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Activation-Induced Depletion of Protein Kinase C Provokes Desensitization of Monocytes/Macrophages in Sepsis¹

Andreas von Knethen^2,3,*, Anja Tautenhahn^*, Hartmut Link, Dirk Lindemann and Bernhard Brüne^2,

^* Department of Cell Biology, University Kaiserslautern, Kaiserslautern, Germany; Department of Internal Medicine I, Westpfalz-Klinikum Kaiserslautern, Kaiserslautern, Germany; Department of Virology, Technical University Dresden, Dresden, Germany; and Institute Biochemistry, Johann-Wolfgang Goethe University, Frankfurt, Germany

	Abstract

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Sepsis accounts for the majority of fatal casualties in critically ill patients, because extensive research failed to significantly improve appropriate therapy strategies. Thus, understanding molecular mechanisms initiating the septic phenotype is important. Symptoms of septic disease are often associated with monocyte/macrophage desensitization. In this study, we provide evidence that a desensitized cellular phenotype is characterized by an attenuated oxidative burst. Inhibition of the oxidative burst and depletion of protein kinase C (PKC) were correlated in septic patients. To prove that PKC down-regulation indeed attenuated the oxidative burst, we set up a cell culture model to mimic desensitized monocytes/macrophages. We show that LPS/IFN--treatment of RAW264.7 and U937 cells lowered PKC expression and went on to confirm these data in primary human monocyte-derived macrophages. To establish a role of PKC in cellular desensitization, we overexpressed PKC in RAW264.7 and U937 cells and tested for phorbolester-elicited superoxide formation following LPS/IFN--pretreatment. Inhibition of the oxidative burst, i.e., cellular desensitization, was clearly reversed in cells overexpressing PKC, pointing to PKC as the major transmitter in eliciting the oxidative burst in monocytes/macrophages. However, PKC inactivation by transfecting a catalytically inactive PKC mutant attenuated superoxide formation. We suggest that depletion of PKC in monocytes from septic patients contributes to cellular desensitization, giving rise to clinical symptoms of sepsis.

	Introduction

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During the pathogenesis of sepsis, monocytes/macrophages are important players due to their ability to produce large amounts of proinflammatory cytokines including, among others, TNF-, IL-1, and IFN- in response to pathogen contact (1, 2). High doses of these cytokines contribute to severe tissue damage, organ failure, and death (3). During the progression of infection, monocytes can switch to a desensitized phenotype characterized by the expression of anti-inflammatory cytokines and a concomitantly attenuated proinflammatory response (4), giving rise to a hampered immune reaction following secondary infections (5, 6). Therefore, the proper regulation of the balance between pro- vs anti-inflammatory cytokine production has been implicated as a key regulating system in the development of sepsis (2).

Generation of superoxide (O

Recently, we showed that treatment of monocytes/macrophages with a combination of LPS and IFN- to simulate septic conditions inhibits their ability to generate ROS in response to PMA (11). PMA, a DAG-homologue, was used as an established activator of the PKC pathway, provoking NADPH oxidase activity and concomitant ROS production (12). A pathophysiological modulation of the oxidative burst as one characteristic activation marker of monocytes/macrophages in septic patients has not been fully understood. In this regard, signaling mechanisms causing an altered ROS production are so far unknown. Therefore, it was our intention to elucidate the capability of monocytes derived from healthy donors compared with cells obtained from septic patients to generate O

In this study, we show that in monocytes derived from septic patients a correlation between an attenuated oxidative burst in response to PMA and depletion of PKC exists. In a cell culture model, we mimicked septic conditions to provide evidence that PKC depletion accounts for a diminished oxidative burst, observed in septic monocytes.

	Materials and Methods

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Materials

PMA and LPS were purchased from Sigma-Aldrich. Hydroethidine (HE), Escherichia coli, and E. coli BioParticles opsonizing reagent were from Molecular Probes. N-nitro L-arginine methyl ester (L-NAME) was from Alexis. Culture supplements, FCS, and AB-positive human serum were ordered from PAA Laboratories. Murine and human recombinant IFN- were from Roche. All other chemicals were of the highest grade of purity and commercially available.

