Interleukin-10 enhances the CD14-dependent phagocytosis of bacteria and apoptotic cells by human monocytes

Human Immunology (2007) 68, 730 –738

Interleukin-10 enhances the CD14-dependent phagocytosis of bacteria and apoptotic cells by human monocytes Marcel Lingnaua, Conny Höflicha*, Hans-Dieter Volka, Robert Sabata,b, and Wolf-Dietrich Döckea a

Institute of Medical Immunology, University Hospital Charité, Berlin, Germany Interdisciplinary Group of Molecular Immunopathology, Dermatology/Medical Immunology, University Hospital Charité, Berlin, Germany

b

Received 20 February 2007; received in revised form 19 June 2007; accepted 19 June 2007

KEYWORDS Human; Monocytes; Phagocytosis; Cytokines; FACS

Summary Monocytes are centrally involved in both specific and nonspecific immunity by secretion of regulatory immune mediators, phagocytosis, and presentation of antigens. Recent work has shown that monocytes can phagocytose bacteria independently from Fc␥, complement, and scavenger receptors via a CD14-mediated process. Furthermore, incorporation of cells undergoing apoptosis is also mediated by CD14. In this study we investigated the regulation of monocytic CD14dependent phagocytosis by the immunoregulatory cytokines interleukin-10 (IL-10), interferon-␥ (IFN-␥) and transforming growth factor-␤1 (TGF-␤1). In this study an in vitro human whole-blood assay was used to test regulation of CD14-dependent phagocytosis of fluorescence-labeled E. coli by IL-10, IFN-␥, and TGF-␤1 in monocytes from healthy donors. Phagocytosis by monocytes from a patient with paroxysmal nocturnal hemoglobinuria (PNH) and its regulation by IL-10 was also investigated. Finally, regulation of monocytic incorporation of apoptotic Jurkat cells by IL-10 was analyzed. For the CD14 blockade, murine anti-CD14 IgG2a antibody RMO52 was used. We observed that IL-10, suggested to be a monocyte-deactivating cytokine, strongly increased the monocytic CD14dependent phagocytosis of E. coli. In contrast, IFN-␥ and TGF-␤1 depressed monocytic CD14 incorporation of E. coli. Compatible with this, IL-10 upregulated CD14 expression on monocytes, whereas IFN-␥ and TGF-␤1 downregulated its expression. IL-10 also increased the monocytic CD14-dependent and -independent phagocytosis of apoptotic cells. As expected, IL-10 strongly increased the CD14independent phagocytosis but had no influence on the CD14-dependent phagocytosis of monocytes from a PNH patient. In conclusion, our data support a general role of IL-10 for activating monocytic scavenger functions, which are at least partly mediated by CD14. This is in line with the fact that IL-10 promotes the development of monocytes to macrophages. The contrasting effects of IL-10 and IFN-␥ on monocytic CD14-dependent phagocytosis may reflect a further mechanism counterbalancing antigen-presentation and nonimmunogenic scavenging of bacterial and cellular debris. TGF-␤, however, may be an inhibitor of both systems. © 2007 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

* Corresponding author. Fax: ⫹49 30 450 524932. E-mail address: conny.hoefl[email protected] (C. Höflich).

0198-8859/$ -see front matter © 2007 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.humimm.2007.06.004

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Interleukin-10 enhances CD14-dependent phagocytosis

ABBREVIATIONS E. coli IFN IL LPS MFI PNH TGF TLR

Escherichia coli interferon interleukin lipopolysaccharide mean fluorescence intensity paroxysmal nocturnal hemoglobinuria transforming growth factor Toll-like receptor

