Blood in Vitro Glucocorticoid-Immunosuppressed

GM-CSF Restores Innate, But Not Adaptive, Immune Responses in Glucocorticoid-Immunosuppressed Human Blood In Vitro This information is current as of September 27, 2021. Jian Xu, Rudolf Lucas, Marcus Schuchmann, Simone Kühnle, Thomas Meergans, Ana P. Barreiros, Ansgar W. Lohse, Gerd Otto and Albrecht Wendel J Immunol 2003; 171:938-947; ; doi: 10.4049/jimmunol.171.2.938 Downloaded from http://www.jimmunol.org/content/171/2/938

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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 © 2003 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

GM-CSF Restores Innate, But Not Adaptive, Immune Responses in Glucocorticoid-Immunosuppressed Human Blood In Vitro1

Jian Xu,* Rudolf Lucas,* Marcus Schuchmann,† Simone Ku¨hnle,* Thomas Meergans,* Ana P. Barreiros,† Ansgar W. Lohse,† Gerd Otto,‡ and Albrecht Wendel2*

Infection remains the major complication of immunosuppressive therapy in organ transplantation. Therefore, reconstitution of the innate immunity against infections, without activation of the adaptive immune responses, to prevent graft rejection is a clinically desirable status in transplant recipients. We found that GM-CSF restored TNF mRNA and protein expression without inducing IL-2 production and T cell proliferation in glucocorticoid-immunosuppressed blood from either healthy donors or liver transplant patients. Gene array experiments indicated that GM-CSF selectively restored a variety of dexamethasone-suppressed, LPS- inducible genes relevant for innate immunity. A possible explanation for the lack of GM-CSF to restore T cell proliferation is its enhancement of the release of IL-1␤R antagonist, rather than of IL-1␤ itself, since exogenously added IL-1␤ induced an IL-2- Downloaded from independent Con A-stimulated proliferation of glucocorticoid-immunosuppressed lymphocytes. Finally, to test the in vivo relevance of our ﬁndings, we showed that GM-CSF restored the survival of dexamethasone- or cyclosporine A-immunosuppressed mice from an otherwise lethal infection with Salmonella typhimurium. In addition to this increased resistance to infection, GM-CSF did not induce graft rejection of a skin allotransplant in cyclosporine A-immunosuppressed mice. The selective restoration potential of GM-CSF suggests its therapeutic use in improving the resistance against infections upon organ transplantation. The Journal of Immunology, 2003, 171: 938–947. http://www.jimmunol.org/

he development of new immunosuppressive drugs with comitant stimulation of the innate immune system leads to increased improved efficacy and decreased toxicity has led to a sub- expression of factors, such as cytokines, that may influence the adap- T stantial improvement in the survival of organ transplant tive immune system to promote the injury of the transplant organ (4). patients and in short-term graft survival for all organs (1). How- Importantly, evidence from animal experiments (8) and clinical stud- ever, under these conditions the inflammatory response to infec- ies (9) implied that restoration of innate immunity without restoration tions is severely impaired, and this represents the most common or with absence of the adaptive immunity might still be beneficial for life-threatening complication of long term immunosuppressive resistance to infection. It is therefore desirable, in immunosuppressed by guest on September 27, 2021 therapy (2). The prevention or effective treatment of infection is organ transplant recipients, to create a status with a preferential re- therefore still a primary goal in organ transplantation. Indeed, a constitution of the innate immune responses, which will contribute to broad range of potential sources of infection as well as the adverse the recognition and control of the infectious agents (10), while keep- effects of antimicrobial drugs used for prophylaxis and therapy ing the adaptive immune responses silent. Such a status requires a represent important challenges to overcome (2). A concerted in- preferential reactivation of the effectors of the innate immune re- terplay between innate and adaptive immune surveillance for graft sponses, i.e., macrophages and/or neutrophils, by pharmacological in- rejection has been indicated (3). A robust innate immune response, tervention without restoring the suppressed adaptive immune re- characterized by macrophage infiltration and up-regulation of mul- sponses, such as the T cell response, which is implicated in graft tiple cytokines, chemokines, and chemokine receptors, has been rejection (11Ð13). Recombinant GM-CSF, a drug approved for he- demonstrated within the first day after transplantation in an alym- matological indications in humans (14), has been indicated in vitro phoid murine model. This innate immune response to the acute and in vivo to enhance the synthesis and release of proinflammatory injury associated with the transplant procedure, however, was not cytokines such as TNF, which is crucial in host defense in various shown to elicit allograft rejection (4Ð6). Moreover, studies using animal infection models (15Ð17). In our laboratory it was previously T cell-deficient mice have shown that prompt rejection can occur shown that GM-CSF potentiates the immune responses to endotoxin only after T cell reconstitution, even when skin or allografts have (18) and restores the impaired immune responses in LPS-desensitized been allowed to recover or heal for Ͼ100 days (7). Rather, con- mice (19) as well as in refractory human monocytes (20). Others found that anergic monocytes from sepsis patients were reactivated *Biochemical Pharmacology, University of Konstanz, Konstanz, Germany; and De- (21, 22), and hyporesponsiveness of whole blood, induced by trauma, partments of †Medicine and ‡Transplantation and Hepatobiliary Surgery, University sepsis, or cardiac surgery, could be overridden in vitro (23) by GM- of Mainz, Mainz, Germany CSF treatment. Therefore, we investigated whether GM-CSF was Received for publication December 9, 2002. Accepted for publication May 6, 2003. able to reconstitute TNF production without activating the adaptive The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance immunity of the T cell response, firstly in dexamethasone-suppressed with 18 U.S.C. Section 1734 solely to indicate this fact. blood from healthy donors, to normalize all conditions required for 1 This work was supported by the Deutsche Forschungsgemeinschaft (Research the study, and secondly in the blood from immunosuppressed liver Group “Endogenous Tissue Injury: Mechanisms of Autodestruction”, Grant We transplant recipients, to confirm and emphasize the potential clinical 686/18). implications in an ex vivo setting. On a molecular basis we used gene 2 Address correspondence and reprint requests to Dr. Albrecht Wendel, Biochemical Pharmacology, University of Konstanz, 78457 Konstanz, Germany. E-mail address: array experiments to investigate a potential selective reconstitution [email protected] capacity of GM-CSF under immunosuppression of genes, in addition

