applied Biosystems, Foster City, CA). The expression levels were GR activation via stimulation of corticosterone production by the quantified by normalization against the mRNA levels of the household gene ABL (30). Primers and probes used are available upon request. adrenal glands. This LQGV-induced corticosterone production is mediated through specific adrenocorticotropic hormone (ACTH) Statistical analysis receptor activation and subsequent cAMP-signaling. Statistical analysis was performed using SPSS version 15 software (SPSS, Chicago, IL). Intergroup differences were analyzed using Mann–Whitney Materials and Methods U test, and p , 0.05 was considered statistically significant. For survival Animals analysis, a Kaplan–Meier analysis followed by a log-rank test was performed. Correlation coefficients were determined with Pearson’s correla- Specific pathogen-free C57BL/6 male mice (Harlan, Horst, The Nether- tion analyses with significance set at p , 0.05. lands), aged 8–12 wk, were used in the experiments, which were approved by the local animal experiments committee. Results LPS-induced shock LQGV reduces LPS-induced mortality via GR activation m Mice were injected i.p. either with 200 l LQGV (50 mg/kg body weight Previously, we demonstrated that LQGV protected BALB/c mice Downloaded from [BW] dissolved in PBS, purity 99.2%; GL Biochem, Shanghai, China) or against LPS-induced mortality (19). In this study, we found that PBS (as control), directly followed by a second i.p. injection with 500 ml of the GR antagonist mifepristone (RU38486; 10 mg/kg BW, dissolved in LQGV administration 24 h prior to LPS injection significantly DMSO; Sigma-Aldrich, Zwijndrecht, The Netherlands) or vehicle. The (p , 0.05) improved the 3-d survival from 20 to 60% in C57BL/6 next day, mice were challenged with an LPS injection (30 mg/kg BW of mice (Fig. 1). This prosurvival effect of LQGV was completely Escherichia coli strain 0111:B4; Sigma-Aldrich), and survival was scored reversed when mice received the GR antagonist mifepristone in every 12 h for 3 d.