Introduction Phagocytosis is a receptor-mediated process performed by scavenger cells to internalize particles such as bacteria and autologous apoptotic cells [1]. In the phagocytic process different receptors are involved: Fc␥ receptors and complement receptors in opsonin-dependent phagocytosis; and various cell surface receptors such as mannose receptors, type A scavenger receptors, and integrins in opsonin-independent phagocytosis [1,2]. Monocytes and macrophages are very potent phagocytic cells. Like other immune functions their phagocytic capacity underlies a stringent regulation by cytokines. Surprisingly, the anti-inflammatory and immunosuppressive cytokine IL-10 leads to the enhancement of Fc␥ and complement receptor mediated phagocytosis [3–5]. In contrast, the “monocyte/ macrophage-activating” lymphokine IFN-␥ depresses phagocytosis of opsonized as well as nonopsonized micropathogens [4,6,7]. For TGF-␤, which also depresses the antigenpresenting and inflammatory activity of mononuclear phagocytes, no significant effects as well as an inhibition of phagocytosis of Mycobacterium tuberculosis have been reported [8,9]. These findings go in parallel with the fact that IL-10 supports the development of monocytes to macrophages, which are the most potent scavenger cells, whereas IFN-␥ and TGF-␤ can promote the differentiation of monocytes and the maturation of these cells [10 –12]. Recently, CD14 was also shown to play a role in the phagocytosis of Gram-positive and -negative bacteria as well as in phagocytosis of apoptotic cells [13–16]. CD14 is a glycosyl phosphatidylinositol–anchored surface molecule on monocytes/macrophages that is crucially involved in their pro-inflammatory function by binding the lipopolysaccharide (LPS)/LPS-binding protein (LBP) complex as well as lipid/ saccharide components from cell walls of Gram-positive bacteria [17]. Soluble CD14 accounts for the LPS presentation to CD14 negative cells [18]. After binding of bacterial constituents by CD14, signal transduction takes place through Toll-like receptors (TLRs) 4 and 2 [19,20]. Recently, Grunwald et al. showed that bacterial phagocytosis via the CD14 receptor is independent from Fc␥ receptors and complement receptors but requires the binding of LBP [21]. They used a human whole-blood assay with opsonized E. coli. Using almost the same assay we investigated the regulation of CD14-dependent incorporation of bacteria by the immunoregulatory cytokines IL-10, IFN-␥, and TGF-␤. Additionally, we analyzed the phagocytosis and its IL-10 regulation in monocytes from a patient with parox-

ysmal nocturnal hemoglobinuria (PNH). Cells from PNH patients do not express glycosyl phosphatidylinositol–anchored proteins like CD14. Finally, we tested the influence of IL-10 on the phagocytosis of apoptotic lymphocytes.

Subjects and methods Cells and culture conditions For whole-blood cultures, heparin-anticoagulated venous blood from healthy participants and the PNH patient was diluted 1:2 with heparinized RPMI 1640 (Biochrom KG, Berlin, Germany) and incubated at 37°C and 5% CO2in 2.0-ml tubes (Eppendorf, Hamburg, Germany) as indicated. All participants and the PNH patient participated in the study on a voluntary basis and signed informed consent forms. For preliminary phagocytosis tests preincubation of whole-blood dilution was conducted with 10 ng/ml of IL-10, IFN-␥, or TGF-␤1 for 1 hour, 4 hours, 24 hours, and 48 hours. Both IL-10 and TGF-␤1 were purchased from R&D Systems (Wiesbaden, Germany). IFN-␥ was purchased from Thomae-Boehringer Ingelheim (Biberach/Riss, Germany). Subsequent experiments to systematically test the influence of the respective cytokines on CD14-dependent phagocytosis were repeated at 24 hours for IL-10 and at 48 hours for IFN-␥ and TGF-␤1.

