Published online before print December 12, 2006
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Departments of
* Microbiology and Immunology and
Physiology, Dartmouth Medical School, Hanover, New Hampshire, USA; and
Department of Microbiology and Immunology, University of Maryland, Baltimore, Maryland, USA
1 Correspondence: Department of Microbiology and Immunology, Dartmouth Medical School, HB7700, 1 Medical Center Drive, Lebanon, NH 03756, USA. E-mail: lehn{at}dartmouth.edu
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(rIFN-) suppresses cell-surface, TLR-mediated IL-10 production as well as CD163 up-regulation. Sustained down-regulation of CD163 mediated by rIFN- can be partially rescued with exogenous rIL-10 but not with exogenous rIL-6. This divergent regulation of CD163 by cytokines demonstrates that human monocytes react differently to infectious signals depending on the cytokine milieu they encounter. Thus, surface CD163 expression on mononuclear phagocytes is a carefully regulated component of the innate immune response to infection.
Key Words: macrophage • inflammation • innate immunity • pattern recognition receptors
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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As CD163 has been proposed to function in the innate immune response and in the resolution of inflammation, this monocyte/macrophage-specific glycoprotein is likely involved in various stages of the inflammatory response [3 ]. CD163 binds haptoglobin-hemoglobin (Hp-Hb) complexes, implicating this receptor as a Hb scavenger receptor [4 ]. Clearance of these complexes is important, as intravascular-free Hb is known to generate reactive oxygen species (ROS) [5 ], activate endothelial cell proinflammatory sequalae [6 ], and oxidize low-density lipoproteins [7 ]. Furthermore, recent studies have revealed that the level of surface CD163 expressed on human macrophages is correlated directly with the uptake of Hp-Hb complexes by these cells [8 ]. Therefore, removal of free Hb from the circulation may be one mechanism by which CD163 exerts an anti-inflammatory function.
In accordance with the hypothesis that CD163 is involved in the resolution of inflammation, anti-inflammatory mediators such as glucocorticoids and IL-10 markedly increase expression of CD163 on the cell surface, and the proinflammatory cytokines TNF-
and IFN- suppress CD163 expression [3
, 9
]. Furthermore, the pleiotropic cytokines IL-6 and TGF-ß, known to exert pro- and anti-inflammatory effects, enhance and suppress CD163 surface expression, respectively [9
, 10
]. However, during an infection, multiple cytokines are likely to influence the expression of surface CD163 concomitantly.
Recent reports demonstrate that CD163 expression on monocytes is regulated during the early innate immune response. Activation of cell-surface TLRs or FcR cross-linking initiates shedding of the extracellular domain of CD163 from the surface of monocytes within 1 h of treatment [11 , 12 ]. CD163 shedding following TLR activation is specific for cell-surface TLRs, as intracellular TLR agonists do not activate shedding of CD163 and do not alter CD163 surface expression after 12–48 h of treatment [13 ]. Soluble CD163, which binds Hp-Hb complexes similarly to the membrane-bound CD163, has been identified in normal human serum and is elevated greatly in conditions of sepsis, pneumonia, and other infections [14 15 16 17 18 ]. Although generation of soluble CD163 is an acute host response to infection, regulation of surface CD163 upon activation of TLRs in relationship to the cytokine response to infection has not been characterized.