Cell culture

The mouse monocyte/macrophage cell line RAW264.7 and the human premonocytic cell line U937 were maintained in RPMI 1640 supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% heat-inactivated FCS (complete RPMI 1640). All experiments were performed using complete RPMI 1640.

Monocyte isolation

We analyzed human cells from peripheral blood of healthy donors and of patients diagnosed with polymicrobial sepsis as defined (13). Septic patients were recruited from the intensive care unit of the Westpfalz-Klinikum Kaiserslautern. Patients were between 42 and 76 years old. The study has been approved by the ethics-committee of the board of physicians in Rhineland-Palatinate, Germany (processing number 837.141.02/3359).

For monocyte enrichment, we isolated PBMCs from donors using Ficoll-Hypaque gradients (PAA Laboratories). We washed PBMCs twice in PBS, and following T cell depletion (CD3⁺) using the MACS technology (Miltenyi Biotec), remaining cells were left to adhere on culture dishes (Primaria 3072; Becton Dickinson) for 60 min at 37°C. Nonadherent cells were removed. Afterward, cells were directly used for the experiments (HE assay or Western blotting) or differentiated to macrophages by culturing them in complete RPMI 1640, containing 10% AB-positive human serum (transient transfection experiments). Flow cytometry confirmed that the monocyte-like population was 90–95% pure (CD14⁺ vs CD14^–).

Vector construction

To overexpress PKC in RAW264.7 macrophages, a retroviral vector was constructed to obtain efficient gene transfer, stable integration of the gene, and to allow FACS-sorting for positive cells as well as detection of PKC translocation. Therefore, the plasmid pPKC-EGFP (BD Clontech, BD Biociences) was used to clone the PKC sequence linked to an enhanced GFP (EGFP) as a fluorescent marker into the retroviral pLXIN vector (BD Clontech, BD Biociences) after an EcoRI cut at positions 1471 and 1510. Consequently, primers were designed to generate a DNA fragment, containing the PKC-EGFP sequence flanked by EcoRI sites. The sequences of the primers were as follows 5' > 3 5'-CCGGAATTCATGGCTGACGTTTTCCCGGG-3', 3' > 5' 5'-GGCGAATTCCGCGGCCGCTTTACTTGTACAG-3'. After PCR giving rise to a 2794-bp fragment, this was EcoRI cut, run on an agarose-gel, eluted, and ligated into the EcoRI cut pLXIN vector. Correct orientation and sequence were verified by restriction analyses and sequencing.

Retroviral transduction

The retroviral infection of RAW264.7 macrophages and U937 cells was performed essentially as described (14, 15). Briefly, after transiently transfecting the packaging plasmids encoding the vesicular stomatitis virus glycoprotein G (16) and the MLV-gag-pol genes (17) together with the retroviral vectors pLXIN-PKC-EGFP into 293T cells, target cells were incubated for 24 h with the infectious supernatant containing 8 µg/ml polybrene. Positive selection based on EGFP-expression was done using the sort option of a FACSCalibur flow cytometer (BD Biosciences). Transduced cell populations containing 100% positive EGFP-expressing cells were used for the experiments.

Flow cytometry of oxygen radical production (HE assay)

Following a prestimulation regime, 5 x 10⁵ cells were incubated for 30 min with 1 µM PMA or 1 µg/ml opsonized E. coli. Opsonization has been performed by incubating E. coli in E. coli BioParticles opsonizing reagent for 30 min, followed by two PBS washing steps, finally diluting opsonized E. coli to 1 µg/µl in PBS. After cell stimulation, 3 µM HE was added and incubations went on for 30 min. Cells were harvested, washed with PBS, and resuspended in 200 µl of PBS. Flow cytometry analysis was performed using a FACSCalibur flow cytometer (BD Biosciences) and HE was measured through a 630-nm long pass filter (FL3). Data from 10,000 cells were collected to reach significance. All incubations were performed at 37°C.