CD14-independent and CD14-dependent phagocytosis of opsonized and nonopsonized E. coli Monocytic CD14-independent and CD14-dependent phagocytosis was investigated in a whole-blood dilution (see above) by means of an assay adapted from the Phagotest kit (Orpegen Pharma, Heidelberg, Germany) according to the recent report from Grunwald et al. [13]. In parallel to FITC-labeled opsonized E. coli (original Phagotest assay) nonopsonized E. coli (Orpegen Pharma) were used. According to the manufacturer’s information, opsonization was performed with immunoglobulin and complement from pooled human sera and E. coli were inactivated by heat before disposal. To block CD14dependent phagocytosis, EDTA (Saarstedt, Nümbrecht, Germany) was supplemented at a final concentration of 1.6 mg/ml. To block CD14-dependent phagocytosis, the whole-blood dilution was preincubated with 25 ␮g/ml of a blocking, murine, anti-CD14 IgG2a monoclonal antibody (mAb) (clone RMO52; Coulter/Immunotech, Krefeld, Hamburg) or a respective isotype control (IgG2a, clone: 20102.1; R&D Systems) for 30 minutes at room temperature. After cooling the whole-blood suspension on ice for 10 min, 20 ␮l of pre-cooled, opsonized or nonopsonized FITC-labeled E. coli bacteria were added. After incubation at 37°C in the dark with periodic mixing for 20 minutes, phagocytosis was stopped on ice. Respective controls incubated at 4°C were always conducted. Afterwards, Quenching Solution (Orpegen Pharma) was added to remove fluorescence of the attached, nonphagocytosed bacteria. Monocytes were then cytofluorometrically analyzed for their ingested FITClabeled E. coli by means of mean fluorescence intensity (MFI) using FACScan machine and Lysys II software (BD Pharmingen, Heidelberg, Germany).

Monocytic CD14 expression For assessment of monocytic CD14 expression on cultured monocytes, 50 ␮l of whole-blood dilution were incubated with 10 ␮l of PE-labeled anti-CD14 mAb (IOM2, Coulter/Immunotech) at 4°C in the dark for 20 minutes. Afterwards the erythrocytes were lysed by FACS Lysing Solution (BD Pharmingen). After washing, monocytes were gated according to their scatter properties and were cytoflu-

732 orometrically analyzed for their MFI of surface CD14 expression using FACScan machine and Lysys II software. For analysis of surface CD14 expression on monocytes and granulocytes from the PNH patient and a healthy control, 50 ␮l of EDTA anticoagulated blood were incubated with saturating amounts of anti–CD14-APC (clone M5E2, BD Pharmingen) and anti-CD45-PerCP (clone 2D1, BD Pharmingen) for 30 minutes at 4°C in darkness. After erythrocyte lysis for 15 minutes in darkness using FACS Lysing Solution, cells were washed and measured on a FACScan machine measuring 50,000 leucocytes. For data analysis Lysys II software was used.

Phagocytosis of apoptotic Jurkat cells, regulation by IL-10 and CD14 dependence For induction of apoptosis, Jurkat cells (ACC 282, DSMZ, Braunschweig, Germany) were irradiated with 40 Gy. Because the flowcytometric apoptosis assay using FITC-labeled recombinant human Annexin V revealed a high proportion of apoptotic cells (⬎70%) 18 hours after irradiation (data not shown), the phagocytosis assay was performed after this time. Nonirradiated Jurkat cells were used as a nonapoptotic control (⬍10% apoptotic cells in Annexin assay). For fluorescence labeling, Jurkat cells were incubated with the greenfluorescent cell linker dye PKH-67 (Sigma, St. Louis, MO, USA) at 5 ␮mol/L for 2 minutes. After extensive washing, 20 ␮l of the irradiated or nonirradiated Jurkat cell suspension (2*107 cells/ml) were given into 100 ␮l of whole-blood dilution and co-incubated at 37°C for 90 minutes in the dark. Phagocytosis was stopped on ice. Then, 100 ␮l EDTA/Trypsin 0.05/0.02% solution (Biochrom KG) was added for 5 minutes before the cell suspension was washed with Ca2⫹- and Mg2⫹-free Dulbecco’s PBS (Life Technologies Ltd, Renfrew, Scotland) for removal of attached cells. After CD14 labeling with anti-CD14-PerCP (clone M⌽P9, BD Pharmingen), monocytes were gated according to CD14 expression and side scatter properties and were then cytofluorometrically analyzed for their MFI of ingested PKH-67–labeled Jurkat cells. To test regulation by IL-10 and CD14 dependence, preincubations with IL-10 and the anti-CD14 mAb RMO52 or a respective isotype antibody were performed as described above.