combination with LQGV (Fig. 1). These data indicate that the http://www.jimmunol.org/ Adrenalectomy prosurvival effect of LQGV in this model is dependent on GR Adrenalectomy (ADX) was performed as described (26), after which mice activation. acclimatized for 7 d before the start of the experiment. All mice received In vivo LQGV treatment reduces the in vitro responsiveness of postoperatively 0.9% NaCl solution as drinking water. splenocytes to L. monocytogenes Ags Ex vivo adrenal gland culture Next, we examined how LQGV treatment activated the GR in im- Complete adrenal glands were isolated and cultured as described (27, 28). mune cells. Splenocytes from untreated mice were in vitro-stimulated Briefly, 300 ml RPMI 1460 medium containing antibiotics, 5% FBS, and with HKLM [which activates multiple pattern-recognition receptors LQGV (50, 5, or 0.5 mg/ml) or PBS was added to the adrenal gland culture for 6 h. The cAMP-blocker H-89 (10 mM; Enzo Life Sciences, Plymouth and evokes a stronger inflammatory response than LPS (21)] in the by guest on September 25, 2021 Meeting, PA), the ACTH-blocker corticotropin-inhibiting peptide (CIP; presence of LQGV (50 mg/ml). HKLM stimulation induced TNF-a 1026 mM, 1027 mM, 1028 mM, or 1029 mM; Phoenix Pharmaceutics, and IL-6 production by splenocytes, which was not affected by ad- Phoenix, AZ), and the luteinizing hormone (LH) receptor antagonist dition of LQGV to the cultures (Fig. 2A,2B). In contrast, splenocytes m Antide (LHR; 100 M; Sigma-Aldrich) were added 5 min before LQGV obtained from mice 18 h after LQGV (50 mg/kg BW) administration stimulation. Culture supernatants and adrenal glands were collected and a , , stored at 280˚C until assayed. produced significantly less TNF- (p 0.01)andIL-6(p 0.05) upon HKLM stimulation than that of splenocytes from PBS-treated In vitro stimulation of LH receptor-transfected Y1-adrenal cells mice (Fig. 2C,2D). This suppressive effect of LQGV was completely The murine LH receptor-transfected Y1-adrenal (LHR-Y1) cells (kindly reversed when LQGV-treated mice also received mifepristone (Fig. provided by Dr. Bill Moyle, University of Medicine and Dentistry of New 2C,2D). These data demonstrate that LQGV does not directly stim- Jersey, Newark, NJ) were cultured as described (29). The cAMP-blocker ulate GR activity in splenocytes and does not directly interfere with H-89 (10 mM), ACTH-blocker CIP (1026 mM), and LH receptor antagonist Antide (100 mM), were added 5 min before addition of recombinant hCG (300 U/ml; Sigma-Aldrich) or LQGV (5 or 50 mg/ml). Cells and supernatants were collected and stored at 280˚C until assayed. In vitro stimulation of splenocytes and cytokine analysis ADX and non-ADX mice were injected i.p. with 200 ml LQGV (50 mg/kg BW) or PBS. Thirty minutes later, 500 ml of the GR antagonist mifepristone or vehicle was administrated i.p. Mice were euthanized 18 h later; blood was collected by cardiac puncture, and splenocytes were isolated and stimulated as described (21). Plasma was obtained by centrifugation (3000 rpm, 10 min), immediately frozen, and stored at 280˚C until assayed. In addition, splenocytes (106 cells/ml) from untreated mice (mice that received neither LQGV nor PBS) were isolated and cultured overnight in the presence of plasma from either LQGV- or PBS-treated mice or with culture media from either LQGV- or PBS-stimulated adrenal glands, with or without 7log10 heat-killed L. monocytogenes (HKLM) (21), and with or without mifepristone (0.3 mg/ml). Culture supernatants were collected, and TNF-a FIGURE 1. Mifepristone injection abolishes the LQGV prosurvival ef- and IL-6 levels were determined by ELISA (R&D Systems Europe, fect. Mice were injected i.p. with either 200 ml LQGV (50 mg/kg BW) or Abingdon, U.K.). PBS, directly followed by a second i.p. injection with 500 ml mifepristone (10 mg/kg BW) or vehicle. The next day, mice were challenged with an Corticosterone quantification LPS injection. Survival was scored every 12 h. Data depicted are from 19 Corticosterone levels in plasma (obtained between 3:00 and 4:00 PM) and mice per group. pp , 0.05 for LQGV + vehicle treatment compared with culture supernatant were determined by ELISA (IBL, Hamburg, Germany). LQGV + mifepristone treatment, or PBS + vehicle. 5068 LQGV STIMULATES ADRENAL GLAND ACTIVATION