FIGURE 1. Reconstitution of TNF, but not IL-1␤, production by GM-CSF in glucocorticoid-immunosuppressed blood. Hepa- rinized whole blood from 10 healthy donors (a and b) and 10 liver transplant patients (c and d) was incubated in complete RPMI 1640 sequentially with dexamethasone (1 ␮M) for 1 h, GM-CSF (50 ng/ml) for 1 h, and LPS (100 ng/ml) for 16 h. ELISA was used to measure the release of TNF-␣ (a and c) and IL-1␤ (b and d), expressed per milliliter of whole blood. Data represent the mean Ϯ SEM of the results. Downloaded from http://www.jimmunol.org/ to TNF, related to innate immunity. To assess the in vivo relevance of endotoxin-free Con A; Sigma-Aldrich, Deisenhofen, Germany). After in- our findings, we investigated whether GM-CSF restored the survival cubation, the cells and cell-free supernatants (by centrifugation at 300 ϫ g, of immunosuppressed mice from an otherwise lethal Salmonella ty- 10 min) were then subjected to different measurements. phimurium infection and assessed its influence on graft rejection of a skin allotransplant in the infected mice. Our findings support the po- ELISA tential clinical value of GM-CSF for the therapeutic improvement of Cytokines in cell-free supernatants were quantified by ELISA using Ab resistance to infections in immunosuppressed organ transplant pairs for TNF, IL-2, IL-1␤, IL-1R antagonist (IL-1ra),3 and IFN-␥ (Endo- patients. gen, Munich, Germany) as well as human rTNF (Bender, Vienna, Austria), IL-1␤, IL-1ra, IL-2, and IFN-␥ (Endogen) standards as previously de- by guest on September 27, 2021 scribed (24). Materials and Methods Human blood sample Cell viability and proliferation assay Blood from 10 healthy donors was used to select the optimal working Cells were washed three times with Dulbecco’s PBS, stained with 1 ␮M concentration of immunosuppressant (dexamethasone) in vitro. We also Calcein AM (Molecular Probes, Leiden, The Netherlands), and kept for 1 h included blood from 10 patients (age, 39Ð69 years; average, 58.6 years), at 37¡C. The fluorescence measurement followed the manufacturer’s man- who all underwent orthotopic liver transplantation at University Hospital ual. Viability and proliferation were presented as a percentage of the pos- (Mainz, Germany). Decompensation of liver function was due to chronic itive control live cells and of the known nonproliferated control live cells, liver diseases, such as primary biliary cirrhosis, chronic viral hepatitis B or respectively. C, autoimmune hepatitis, or acute liver failure. All these patients were treated with methylprednisolone (12 mg for nine patients and 36 mg for one patient) combined with tacrolimus. Blood was taken from these pa- Two-way MLR assay tients at the lowest level of immunosuppression by tacrolimus (Cmin) A two-way MLR assay was performed according to a procedure similar to within 1 mo after transplantation. All patients gave written informed con- that previously described on human PBMC (25), using the Cell Prolifera- sent to transplantation and follow-up examinations. tion ELISA kit (Roche Applied Science, Mannheim, Germany). Briefly, ␮ ϫ 6 Preparation of human PBMC 50- l aliquots of cells (2.5 10 /ml) from each of two allogeneic donors were added to wells of a 96-well, flat-bottom plate in the absence or the PBMC were prepared in cell preparation tubes (Vacutainer CPT; BD Bio- presence of dexamethasone and/or GM-CSF. The cell cultures were incu- sciences, Franklin Lakes, NJ) according to the manufacturer’s instructions. bated at 37¡C for 5 days in RPMI 1640 supplemented with 2.5 IU/ml Cell numbers were adjusted to 5 ϫ 106 cells/ml in RPMI 1640 (Invitrogen, heparin and 100 IU/ml penicillin/streptomycin. Bromodeoxyuridine Karlsruhe, Germany) before further incubation. Lymphocytes were pre- (BrdU) was added 24 h before fixation of the cells. After 5 days of incu- pared as the nonadherent fraction from PBMCs upon growth adherence for bation, the culture medium was removed, the cells were fixed, and the 2 h (nonadherent fraction of PBMC). DNA was denatured for 0.5 h. Additional nonradioactive detection steps, using an anti-BrdU Ab-peroxidase complex, were performed according to Whole blood or PBMC/lymphocyte incubations the instructions of the manufacturer. Heparinized whole blood or PBMC in complete RPMI 1640, which is supplemented with 2.5 IU/ml heparin (Liquemin; Hoffmann-La Roche, Human TNF bioassay Grenzach-Whylen, Germany) and 100 IU/ml penicillin/streptomycin (Bio- The bioactivity of TNF, assessed as cytotoxicity in WEHI 164 subclone 13 chrom, Germany), were added to a 96-well plate and incubated with or fibrosarcoma cells, was evaluated using the ethidium homodimer-1 incor- without dexamethasone (1 ␮M; Dexa-Allvoran; TAD Pharmaceuticals, poration assay (Molecular Probes), as described previously (26). Cuxhaven, Germany) for 1 h and with or without GM-CSF (50 ng/ml; LEUCOMAX 400, Molgramostim; Essex Pharma, Munich, Germany) for another 1 h. Plates were incubated for 1 (cDNA expression array) and 16 h 3 (LPS model) or 72 h (Con A model) at 37¡Cin5%CO2 after addition of Abbreviations used in this paper: IL-1ra, IL-1R antagonist; BrdU, bromodeoxyuri- the stimuli (100 ng/ml of LPS from Salmonella abortus equi or 5 ␮g/ml dine; Cdk, cyclin-dependent kinase; CsA, cyclosporine A. 940 GM-CSF RESTORES INNATE RESPONSES IN GLUCOCORTICOID-IMMUNOSUPPRESSED BLOOD