Effect of the anti-CD14 mAb RMO52 on LPS-induced TNF-␣ production For analyzing the effect of the anti-CD14 mAb RMO52 on LPS-induced TNF-␣ production, whole blood was diluted 1:2 with heparinized RPMI 1640. After 30 minutes preincubation of the whole-blood dilution with 25 ␮g/ml of the anti-CD14 mAb RMO52 or a respective isotype control, the cells were stimulated with 500 pg/ml LPS (Escherichia coli 0127:B8, Sigma-Aldrich, Heidelberg, Germany) in Eppendorf tubes for 4 hours at 37°C and 5% CO2. Culture supernatants were analyzed for TNF-␣ using the IMMULITE semi-automatic immunoassay system (DPC Biermann Inc., Bad Nauheim, Germany), a solid-phase, two-site chemiluminescence immunoassay with a calibration range of 1.7 to 1,000 pg/ml. In parallel, TNF-␣ induction by FITC-labeled E. coli was analyzed using the same test system as described above but with 20 ␮l of FITC-labeled E. coli from the Phagotest assay instead of LPS.

Statistical analysis Data are given as the mean ⫾ standard error of the mean. Statistical analyses were performed by Friedman’s two-tailed analysis of variance and Wilcoxon matched-pairs, signed-ranks test using SPSS software (SPSS Inc., Chicago, IL, USA).

M. Lingnau et al.

Results Validation of phagocytosis assay in human whole-blood dilution For analysis of phagocytosis in human whole blood we used an assay adapted from the commercially available Phagotest kit. The test is based on cytofluocytometrical measurement of the uptake of fluorescence-labeled E. coli by respective cells of interest. As demonstrated in Figure 1A, to analyze the measured data we first performed the leucocyte gating by propidium iodide properties to exclude cell debris and cell aggregates. Then we did the monocyte gating by forward and side scatter properties to exclude lymphocytes and granulocytes. As shown in Figure 1B, the use of FITC-labeled E. coli and subsequent FITC quenching enabled an accurate measurement of monocytic incorporation of E. coli. In fact, no relevant monocytic FITC fluorescence intensity was detected in either samples without bacteria or in samples that were incubated at 4°C with FITC-labeled E. coli. FITC quenching was needed to exclude monocytic FITC fluorescence by E. coli not phagocytosed but attached to the monocyte surface.

Phagocytosis and its regulation by IL-10 in monocytes from a PNH patient The aim of our study was to characterize the regulation of CD14-dependent phagocytosis in monocytes. To investigate this phagocytosis pathway, it was necessary to inhibit CD14dependent incorporation. To check whether EDTA, used for this purpose in previous studies, actually blocks all CD14dependent phagocytosis pathways, we determined the incorporation of FITC-labeled E. coli by CD14-deficient monocytes from a PNH patient. As shown in Figure 2A, monocytes from the PNH patient expressed no CD14 on their surface. In contrast to control monocytes, the incubation of monocytes from the PNH patient with EDTA completely inhibited the phagocytosis of E. coli (Figure 2B). This demonstrated that EDTA completely blocked all CD14-dependent pathways of phagocytosis. Importantly, a 24 hours preincubation of whole blood with IL-10 strongly increased the CD14-independent phagocytosis but had no influence on the CD14-dependent phagocytosis of monocytes from the PNH patient (Figure 2B).

Characterization of CD14-independent and CD14-dependent phagocytosis of opsonized and nonopsonized E. coli Afterwards we tested the phagocytosis of opsonized and nonopsonized E. coli in the presence of EDTA and/or antiCD14 mAb RMO52 or a respective isotype antibody by monocytes from healthy participants. As shown in Figure 3, neither the selective blockade of CD14-independent phagocytosis by EDTA nor the selective blockade of CD14-dependent phagocytosis by anti-CD14 mAb was sufficient to block bacterial uptake by these cells. Only the simultaneous blockade of both pathways was able to completely inhibit bacterial phagocytosis independent from opsonization. In fact, the addition of anti-CD14 mAb to EDTA samples reduced the phagocytosis of

