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The Journal of Immunology, 2003, 171: 938-947.
Copyright © 2003 by The American Association of Immunologists

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

Jian Xu*, Rudolf Lucas*, Marcus Schuchmann{dagger}, Simone Kühnle*, Thomas Meergans*, Ana P. Barreiros{dagger}, Ansgar W. Lohse{dagger}, Gerd Otto{ddagger} and Albrecht Wendel2,*

* Biochemical Pharmacology, University of Konstanz, Konstanz, Germany; and Departments of {dagger} Medicine and {ddagger} Transplantation and Hepatobiliary Surgery, University of Mainz, Mainz, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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{beta}R antagonist, rather than of IL-1{beta} itself, since exogenously added IL-1{beta} induced an IL-2-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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The development of new immunosuppressive drugs with improved efficacy and decreased toxicity has led to a substantial improvement in the survival of organ transplant patients and in short-term graft survival for all organs (1). However, under these conditions the inflammatory response to infections is severely impaired, and this represents the most common life-threatening complication of long term immunosuppressive therapy (2). The prevention or effective treatment of infection is therefore still a primary goal in organ transplantation. Indeed, a broad range of potential sources of infection as well as the adverse effects of antimicrobial drugs used for prophylaxis and therapy represent important challenges to overcome (2). A concerted interplay between innate and adaptive immune surveillance for graft rejection has been indicated (3). A robust innate immune response, characterized by macrophage infiltration and up-regulation of multiple cytokines, chemokines, and chemokine receptors, has been demonstrated within the first day after transplantation in an alymphoid murine model. This innate immune response to the acute injury associated with the transplant procedure, however, was not shown to elicit allograft rejection (4, 5, 6). Moreover, studies using T cell-deficient mice have shown that prompt rejection can occur only after T cell reconstitution, even when skin or allografts have been allowed to recover or heal for >100 days (7). Rather, concomitant stimulation of the innate immune system leads to increased expression of factors, such as cytokines, that may influence the adaptive immune system to promote the injury of the transplant organ (4). Importantly, evidence from animal experiments (8) and clinical studies (9) implied that restoration of innate immunity without restoration or with absence of the adaptive immunity might still be beneficial for resistance to infection. It is therefore desirable, in immunosuppressed organ transplant recipients, to create a status with a preferential reconstitution of the innate immune responses, which will contribute to the recognition and control of the infectious agents (10), while keeping the adaptive immune responses silent. Such a status requires a preferential reactivation of the effectors of the innate immune responses, i.e., macrophages and/or neutrophils, by pharmacological intervention without restoring the suppressed adaptive immune responses, such as the T cell response, which is implicated in graft rejection (11, 12, 13). Recombinant GM-CSF, a drug approved for hematological indications in humans (14), has been indicated in vitro and in vivo to enhance the synthesis and release of proinflammatory cytokines such as TNF, which is crucial in host defense in various animal infection models (15, 16, 17). In our laboratory it was previously shown that GM-CSF potentiates the immune responses to endotoxin (18) and restores the impaired immune responses in LPS-desensitized mice (19) as well as in refractory human monocytes (20). Others found that anergic monocytes from sepsis patients were reactivated (21, 22), and hyporesponsiveness of whole blood, induced by trauma, sepsis, or cardiac surgery, could be overridden in vitro (23) by GM-CSF treatment. Therefore, we investigated whether GM-CSF was able to reconstitute TNF production without activating the adaptive immunity of the T cell response, firstly in dexamethasone-suppressed blood from healthy donors, to normalize all conditions required for the study, and secondly in the blood from immunosuppressed liver transplant recipients, to confirm and emphasize the potential clinical implications in an ex vivo setting. On a molecular basis we used gene array experiments to investigate a potential selective reconstitution capacity of GM-CSF under immunosuppression of genes, in addition to TNF, related to innate immunity. To assess the in vivo relevance of our findings, we investigated whether GM-CSF restored the survival of immunosuppressed mice from an otherwise lethal Salmonella typhimurium infection and assessed its influence on graft rejection of a skin allotransplant in the infected mice. Our findings support the potential clinical value of GM-CSF for the therapeutic improvement of resistance to infections in immunosuppressed organ transplant patients.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Human blood sample