Complementary DNA expression array Equal numbers of PBMC obtained from different healthy donors in the same setting were pooled after incubation and subjected to total RNA isolation. mRNA expression was analyzed using Atlas Human Arrays 1.2 (Clontech, Palo Alto, CA), which contained 1176 human cDNAs encoding proteins with a wide range of functions. The assay followed the manufacturer’s manual. Signal intensity was quantitated with the ImageMaster VDS software package (Amersham Pharmacia Biotech, San Francisco, CA). The levels of expression were normalized using several highly expressed housekeeping genes, including genes coding for ubiquitin, GAPDH, HLA class I histocompatibility Ag C-4 ␣ subunit (HLA C-4), ␤-actin, 60S ribosomal protein L13A, and 40S ribosomal protein S9.

Western blotting Cell extracts (20 ␮g protein) were separated on a 12% polyacrylamide gel and transferred onto a nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany). The membrane was probed with anti-p27kip1 Ab (BD PharMingen, San Diego, CA) and anti-cyclin-dependent kinase 2 (anti- Cdk2) Ab (Santa Cruz Biotechnology, Santa Cruz, CA), respectively. Im- munoblots were detected by a HRP-conjugated secondary Ab and ECL

(Amersham Pharmacia Biotech, Piscataway, NJ). Downloaded from

Infection model under immunosuppression CBA/Ca mice were immunosuppressed for 2 days with dexamethasone (1 mg/kg i.p., six mice per group) or for 7 days with cyclosporine A (30 mg/kg, three mice per group) and were then infected with S. typhimurium (5 ϫ 105 bacteria/kg i.p.). The recombinant murine GM-CSF (provided by

Dr. F. R. Seiler, Behring-Werke, Marburg, Germany; 50 ␮g/kg i.v.) was http://www.jimmunol.org/ given once on day 2 (dexamethasone immunosuppression groups) or once on days 7, 8, 9, and 10 (cyclosporine A (CsA) suppression group). Survival was then monitored over 72 h and followed for 3 wk. Aerobic CFU were counted after overnight incubation (37¡C) of the Columbia blood agar plates (Heipha; Biotest, Heidelberg, Germany) spread with 100 ␮l of whole blood. All animals received humane care according to European Council Directive 86/609/EEC and the national German regulations. The directives of the ethical committee of University of Konstanz were followed.

Skin allotransplantation by guest on September 27, 2021 CBA/Ca mice (four per group) in transplantation experiments were immunosuppressed with a daily injection of 30 mg/kg CsA i.p. After anesthe- tizing and shaving the recipient, a patch of skin, 1 ϫ 1 cm, was removed from the back to one side of the spine. Then donor tail skin of matching size was embedded in the graft bed. The graft was sutured with 8Ð12 single stitches with a 5-0 filament. Vaseline gauze was put on the graft, followed by a soft cotton gauze and a Band-Aid. The bandage and the sutures were removed on day 7 after surgery. Rejection of the graft was assessed from day 7 on by morphological changes. The time of rejection was defined as complete necrosis of the graft. Bacterial infection with S. typhimurium FIGURE 2. GM-CSF susceptible reconstitution of gene expression in (5 ϫ 105/kg i.p.) in transplanted CBA/Ca mice was induced on day 7, and dexamethasone-suppressed human PBMC. PBMC (5 ϫ 106 cells/ml) pre- survival was monitored over a period of up to 4 wk. Grafts and surrounding pared from healthy donors were incubated sequentially with dexametha- native skin of some transplanted mice were photographed regularly, and sone (1 ␮M) for 1 h, GM-CSF (50 ng/ml) for 1 h, and LPS (100 ng/ml) for samples were excised for histological examination. To determine the 1 h. Equal numbers of PBMC obtained from different donors in the same course of infection, some mice were sacrificed at different times for the setting were pooled after incubation and subjected to total RNA isolation. CFU measurement in liver, spleen, blood, and peritoneum. mRNA expression was analyzed using Atlas Human Arrays 1.2. Presented Determination of the working concentrations are the selected grids from the original arrays. The six detected housekeeping genes included ubiquitin, GAPDH, HLA C-4␣, ␤-actin, L13A, and S9 The optimal working concentrations of GM-CSF (50 ng/ml) and LPS (100 genes. The arrows indicate the location of the corresponding genes that ng/ml) for the priming or stimulation of TNF production were based on were restored, except for IL-1␤, by GM-CSF. Independent experiments what was previously reported (20). Since dexamethasone has been reported with cDNA expression array were performed twice, and an up-/down-reg- to cause cell death or apoptosis of monocytes (27) or lymphocytes (28), we ulation of 2-fold or more was considered significant. first examined the viability of the dexamethasone-treated PBMC. We found that in the range from 0.1 to 10 ␮M dexamethasone, cells maintained their viability within 16 h. At 72 h after treatment a viability loss occurred at dexamethasone concentrations Ͼ1 ␮M. Since 1 ␮M dexamethasone was sufficient for almost complete inhibition of LPS-induced TNF production Results without inducing significant cytotoxicity, this concentration was chosen for GM-CSF restores the release of bioactive TNF, but not IL-1␤, all additional experiments. in glucocorticoid-immunosuppressed blood Statistical analysis To investigate the potential of GM-CSF, under immunosuppression, to restore TNF production upon LPS stimulation, we first Data were expressed as the mean Ϯ SEM, and datasets were subjected to one-way ANOVA, followed by Bonferroni’s multiple comparison tests used an experimental model in which whole blood from 10 healthy (PRISM; GraphPad, San Diego, CA). A value of p Ͻ 0.05 (indicated as an donors was exposed to dexamethasone and then stimulated with asterisk in figures) was considered significant. LPS. The results show that in dexamethasone-suppressed whole The Journal of Immunology 941