Interleukin-10 enhances CD14-dependent phagocytosis

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Figure 1. Validation of phagocytosis assay. Monocytic incorporation of FITC-labeled Escherichia coli (E. coli) was investigated in whole-blood dilution by means of an assay adapted from the Phagotest kit. (A) Black dots represent the monocyte population after gating of leucocytes using side scatter and propidium iodide properties to exclude debris and cellular aggregates and subsequent gating of monocytes using forward and side scatter properties to exclude lymphocytes and granulocytes. (B) Monocytic FITC fluorescence intensity after preincubation with or without FITC-labeled E. coli and subsequent incubation with or without quenching solution is demonstrated. Phagocytosis only occurs at 37°C and FITC quenching solution does differentiate between internalized and surface bound FITC-labeled E. coli.

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opsonized bacteria to 1.5% and of nonopsonized bacteria to 2.3%.

Cytokine regulation of CD14-dependent phagocytosis of nonopsonized E. coli and of monocytic CD14 expression Next we tested the influence of IL-10, IFN-␥, and TGF-␤1 on monocytic CD14-dependent bacterial uptake. Our preliminary experiments showed that IL-10 preincubation for 24 hours was most effective in increasing CD14-dependent phagocytosis (IL-10 –induced increase of CD14-dependent phagocytosis relative to respective controls: at 24 hours to 190% ⫾ 17.6%, at 48 hours to 153% ⫾ 37.8%, no effects at 1 hour and 4 hours; n ⫽ 2). Both IFN-␥ and TGF-␤1 exhibited

opsonized E. coli

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their maximal suppressive effects after 48 hours (IFN-␥– induced inhibition of CD14-dependent phagocytosis relative to respective controls: at 4 hours to 88% ⫾ 4%, at 24 hours to 85% ⫾ 12%, at 48 hours to 41% ⫾ 1.5%, no effect at 1 hour; n ⫽ 2) (TGF-␤1–induced inhibition of CD14-dependent phagocytosis relative to respective controls: at 24 hours to 85% ⫾ 13%, at 48 hours to 35% ⫾ 1%, no effects at 1 hour and 4 hours; n ⫽ 2). Therefore, for IL-10 24 hours, and for IFN-␥ and TGF-␤1 48 hours were used for systematic assessment of their effects on CD14-dependent phagocytosis in the wholeblood assay. As show in Figure 4A, CD14-dependent phagocytosis was significantly increased by IL-10 at 24 hours and significantly decreased by IFN-␥ and TGF-␤1 at 48 hours (IL-10induced increase of CD14-dependent phagocytosis relative to respective controls: to 146.5% ⫾ 18.39%; IFN-␥–/ TGF-␤1–induced inhibition of CD14-dependent phagocytosis relative to respective controls: to 41.8% ⫾ 10.13%/43.2% ⫾ 7.40%; n ⫽ 6, p ⬍ 0.05, Wilcoxon test). For all cytokine preincubations, stringent CD14 dependence of phagocytosis was proven by blocking with the anti-CD14 mAb RMO52 in comparison to a respective isotype mAb (phagocytosis of FITC-labeled E. coli by adding anti-CD14 mAb: always ⬍15%; Figure 4). Subsequently we tested whether monocytic CD14 expression was affected in the same way as CD14-dependent phagocytosis after preincubation with IL-10 for 24 hours or with IFN-␥ or TGF-␤1 for 48 hours. IL-10 upregulated and IFN-␥ and TGF-␤1 downregulated CD14 expression (monocytic CD14 expression [MFI] after IL-10 preincubation for 24

phagocytosis of E. coli (MFI)

Figure 2. Phagocytosis of Escherichia coli (E. coli) and its regulation by interleukin-10 (IL-10) in monocytes from a patient with paroxysmal nocturnal hemoglobinuria (PNH). (A) Black dots represent the monocyte population after gating using forward and side scatter properties and CD45 expression. In contrast to a healthy control, monocytes from the PNH patient did not express surface CD14 protein. (B) The whole-blood dilution was preincubated with or without IL-10 for 24 hours. Afterward EDTA was added to the samples as indicated and the monocytic phagocytosis of FITC-labeled nonopsonized E. coli was cytofluorometrically assessed. In contrast to monocytes from healthy controls, EDTA completely blocked the incorporation of E. coli in monocytes from the PNH patient through inhibition of the CD14-independent pathway. (Black bars: PNH patient; white bars: healthy control.)