Herein, we demonstrate that in addition to acute shedding in response to activation of cell-surface TLRs, surface CD163 expression on monocytes recovers to levels that are significantly higher than those observed on untreated monocytes by 24–72 h post-treatment. We also show that the TLR-induced production of IL-6 and IL-10, cytokines shown to up-regulate CD163 previously, occurs concomitantly with CD163 up-regulation, and up-regulation of CD163 can be blocked with neutralizing antibodies to IL-6 and/or IL-10, indicating their role as intermediates in this process. In addition, recombinant (r)IFN- inhibits TLR-mediated IL-10 production and CD163 up-regulation. The addition of exogenous rIL-10, but not rIL-6, partially rescues the rIFN--mediated suppression of CD163 up-regulation. Collectively, these data support the hypothesis that Hb uptake by monocytes and macrophages is a tightly regulated component of the innate immune response.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Determination of surface CD163 expression
PBMC were cultured as described above and treated for 0–72 h as indicated with medium alone, 1 ng/mL phenol-water-extracted (protein-free) Escherichia coli K235 LPS (TLR4 agonist) [20
, 21
], 10 ng/ml S-[2,3-Bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-(R)-Cys-(S)-Ser-Lys4-OH, trihydrochloride (Pam3Cys; EMC Microcollections GmbH, Tübingen, Germany; TLR2/1 agonist), 200 ng/mL Salmonella typhimurium flagellin (TLR5 agonist, Invivogen, San Diego, CA, USA), 200 ng/mL protease-digested S. typhimurium flagellin, 20 ng/mL Actimmune human rIFN--1b (Intermune, Brisbane, CA, USA), and 10 ng/mL human rIL-10 and/or 10 ng/mL human rIL-6 (Peprotech, Rocky Hill, NJ, USA). Extensive dose-response experiments were performed with all of the purified PAMPs used in this manuscript. Concentrations of PAMPs producing maximum shedding responses were used in the subsequent experiments, as an indication of TLR activation. Digested flagellin was prepared as described previously [13
].
Following treatment, surface expression of CD163 was determined as described previously with the purified mouse IgG1 mAb Mac 2-158 (anti-CD163, Maine Biotechnology, Portland, ME, USA) [13 ]. Mean fluorescent intensity (MFI) was determined by the geometric mean of the fluorescence of gated monocytes.
Inhibition of LPS-mediated CD163 shedding and up-regulation
To inhibit TLR4-mediated signaling, TLR agonists were preincubated with 15 µg/mL polymyxin B sulfate (PMX; Gibco, Grand Island, NY, USA) for 30 min at room temperature prior to addition to cells. PMX remained in the culture throughout the duration of the experiment.
IL-6 and IL-10 ELISAs
Following treatment, cells were centrifuged at 450 g for 1 min. Supernatants were collected and analyzed for IL-6 and/or IL-10 levels. IL-6 concentrations were determined using an IL-6 sandwich ELISA (Peprotech), and IL-10 concentrations were determined using an IL-10 sandwich ELISA (Biosource, Camarillo, CA, USA) as described by the manufacturer.
Inhibition of TLR-mediated CD163 up-regulation by anticytokine neutralizing antibodies
To inhibit IL-6 and IL-10, PBMC were treated as described above without or with 0.5 µg/mL rat antihuman IL-6-neutralizing antibody, 0.5 µg/mL rat antihuman IL-10-neutralizing antibody, or a rat IgG1 isotype control antibody. All cytokine-neutralizing antibodies were obtained from BD PharMingen (San Diego, CA, USA). Antibodies remained in culture for the duration of the experiment.
Limulus amoebocyte lysate (LAL) assay for detection of LPS contamination
The QCL-1000® chromogenic LAL endpoint assay (Cambrex, Cottonwood, AZ, USA) was used to detect endotoxin contamination of all TLR agonists following the manufacturer’s protocol. Briefly, protein-free, phenol-water-extracted E. coli K235 LPS was used to create a linear standard curve from 10 to 100 pg/ml. TLR agonists were serially diluted to determine a concentration of agonist within the standard curve of the assay. Pam3Cys was determined to have less than 1 pg/mL LPS contamination, and S. typhimurium flagellin was determined to have 135 ± 11.3 pg/mL LPS contamination.
Statistical analysis
One-way or two-way ANOVA statistical analyses were performed as indicated in the figure legends followed by a Bonferroni post-test analysis. An unpaired Student’s t-test was used to compare flagellin versus flagellin + PMX treatments, as indicated in Results. A P value <0.05 was accepted as statistically significant. Experimental values were expressed as means ± SD (unless otherwise indicated) and are representative of greater than or equal to three separate experiments from greater than or equal to three donors. All statistics and graphs were performed using GraphPad Prism Version 4.0c for Macintosh (GraphPad Software, San Diego, CA, USA).