Fluorescence microscopy

To follow PKC translocation, PKC-EGFP-positive cells were seeded in 6-cm petri dishes. After stimulation 1 µM PMA was added. Translocation of PKC-EGFP was analyzed using an Axioscope (Carl Zeiss), documented by a CCD camera (Visitron Systems) and the MetaFluor software (Visitron Systems).

Transient transfection

The plasmid, encoding for a catalytically inactive PKC protein (pEF-PKC K368R), kindly provided by G. Baier (Institute of Medical Biology and Human Genetics, University of Innsbruck, Innsbruck, Austria) (18), was transfected into primary human macrophages using jetPEI (Biomol) according to the manufacturer’s instructions. Transfection of the empty pEF-vector was used as a control. Selection of positive clones was performed by 1-wk treatment with the antibiotic G418. Surviving cells were used for the HE assay.

Western blot analysis

Cell lysis was achieved with lysis buffer (50 mM Tris, 5 mM EDTA, 150 mM NaCl, 0.5% Nonidet-40, 1 mM PMSF (pH 8.0)) and sonication (Branson sonifier; 20 s, duty cycle 100%, output control 60%). Whole cell lysates were cleared by centrifugation (10,000 x g, 5 min), and protein concentration was determined with the Lowry method. Protein (50 µg) was resolved on 10% polyacrylamide gels and blotted onto nitrocellulose sheets, basically following standard methodology. Equal loading and correct protein transfer to nitrocellulose was routinely quantitated by Ponceau S staining. Filters were incubated with the anti-PKC, -PKC, -PKC, and -PKC Abs (1:500; Transduction Laboratories, BD Biosciences) or anti-actin Ab (1:2000; Amersham Biosciences) overnight at 4°C. HRP-conjugated polyclonal Abs (1:5000; Amersham Biosciences) were used for ECL detection.

Statistical analysis

Due to small sample volumes some experiments were only performed twice. Otherwise each experiment was performed at least three times. Statistical analysis was performed using the paired t test. We considered p 0.05 as significant. Otherwise representative data are shown.

	Results

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Desensitized monocytes/macrophages derived from septic patients

The respiratory burst is an early activation marker in response to pathogen contact and constitutes a pivotal role in killing microorganisms (19). Therefore, we first established a test system to follow oxygen radical formation of primary monocytes derived from septic patients compared with healthy donors in response to PMA as described (11). In this study, we provide evidence that in monocytes of 7 of 12 preparations from septic patients the ability to generate superoxide radicals in response to PMA was abolished (Fig. 1A, left panel). Activation of all 13 control samples evoked ROS production (Fig. 1A, right panel). Statistics of flow cytometric results revealed no significant induction of superoxide radical formation in monocytes derived from septic patients compared with a significant increase in healthy donors (Fig. 1B).

View larger version (12K): [in this window] [in a new window]   FIGURE 1. Oxygen radical production in monocytes derived from septic patients. A, ROS production in response to 1 µM PMA was analyzed by flow cytometry using 3 µM HE as the redox-sensitive dye. Experiments were performed in duplicate. B, Statistical evaluation of flow cytometric data (*, p 0.05). For details see Materials and Methods.

PKC depletion in monocytes derived from septic patients

To correlate diminished ROS formation in monocytes of septic patients in response to PMA with the expression of PKC, Western blot analyses were performed. Protein expression revealed a constitutive PKC expression in monocytes from healthy individuals (n = 11), and in line with our hypothesis, ROS formation in response to PMA as shown for four samples in Fig. 2 (upper panel, ROS formation; lower panel, PKC expression). Interestingly, we observed no PKC expression in monocytes derived from septic patients where ROS formation was inhibited (n = 7). Four individual preparations are depicted in Fig. 2. These data support the assumption that changes in PKC expression might cause septic desensitization of monocytes. To elucidate whether down-regulation of PKC indeed causes an attenuated oxidative response, we setup a cell culture model to mimic septic monocytes/macrophages.