Blood from 10 healthy donors was used to select the optimal working concentration of immunosuppressant (dexamethasone) in vitro. We also included blood from 10 patients (age, 39–69 years; average, 58.6 years), who all underwent orthotopic liver transplantation at University Hospital (Mainz, Germany). Decompensation of liver function was due to chronic liver diseases, such as primary biliary cirrhosis, chronic viral hepatitis B or 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 patients at the lowest level of immunosuppression by tacrolimus (Cmin) within 1 mo after transplantation. All patients gave written informed consent to transplantation and follow-up examinations.

Preparation of human PBMC

PBMC were prepared in cell preparation tubes (Vacutainer CPT; BD Biosciences, Franklin Lakes, NJ) according to the manufacturer’s instructions. Cell numbers were adjusted to 5 x 106 cells/ml in RPMI 1640 (Invitrogen, Karlsruhe, Germany) before further incubation. Lymphocytes were prepared as the nonadherent fraction from PBMCs upon growth adherence for 2 h (nonadherent fraction of PBMC).

Whole blood or PBMC/lymphocyte incubations

Heparinized whole blood or PBMC in complete RPMI 1640, which is supplemented with 2.5 IU/ml heparin (Liquemin; Hoffmann-La Roche, Grenzach-Whylen, Germany) and 100 IU/ml penicillin/streptomycin (Biochrom, Germany), were added to a 96-well plate and incubated with or without dexamethasone (1 µM; Dexa-Allvoran; TAD Pharmaceuticals, 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 (LPS model) or 72 h (Con A model) at 37°C in 5% CO2 after addition of the stimuli (100 ng/ml of LPS from Salmonella abortus equi or 5 µg/ml endotoxin-free Con A; Sigma-Aldrich, Deisenhofen, Germany). After incubation, the cells and cell-free supernatants (by centrifugation at 300 x g, 10 min) were then subjected to different measurements.

ELISA

Cytokines in cell-free supernatants were quantified by ELISA using Ab pairs for TNF, IL-2, IL-1{beta}, IL-1R antagonist (IL-1ra),3 and IFN-{gamma} (Endogen, Munich, Germany) as well as human rTNF (Bender, Vienna, Austria), IL-1{beta}, IL-1ra, IL-2, and IFN-{gamma} (Endogen) standards as previously described (24).

Cell viability and proliferation assay

Cells were washed three times with Dulbecco’s PBS, stained with 1 µM Calcein AM (Molecular Probes, Leiden, The Netherlands), and kept for 1 h at 37°C. The fluorescence measurement followed the manufacturer’s manual. Viability and proliferation were presented as a percentage of the positive control live cells and of the known nonproliferated control live cells, respectively.

Two-way MLR assay

A two-way MLR assay was performed according to a procedure similar to that previously described on human PBMC (25), using the Cell Proliferation ELISA kit (Roche Applied Science, Mannheim, Germany). Briefly, 50-µl aliquots of cells (2.5 x 106/ml) from each of two allogeneic donors were added to wells of a 96-well, flat-bottom plate in the absence or the presence of dexamethasone and/or GM-CSF. The cell cultures were incubated at 37°C for 5 days in RPMI 1640 supplemented with 2.5 IU/ml heparin and 100 IU/ml penicillin/streptomycin. Bromodeoxyuridine (BrdU) was added 24 h before fixation of the cells. After 5 days of incubation, the culture medium was removed, the cells were fixed, and the DNA was denatured for 0.5 h. Additional nonradioactive detection steps, using an anti-BrdU Ab-peroxidase complex, were performed according to the instructions of the manufacturer.