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Figure 1. Up-regulation of CD163 follows cell-surface, TLR-mediated CD163 shedding. PBMC from (A) Donor 1 and (B) Donor 2 were treated for 0–72 h with media alone or 1 ng/mL LPS, pretreated without or with 15 µg/mL PMX for 30 min. PBMC from (C) Donor 1 and (D) Donor 2 were treated for 0–72 h with media alone, 10 ng/mL Pam3Cys, 200 ng/mL S. typhimurium flagellin, and 200 ng/mL protease-digested flagellin, pretreated without or with 15 µg/mL PMX for 30 min. Surface expression of CD163 was analyzed by flow cytometry. Representative data shown are from two of six experiments using six different donors, and values are mean MFI ± SD of triplicates. An unpaired Student’s t-test was used to calculate significance between flagellin and flagellin + PMX treatments (*, P<0.05).
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PBMC treatment with TLR2 and TLR5 agonists causes up-regulation of CD163
The TLR2 and TLR5 agonists Pam3Cys and S. typhimurium flagellin, respectively, have been shown to activate monocyte shedding of CD163 [13
]. To determine if these agonists also cause CD163 up-regulation, PBMC were treated with Pam3Cys and Salmonella flagellin for 0–72 h, which resulted in a decrease in surface CD163 by 1 h, indicative of CD163 shedding and up-regulation of CD163 by 24 h (Fig. 1C
and 1D)
. To determine if LPS contamination of Pam3Cys and flagellin contributed to CD163 up-regulation in these treatment groups, TLR2 and TLR5 agonists were pretreated with PMX for 30 min prior to PBMC treatment. PMX-pretreated Pam3Cys and Salmonella flagellin still resulted in CD163 up-regulation, indicating that the effects of these ligands cannot be attributed to contamining LPS. Thus, specific stimulation of human monocytes through the cell surface molecules TLR2, TLR4, or TLR5 results in CD163 up-regulation (Fig. 1)
. However, up-regulation of CD163 by flagellin was reduced significantly in the presence of PMX (Fig. 1C
and 1D)
. Given the extent to which flagellin was contaminated with LPS, it is likely that dual signaling through TLR4 and TLR5 accounted for the higher level of CD163 in the absence of PMX. Therefore, in all subsequent experiments, flagellin preparations were pretreated with PMX to block the effect of contaminating LPS. To evaluate the specificity of flagellin-mediated CD163 up-regulation more critically, the flagellin preparation was protease-digested, which completely eliminated the ability of this TLR5 ligand to up-regulate CD163 in the presence of PMX (Fig. 1C
and 1D)
. Thus, the simultaneous engagement of two TLRs, i.e., TLR5 and TLR4, augmented CD163 up-regulation when compared with activation through a single TLR.
Up-regulation of CD163 is enhanced following simultaneous activation of TLR2 and TLR4
To confirm further that the simultaneous activation of multiple TLRs results in enhanced up-regulation of CD163 when compared with activation of a single TLR, monocytes were treated with two separate, highly purified TLR2 and TLR4 agonists, alone or in combination. Figure 2A
demonstrates that activation of TLR2 and/or TLR4 decreased surface expression of CD163 at 1 h post-treatment. This down-regulation of surface CD163 is not a result of global suppression of monocyte surface markers by TLR activation, as CD45 is unchanged by TLR activation in comparison with untreated controls (Fig. 2A
, inset). Furthermore, at 48 h post-treatment, surface expression of CD163 rebounded to levels as high or greater than untreated controls in all treatment groups (Fig. 2B)
. It is interesting that activation of TLR2 and TLR4 on monocytes resulted in enhanced up-regulation of surface CD163 in comparison with activation of TLR2 or TLR4 alone. This enhanced up-regulation of surface CD163 was reduced by PMX to levels comparable with those on cells treated with Pam3Cys alone, indicating that dual activation of multiple TLRs results in enhanced up-regulation of CD163. In contrast, TLR activation did not alter the expression of CD45 in comparison with untreated controls, demonstrating that CD163 up-regulation is a specific monocyte response following TLR activation (Fig. 2B
, inset). Our data demonstrate that activation of multiple TLRs enhances the ability of monocytes to up-regulate CD163 in comparison with activation of a single TLR.