View larger version (18K): [in this window] [in a new window]   FIGURE 2. ROS production and PKC expression in monocytes of septic patients. ROS production in monocytes is shown as mean HE fluorescence (log FL3) of four individual sepsis patients and four healthy donors untreated and in response to 1 µM PMA (upper panel). The corresponding PKC expression was detected by Western blot analysis as described in Materials and Methods (lower panel). Individual experiments were performed twice.

LPS/IFN-/L-NAME treatment promoted down-regulation of PKC

We treated monocytes/macrophages for 15 h with a combination of LPS and IFN-, two established mediators of the septic disease to obtain a desensitized phenotype (21, 22). Because these agents are known activators of inducible NO synthase, at least in murine cells (23), we routinely used L-NAME to suppress NO formation and thereby exclude any possible interference of reactive nitrogen species during ROS determination. Subsequently, we performed Western blot experiments to follow expression of classical, novel, and atypical PKC isoforms to examine their contribution on monocyte/macrophage desensitization. Resting RAW264.7 macrophages and U937 monocytes express PKC, , , and (Fig. 3, A and B). Expression of the PKC, , and are down-regulated in response to increasing concentrations of PMA added for 15 h. This can be explained based on their structural organization, containing a DAG-binding site. In contrast, the expression of PKC, an atypical PKC family member, did not change after PMA addition, again consistent with the absence of a DAG-responsive site. Treatment of the cells for 15 h with a combination of LPS/IFN-/L-NAME suppressed expression of PKC only. To verify the significance of this effect for primary cells, we treated primary macrophages derived from peripheral blood monocytes, with LPS/IFN-/L-NAME for different times. PKC expression declined 1 h after LPS/IFN-/L-NAME treatment. As shown in Fig. 3C, depletion of PKC was not as complete, although clearly visible.

View larger version (23K): [in this window] [in a new window]   FIGURE 3. PKC expression in monocytes/macrophages under the influence of LPS/IFN-/L-NAME. RAW264.7 macrophages (A) and U937 cells (B) were stimulated for 15 h with the indicated concentrations of PMA, with a combination of LPS (10 µg/ml), IFN- (100 U/ml), and L-NAME (1 mM), or remained as controls. PKC, , , and expression were detected by Western blot analysis as described in Materials and Methods. Experiments were performed three times. C, Primary monocyte-derived macrophages were treated for 0.5, 1, and 4 h with a combination of LPS (10 µg/ml), IFN- (100 U/ml), and L-NAME (1 mM), or remained as controls. PKC expression was detected by Western blot analysis as described in Materials and Methods. Experiments were performed three times.

View larger version (20K): [in this window] [in a new window]   FIGURE 4. Overexpression of a PKC kinase-inactive mutant attenuated ROS production. ROS production in response to 1 µM PMA in primary monocyte-derived macrophages, transfected with a kinase inactive PKC mutant (pEF-PKC K368R) or an empty control vector (pEF-control), was analyzed by flow cytometry using 3 µM HE as the redox-sensitive dye. Experiments were performed three times.

Overexpression of PKC allowed to regain HE oxidation after PMA stimulation

After retroviral infection with the pLXIN-PKC-EGFP construct, RAW264.7 expressed endogenous PKC and an 30 kDa larger PKC form, which corresponds to its EGFP tag as determined by Western blot analysis (Fig. 5A, left panel). Similar results were obtained in U937 cells (Fig. 5A, right panel). Functionality of the PKC-EGFP protein was verified by analyzing its membrane translocation in response to PMA treatment by fluorescence microscopy (data not shown). Subsequently, cells were treated for 15 h with LPS/IFN-/L-NAME and, finally, PKC was analyzed in cell lysates. Only endogenous PKC is down-regulated, whereas the level of exogenous EGFP-linked PKC remained unchanged (Fig. 5B). Based on these Western blot results, we performed a final set of experiments to characterize the influence of PKC overexpression in monocyte/macrophage desensitization.