Human TNF bioassay

The bioactivity of TNF, assessed as cytotoxicity in WEHI 164 subclone 13 fibrosarcoma cells, was evaluated using the ethidium homodimer-1 incorporation assay (Molecular Probes), as described previously (26).

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 {alpha} subunit (HLA C-4), {beta}-actin, 60S ribosomal protein L13A, and 40S ribosomal protein S9.

Western blotting

Cell extracts (20 µg 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. Immunoblots were detected by a HRP-conjugated secondary Ab and ECL (Amersham Pharmacia Biotech, Piscataway, NJ).

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 x 105 bacteria/kg i.p.). The recombinant murine GM-CSF (provided by Dr. F. R. Seiler, Behring-Werke, Marburg, Germany; 50 µg/kg i.v.) was 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 µl 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

CBA/Ca mice (four per group) in transplantation experiments were immunosuppressed with a daily injection of 30 mg/kg CsA i.p. After anesthetizing and shaving the recipient, a patch of skin, 1 x 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 8–12 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 (5 x 105/kg i.p.) in transplanted CBA/Ca mice was induced on day 7, and survival was monitored over a period of up to 4 wk. Grafts and surrounding native skin of some transplanted mice were photographed regularly, and samples were excised for histological examination. To determine the course of infection, some mice were sacrificed at different times for the CFU measurement in liver, spleen, blood, and peritoneum.

Determination of the working concentrations

The optimal working concentrations of GM-CSF (50 ng/ml) and LPS (100 ng/ml) for the priming or stimulation of TNF production were based on what was previously reported (20). Since dexamethasone has been reported to cause cell death or apoptosis of monocytes (27) or lymphocytes (28), we first examined the viability of the dexamethasone-treated PBMC. We found that in the range from 0.1 to 10 µM dexamethasone, cells maintained their viability within 16 h. At 72 h after treatment a viability loss occurred at dexamethasone concentrations >1 µM. Since 1 µM dexamethasone was sufficient for almost complete inhibition of LPS-induced TNF production without inducing significant cytotoxicity, this concentration was chosen for all additional experiments.

Statistical analysis

Data were expressed as the mean ± SEM, and datasets were subjected to one-way ANOVA, followed by Bonferroni’s multiple comparison tests (PRISM; GraphPad, San Diego, CA). A value of p < 0.05 (indicated as an asterisk in figures) was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
GM-CSF restores the release of bioactive TNF, but not IL-1{beta}, in glucocorticoid-immunosuppressed blood

To investigate the potential of GM-CSF, under immunosuppression, to restore TNF production upon LPS stimulation, we first used an experimental model in which whole blood from 10 healthy donors was exposed to dexamethasone and then stimulated with LPS. The results show that in dexamethasone-suppressed whole blood, the LPS-induced TNF release is significantly reduced compared with the LPS only setting, but is significantly restored in the presence of GM-CSF, although not to the level of GM-CSF-primed naive cells from healthy donors (Fig. 1a). In contrast, under immunosuppression, IL-1{beta} release is not enhanced by GM-CSF, although this is the case in the nonsuppressed setting (Fig. 1b). These observations also hold true in clinical samples ex vivo, since exogenous GM-CSF restores LPS-induced TNF (Fig. 1c), but not IL-1{beta} (Fig. 1d) release in blood taken from 10 immunosuppressed liver transplant patients.



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  		FIGURE 1. Reconstitution of TNF, but not IL-1{beta}, production by GM-CSF in glucocorticoid-immunosuppressed blood. Heparinized 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 µM) 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-{alpha} (a and c) and IL-1{beta} (b and d), expressed per milliliter of whole blood. Data represent the mean ± SEM of the results.