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Figure 2. Up-regulation of CD163 is enhanced following simultaneous activation of TLR2 and TLR4. PBMC were treated for 1 or 48 h with media alone, 1 ng/mL LPS, and/or 10 ng/mL Pam3Cys, pretreated without or with 15 µg/mL PMX for 30 min. Surface expression of CD163 and CD45 was analyzed by flow cytometry. Representative data shown are from one of four experiments using four different donors, and values are mean MFI ± SD of triplicates. A two-way ANOVA was used to calculate the significance between treatment and controls (*, P<0.001) and treatment and treatment + PMX (#, P<0.001).
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Figure 3. IL-6 and IL-10 production following stimulation of PBMC with cell-surface TLR agonists is detected concomitant with CD163 up-regulation. PBMC were treated for 24 h with 1 ng/mL LPS, 10 ng/mL Pam3Cys, 200 ng/mL S. typhimurium flagellin, and 200 ng/mL protease-digested flagellin, without or with PMX. Supernatants from these cultures were collected and analyzed by a sandwich ELISA for (A) IL-6 or (B) IL-10. Data shown are pooled data from five of five experiments using five different donors, and values are mean ± SE. A two-way ANOVA was used to calculate the significance between treatment and treatment + rIFN- (*, P<0.001). BD, Below detection; ND, not done.
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Figure 4. Neutralizing antibodies to IL-6 and IL-10 inhibit the cell-surface, TLR-mediated up-regulation of CD163. PBMC were treated with 1 ng/mL LPS, 10 ng/mL Pam3Cys, or 200 ng/mL S. typhimurium flagellin, without or with neutralizing antibodies to IL-6, IL-10, or an isotype antibody for 72 h, as described in Materials and Methods. Surface expression of CD163 was analyzed by flow cytometry. Representative data shown are from one of three experiments using three different donors, and values are mean MFI ± SD of triplicates. Two-way ANOVA was used to calculate the significance between all treatment groups (+, P<0.01; *, P<0.001).
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blocks CD163 up-regulation [27
]. Newly infiltrating monocytes can then respond to TLR agonists in combination with IFN-. This classical activation of macrophages results in enhanced production of ROS, reactive nitrogen species, and proinflammatory cytokines and helps to defend against infection [28
]. To determine whether classically activated monocytes regulate CD163 differently from monocytes treated with TLR agonists alone, PBMC were treated with TLR agonists in the absence or presence of rIFN-. Although activation of TLR2, TLR4, and TLR5 led to up-regulation of CD163, the concurrent presence of rIFN- resulted in sustained down-regulation of CD163 following TLR-mediated shedding (Fig. 5
). These data are in accordance with previous reports showing that rIFN- alone does not alter CD163 expression by 1 h but is able to suppress surface levels of CD163 by 24 h post-treatment [9
]. Furthermore, we show that rIFN- does not activate or inhibit CD163 shedding, although it does dramatically suppress the TLR-mediated up-regulation of CD163.
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Figure 5. Down-regulation of surface CD163 is sustained following treatment with cell-surface TLR agonists and rIFN-. PBMC were treated with 1 ng/mL LPS, 10 ng/mL Pam3Cys, or 200 ng/mL S. typhimurium flagellin, without or with 20 ng/mL rIFN- for 0–72 h before measuring expression of CD163 by flow cytometry. Representative data shown are from one of six experiments using six different donors, and values are mean MFI ± SD of triplicates.