View larger version (31K): [in this window] [in a new window]   FIGURE 5. Overexpression of PKC attenuated LPS/IFN-/L-NAME-mediated macrophage desensitization. A, Overexpression of PKC-EGFP was detected in RAW264.7 macrophages and U937 cells by Western blot analysis as described in Materials and Methods. B, PKC-overexpressing RAW264.7 macrophages and U937 cells were stimulated for 15 h with a combination of LPS (10 µg/ml), IFN- (100 U/ml), and L-NAME (1 mM), or remained as controls. PKC expression was detected by Western blot analysis as described in Materials and Methods. C, PKC-overexpressing RAW264.7 macrophages and U937 cells were pretreated for 15 h with a combination of LPS, IFN-, and L-NAME. PKC translocation in response to 1 µM PMA was analyzed by fluorescence microscopy. For details, see Materials and Methods. D, PKC-overexpressing RAW264.7 macrophages and control RAW264.7 cells were stimulated for 15 h with a combination of LPS (10 µg/ml), IFN- (100 U/ml), and L-NAME (1 mM), or remained as controls. ROS production in response to 1 µM PMA was analyzed by flow cytometry using 3 µM HE as the redox-sensitive dye. E, Statistical evaluation of flow cytometric data for RAW264.7 macrophages (left panel) and U937 cells (right panel) (*, p 0.05). For details, see Materials and Methods.

To exclude the possibility that our data are restricted to the use of PMA we performed a final set of experiments using opsonized E. coli to provoke ROS formation in U937 cells. As shown in Fig. 6, LPS/IFN-/L-NAME pretreatment of U937 cells inhibited the E. coli-initiated oxidative burst, while overexpression of PKC-EGFP restored ROS formation. These data point to PKC as one important component in the signaling cascade leading to ROS production in response to pathogen contact. Its depletion observed in monocytes derived from septic patients might therefore be responsible for cellular desensitization.

View larger version (18K): [in this window] [in a new window]   FIGURE 6. LPS/IFN-/L-NAME-prestimulation attenuated ROS production in response to opsonized E. coli. PKC-overexpressing U937 cells and control U937 cells were stimulated for 15 h with a combination of LPS (10 µg/ml), IFN- (100 U/ml), and L-NAME (1 mM), or remained as controls. ROS production in response to 1 µg/ml opsonized E. coli was analyzed by flow cytometry using 3 µM HE as the redox-sensitive dye. Experiments were performed three times. Statistical evaluation of flow cytometric data (*, p 0.05). For details, see Materials and Methods.

	Discussion

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We provide evidence that in monocytes of 7 of 12 preparations from septic patients the ability to generate superoxide radicals in response to PMA was abolished. Because it is known that monocytes of sepsis patients often show a desensitized phenotype (6, 20), we elucidated the underlying mechanism. An attenuated respiratory burst in monocytes/macrophages has been described in response to IL-4, causing LPS-receptor (CD14) and TLR-4 down-regulation (24). These transcriptional mechanisms prevent signal transmission provoking cellular activation following LPS binding. Inhibition of superoxide radical formation in chronic granulomatous disease has also been observed as a consequence of the deletion or attenuation of expression of a component of the NADPH oxidase system, not allowing functional assembly of this multienzyme complex (25). Furthermore, inhibition of PKC, an established activator of the O

To establish a cause-effect relation between down-regulation of PKC in monocytes derived from septic patients and an attenuated oxidative response, we established a cell culture model to mimic desensitized monocytes/macrophages. Therefore, immunological activation of macrophages is achieved by the T_H1 cytokine IFN- and LPS, the outer membrane-constituent of Gram-negative bacteria (30). Using this system, we verified the role of PKC in ROS production, illustrating the LPS/IFN-/L-NAME pretreatment-dependent depletion of PKC and concomitant inhibited O