 
Since the bioactivity of TNF can be neutralized by its soluble receptors sTNF-R1 and sTNF-R2, the latter of which is cleaved by the same convertase (TNF-{alpha}-converting enzyme) that mediates the shedding of TNF from its membrane-bound pro form (29), we also performed a TNF cytotoxicity assay and found that the TNF restored by GM-CSF in the blood from immunosuppressed liver transplant patients is fully bioactive (bioassay: restoration from 530 ± 50 to 6870 ± 1440 pg/ml; comparable to ELISA: restoration from 640 ± 170 to 7080 ± 2350 pg/ml; data not shown).

GM-CSF differentially up-regulates LPS-induced gene expression in dexamethasone-suppressed human PBMC

To investigate to which genes in humans the differential reconstitution potential of GM-CSF extends, we have analyzed the gene expression profile altered by GM-CSF under immunosuppression. As is apparent in the selected grids from the cDNA expression array (Fig. 2), TNF mRNA expression is suppressed by dexamethasone, but can be significantly up-regulated by GM-CSF, almost reaching the level of the LPS-treated setting. A variety of other genes, produced mainly by monocytes/macrophages in PBMC, such as IL-8, IL-6, and monocyte-specific platelet-activating factor receptor, are also restored by GM-CSF to at least 90% of the LPS-inducible signal, similar to the gene for TNF, except for IL-1{beta} (Table I). In contrast, the expression of genes implicated in the LPS-inducible lymphocyte responses, such as CD27 and T cell-specific RANTES, are not up-regulated by GM-CSF in dexamethasone-suppressed PBMC (Table I). Taken together, these results indicate a preferential restoring potential of GM-CSF on dexamethasone-suppressed genes related to the activation of monocytes/macrophages, the major players in innate immunity.



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  		FIGURE 2. GM-CSF susceptible reconstitution of gene expression in dexamethasone-suppressed human PBMC. PBMC (5 x 106 cells/ml) prepared from healthy donors were incubated sequentially with dexamethasone (1 µM) for 1 h, GM-CSF (50 ng/ml) for 1 h, and LPS (100 ng/ml) for 1 h. Equal numbers of PBMC obtained from different 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. Presented are the selected grids from the original arrays. The six detected housekeeping genes included ubiquitin, GAPDH, HLA C-4{alpha}, {beta}-actin, L13A, and S9 genes. The arrows indicate the location of the corresponding genes that were restored, except for IL-1{beta}, by GM-CSF. Independent experiments with cDNA expression array were performed twice, and an up-/down-regulation of 2-fold or more was considered significant.

 

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

 
GM-CSF does not restore T cell response and T cell proliferation

Since the rationale of our pharmacological approach is to preferentially restore the innate immune defense without reactivating the adaptive immune system, we have examined whether GM-CSF activates T cell responses (typically IL-2 release and T cell proliferation) in glucocorticoid-immunosuppressed blood. GM-CSF does not induce IL-2 release in dexamethasone-suppressed, Con A-stimulated blood from healthy donors and in blood from liver transplant patients, but does so in the controls (Fig. 3, a and b). Consistently, under these conditions GM-CSF does not restore the Con A-stimulated proliferation of immunosuppressed PBMC (Fig. 3, c and d). To further confirm the lack of activation by GM-CSF of lymphocytes under immunosuppression, a two-way MLR assay was performed in which the proliferation of mixed PBMC from two allogeneic donors was assessed by measuring BrdU incorporation into the proliferating cells. In parallel, IFN-{gamma} release in the supernatant from the 5-day cultured PBMC was measured. The results (Fig. 4) clearly show that in the absence of dexamethasone, GM-CSF enhances T cell proliferation in a two-way MLR of allogeneic PBMC, but fails to restore such an effect when PBMC are treated with dexamethasone. Moreover, the release of IFN-{gamma}, a potent proinflammatory cytokine produced by activated T lymphocytes, can be suppressed by glucocorticoids (30), but GM-CSF cannot restore the activation of T cells to release IFN-{gamma} (Fig. 4). These results are in line with those obtained from the Con A model.



