The cytokine IL-6 plays a protective role in immune responses against bacterial infections. However, the mechanisms of IL-6–mediated protection are only partially understood. IL-6 can signal via the IL-6R complex composed of membrane-bound IL-6Rα (mIL-6Rα) and gp130. Owing to the restricted expression of mIL-6Rα, classical IL-6 signaling occurs only in a limited number of cells such as hepatocytes and certain leukocyte subsets. IL-6 also interacts with soluble IL-6Rα proteins and these IL-6/soluble IL-6Rα complexes can subsequently bind to membrane-bound gp130 proteins and induce signaling. Because gp130 is ubiquitously expressed, this IL-6 trans-signaling substantially increases the spectrum of cells responding to IL-6. In this study, we analyze the role of classical IL-6 signaling and IL-6 trans-signaling in the innate immune response of mice against Listeria monocytogenes infection. We demonstrate that L. monocytogenes infection causes profound systemic IL-6 production and rapid loss of IL-6Rα surface expression on neutrophils, inflammatory monocytes, and different lymphocyte subsets. IL-6–deficient mice or mice treated with neutralizing anti–IL-6 mAb displayed impaired control of L. monocytogenes infection accompanied by alterations in the expression of inflammatory cytokines and chemokines, as well as in the recruitment of inflammatory cells. In contrast, restricted blockade of IL-6 trans-signaling by application or transgenic expression of a soluble gp130 protein did not restrain the control of infection. In summary, our results demonstrate that IL-6Rα surface expression is highly dynamic during the innate response against L. monocytogenes and that the protective IL-6 function is dependent on classical IL-6 signaling via mIL-6Rα.
The cytokine IL-6 is a central regulator of inflammatory processes. In response to infection but also during chronic inflammation, IL-6 is produced by variety of cells and has pro- as well as anti-inflammatory activities (1, 2). IL-6 is a major inductor of acute phase proteins and is involved in the control of neutrophil and monocyte responses following infection. IL-6 is also a survival factor for lymphocytes and it promotes Ab production by B cells (1–3). More recently, IL-6 has been identified as a decisive cytokine for the differentiation of CD4+ T cells into Th17 cells, which are considered to be a major proinflammatory T cell population (4, 5). As a consequence, blockade of IL-6 or IL-6 signaling has a profound impact on innate and acquired immune responses. Impaired IL-6 function causes enhanced susceptibility of mice to infection with various pathogens (6–15) but also results in protection in several mouse models for chronic inflammatory diseases (16–18). Not surprisingly, IL-6 signaling is also an important target of intervention in chronic inflammatory human diseases such as rheumatoid arthritis and Crohn’s disease (19).
The IL-6 receptor consists of membrane-bound IL-6Rα (mIL-6Rα, CD126) and the gp130 chain (CD130). mIL-6Rα binds IL-6 but has no intrinsic signaling capacity. Upon IL-6 binding, mIL-6Rα interacts with gp130, which subsequently initiates IL-6 signal transduction (2). In contrast to the ubiquitous expression of gp130, expression of mIL-6Rα is restricted to hepatocytes and certain subsets of leukocytes (20). Thus, IL-6 can only activate a limited number of cells via classical IL-6R signaling. However, a soluble form of IL-6Rα (sIL-6Rα) is constitutively found in the serum, and following inflammation mIL-6Rα is actively shed from inflammatory cells, resulting in enhanced local and systemic sIL-6Rα concentrations (21, 22). sIL-6Rα still binds IL-6, and the IL-6/sIL-6Rα complexes can interact with membrane-bound gp130 and thereby provoke signal transduction (23). This process, which is called IL-6 trans-signaling, allows activation of mIL-6Rα–negative cells by IL-6 and thereby substantially expands the number of IL-6–responsive cells (2, 3). Under normal conditions, IL-6 levels in human blood are in the range of 1–5 pg/ml. sIL-6Rα and soluble gp130 (sgp130) levels, however, are in the range of 50–100 and 100–200 ng/ml, respectively. Secreted IL-6 therefore will bind to sIL-6Rα and the complex of IL-6/sIL-6Rα will bind to sgp130 and thereby be neutralized. Hence, sIL-6Rα and sgp130 constitute a buffer for systemically active IL-6. Only when IL-6 concentrations exceed those of sIL-6Rα and sgp130 can IL-6 act systemically (24, 25).
IL-6/sIL-6Rα complexes also offer a target for therapeutic intervention. Soluble gp130 proteins have been designed that neutralize IL-6/sIL-6Rα complexes but do not interfere with signaling via mIL-6Rα (24, 25). These proteins have been used to dissect the role of classical IL-6 signaling and IL-6 trans-signaling in different mouse models for infection and inflammatory diseases (22, 26, 27). Because sgp130 proteins show beneficial effects in models of chronic inflammation (16, 28, 29), they might also offer a new option for the treatment of chronic inflammatory diseases in humans (30).