FIGURE 2. In vivo LQGVadministration reduces HKLM-induced TNF-a and IL-6 production by splenocytes. A and B, Splenocytes from untreated mice were stimulated overnight with 7log10 HKLM in the presence of 50 mg/ml LQGV, after which TNF-a (A) and IL-6 (B) levels in culture supernatants were determined by ELISA. C and D, In other experiments, mice were treated with LQGV (50 mg/kg BW) or PBS, in combination with or without mifepristone, and 18 h later splenocytes were isolated and cultured overnight with 7log10 HKLM, after which TNF-a (C) and IL-6 (D) levels in the culture supernatants were determined by ELISA. Data depicted are from five mice per group. pp , 0.05; ppp , 0.01. US, unstimulated. Downloaded from cytokine production and secretion but suggest that the immunosup- rone secretion (Fig. 5B). Recombinant hCG and the irrelevant pressive effect of LQGV is established through an in vivo-released tetrapeptide glutamic acid-proline-proline-glutamic acid (EPPE) secondary mediator that stimulates the GR. did not stimulate adrenal corticosterone secretion (data not shown). Next, we examined whether the adrenal gland culture media af- LQGV induces the systemic release of an immunosuppressive fected HKLM-induced TNF-a and IL-6 production by splenocytes. mediator Splenocytes were isolated from untreated mice and stimulated http://www.jimmunol.org/ Next, we determined whether LQGV induced the systemic release of with HKLM in the presence of culture media from adrenal glands an immunosuppressive mediator that acts through the GR. Plasma stimulated with either LQGVor PBS. Culture media obtained from was obtained from mice 18 h after PBS or LQGV injection. Plasma LQGV-stimulated adrenal glands inhibited HKLM-induced TNF-a from both LQGV- and PBS-treated mice inhibited HKLM-induced production in a dose-dependent manner (Fig. 5C). When a total of TNF-a production by naive splenocytes in a dose-dependent man- 8% (v/v) adrenal gland culture medium was added to the culture, ner (Fig. 3A). When a total volume of 30% (v/v) plasma was added media from LQGV-stimulated adrenal glands reduced HKLM- to the culture, plasma from LQGV-treated mice reduced HKLM- induced TNF-a (p , 0.05) (Fig. 5D) and IL-6 (p , 0.05) (Fig. induced TNF-a (p , 0.01) and IL-6 (p , 0.05) production to sig- 5E) production to significantly lower levels than observed with by guest on September 25, 2021 nificantly lower levels than observed with plasma from PBS-treated media from PBS-stimulated adrenal glands. This effect was com- mice (Fig. 3B,3C). Addition of mifepristone to the cultures completely abolished when mifepristone was co-added to the cultures pletely reversed the immunosuppressive effect of plasma from (Fig. 5D,5E). These data suggest that LQGV activates the adrenal LQGV-treated mice (Fig. 3B,3C). Collectively, these data suggest glands to synthesize and secrete corticosterone, which reduces the that the reduced capacity of splenocytes to respond to HKLM Ags, in vitro responsiveness of splenocytes to Listeria Ags. as displayed after in vivo LQGV administration, is established LQGV activates the adrenal ACTH receptor through the systemic release of a GR activating factor. Next, we determined the mRNA expression levels of 11-b-hydro- The immunosuppressive effect of LQGV is abolished by ADX xylase, the enzyme that converts deoxycorticosterone to cortico- To examine the role of the adrenal glands in LQGV-induced sterone. LQGV dose-dependently enhanced the mRNA expression immunosuppression, splenocytes from both LQGV- and PBS- levels of 11-b-hydroxylase (Fig. 6A), which correlated positively treated ADX mice were stimulated with HKLM. Removal of and significantly (r = 0.426; p , 0.05) with the corticosterone levels the adrenal glands completely abolished the immunosuppressive detected in the culture media (Fig. 6B). The cAMP-blocker H-89 effect of LQGV (Fig. 4A), as reflected by the TNF-a and IL-6 and ACTH receptor-blocker CIP completely abolished the LQGV- levels detected in culture supernatants. Also, plasma from LQGV- induced corticosterone release, whereas this was not observed treated ADX mice did not reduce the HKLM-induced TNF-a and when the LH receptor antagonist Antide was added to the organ IL-6 production by splenocytes from untreated mice (Fig. 4B). culture (Fig. 6C). Moreover, CIP inhibited the LQGV-induced in- These data demonstrate that the adrenal glands are involved in crease of 11-b-hydroxylase mRNA expression in a dose-dependent establishing the immunosuppressive effect of LQGV. manner (Fig. 6D). H-89, LH receptor antagonist, and CIP alone did not affect the basal adrenal 11-b-hydroxylase mRNA expression LQGV stimulates adrenal corticosterone production levels (data not shown). These data suggest that LQGV activates The previous experiments suggest that LQGV induces the release the adrenal glands to actively synthesize and secrete corticosterone of an adrenal-derived immunosuppressive mediator that exerts its through an ACTH receptor-activated cAMP-signaling cascade. action through the GR. Therefore, we determined plasma corticosterone levels at different time points after LQGV administration. LQGV activates the ACTH receptor on the murine adrenal cell Corticosterone plasma levels were significantly higher at 1 h (0.265 line LHR-Y1 mM/l versus 0.074 mM/l; p , 0.05), at 6 h (0.664 mM/l versus 0.055 To study further the specificity of LQGV for the ACTH receptor, mM/l; p , 0.05), and at 24 h (0.406 mM/l versus 0.050 mM/l; p , we examined the effects of LQGV on ACTH receptor and LH 0.05) in LQGV-treated mice than in PBS-treated mice (Fig. 5A). receptor activation by using the adrenal cell line LHR-Y1 [in which Ex vivo stimulation of complete adrenal glands revealed that LQGV the LH receptor has been cloned (29)]. These cells were stimulated dose-dependently and significantly (p , 0.05) induced corticoste- with recombinant hCG or LQGVin the presence or absence of H-89, The Journal of Immunology 5069 Downloaded from