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  		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 µM) for 1 h, GM-CSF (50 ng/ml) for 1 h, and Con A (5 µg/ml) for 72 h. IL-2 release was measured by ELISA (a and b). The proliferation of PBMC was assessed by measuring Calcein-AM fluorescence (c and d) and is presented as a percentage of the nonproliferated control cells. Data represent the mean ± SEM of the healthy donor group (n = 10; a and c) and the transplant recipient group (n = 10; b and d).

 


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  		FIGURE 4. The inability of GM-CSF to activate the two-way MLR and IFN-{gamma} generation in dexamethasone-suppressed human PBMC. PBMC prepared from two healthy allogeneic donors were adjusted to 2.5 x 106 cells/ml in complete RPMI 1640. The allogeneic PBMC were incubated sequentially with dexamethasone (1 µM) 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. Twenty-four hours before fixation of the cells, BrdU was added to label the cells. At 5 days of incubation, IFN-{gamma} 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 (A450 - A690). Results are representative of three different experiments.

 
IL-1{beta} restores Con A-induced proliferation of lymphocytes in immunosuppressed PBMC independently of IL-2, involving regulation of p27kip1 and Cdk2

As indicated above, exogenous GM-CSF cannot induce Con A-stimulated proliferation of lymphocytes in immunosuppressed PBMC. Moreover, in the same setting, GM-CSF did not up-regulate IL-1{beta}, but, rather, increased the expression of IL-1ra (Table II), which is known to neutralize IL-1{beta} bioactivity. Therefore, we next investigated whether IL-1{beta} can restore Con A-stimulated proliferation during immunosuppression. The results indicate that exogenous IL-1{beta} indeed partially restores Con A-stimulated proliferation of lymphocytes both in vitro (Fig. 5a) and ex vivo (Fig. 5b), probably by means of an IL-2-independent mechanism, since this IL-1{beta} effect cannot be blocked by a neutralizing anti-human IL-2 mAb (Fig. 5b). Since p27kip1, which inhibits the activation of Cdk2, plays an important role in IL-2-induced T cell proliferation (31, 32, 33), we wondered whether these proteins are implicated in the IL-1{beta}-restored, IL-2-independent proliferation of the immunosuppressed PBMC. We observed a down-regulation of p27kip1 and an up-regulation of Cdk2 protein expression, correlating with proliferation, in Con A-stimulated healthy control and IL-1{beta}-treated immunosuppressed Con A-stimulated settings both in vitro and ex vivo, suggesting the involvement of these cell cycle proteins (Fig. 5c).


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

 


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  		FIGURE 5. Induction by IL-1{beta} 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 µM) for 1 h, GM-CSF (50 ng/ml) for 1 h, and Con A (5 µg/ml) for 72 h at 37°C in 5% CO2. Proliferation of PBMC, assessed by measuring Calcein-AM fluorescence, is presented as percentage of the nonproliferated control cells. Data represent the mean ± SEM of the healthy donor group (n = 10; a) and the transplant patient group (n = 10; b). The neutralizing anti-human IL-2 mAb (R&D Systems) was applied at a final concentration of 0.5 µg/ml. c, Western blot analysis of p27kip1 and Cdk2 protein in lymphocytes from healthy PBMC untreated (control) or treated with dexamethasone (+dex) and from liver transplant recipients (ex vivo) at the indicated times after Con A stimulation. Results are representative of three different experiments.