Table I. Genes restored by GM-CSF in immunosuppressed, LPS-stimulated human PBMC

IDa Gene Code Protein/Gene Foldb

L25080 A01g Transforming protein RhoA H12 3.3 M90813 A04h Cyclin D2 4.0 U09579 A07k Cdk inhibitor 1 2.2 L36645 B10e Ephrin A receptor 4 precursor 3.2 M33336 B14g cAMP-dependent protein kinase I ␣ regulatory subunit 3.2 L35253 B07h Mitogen-activated protein kinase p38 3.0 L31951 B05j c-Jun N-terminal kinase 2 2.0 X08004 B02n Ras-related protein RAP-1B 2.0 L19067 C12c NF-␬B transcription factor p65 subunit 2.0c X01394 C08f TNF precursor 4.7 Y09392 C02g WSL protein 2.0 U28014 C06h Caspase-4 precursor 2.0 X04106 C02i Calcium-dependent protease small (regulatory) subunit; calpain; 2.0 M74524 D05a Ubiquitin-conjugating enzyme E2 17-kDa 2.6 M60974 D01b Growth arrest and DNA damage-inducible protein 3.3 U12979 D14j Activated RNA polymerase II transcriptional coactivator p15 2.1 X78710 D04k Metal regulatory transcription factor 3.4 X61498 D05m NF-␬-B p100 subunit 2.0c M68891 D08m Endothelial transcription factor GATA2 8.0 Downloaded from X52541 E10a Early growth response protein 1 2.7 M62831 E11a Transcription factor ETR101 2.0 U30504 E03d Transcription initiation factor TFIID 31-kDa subunit 2.1 M59079 E13d CCAAT-binding transcription factor subunit B 2.1 L11672 E07e Zinc ﬁnger protein 91 2.0 M33374 E05h Cell adhesion protein SQM1 2.3

X07979 E09i Fibronectin receptor ␤ subunit; CD29 Ag 2.5 http://www.jimmunol.org/ D10202 E09j Platelet-activating factor receptor 3.5 X72304 E10k Corticotropin-releasing factor receptor 1 precursor 3.5 M29696 E14k IL-7R ␣ subunit precursor 2.1 Y00371 F02a 70-kDa heat shock protein 2.1 X07270 F04b Heat shock 90-kDa protein A 2.0 M23452 F1le Macrophage inflammatory protein 1 ␣ precursor 2.0 X53799 F10g Macrophage inflammatory protein 2 ␣ (MIP2-␣) 2.0 X78686 F12g Granulocyte chemotactic protein 2 2.0 Y00787 F14g IL-8 precursor 3.7 J04130 F03h Macrophage inflammatory protein 1␤ precursor 3.9 X04602 F13i IL-6 precursor 2.6 by guest on September 27, 2021 U29607 F12k Methionine aminopeptidase 2 2.3 D00759 F011 Proteasome component C2 2.0 D00760 F021 Proteasome component C3 2.0 D00761 F031 Proteasome component C5 2.0 D00762 F041 Proteasome component C8 2.1 D26512 F09m Matrix metalloproteinase 14 precursor 2.4 AF036906 C03e Linker for activation of T cells 1.1 M63928 C03g T cell activation CD27 antigen 1.1 M15796 C061 Proliferating cyclic nuclear antigen 1.0 X55122 E08c Trans-acting T cell-specific transcription factor GATA3 1.0 J03132 E14g ICAM1 precursor 1.2 M37435 F03e Macrophage-specific CSF-1 1.2 M21121 F10e T cell-specific RANTES protein precursor 1.1 K02770 F10i IL-1␤ precursor 1.0