FIGURE 4. ADX abolishes the immunosuppressive effect of LQGV. A, ADX mice were treated with LQGV (50 mg/kg BW) or PBS, and 18 h later

splenocytes were obtained and cultured overnight with 7log10 HKLM, and TNF-a and IL-6 levels in culture supernatants were determined by ELISA.

B, In other experiments, ADX mice were treated with LQGV or PBS, and http://www.jimmunol.org/ 18 h later plasma was isolated, and 30% (v/v) was used in an overnight

stimulation of splenocytes from untreated non-ADX mice with 7log10 HKLM, and TNF-a and IL-6 levels in culture supernatants were determined by ELISA. Data depicted are from ﬁve mice per group. pp , 0.05. US, unstimulated.

25, 33). In this study, we demonstrate that the hCG-related tetrapeptide LQGV, which reduces immune activation in response to by guest on September 25, 2021 FIGURE 3. LQGV induces the release of an immunosuppressive me- hemorrhagic shock and resuscitation, L. monocytogenes infec- diator in plasma. A, Mice were treated with LQGV (50 mg/kg BW) or tion, and LPS injection (19–21), exerts anti-inflammatory effects PBS, and 18 h later plasma was obtained and used in increasing amounts through the induction of corticosterone production and secretion (v/v) in overnight cultures of splenocytes from untreated mice stimulated by the adrenal glands. In addition, we show that the LQGV-induced a with 7log10 HKLM. Thereafter, TNF- levels in culture supernatants were corticosterone production and secretion is mediated through ACTH determined by ELISA. Data depicted are from four mice per group. B and receptor activation and subsequent cAMP signaling. C, In other experiments, mice were treated with LQGV or PBS, and 18 h Inflammation is a physiological reaction to infection and tissue later plasma was isolated, and 30% (v/v) was used in an overnight stim- injury (34). However, an uncontrolled inflammatory response can ulation of splenocytes from untreated mice with 7log HKLM in com- 10 culminate in SIRS and finally multiple organ dysfunction syn- bination with or without mifepristone. TNF-a (B) and IL-6 (C)levelsin culture supernatants were determined by ELISA. Data depicted are from drome, which is a major cause of in-hospital deaths worldwide seven mice per group. pp , 0.05; ppp , 0.01. US, unstimulated. (35). LPS challenge of mice is a TLR4 driven in vivo SIRS model that can result in multiple organ dysfunction syndrome and even- CIP, or LH receptor antagonist. In line with previous observa- tually death (35). In line with our previous study (19), we found tions (31, 32), recombinant hCG increased the corticosterone level that LQGV (50 mg/kg BW) can inhibit LPS-induced mortality in and 11-b-hydroxylase mRNA expression level. This increase was mice. This effect was completely reversed by mifepristone admin- inhibited by H-89 and the LH receptor antagonist (Fig. 7A,7B). istration, demonstrating that LQGV exerts its in vivo prosurvival LQGV dose-dependently increased 11-b-hydroxylase mRNA ex- effect through GR activation. Our observations are in line with those pression levels and corticosterone levels, which were reduced by of others who have demonstrated that GR-signaling blockade in- H-89 and CIP, but not by the LH receptor antagonist (LHR) (Fig. creases the vulnerability of organisms to tissue injury and LPS- 7A,7B). In addition, mRNA expression levels of 11-b-hydroxylase induced inflammation (23, 36). correlated positively and significantly with corticosterone levels Glucocorticosteroids exert their immunosuppressive effect through detected in culture media (Fig. 7C). The irrelevant tetrapeptide binding to the cytoplasmic GR (22, 33). Upon ligand binding, GR glutamic acid-proline-proline-glutamic acid (EPPE) did not increase translocates into the nucleus, where it interacts with glucocorticoid the 11-b-hydroxylase mRNA expression (data not shown). These responsive elements in the promoter region of target genes and reg- results demonstrate that LQGV induces 11-b-hydroxylase mRNA ulates their expression (22). The activated GR can also regulate gene expression through ACTH receptor-induced cAMP signaling. expression through direct interaction with transcription factors, such as AP-1, NF-kB, and STATs (37). We found that splenocytes from Discussion LQGV-treated mice produced significantly less of the AP-1 and NF- Endogenous glucocorticosteroid production and function are cru- kB controlled cytokines IL-6 and TNF-a when stimulated with cial in the control and resolution of inflammatory responses (22, HKLM in vitro, which was reversed when the GR-blocker mife- 5070 LQGV STIMULATES ADRENAL GLAND ACTIVATION