M. Lingnau et al.

non-opsonized E. coli

Figure 3. Assessment of CD14-independent and CD14-dependent monocytic phagocytosis of Escherichia coli (E. coli) in whole-blood dilution. Unrestricted phagocytosis was assessed using opsonized or nonopsonized E. coli in calciumcontaining culture medium (“control”) by means of flow cytometry. CD14-independent phagocytosis was inhibited by addition of EDTA (“⫹ EDTA”). CD14-dependent phagocytosis was prohibited by an anti-CD14 mAb (“⫹ anti-CD14 mAb”). Neither the selective inhibition of the CD14-independent nor the CD14-dependent pathway deteriorated the monocytic phagocytosis of E. coli, but blocking of both pathways resulted in almost complete abrogation. The results of three independent experiments are given as mean ⫾ standard error of the mean of monocytic FITC MFI.

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Interleukin-10 enhances CD14-dependent phagocytosis

Effects of the anti-CD14 antibody RMO52 on LPS-induced TNF-␣ production

Figure 4. Cytokine regulation of CD14-dependent phagocytosis of Escherichia coli (E. coli). The whole-blood dilution was preincubated with or without interleukin-10 (IL-10) for 24 hours (10 ng/ml) (A) and IFN-␥ or TGF-␤1 (10 ng/ml each) for 48 hours (B). Afterwards, EDTA was added to each sample to block the CD14-independent incorporation of bacteria. Additionally, one of the three samples from each culture was incubated with a blocking murine anti-CD14 IgG2a mAb (RMO52) and another one with an isotype control mAb (each at 25 ␮g/ml), respectively, to prove CD14 dependence on the subsequent phagocytosis assay. Monocytic phagocytosis of FITC-labeled nonopsonized E. coli was assessed by means of flow cytometry. For each cytokine the results of six independent experiments are given as mean ⫾ standard error of the mean in relation to monocytic FITC MFI of the respective cytokine-untreated, EDTA-supplemented control (* p ⬍ 0.05 vs respective cytokine-untreated, EDTA-supplemented control, # p ⬍ 0.05 vs untreated and isotype control mAb-treated culture with the same cytokine, Wilcoxon test).

hours: 1,197 ⫾ 148 vs 855 ⫾ 93 in controls; monocytic CD14 expression [MFI] after IFN-␥ and TGF-␤1 preincubation for 48 hours: 711 ⫾ 302 and 483 ⫾ 55, respectively, vs 1,047 ⫾ 81 in controls; n ⫽ 12, p ⬍ 0.001, Wilcoxon test).

IL-10 effect on the CD14-dependent phagocytosis of apoptotic Jurkat cells We then investigated whether IL-10 could also increase CD14-dependent incorporation of apoptotic cells. Although the ingestion of nonapoptotic, PKH-67-labeled Jurkat cells was insignificant, a strong increase in phagocytosis of apoptotic cells by control monocytes was observed (monocytic MFI after incubation with nonapoptotic/apoptotic Jurkat cells: 17.5/195.9; n ⫽ 2; Figure 5A), which could be largely inhibited by an anti-CD14 mAb but not by the isotype control mAb (monocytic MFI after incubation with apoptotic Jurkat cells and anti-CD14 mAb/isotype control mAb: 41.7/208.6; n ⫽ 2; Figure 5A). The IL-10 preincubation of monocytes nearly doubled the PKH-67-related monocytic MFI demonstrating enhanced ingestion (Figures 5A and 5B). Interestingly, CD14 blockade only partially inhibited the IL-10 induced increase of apoptotic Jurkat cell incorporation (Figure 5A and 5B).