 
GM-CSF restores the survival of immunosuppressed mice against Salmonella infection

Since immunosuppressive treatment with corticosteroids can abrogate the resistance of CBA/Ca mice to S. typhimurium infection, thus causing the death of the animals (34), we used this model to test whether the presence of GM-CSF indeed protected the immunosuppressed host against infection. In the control group, Salmonella-resistant CBA/Ca mice survived a period of 3 wk after infection without symptoms of disease and without relevant numbers of blood-borne bacteria 4 days after infection (data not shown). In the dexamethasone-immunosuppressed group, significant numbers of bacteria were measurable in blood (310 ± 30 CFU/100 µl of whole blood), and all animals in this group died within 2 wk (with two mice dying within 1 wk). In contrast, when immunosuppressed mice were pretreated with GM-CSF, low or no bacterial counts were detectable on day 4 after infection (20 ± 5 CFU/100 µl of whole blood). After a transient phase of infectious disease, these mice recovered, and all survived the time interval chosen, except for one that died 2 wk later (Fig. 6a). Like others, we were unable to detect TNF in the serum of Salmonella-infected CBA/Ca mice (35, 36), although this cytokine is clearly involved in innate resistance to S. typhimurium in this model (35, 36). Since CsA is a widely used immunosuppressive drug that also suppresses T lymphocyte-dependent immune responses (37), which are implicated in graft rejection (11, 12, 13), we further tested the infection model under CsA immunosuppression. Consistent with the dexamethasone suppression model, in the CsA-immunosuppressed group all Salmonella-infected mice died within 1 wk. However, when the immunosuppressed mice were pretreated with GM-CSF, all survived the infection (Fig. 6b). It should be noted that GM-CSF is able to restore LPS-induced TNF release ex vivo in the peritoneal macrophages from CsA-immunosuppressed mice (restored to 333.2 ± 21.1 pg/ml, compared with the LPS alone setting of 361.2 ± 4.2 pg/ml; data not shown). We thus conclude that the restoration of the innate immune responses in these murine immunosuppression models upon GM-CSF treatment is sufficient to overcome an otherwise lethal bacterial infection.



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  		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 x 105 bacteria/kg i.p.). Where indicated, recombinant murine GM-CSF (50 µg/kg i.v.) 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 analyzed using the log-rank test. ***, p < 0.003 (p < 0.05 was considered significant).

 
GM-CSF does not promote skin graft rejection, but enables the survival of infected immunosuppressed and transplanted mice

We have shown that GM-CSF can boost immunity against infection in immunosuppressed animals. However, it was not known whether intervention with GM-CSF might have negative consequences on the outcome of transplant acceptance. To test this, we used a combined transplant/infection model in which we transplanted mice after 7 days of daily immunosuppression with CsA (30 mg/kg i.p.) and then infected these mice on day 7 with S. typhimurium. Under continued immunosuppression, mice received 50 µg/kg GM-CSF daily from day 7 until day 10. The survival of the animals and the acceptance of the graft were assessed daily over a period of 4 wk. The immunosuppressed and transplanted animals pretreated with GM-CSF all survived the observation period of 28 days (data not shown). The study drug efficiently reduced the bacterial load from 300 ± 40 CFU/100 µl of whole blood in CsA-treated infected mice to 10 ± 5 CFU/100 µl in CsA/GM-CSF-treated mice. Furthermore, all surviving animals pretreated with GM-CSF accepted the skin grafts without any signs of rejection within 4 wk (Fig. 7b). All skin grafts were successfully integrated within the naive tissue (Fig. 7d) and showed no overt morphological changes compared with control skin (Fig. 7c). Whereas immunosuppressed mice in the infection control group (S. typhimurium/CsA) all died within 7 days of infection without graft rejection, mice in the transplantation control group, i.e., without immunosuppression, lost all grafts within 7 days of surgery (Fig. 7a), but survived bacterial infection due to this strain’s inherent Salmonella resistance. These experiments demonstrate that GM-CSF treatment allows successful handling of the infection by reactivation of the experimentally suppressed immune system and at the same time does not negatively interfere with skin transplant acceptance.