a GenBank accession number. b Fold increase is defined as the factor that is added by GM-CSF on top of the increase found by LPS alone of the dexamethasone-suppressed response. c Restoration rate (compared to the purely LPS-treated) was Ͻ50%. blood, the LPS-induced TNF release is significantly reduced com- performed a TNF cytotoxicity assay and found that the TNF re- pared with the LPS only setting, but is significantly restored in the stored by GM-CSF in the blood from immunosuppressed liver presence of GM-CSF, although not to the level of GM-CSF- transplant patients is fully bioactive (bioassay: restoration from primed naive cells from healthy donors (Fig. 1a). In contrast, under 530 Ϯ 50 to 6870 Ϯ 1440 pg/ml; comparable to ELISA: restora- immunosuppression, IL-1␤ release is not enhanced by GM-CSF, tion from 640 Ϯ 170 to 7080 Ϯ 2350 pg/ml; data not shown). although this is the case in the nonsuppressed setting (Fig. 1b). These observations also hold true in clinical samples ex vivo, since GM-CSF differentially up-regulates LPS-induced gene exogenous GM-CSF restores LPS-induced TNF (Fig. 1c), but not expression in dexamethasone-suppressed human PBMC IL-1␤ (Fig. 1d) release in blood taken from 10 immunosuppressed To investigate to which genes in humans the differential reconsti- liver transplant patients. tution potential of GM-CSF extends, we have analyzed the gene Since the bioactivity of TNF can be neutralized by its soluble expression profile altered by GM-CSF under immunosuppression. receptors sTNF-R1 and sTNF-R2, the latter of which is cleaved by As is apparent in the selected grids from the cDNA expression the same convertase (TNF-␣-converting enzyme) that mediates the array (Fig. 2), TNF mRNA expression is suppressed by dexameth- shedding of TNF from its membrane-bound pro form (29), we also asone, but can be significantly up-regulated by GM-CSF, almost 942 GM-CSF RESTORES INNATE RESPONSES IN GLUCOCORTICOID-IMMUNOSUPPRESSED BLOOD

FIGURE 3. The lack of activation by GM-CSF of T cell response in immunosuppressed human blood. Heparinized whole blood or PBMC from 10 healthy donors and 10 liver transplant patients was incubated in complete RPMI 1640 sequentially with dexamethasone (1 ␮M) for 1 h, GM-CSF (50 ng/ml) for 1 h, and Con A (5 ␮g/ml) for 72 h. IL-2 release was measured by ELISA (a and b). The proliferation of PBMC was assessed by measuring Cal- cein-AM fluorescence (c and d) and is presented as a percentage of the nonproliferated control cells. Data represent the mean Ϯ SEM of the healthy donor group (n ϭ 10; a and c) and the transplant recipient group (n ϭ 10; b and d). Downloaded from http://www.jimmunol.org/ reaching the level of the LPS-treated setting. A variety of other liferation) in glucocorticoid-immunosuppressed blood. GM-CSF genes, produced mainly by monocytes/macrophages in PBMC, does not induce IL-2 release in dexamethasone-suppressed, Con such as IL-8, IL-6, and monocyte-specific platelet-activating factor A-stimulated blood from healthy donors and in blood from liver receptor, are also restored by GM-CSF to at least 90% of the LPS- transplant patients, but does so in the controls (Fig. 3, a and b). inducible signal, similar to the gene for TNF, except for IL-1␤ Consistently, under these conditions GM-CSF does not restore the (Table I). In contrast, the expression of genes implicated in the Con A-stimulated proliferation of immunosuppressed PBMC (Fig. LPS-inducible lymphocyte responses, such as CD27 and T cell- 3, c and d). To further confirm the lack of activation by GM-CSF specific RANTES, are not up-regulated by GM-CSF in dexa- of lymphocytes under immunosuppression, a two-way MLR assay methasone-suppressed PBMC (Table I). Taken together, these re- was performed in which the proliferation of mixed PBMC from by guest on September 27, 2021 sults indicate a preferential restoring potential of GM-CSF on two allogeneic donors was assessed by measuring BrdU incorpo- dexamethasone-suppressed genes related to the activation of ration into the proliferating cells. In parallel, IFN-␥ release in the monocytes/macrophages, the major players in innate immunity. supernatant from the 5-day cultured PBMC was measured. The results (Fig. 4) clearly show that in the absence of dexamethasone, GM-CSF does not restore T cell response and T cell GM-CSF enhances T cell proliferation in a two-way MLR of al- proliferation logeneic PBMC, but fails to restore such an effect when PBMC are Since the rationale of our pharmacological approach is to prefer- treated with dexamethasone. Moreover, the release of IFN-␥,a entially restore the innate immune defense without reactivating the potent proinflammatory cytokine produced by activated T lympho- adaptive immune system, we have examined whether GM-CSF cytes, can be suppressed by glucocorticoids (30), but GM-CSF activates T cell responses (typically IL-2 release and T cell pro- cannot restore the activation of T cells to release IFN-␥ (Fig. 4).

FIGURE 4. The inability of GM-CSF to activate the two-way MLR and IFN-␥ generation in dexamethasone-suppressed human PBMC. PBMC prepared from two healthy allogeneic donors were adjusted to 2.5 ϫ 106 cells/ml in complete RPMI 1640. The allogeneic PBMC were incubated sequentially with dexamethasone (1 ␮M) for 1 h and GM-CSF (50 ng/ml) for 1 h. Fifty-microliter aliquots of cells from each of two allogeneic donors were mixed and incubated at 37¡C for 5 days. Twen- ty-four hours before ﬁxation of the cells, BrdU was added to label the cells. At 5 days of incubation, IFN-␥ in the supernatant was measured by ELISA; the remaining cells were subjected to a proliferation assay by measuring BrdU incorporation, using Ab anti-BrdU- POD. Proliferation is presented as absorbance Ϫ (A450 A690). Results are representative of three different experiments. The Journal of Immunology 943

Table II. Enhancement of IL-1ra release by GM-CSF in immunosuppressed, LPS- or Con A-stimulated human PBMC from liver transplant patients (n ϭ 10)a

IL-1ra Release (ng/ml Ϯ SEM)

LPS model (16 h) Con A model (72 h)

Untreated GM-SCF LPS GM-CSF/LPS Untreated GM-SCF Con A GM-CSF/Con A

0.06 Ϯ 0.04 4.90 Ϯ 0.63 2.99 Ϯ 0.61 16.48 Ϯ 2.45b 0.95 Ϯ 0.35 6.88 Ϯ 2.00 9.89 Ϯ 1.72 22.57 Ϯ 3.62b

a Whole blood was treated and the release of IL-1ra (nanograms per milliliter Ϯ SEM) was measured as described. b Signiﬁcant difference (n ϭ 10; p Ͻ 0.01) compared to the purely Con A- or LPS-treated setting.