Finally, we questioned whether the anti-CD14 antibody RMO52 also inhibits CD14-dependent TNF-␣ production. Preliminary tests revealed that only LPS but not FITC-labeled E. coli induced significant TNF-␣ production (LPS-induced TNF-␣ production: ⬎1000 pg/ml; E. coli induced TNF-␣ production: ⬍11 pg/ml; n ⫽ 2). Therefore, the following experiments were performed with LPS. Our results showed that the RMO52 antibody not only inhibited the phagocytosis of bacteria and apoptotic cells (Figures 2, 3, 4, and 5), but also prevented the LPS-induced monocytic TNF-␣ production (6283 ⫾ 916 pg/ml TNF-␣ secretion after LPS stimulation versus 102 ⫾ 19 pg/ml after preincubation with anti-CD14 antibody RMO52 or 8446 ⫾ 939 pg/ml after isotype antibody preincubation; n ⫽ 3).

Discussion In this study, we addressed the cytokine-mediated regulation of monocytic CD14-dependent phagocytosis. In doing so we observed that IL-10, suggested to be a monocyte-deactivating cytokine, strongly increased the CD14-dependent phagocytosis of nonopsonized, Gram-negative bacteria. In contrast, the monocyte-activating cytokines IFN-␥ and TGF-␤1 depressed CD14-dependent phagocytosis. These findings were in line with the regulation of CD14 surface expression by IL-10, IFN-␥, and TGF-␤1 ([22–24] and our own data). As expected IL-10 strongly increased the CD14-independent phagocytosis but had no influence on the CD14-dependent phagocytosis of monocytes from a PNH patient. Furthermore, monocytes from the PNH patient demonstrated increased CD14-independent phagocytosis compared with respective control monocytes. This is in line with in vitro data presented by Grundwald et al. and with own experiments showing that blockage of CD14-dependent phagocytosis strengthened CD14-independent phagocytosis and vice versa ([13] and our own unpublished data). As far as we know, the mechanisms behind this phenomenon are not yet analyzed. Furthermore, IL-10 increased the CD14-dependent and -independent phagocytosis of apoptotic cells. Recently this was also demonstrated by Ogden et al [25]. Considerable effects on monocytic CD14-dependent phagocytosis could only be seen at preincubation periods longer than 4 hours and they were paralleled by corresponding regulations of monocytic surface CD14 expression. This suggests that regulation of CD14 may be responsible for the effects seen on CD14-dependent phagocytosis but does not exclude other effects mediated by IL-10, IFN-␥ and TGF-␤. We recently analyzed the gene expression profile in peripheral blood mononuclear cells from psoriatic patients treated with IL-10 [26]. We could show that apart from the induction of genes coding for cell surface receptors associated with phagocytosis genes involved in phagocytosis signaling were induced [26]. In fact, among the cell surface receptors associated with phagocytosis the Fc␥ receptors CD64, CD32, and CD16, the scavenger receptor MARCO and two proteins associated with the uptake of auto-antigens— c-mer and Gas6 —were upregulated [26]. The latter in particular could be responsible for the only partial CD14 dependence of IL-10

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Figure 5. Effects of interleukin-10 (IL-10) on monocytic phagocytosis of apoptotic cells. Whole-blood dilution was preincubated with or without IL-10 (10 ng/ml) for 18 hours. Afterwards a blocking murine anti-CD14 IgG2a mAb (RMO52) or an isotype control mAb (each at 25 ␮g/ml) was added for 30 minutes as indicated. After 90 minutes of co-incubation with PKH-67-labeled nonapoptotic and apoptotic Jurkat cells, monocytic incorporation of nonapoptotic and apoptotic cells was assessed by means of flow cytometry. (A) Results of two independent experiments are given as mean ⫾ standard error of the mean in relation to monocytic PKH-67-dependent MFI of control. (B) Histograms show the PKH-67-dependent MFI of monocytes as the measure of apoptotic cell phagocytosis after different preincubation protocols. The numbers top right from each histogram represent the percentage and fluorescence intensity (in brackets) of positive monocytes. Representative results from one of two independent experiments are shown.