FIGURE 5. LQGV activates the adrenal glands to secrete corticosterone. A, Mice were treated with LQGV (50 mg/kg BW) or PBS, and plasma corticosterone levels were determined at 0, 1, 6, and 24 h after treatment. Plasma corticosterone levels in LQGV- treated mice increased and peaked at 6 h, after which the levels slowly declined. Data depicted are from eight mice per time point. B, Adrenal glands from naive mice were ex vivo-stimulated with either 0.5, 5, or 50 mg/ ml LQGV, and corticosterone levels in supernatant were determined. C, Culture supernatant collected from ex vivo adrenal gland stimulation was used in increasing amounts (v/v) in overnight stimulation of splenocytes from untreated mice with 7log10 HKLM. Downloaded from Thereafter, TNF-a levels in culture supernatants were determined by ELISA. Data depicted are from four mice per group. D and E, Culture supernatant (8%, v/v) was added to an overnight stimulation of splenocytes from untreated mice with 7log10 HKLM, and

TNF-a (D) and IL-6 (E) concentrations in http://www.jimmunol.org/ culture supernatants were measured by ELISA. Data depicted are from seven mice per group. pp , 0.05; ppp , 0.01. US, unstimulated.

pristone was coadministered with LQGV to the mice. Also, in vitro by itself. Together, these data indicate that LQGV renders cells less by guest on September 25, 2021 blockage of GR activity reversed the inhibitory activity of plasma susceptible to multiple TLR activation through corticosterone- from LQGV-treated mice with regard to HKLM-induced IL-6 and induced GR activity. We cannot exclude that other steroids, such as TNF-a production by splenocytes. Furthermore, LQGV administra- progesterone, are also produced and secreted upon LQGV-induced tion to mice resulted in increased corticosterone levels in plasma. Our ACTH receptor activation. However, corticosterone has a 5 to 10 study also shows that in vitro administration of LQGV to splenocyte times higher afﬁnity for the GR than that of progesterone (38, 39). cultures did not inhibit HKLM-induced IL-6 and TNF-a production Therefore, we believe that corticosterone is the major contributing