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suppresses TLR-mediated up-regulation of CD163, we tested whether rIFN- reduced production of IL-10 and/or IL-6 by TLR-activated PBMC. Stout et al. [29
] showed recently that LPS stimulation of IL-10 release is eliminated when murine macrophages are treated simultaneously with LPS and rIFN-, and Hu et al. [30
] demonstrated that IFN- abrogates human macrophage production of IL-10 upon treatment with Pam3Cys. Here, we extend these findings using human PBMC to demonstrate that rIFN- inhibited the production of IL-10 induced by TLR2, TLR4, and TLR5 agonists (Fig. 6A
), without reducing IL-6 production (Fig. 6B)
. Production of IL-10 was reduced by rIFN- in all six donors tested, whereas IL-6 was enhanced by rIFN- in two donors and unchanged in four following stimulation of PBMC with cell-surface TLR agonists. These data imply that rIFN- may suppress TLR-mediated up-regulation of CD163 by inhibiting the production of IL-10 following activation of cell-surface TLRs. Moreover, these data further support the hypothesis that IL-10 likely plays a more significant role in mediating CD163 up-regulation than does IL-6, as rIFN- treatment does not reduce IL-6 production following TLR activation.
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Figure 6. rIFN- alters the cytokine response of human PBMC upon stimulation with cell-surface TLR agonists. PBMC were treated with 1 ng/mL LPS, 10 ng/mL Pam3Cys, or 200 ng/mL S. typhimurium flagellin, without or with 20 ng/mL rIFN- for 24 h. Supernatants from these cultures were collected and analyzed by a sandwich ELISA for (A) IL-10 or (B) IL-6. Data shown are pooled data from six of six experiments using six different donors, and values are mean ± SE. A two-way ANOVA was used to calculate the significance between treatment and treatment + rIFN- (*, P<0.001).
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suppresses production of IL-10 following TLR activation, we tested if exogenous rIL-10 or rIL-6 could rescue suppression of the TLR-mediated CD163 up-regulation by rIFN-. Figure 7A
demonstrates that the addition of exogenous rIL-10 partially rescued the rIFN--mediated suppression of CD163 up-regulation (*, P<0.001). These data indicate that although suppression of CD163 expression was, in part, caused by the inhibition of TLR-mediated IL-10 production by rIFN-, rIFN- also reduced monocyte responsiveness to exogenous rIL-10 (Fig. 7A
; +, P<0.001). Thus, at least two mechanisms account for rIFN- suppression of TLR-mediated CD163 up-regulation: rIFN- mediates inhibition of IL-10 production from TLR-stimulated PBMC and also reduces the responsiveness of monocytes to exogenous, rIL-10-mediated CD163 induction. In contrast, rIL-6 was not able to rescue CD163 up-regulation following classical activation of monocytes (Fig. 7B
; 
, P<0.001). Furthermore, activation of TLRs or rIFN- treatment reduced monocyte responsiveness to exogenous rIL-6 induction of CD163 (Fig. 7B
; 
, P<0.001).
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Figure 7. CD163 surface expression is rescued partially with exogenous rIL-10 but not rIL-6 following classical activation. PBMC were treated without or with 20 ng/mL rIFN- and/or 10 ng/mL rIL-10 (A) and/or 10 ng/mL rIL-6 (B) in combination with 1 ng/mL LPS, 10 ng/mL Pam3Cys, or 200 ng/mL S. typhimurium flagellin for 72 h. Surface expression of CD163 was analyzed by flow cytometry after 72 h. Representative data shown are from one of three experiments using three different donors, and values are mean MFI ± SD of triplicates. Two-way ANOVA was used to calculate the significance between treatment groups (+, P<0.001, for treatment+rIFN- vs. treatment+rIFN-+rIL-10; *, P<0.001, for treatment+rIL-10 versus treatment+rIFN-+rIL-10; , P<0.001, for treatment versus treatment+rIFN-+rIL-6; , P<0.001, for control+rIL-6 versus TLR agonist+rIL-6).