These results are in line with those obtained from the Con A lation of Cdk2 protein expression, correlating with proliferation, in model. Con A-stimulated healthy control and IL-1␤-treated immunosuppressed Con A-stimulated settings both in vitro and ex vivo, suggest- ␤ IL-1 restores Con A-induced proliferation of lymphocytes in ing the involvement of these cell cycle proteins (Fig. 5c). immunosuppressed PBMC independently of IL-2, involving regulation of p27kip1 and Cdk2 GM-CSF restores the survival of immunosuppressed mice As indicated above, exogenous GM-CSF cannot induce Con A- against Salmonella infection Downloaded from stimulated proliferation of lymphocytes in immunosuppressed Since immunosuppressive treatment with corticosteroids can ab- PBMC. Moreover, in the same setting, GM-CSF did not up-regu- rogate the resistance of CBA/Ca mice to S. typhimurium infection, late IL-1␤, but, rather, increased the expression of IL-1ra (Table thus causing the death of the animals (34), we used this model to II), which is known to neutralize IL-1␤ bioactivity. Therefore, we test whether the presence of GM-CSF indeed protected the immu- next investigated whether IL-1␤ can restore Con A-stimulated pro- nosuppressed host against infection. In the control group, Salmo- liferation during immunosuppression. The results indicate that ex- nella-resistant CBA/Ca mice survived a period of 3 wk after in- http://www.jimmunol.org/ ogenous IL-1␤ indeed partially restores Con A-stimulated prolif- fection without symptoms of disease and without relevant numbers eration of lymphocytes both in vitro (Fig. 5a) and ex vivo (Fig. of blood-borne bacteria 4 days after infection (data not shown). In 5b), probably by means of an IL-2-independent mechanism, since the dexamethasone-immunosuppressed group, signiﬁcant numbers this IL-1␤ effect cannot be blocked by a neutralizing anti-human IL-2 of bacteria were measurable in blood (310 Ϯ 30 CFU/100 ␮lof mAb (Fig. 5b). Since p27kip1, which inhibits the activation of Cdk2, whole blood), and all animals in this group died within 2 wk (with plays an important role in IL-2-induced T cell proliferation (31Ð33), two mice dying within 1 wk). In contrast, when immunosup- we wondered whether these proteins are implicated in the IL-1␤- pressed mice were pretreated with GM-CSF, low or no bacterial restored, IL-2-independent proliferation of the immunosuppressed counts were detectable on day 4 after infection (20 Ϯ 5 CFU/100 by guest on September 27, 2021 PBMC. We observed a down-regulation of p27kip1 and an up-regu- ␮l of whole blood). After a transient phase of infectious disease,

FIGURE 5. Induction by IL-1␤ of Con A-stimulated IL-2-independent proliferation of lymphocytes from glucocorticoid-immunosuppressed blood, regulated by p27kip1 and Cdk2. PBMC were incubated in complete RPMI 1640 sequentially with dexamethasone (1 ␮M) for 1 h, GM-CSF (50 ng/ml) for 1 h, and ConA(5␮g/ml) for 72 h at 37¡Cin5%

CO2. Proliferation of PBMC, assessed by measuring Calcein-AM ﬂuorescence, is presented as percentage of the nonproliferated control cells. Data represent the mean Ϯ SEM of the healthy donor group (n ϭ 10; a) and the transplant patient group (n ϭ 10; b). The neutralizing anti- human IL-2 mAb (R&D Systems) was applied at a ﬁnal concentration of 0.5 ␮g/ ml. c, Western blot analysis of p27kip1 and Cdk2 protein in lymphocytes from healthy PBMC untreated (control) or treated with dexamethasone (ϩdex) and from liver transplant recipients (ex vivo) at the indicated times after Con A stimulation. Results are representative of three different experiments. 944 GM-CSF RESTORES INNATE RESPONSES IN GLUCOCORTICOID-IMMUNOSUPPRESSED BLOOD

FIGURE 6. Survival of a lethal S. typhimurium infection of immunosuppressed mice when treated with GM-CSF. CBA/Ca mice were immunosuppressed with dexamethasone (a; 1 mg/kg i.p., six mice per group) for 2 days or with CsA (b; 30 mg/kg, three mice per group) for 7 days. All mice were then infected with S. typhimurium (5 ϫ 105 bacteria/kg i.p.). Where indicated, re-

combinant murine GM-CSF (50 ␮g/kg i.v.) Downloaded from was given once on day 2 (a) or once on days 7, 8, 9, and 10 (b). Survival was then monitored over 72 h and followed for 3 wk in S. typhimurium-infected native, immunosuppressed, and immunosuppressed plus GM- CSF-treated animals. Survival curves were