FIGURE 6. LQGVactivates the adrenal ACTH receptor. A, Adrenal glands were ex vivo-stimulated with either 0.5, 5, or 50 mg/ml LQGV or with a combination of 50 mg/ml LQGV with either 10 mM cAMP-blocker H-89, 100 mMLHR, or 1026 mM ACTH receptor antagonist CIP, and 11-b-hydroxylase mRNA expression levels were determined. B, Correlation analysis between adrenal 11-b-hydroxylase mRNA expression levels and corticosterone levels with different LQGV stimuli. Statistical signiﬁcance was determined by Pearson’s correlation analyses. C, Adrenal glands were ex vivo-stimulated with 50 mg/ml LQGV alone or in combination with either 10 mMcAMP- blocker H-89, 100 mMLHR,or1026 mM ACTH receptor antagonist (CIP), and corticosterone levels in supernatant were determined. D, Adrenal glands were ex vivo-stimulated with PBS or 50 mg/ml LQGV in combination with either 1029 mM, 1028 mM, 1027 mM, or 1026 mM CIP, and 11-b-hydroxylase mRNA expression levels were determined. Data depicted are from ﬁve or six ex vivo-stimulated adrenal glands from different naive mice per group. pp , 0.05. The Journal of Immunology 5071 Downloaded from http://www.jimmunol.org/