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, IL-10, and IL-6. We establish that stimulation of TLR4 alone by LPS is sufficient for activation of monocyte CD163 up-regulation following shedding. We extend these findings to demonstrate that TLR2 and TLR5 agonists Pam3Cys and S. typhimurium flagellin, respectively, also cause CD163 up-regulation subsequent to decreased surface CD163 induced by cell-surface, TLR-mediated shedding. CD163 up-regulation caused by flagellin was blocked partially by the endotoxin inhibitor PMX, indicating that contaminating LPS, signaling through TLR4, in combination with flagellin activation of TLR5, mediates more robust up-regulation than stimulation with the individual TLR agonists alone. This enhanced up-regulation by LPS-contaminated flagellin was consistent with increased production of IL-6 and IL-10. Furthermore, the purified TLR agonists Pam3Cys and LPS also caused enhanced up-regulation of surface CD163 when used to activate TLR2 and TLR4 concomitantly in comparison with activation of the individual TLRs alone. These data indicate that simultaneous signaling through two different TLRs results in enhanced monocyte cytokine production and up-regulation of CD163. Stimulation of PBMC with cell-surface TLR2, TLR4, and TLR5 agonists results in the release of IL-6 and IL-10, which is coordinated with CD163 up-regulation. The contribution of these cytokines to the up-regulation of CD163 was tested using neutralizing antibodies to IL-6 and IL-10. Up-regulation of CD163 by LPS and Pam3Cys was abrogated completely by IL-10-neutralizing antibodies, and blockade of IL-6 and IL-10 was required to inhibit flagellin-mediated CD163 up-regulation. Previous reports have indicated that IL-10 increases CD163 mRNA directly [12 ]. In contrast, Buechler et al. [9 ] demonstrated that message levels of CD163 are decreased following activation of monocytes with LPS as early as 8 h post-treatment and remain suppressed at least until 32 h post-treatment. This is in agreement with our own published data demonstrating that message levels of CD163 are decreased following an 18-h treatment of purified monocytes with LPS [12 ]. Thus, there is a complex regulatory network by which surface expression of CD163 is altered following activation of monocytes with LPS without a direct correlation to CD163 message levels.
Our data highlight a link between IL-10 production and CD163 up-regulation, two proposed anti-inflammatory sequelae. The link between anti-inflammatory events and TLR activation is perplexing, as recognition of bacterial products would be predicted to elicit a strong, proinflammatory response to stimulate clearance of the infection. A partial anti-inflammatory response elicited by TLR activation could limit the maturation and migration of macrophages from sites of infection prior to their acquisition of high levels of antigen [2 ]. Induction of scavenger and opsoninic receptors by IL-10 may facilitate antigen loading and initiate antigen presentation and adaptive immune responses to the infectious agent [2 ]. Furthermore, production of anti-inflammatory mediators via TLR activation upon contact with avirulent commensals would allow clearance of the infection and prevention of unnecessary tissue damage. This could be important in the regulation of inflammation at mucosal sites where contact with commensal bacteria is common. Thus, the initial innate immune response to infection includes the release of proinflammatory cytokines to recruit new leukocytes to areas of inflamed tissue, in addition to the production of anti-inflammatory mediators such as IL-10, which help to limit damage to the local tissue environment [25 , 26 ].
If an infection is not cleared by an acute immune response, newly infiltrated NK cells and antigen-specific CD4+ T cells will be activated within the inflamed tissue to produce IFN- and/or up-regulate CD40 ligand [27
]. Simultaneous treatment with IFN- and TLR agonists stimulates macrophages to release much higher levels of proinflammatory cytokines, reactive oxygen radicals, and reactive nitrogen species in comparison with macrophages activated with TLR agonists or IFN- alone [28
]. The susceptibility of IFN-–/– and IFN- receptor–/– mice to infection with intracellular pathogens indicates the importance of the IFN- signal to macrophages [33
], which from these knockout mice, are unable to mount the robust, inflammatory response needed to kill intracellular pathogens [33
]. Here, we report that IFN- inhibits CD163 recovery and up-regulation, which would otherwise occur after acute TLR2-, TLR4- or TLR5-induced CD163 shedding.