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  		FIGURE 7. Retention of skin transplants in GM-CSF-treated, Salmonella-infected immunosuppressed mice. CBA/Ca mice (n = 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 x 105 S. typhimurium. Under continued immunosuppression, mice received 50 µg/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 µm) 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.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Based on our previous finding that GM-CSF potentiates or reactivates the impaired immune responses in immunocompromised mice or human cells (18, 19, 20, 23), the study reported here translates this property of GM-CSF in a clinically relevant ex vivo setting, using glucocorticoid-immunosuppressed blood from liver transplant patients and an in vivo murine immunosuppressed infection model. In these in vitro and ex vivo approaches, we have observed a recovery of the proinflammatory TNF response, without reactivation of the T cell response, in terms of IL-2 production and proliferation. As a mechanistic rationale, the restoration of TNF production by GM-CSF may result from the restored mRNA expression of the cytokine.

Although we presented no direct evidence in humans that GM-CSF treatment will protect transplant patients from bacterial infection under immunosuppression, we have shown in vivo both in an infection and an infection/transplantation mouse model that GM-CSF restores innate immunity against infections to strengthen this hypothesis. In addition, TNF is known to be crucial in the host defense against infections (15, 16, 17). Indeed, the neutralization of TNF bioactivity, as recently approved for the treatment of Crohn’s disease or rheumatoid arthritis, was associated with the development of active tuberculosis in some patients (38). Moreover, at the protein level, excessive inhibition of TNF production due to routine immunosuppression was suggested to lead to an insufficient response to infectious stimuli in kidney (39) as well as liver (40) transplant recipients. Bacterial infection occurs in up to 68% of liver transplant recipients, commonly with infection of the liver, biliary tract, peritoneal cavity, bloodstream, and surgical wound. Most such infections occur in the first 2 mo following transplantation. The results from additional experiments using later stage post-transplantation blood (but within 12 mo) demonstrated a similar reconstitution of TNF release by GM-CSF to early stage post-transplantation blood (data not shown). Therefore, the GM-CSF-restored TNF response could be of benefit for the liver transplant recipient in overcoming bacterial infection in both early and late stage post-transplantation. In addition to bacterial infection, viral infection remains the major complication in the later stage of post-transplantation (2, 41). There are reports demonstrating that TNF protects against virus infection in vitro and in vivo by means of improving viral clearance or inhibiting viral replication (42), including CMV, the fatal agent for immunocompromised individuals, such as organ transplant patients (2, 41). It was also found that TNF may inhibit early transgenic expression by CMV promoters in vivo, a mechanism that is independent of adaptive immunity and is probably secondary to innate immune responses to virus infection (43).

The possibility that GM-CSF treatment might affect organ rejection is unlikely based on our results in the mouse transplant model, in which the CsA-suppressed mice survived an otherwise lethal bacterial infection when pretreated with GM-CSF, without inducing graft rejection after skin allo-transplantation. Our experimental findings are in agreement with the observations of a pilot study in children treated with GM-CSF after orthotopic liver transplantation, where the study drug was well tolerated, and no rejection episode was induced. Rather, GM-CSF was shown to be beneficial in patients with severe bacterial infection (44).

Concerning the possible role of TNF in graft rejection in organ transplant patients, only a few reports are published. In fact, in neither renal nor liver transplant patients was an association between TNF producer genotype and rejection found at the mRNA level (45). In addition, GM-CSF, the TNF-reconstituting agent in our model, has been safely applied following liver transplantation in the treatment of neutropenia (44).

The gene array experiment demonstrated that in addition to the gene for TNF, the reconstitution potential of GM-CSF extends to more genes encoding transcription factors, such as NF-{kappa}B (p65 subunit), a critical regulator of many cytokine genes and one of the important targets of the suppressive action of corticosteroids (46). It seems likely that the partial reconstitution of NF-{kappa}B expression by GM-CSF may contribute to the restored expression of other cytokine genes as well. Factors involved in the GM-CSF restoration effect may also involve the immediate early gene Egr-1, which, when induced by LPS, is required for maximal induction of TNF gene expression (47). The observation that various other transcription factors are up-regulated by GM-CSF (Table I) points to a general mechanism by which GM-CSF restores the transcription process.