FIGURE 7. LQGV activates the ACTH receptor on the murine adrenal cell line LHR-Y1. A and B, LHR-Y1 cells were stimulated with either 300 U recombinant hCG, 5 or 50 mg/ml LQGV alone or in combination with either 10 mM cAMP-blocker H-89, 100 mM LHR, or 1026 mM ACTH receptor antagonist (CIP). Subsequently, 11-b-hydroxylase mRNA expression levels (A) and corticosterone levels in supernatant (B) were determined in the cultured cells. C, Correlation analysis between 11-b-hydroxylase mRNA expression levels and corticosterone levels after 6 h stimulation with either 300 U by guest on September 25, 2021 recombinant hCG, 5 mg/ml LQGV, or 50 mg/ml LQGV in combination with either the cAMP-blocker H-89, LHR, or ACTH receptor antagonist CIP. Data depicted are from four single experiments. pp , 0.05. anti-inflammatory glucocorticosteroid induced by LQGV treatment sterone levels detected in adrenal gland culture supernatants. in our studies (21, 40). These data suggest that LQGV indeed stimulates corticosterone Glucocorticosteroid production is regulated by 11-b-hydroxylase production and secretion by adrenal cells. Alternatively, the in- within the zona fascilata of the adrenal cortex (41). In rodents, 11- crease in plasma corticosterone level after LQGV administration b-hydroxylase converts deoxycorticosterone to corticosterone, and in could be due to an increased level of corticosteroid-binding humans it converts deoxycortisol to cortisol (41). The expression of globulin (CBG), which upon binding enhances the corticoste- 11-b-hydroxylase is dependent on ACTH-induced ACTH receptor rone half-life from 30 to 60 min to ∼5 d (47). We regard this as activation and subsequent cAMP signaling (41–43). LQGV increased unlikely because CBG is produced in the liver (48) and therefore 11-b-hydroxylase mRNA expression levels and stimulated cortico- could not have influenced the increase in corticosterone levels sterone production in ex vivo adrenal gland cultures. These effects detected in our adrenal gland culture supernatants. Furthermore, were abolished when cAMP signaling or ACTH receptor activation glucocorticoids bound to CBG are biologically inactive (49), were blocked. While, LQGV is part of the amino acid sequence of whereas we here show that LQGV stimulation results in the re- loop 2 of b-hCG (13), it exerts its effects independent of the LH/hCG lease of bioactive corticosterone, as reflected by the fact that receptor as is evident from our LH/hCG receptor blocking studies in mifepristone abolishes the LQGV effects. All together, these data adrenal gland cultures and LHR-Y1 cells. This notion is strengthened indicate that LQGV can stimulate de novo corticosterone pro- by our observation that LQGV did not directly inhibit the response of duction and secretion by murine adrenal glands. splenocytes to HKLM, despite the fact that immune cells can express Previously, we demonstrated that LQGV reduced inflammation the LH/hCG receptor and do respond to recombinant hCG (8, 9, 44, associated with hemorrhagic shock and resuscitation and reduced 45). Thus, LQGV actively stimulates the ACTH receptor of adrenal LPS-induced septic shock, but LQGV enhanced the susceptibility cells, but more studies are needed to fully explain the mechanism by to L. monocytogenes infection (19–21). GR blockade has been which LQGV activates the ACTH receptor. shown to increase disease severity during hemorrhagic shock and The in vivo corticosterone plasma levels were increased from at resuscitation as well as LPS-induced septic shock (25, 46, 50), least 1 h up to 24 h after LQGV administration. These kinetics whereas synthetic glucocorticosteroids are protective in these clearly differ from the in vivo stress-induced release of pre-existing models (51, 52). High corticosterone levels also render mice more corticosterone, which typically rises and declines again within 30– susceptible to L. monocytogenes infection (46, 53–55). Therefore, 60 min (46). Also, the ex vivo LQGV-induced 11-b-hydroxylase we suggest that LQGV-stimulated adrenal glucocorticosteroid pro- mRNA expression levels correlated positively with the cortico- duction and subsequent GR activation contributed to the immuno- 5072 LQGV STIMULATES ADRENAL GLAND ACTIVATION suppressive effects that we found in our previous studies (14, 19– monocytes in the pathogenesis of sepsis: consequences for immunomonitoring and treatment. Neth. J. Med. 55: 132–141. 21). However, we do not exclude the possibility that LQGV exerts 17. Rittirsch, D., M. A. Flierl, and P. A. Ward. 2008. Harmful molecular mechanisms other effects as well. in sepsis. Nat. Rev. Immunol. 8: 776–787. Synthetic glucocorticosteroids are the most frequently used 18. Osborn, T. M., J. K. Tracy, J. R. Dunne, M. Pasquale, and L. M. Napolitano. 2004. Epidemiology of sepsis in patients with traumatic injury. Crit. Care Med. drugs to treat autoimmune and inflammatory diseases. Long-term 32: 2234–2240. glucocorticosteroid treatment can lead to several severe side ef- 19. Khan, N. A., M. P. Vierboom, C. van Holten-Neelen, E. Breedveld, E. Zuiderwijk- fects, such as atrophy of the skin and muscles, osteoporosis, and Sick, A. Khan, I. Kondova, G. Braskamp, H. F. Savelkoul, W. A. Dik, et al. 2010. Mitigation of septic shock in mice and rhesus monkeys by human chorionic adrenal gland insufficiency. In the clinic, glucocorticosteroid treat- gonadotrophin-related oligopeptides. Clin. Exp. Immunol. 160: 466–478. ment has to be tapered during a period of weeks to several months to 20. van den Berg, H. 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Corrections van der Zee, M., J. W. van den Berg, C. van Holten-Neelen, and W. A. Dik. 2010. The b-human chorionic gonadotropin-related peptide LQGV exerts anti-inﬂammatory effects through activation of the adrenal gland and glucocorticoid receptor in C57BL/6 mice. J. Immunol. 185: 5066–5073.

The authors refer to Antide as being a luteinizing hormone (LH)-receptor antagonist in this work. Antide is, however, not an LH-receptor antagonist but an LH-releasing hormone antagonist (Ljungqvist, A., D. M. Feng, W. Hook, Z. X. Shen, C. Bowers, and K. Folkers. 1988. Antide and related antagonists of luteinizing hormone release with long action and oral activity. Proc. Natl. Acad. Sci. USA 85: 8236– 8240), meaning that Antide does not block the LH receptor itself. Therefore, from the experiments, the authors can not exclude the possibility that the b-human chorionic gonadotropin-related peptide LQGV did (to a certain extent) interact with and activate LH-receptor expressed on glucocorticosteroid producing cells. This does not influence the major finding of the study, namely, that LQGV exerts anti- inflammatory action in C57BL/6 mice through ACTH receptor-driven adrenal gland and glucocorticoid receptor activation. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1090122