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Interleukin-10 enhances CD14-dependent phagocytosis increased phagocytosis of apoptotic Jurkat cells observed in our test system. The studies of CD14-dependent LPS internalization suggested that monocytes take up aggregated LPS predominantly via plasma membrane invaginations. Interestingly, this process was neither attenuated in macrophages from LPS-hyporesponsive mice (C3H/HeJ) nor after in vitro LPS desensitization [31]. Furthermore, it is likely that the region of the CD14 molecule, which mediates the binding of E. coli and apoptotic Jurkat cells, is the same as or may overlap the region that mediates the binding of the LPS-LBP complex, leading to the production of pro-inflammatory cytokines [32]. This indicates that the intracellular signaling pathway of CD14-dependent phagocytosis is different from that of the inflammatory CD14 mediated LPS response. This would also explain how IL-10 could act on two CD14-related mechanisms in a contrasting manner, by depressing the CD14 conveyed inflammatory cytokine response and by largely increasing the CD14-dependent phagocytosis. At this time, we can only speculate about the signaling molecules involved in these antagonistic immune responses mediated by CD14. So it is known that CD14 can activate different TLRs and that different TLRs can induce different signaling pathways [33]. Further experiments are needed to rule out whether TLRs are responsible for the antagonistic effects seen in our model. Data underlining a physiologic role for CD14-dependent phagocytosis come from different research groups. Wenneras et al. tested the influence of a CD14 blockade on Shigella induced disease severity in a rabbit model [27]. They observed that anti-CD14 treatment interfered with host defense mechanisms involved in removal/eradication of Shigella. Frevert et al. used an E. coli pneumonia model in rabbits to study the influence of a CD14 blockade on disease development [28]. The blockade of CD14 improved the mean arterial blood pressure and decreased the i.v. fluid requirements but led to increased bacterial burdens in the bronchoalveolar lavage fluid and widened the alveolar-arterial oxygen difference. Liu et al. indicated a role of CD14 in the phagocytosis of Alzheimer’s amyloid peptide [29]. Walter et al. presented data suggesting a protective role for CD14dependent phagocytosis in experimental autoimmune encephalomyelitis and multiple sclerosis [30]. With respect to a physiologic role of IL-10 for (CD14dependent) phagocytosis, our data indicate a general role of this cytokine for activating monocytic scavenger functions, which are at least partly mediated by CD14. However, with respect to intracellular killing it has been reported that both free and surface IL-10 inhibit the killing of uptaken micropathogens by monocytes/macrophages [34]. It is possible that the main physiologic importance of IL-10-induced phagocytosis is the nonharmful scavenging of autologous apoptotic cells and of cellular debris from autologous and bacterial sources. For this it is important to enlarge the phagocytic capacity on the one side but to depress the inflammatory response as well as antigen presentation on the other side in order to prevent further tissue injury as well as autoimmunity. The antithetical effects of IL-10 on cellular immunity and monocytic scavenging function fulfill these requirements. In this context it is notable that: (1) cells undergoing apoptosis can release IL-10, and (2) that ingested peptides susceptible to lysosomal proteolysis are nonimmu-

nogenic in contrast to peptides less susceptible to lysosomal proteolysis [35,36]. Remarkably, Ronchetti et al. very recently provided direct evidence for a role of IL-10 in preventing the immunogenicity of apoptotic material [37]. According to the authors, the lowered CTL response to apoptotic in comparison to nonapoptotic tumor cells was largely enhanced in IL-10 knockout mice. In contrast, treatment of rats with GM-CSF, which stimulates phagocytosis and macrophage development like IL-10, decreased the in vivo immunogenicity of apoptotic cells [38]. A role of IL-10 for the nonimmunogenic scavenging of autologous tissues with high turnover could also be hypothesized from the selective development of autoimmune enterocolitis in IL-10 knockout mice [39].

Acknowledgments The authors thank M.S. for donating blood to analyze the influence of interleukin-10 on phagocytosis of CD14-deficient monocytes. The authors also thank Brigitte Ketel for excellent technical assistance and Elizabeth Wallace for accurate proofreading of the manuscript.

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