/p Ͻ http://www.jimmunol.org ,ءءء .analyzed using the log-rank test 0.003 (p Ͻ 0.05 was considered signiﬁcant). by guest on September 27, 2021

these mice recovered, and all survived the time interval chosen, quences on the outcome of transplant acceptance. To test this, we except for one that died 2 wk later (Fig. 6a). Like others, we were used a combined transplant/infection model in which we trans- unable to detect TNF in the serum of Salmonella-infected CBA/Ca planted mice after 7 days of daily immunosuppression with CsA mice (35, 36), although this cytokine is clearly involved in innate (30 mg/kg i.p.) and then infected these mice on day 7 with S. resistance to S. typhimurium in this model (35, 36). Since CsA is typhimurium. Under continued immunosuppression, mice received a widely used immunosuppressive drug that also suppresses T 50 ␮g/kg GM-CSF daily from day 7 until day 10. The survival of lymphocyte-dependent immune responses (37), which are impli- the animals and the acceptance of the graft were assessed daily cated in graft rejection (11Ð13), we further tested the infection over a period of 4 wk. The immunosuppressed and transplanted model under CsA immunosuppression. Consistent with the dexa- animals pretreated with GM-CSF all survived the observation pe- methasone suppression model, in the CsA-immunosuppressed riod of 28 days (data not shown). The study drug efﬁciently re- group all Salmonella-infected mice died within 1 wk. However, duced the bacterial load from 300 Ϯ 40 CFU/100 ␮l of whole when the immunosuppressed mice were pretreated with GM-CSF, blood in CsA-treated infected mice to 10 Ϯ 5 CFU/100 ␮lin all survived the infection (Fig. 6b). It should be noted that GM- CsA/GM-CSF-treated mice. Furthermore, all surviving animals CSF is able to restore LPS-induced TNF release ex vivo in the peritoneal macrophages from CsA-immunosuppressed mice (re- pretreated with GM-CSF accepted the skin grafts without any stored to 333.2 Ϯ 21.1 pg/ml, compared with the LPS alone setting signs of rejection within 4 wk (Fig. 7b). All skin grafts were suc- of 361.2 Ϯ 4.2 pg/ml; data not shown). We thus conclude that the cessfully integrated within the naive tissue (Fig. 7d) and showed restoration of the innate immune responses in these murine immuno overt morphological changes compared with control skin (Fig. nosuppression models upon GM-CSF treatment is sufﬁcient to 7c). Whereas immunosuppressed mice in the infection control overcome an otherwise lethal bacterial infection. group (S. typhimurium/CsA) all died within 7 days of infection without graft rejection, mice in the transplantation control group, GM-CSF does not promote skin graft rejection, but enables the i.e., without immunosuppression, lost all grafts within 7 days of survival of infected immunosuppressed and transplanted mice surgery (Fig. 7a), but survived bacterial infection due to this We have shown that GM-CSF can boost immunity against infec- strain’s inherent Salmonella resistance. These experiments dem- tion in immunosuppressed animals. However, it was not known onstrate that GM-CSF treatment allows successful handling of the whether intervention with GM-CSF might have negative conse- infection by reactivation of the experimentally suppressed immune The Journal of Immunology 945

FIGURE 7. Retention of skin transplants in GM-CSF-treated, Salmonella-infected immunosuppressed mice. CBA/Ca mice (n ϭ 4) were transplanted with allogeneic skin. Either untreated (a) or after 7 days of daily immunosuppression with CsA (30 mg/kg i.p.; b and d), these mice were infected with 5 ϫ 105 S. typhimurium. Downloaded from Under continued immunosuppression, mice received 50 ␮g/kg GM-CSF daily from day 7 until day 10. Presented are macrophotographs of graft from skin-transplanted, infected, but untreated, mice on day 7 (a) and CsA-suppressed, skin- transplanted, infected, GM-CSF-treated mice on ␮

day 18 (b), as well as H&E-stained slices (2 m) http://www.jimmunol.org/ from uninfected, untreated, and nontransplanted controls (c) or mice with the same setting as b but on day 21 after skin transplantation (d). Black arrows indicate revascularization. by guest on September 27, 2021