NK cells (CD56+) are innate immune lymphocytes critical to host defense against invading infectious pathogens and malignant transformation through elaboration of cytokines and cytolytic activity (48). The role of NK cells in allograft rejection is uncertain. The NK cells by themselves cannot reject allografts, but they are in the graft-infiltrating cell population and may therefore contribute to graft damage (49). Under standard immunosuppressive therapy, an increase in NK cell activity (50) and NK cell traffic into the liver graft has been observed immediately after liver transplantation (51). Despite the continued administration of conventional immunosuppressive agents, NK cell function has been found to recover in renal transplantation (52). However, NK allo-specific constellations did not seem to play a major role in acute hepatic allograft rejection (53); rather, they were shown to play a role in the recovery from CMV infection in a substantial number of renal transplant patients (54). Although GM-CSF significantly suppressed the generation of NK cells from bone marrow in vitro (55) and in humans (56), it has been found to support the growth of NK cell progenitors from CD38+CD34++lineage- cells (57). Consistent with this, the combined low dose IL-2/GM-CSF therapy results in a marked expansion of human cellular subsets, including NK cells, which play a critical role in the prevention of EBV-associated lymphoproliferative disease in vivo (58). Functionally, GM-CSF has been shown to enhance IL-2-activated NK cell lysis of clonogenic acute myeloid leukemia cells by up-regulating target cell expression of ICAM-1 (59). Coexpression of GM-CSF and the costimulatory molecule B70 has been reported to enhance NK-mediated cytotoxicity and induce the antitumor immunity in hepatoma transplanted into nude mice (60). Based on the above findings, we believe that an activation (if any) of NK cells by GM-CSF under immunosuppression may be beneficial to the host defense against infections rather than harmful to the graft.

In agreement with other results (61, 62), we found that GM-CSF enhanced IL-1ra release not only after an LPS but also after a Con A stimulus in immunosuppressed human blood (Table II). It is striking that exogenous IL-1{beta} restored the Con A-induced T cell proliferation in glucocorticoid-immunosuppressed blood independently of IL-2. Since GM-CSF did not restore the release of IL-1{beta}, but, rather, led to its neutralization, these results might explain why GM-CSF did not restore the proliferation of immunosuppressed PBMC in view of the observed effect of IL-1{beta} on T cell proliferation. In agreement with the clinical experience that steroid therapy of graft rejection fails when IL-1ra production is defective (63), the inability of GM-CSF to restore the release of IL-1{beta} combined with the enhancement of the release of IL-1ra is a favorable property for a drug supposed not to interfere with graft acceptance. Further supportive arguments for such a use come from in vivo studies (64) as well as from clinical studies showing that GM-CSF increases the respiratory burst of neutrophils and/or increases neutrophil count after liver transplantation (44, 65) and can be used in the management of microbial diseases by means of enhancing macrophage functions (66). Therefore, our results indicate the potential therapeutic use of GM-CSF in improving the resistance of immunosuppressed organ transplant patients against bacterial infections.


   Acknowledgments
 
We thank Drs. Juerg Hamacher, Jutta Schlepper-Schaefer, and Gerald Kuenstle for critically reading the manuscript.


   Footnotes
 
1 This work was supported by the Deutsche Forschungsgemeinschaft (Research Group "Endogenous Tissue Injury: Mechanisms of Autodestruction", Grant We 686/18). Back

2 Address correspondence and reprint requests to Dr. Albrecht Wendel, Biochemical Pharmacology, University of Konstanz, 78457 Konstanz, Germany. E-mail address: albrecht.wendel{at}uni-konstanz.de Back

3 Abbreviations used in this paper: IL-1ra, IL-1R antagonist; BrdU, bromodeoxyuridine; Cdk, cyclin-dependent kinase; CsA, cyclosporine A. Back

Received for publication December 9, 2002. Accepted for publication May 6, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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