To finally verify the impact of PKC in ROS formation we overexpressed PKC to restore the desensitized cellular phenotype. To achieve high gene transfer efficiency and the possibility to sort positive cells we used a retroviral system to transduce RAW264.7 and U937 cells, giving rise to 100% positive clones for PKC-EGFP. A similar fluorescent vector system was used by Maasch et al. (31) to elucidate the role of intracellular free calcium in targeting PKC in vascular smooth muscle cells. In our experiments, PKC-EGFP overexpression was functional and, in contrast to the endogenous form of PKC, was not altered in response to LPS/IFN-/L-NAME prestimulation. This is in some analogy to the work of Gschwend et al. (32), when in the human prostate cancer cell line LNCaP, bryostatin 1 led to a time-dependent depletion of endogenous PKC. This depletion was attenuated in cells overexpressing exogenous PKC. In our system, overexpression of PKC is controlled by a Moloney murine sarcoma virus long terminal repeat, provoking a constitutive level of PKC expression, which explains high PKC-EGFP expression in response to LPS/IFN-/L-NAME prestimulation. Consequently, incorporating these cells into our experimental approach, we tested their capability to generate superoxide radicals after the LPS/IFN-/L-NAME pretreatment regime in response to PMA. As expected, the PMA-initiated oxidative burst after LPS/IFN-/L-NAME prestimulation is restored in monocytes/macrophages overexpressing PKC. We noticed similar effects in PKC-overexpressing cells in response to opsonized E. coli demonstrating the pathophysiological significance of our data.

Our results support a role of PKC in ROS production, suggesting a likely involvement of PKC expression regulation in desensitizing monocytes in sepsis. It will be challenging to define molecular mechanisms leading to PKC depletion in sepsis, thus attenuating the oxidative burst in one part of septic patients while not modulating PKC expression and causing ROS formation in the other part.

	Disclosures

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The authors have no financial conflict of interest.

	Acknowledgments

 
We thank J. Zimmerman and N. Wallner for expert technical assistance, and Drs. T. Huber and R. Wendler for providing the septic material and supporting the project.

	Footnotes

 
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 with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by a grant from the Deutsche Forschungsgemeinschaft (Br 999).

² A.v.K. and B.B. shared senior authorship.

³ Address correspondence and reprint requests to Dr. Andreas von Knethen, Department of Cell Biology, University of Kaiserslautern, Erwin-Schroedinger-Strausse 13/420A, 67663 Kaiserslautern, Germany. E-mail address: aknethen{at}rhrk.uni-kl.de

⁴ Abbreviations used in this paper: ROS, reactive oxygen species; PKC, protein kinase C; DAG, diacylglycerol; HE, hydroethidine; L-NAME, N-nitro L-arginine methyl ester; EGFP, enhanced GFP.

Received for publication March 22, 2004. Accepted for publication February 1, 2005.

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Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

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				A. von Knethen, M. Soller, N. Tzieply, A. Weigert, A. M. Johann, C. Jennewein, R. Kohl, and B. Brune PPAR{gamma}1 attenuates cytosol to membrane translocation of PKC{alpha} to desensitize monocytes/macrophages J. Cell Biol., February 26, 2007; 176(5): 681 - 694. [Abstract] [Full Text] [PDF]

				A. Weigert, A. M. Johann, A. von Knethen, H. Schmidt, G. Geisslinger, and B. Brune Apoptotic cells promote macrophage survival by releasing the antiapoptotic mediator sphingosine-1-phosphate Blood, September 1, 2006; 108(5): 1635 - 1642. [Abstract] [Full Text] [PDF]

				R. Kohl, S. Preiss, A. von Knethen, and B. Brune Oxidized low-density lipoprotein depletes PKC{alpha} and attenuates reactive oxygen species formation in monocytes/macrophages Cardiovasc Res, August 1, 2006; 71(3): 574 - 585. [Abstract] [Full Text] [PDF]

				V. Fronhofer, M. R. Lennartz, and D. J. Loegering Role of PKC isoforms in the Fc{gamma}R-mediated inhibition of LPS-stimulated IL-12 secretion by macrophages J. Leukoc. Biol., February 1, 2006; 79(2): 408 - 415. [Abstract] [Full Text] [PDF]

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