system and at the same time does not negatively interfere with skin this hypothesis. In addition, TNF is known to be crucial in the host transplant acceptance. defense against infections (15Ð17). Indeed, the neutralization of TNF bioactivity, as recently approved for the treatment of Crohn’s Discussion disease or rheumatoid arthritis, was associated with the develop- Based on our previous finding that GM-CSF potentiates or reac- ment of active tuberculosis in some patients (38). Moreover, at the tivates the impaired immune responses in immunocompromised protein level, excessive inhibition of TNF production due to rou- mice or human cells (18Ð20, 23), the study reported here translates tine immunosuppression was suggested to lead to an insufficient this property of GM-CSF in a clinically relevant ex vivo setting, response to infectious stimuli in kidney (39) as well as liver (40) using glucocorticoid-immunosuppressed blood from liver trans- transplant recipients. Bacterial infection occurs in up to 68% of plant patients and an in vivo murine immunosuppressed infection liver transplant recipients, commonly with infection of the liver, model. In these in vitro and ex vivo approaches, we have observed biliary tract, peritoneal cavity, bloodstream, and surgical wound. a recovery of the proinflammatory TNF response, without reacti- Most such infections occur in the first 2 mo following transplan- vation of the T cell response, in terms of IL-2 production and tation. The results from additional experiments using later stage proliferation. As a mechanistic rationale, the restoration of TNF post-transplantation blood (but within 12 mo) demonstrated a sim- production by GM-CSF may result from the restored mRNA ex- ilar reconstitution of TNF release by GM-CSF to early stage post- pression of the cytokine. transplantation blood (data not shown). Therefore, the GM-CSF- Although we presented no direct evidence in humans that GM- restored TNF response could be of benefit for the liver transplant CSF treatment will protect transplant patients from bacterial in- recipient in overcoming bacterial infection in both early and late fection under immunosuppression, we have shown in vivo both in stage post-transplantation. In addition to bacterial infection, viral an infection and an infection/transplantation mouse model that infection remains the major complication in the later stage of post- GM-CSF restores innate immunity against infections to strengthen transplantation (2, 41). There are reports demonstrating that TNF 946 GM-CSF RESTORES INNATE RESPONSES IN GLUCOCORTICOID-IMMUNOSUPPRESSED BLOOD protects against virus infection in vitro and in vivo by means of cell expression of ICAM-1 (59). Coexpression of GM-CSF and the improving viral clearance or inhibiting viral replication (42), in- costimulatory molecule B70 has been reported to enhance NK- cluding CMV, the fatal agent for immunocompromised individu- mediated cytotoxicity and induce the antitumor immunity in hep- als, such as organ transplant patients (2, 41). It was also found that atoma transplanted into nude mice (60). Based on the above find- TNF may inhibit early transgenic expression by CMV promoters ings, we believe that an activation (if any) of NK cells by GM-CSF in vivo, a mechanism that is independent of adaptive immunity and under immunosuppression may be beneficial to the host defense is probably secondary to innate immune responses to virus infec- against infections rather than harmful to the graft. tion (43). In agreement with other results (61, 62), we found that GM-CSF The possibility that GM-CSF treatment might affect organ re- enhanced IL-1ra release not only after an LPS but also after a Con jection is unlikely based on our results in the mouse transplant A stimulus in immunosuppressed human blood (Table II). It is model, in which the CsA-suppressed mice survived an otherwise striking that exogenous IL-1␤ restored the Con A-induced T cell lethal bacterial infection when pretreated with GM-CSF, without proliferation in glucocorticoid-immunosuppressed blood indepen- inducing graft rejection after skin allo-transplantation. Our exper- dently of IL-2. Since GM-CSF did not restore the release of IL-1␤, imental findings are in agreement with the observations of a pilot but, rather, led to its neutralization, these results might explain why study in children treated with GM-CSF after orthotopic liver trans- GM-CSF did not restore the proliferation of immunosuppressed plantation, where the study drug was well tolerated, and no rejec- PBMC in view of the observed effect of IL-1␤ on T cell prolifer- tion episode was induced. Rather, GM-CSF was shown to be ben- ation. In agreement with the clinical experience that steroid ther- eficial in patients with severe bacterial infection (44). apy of graft rejection fails when IL-1ra production is defective

Concerning the possible role of TNF in graft rejection in organ (63), the inability of GM-CSF to restore the release of IL-1␤ com- Downloaded from transplant patients, only a few reports are published. In fact, in bined with the enhancement of the release of IL-1ra is a favorable neither renal nor liver transplant patients was an association be- property for a drug supposed not to interfere with graft acceptance. tween TNF producer genotype and rejection found at the mRNA Further supportive arguments for such a use come from in vivo level (45). In addition, GM-CSF, the TNF-reconstituting agent in studies (64) as well as from clinical studies showing that GM-CSF our model, has been safely applied following liver transplantation increases the respiratory burst of neutrophils and/or increases neu-

in the treatment of neutropenia (44). trophil count after liver transplantation (44, 65) and can be used in http://www.jimmunol.org/ The gene array experiment demonstrated that in addition to the the management of microbial diseases by means of enhancing gene for TNF, the reconstitution potential of GM-CSF extends to macrophage functions (66). Therefore, our results indicate the po- more genes encoding transcription factors, such as NF-␬B (p65 tential therapeutic use of GM-CSF in improving the resistance of subunit), a critical regulator of many cytokine genes and one of the immunosuppressed organ transplant patients against bacterial important targets of the suppressive action of corticosteroids (46). infections. It seems likely that the partial reconstitution of NF-␬B expression by GM-CSF may contribute to the restored expression of other Acknowledgments cytokine genes as well. Factors involved in the GM-CSF restora- We thank Drs. Juerg Hamacher, Jutta Schlepper-Schaefer, and Gerald tion effect may also involve the immediate early gene Egr-1, Kuenstle for critically reading the manuscript. by guest on September 27, 2021 which, when induced by LPS, is required for maximal induction of References TNF gene expression (47). The observation that various other tran- 1. Sayegh, M. H., and L. A. Turka. 1998. The role of T-cell costimulatory activation scription factors are up-regulated by GM-CSF (Table I) points to pathways in transplant rejection. N. Engl. J. Med. 338:1813. a general mechanism by which GM-CSF restores the transcription 2. Fishman, J. A., and R. H. Rubin. 1998. Infection in organ-transplant recipients. process. N. Engl. J. Med. 338:1741. ϩ 3. Maier, S., C. Tertilt, N. Chambron, K. Gerauer, N. Huser, C. D. Heidecke, and NK cells (CD56 ) are innate immune lymphocytes critical to K. Pfeffer. 2001. Inhibition of natural killer cells results in acceptance of cardiac Ϫ Ϫ host defense against invading infectious pathogens and malignant allografts in CD28 / mice. Nat. Med. 7:557. 4. Baldwin, W. M., C. P. Larsen, and R. L. Fairchild. 2001. Innate immune re- transformation through elaboration of cytokines and cytolytic ac- sponses to transplants: a significant variable with cadaver donors. Immunity 14: tivity (48). The role of NK cells in allograft rejection is uncertain. 369. The NK cells by themselves cannot reject allografts, but they are 5. He, H., J. R. Stone, and D. L. Perkins. 2002. Analysis of robust innate immune response after transplantation in the absence of adaptive immunity. Transplan- in the graft-infiltrating cell population and may therefore contrib- tation 73:853. ute to graft damage (49). Under standard immunosuppressive ther- 6. Christopher, K., T. F. Mueller, C. Ma, Y. Liang, and D. L. Perkins. 2002. 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