As IL-10 and IL-6 are induced by TLR activation and are known to induce surface CD163 expression, we postulated that IFN- might inhibit their production or action. As shown in Figure 6
, IFN- abrogated the production of IL-10 completely by human PBMC in response to TLR2, TLR4, and TLR5 agonists. This confirms and extends a recent report by Hu and coworkers [30
], who showed that IFN- treatment of human macrophages suppressed the synthesis and release of IL-10 following TLR2 activation. They demonstrated that IFN- increased the activity of glycogen synthase kinase-3, which correlated with inhibition of AP-1-mediated DNA binding, an important transcription factor involved in IL-10 gene induction [30
]. In striking contrast to IL-10, our data show that IFN- treatment led to a significant increase in IL-6 production in two out of six donors tested after cell-surface TLR ligation and did not alter IL-6 production in four donors. These observations suggest that blockade of IL-10 production is largely responsible for the inhibitory effect of IFN- on CD163 up-regulation.
It is clear from our data that the responsiveness of monocytes to IL-10 is also decreased by IFN-, as exogenous rIL-10 only partially reversed the inhibitory effects of rIFN- on up-regulation of CD163 (Fig. 7)
. This is supported by a recent study showing that rIFN- inhibits IL-10-mediated changes in the gene expression profile of human macrophages without altering surface expression of the IL-10 receptor [34
]. In contrast to rIL-10, which partially reversed the inhibitory effect of IFN- on up-regulation of CD163, exogenous rIL-6 was completely ineffective. In addition, TLR-stimulated monocytes are less responsive to exogenous rIL-6 induction of surface CD163 compared with IL-6 alone. This TLR-mediated decrease in monocyte responsiveness to rIL-6 supports a recent study demonstrating reduced Stat1 and Stat3 activation in LPS-stimulated human macrophages treated with rIL-6, although the direct mechanism of IL-6 signaling inhibition is unknown [35
]. This may be a result of changes in surface expression of the IL-6 receptor following stimulation of monocytes with TLR agonists or IFN- treatment, which have been reported in the literature for rat Kupffer cells and human monocytic cell lines, although with conflicting results [36
37
38
]. Regardless of the mechanism of action, these data indicate that the monocyte activation profile following stimulation of TLRs is influenced dramatically by the cytokine milieu encountered by these cells during an innate immune response to infection.
This report identifies clearly how the monocyte response to infection is dependent on activation of PRRs and the local cytokine environment in which the monocyte resides. These findings highlight the remarkable plasticity of monocytes and the coordinated regulation of their activation by cytokines, two properties that allow these cells to modulate the progression of inflammatory responses. Furthermore, these studies emphasize the potential importance of down-regulating surface CD163 upon classical activation of macrophages. We propose that IFN--mediated suppression of CD163 is an antimicrobial response, which limits pathogen replication by an unknown mechanism. One possibility is that decreased surface expression of CD163 may reduce the contribution of Hb internalization to intracellular iron pools, thereby limiting the growth of intracellular pathogens dependent on iron for replication. Future experiments are needed to determine if CD163-mediated Hb internalization contributes to the intracellular pool of iron during intracellular infection. If our hypothesis is supported by such studies, therapies designed to decrease CD163 expression could be used to limit intracellular replication of pathogens known to infect macrophages.
Received July 4, 2006; revised August 5, 2006; accepted November 2, 2006.
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R triggers shedding of the hemoglobin-haptoglobin scavenger receptor CD163 J. Leukoc. Biol. 76,271-277-primed, C3H/HeJ macrophages to a fully tumoricidal state J. Immunol. 139,3697-3702[Abstract] suppresses IL-10 production and synergizes with TLR2 by regulating GSK3 and CREB/AP-1 proteins Immunity 24,563-574[CrossRef][Medline] in the outcome of chlamydial infection Curr. Opin. Immunol. 14,444-451[CrossRef][Medline] J. Immunol. 171,5034-5041 and tumor necrosis factor- in human monocytic cell line (THP-1). Priming effect of IFN- J. Immunol. 147,2630-2637This article has been cited by other articles:
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