The Gram-positive bacterium Listeria monocytogenes can cause severe disease in immune-suppressed individuals, and in pregnant women, infection of the fetus can lead to abortion or to high fatality rates in neonates (31). In mice, infection with L. monocytogenes provokes a rapid activation of the innate immune system, which is essential for the restriction of bacteria replication. In particular, the production of proinflammatory cytokines such as TNF-α and IFN-γ and the recruitment of inflammatory monocytes to sites of infection are crucial for the early control of L. monocytogenes (32, 33). Infection also leads to robust production of IL-6, and IL-6 deficiency or IL-6 neutralization with Abs results in enhanced susceptibility to L. monocytogenes (6, 34–41); however, mechanisms of IL-6–mediated protection are so far unclear.
In this study, we characterize the role of different IL-6 signaling pathways for the innate control of L. monocytogenes. We demonstrate that following infection, mIL-6Rα is rapidly lost from the surface of all leukocytes analyzed. In parallel, high serum levels of IL-6 can be detected, providing the basis for classical IL-6 signaling and IL-6 trans-signaling. Using neutralizing anti–IL-6 mAbs, which block IL-6 classical and trans-signaling, and a sgp130 Fc fusion protein (sgp130Fc), which only blocks IL-6 trans-signaling, as well as IL-6–deficient mice and mice transgenic (Tg) for sgp130Fc, we can show that classical IL-6 signaling but not IL-6 trans-signaling is crucial for the IL-6–mediated control of L. monocytogenes.
Materials and Methods
Abs and reagents
A soluble fusion protein of the extracellular domains of gp130 and the Fc part of human IgG1 (sgp130Fc) was constructed and purified as described in Jostock et al. (25). As demonstrated before, human sgp130Fc can bind and neutralize mouse IL-6/IL-6Rα complexes with similar efficacy as mouse sgp130Fc (25). Purified human IgG was purchased from Dianova (Hamburg, Germany).
Ligand binding capacity and biological activity of sgp130Fc were measured by a Ba/F3-gp130 cell proliferation assay. Ba/F3 cells (murine pre-B cells) stably transfected with human gp130 (Ba/F3-gp130 cells) have been described previously (25). Ba/F3-gp130 cells were seeded with 5 × 103 cells per well in 96-well plates. Proliferation was induced by adding 100 ng/ml IL-6 plus 50 ng/ml sIL-6Rα and different concentrations of sgp130Fc. Proliferation was measured by the cell titer colorimetric assay (Promega) as described previously (42). The activity assay for the batch of sgp130Fc used for the present study is shown in Supplemental Fig. 1.
Mice and animal experiments
C57BL/6 mice, IL-6 knockout (IL-6KO) mice (6), sgp130FcTg mice (24), and CCR2KO mice (43) were bred at the Central Animal Facilities of the University Medical Center Hamburg–Eppendorf, at the House for Experimental Therapy in Bonn, or at the Victor Hensen Facility of the University of Kiel. IL-6KO and sgp130Tg mice were on the C57BL/6N background. In experiments with these mouse strains, matched C57BL/6N mice were used as controls. All other experiments were done with C57BL/6J mice. Animal experiments were conducted according to the German Animal Protection Law.
Mice were i.v. infected via the lateral tail vein with 2 × 104 L. monocytogenes strain EGD in 200 μl PBS. Bacterial inoculi were always controlled by plating serial dilutions on tryptic soy broth agar plates. For determination of bacterial burdens in spleens and livers, mice were killed, organs were homogenized in PBS containing 0.5% Triton X-100, serial dilutions of homogenates were plated on tryptic soy broth agar, and colonies were counted after 48 h incubation at 37°C.
For blocking of IL-6 signaling or trans-signaling, mice were treated with 500 μg neutralizing anti–IL-6 mAb (clone MP5-20F3) (44) or 250 μg sgp130Fc. Concentrations of anti–IL-6 mAb and sgp130Fc have been shown to be effective in blocking global IL-6 signaling and trans-signaling (26). Control mice received 250 μg purified human IgG. Abs and fusion proteins were injected i.p. in a volume of 200 μl PBS 1 d prior to L. monocytogenes infection. Abs and sgp130Fc proteins were controlled for low endotoxin content using a commercial Limulus amebocyte lysate assay (Lonza, Basel, Switzerland).
Purification and staining of cells
For characterization of different leukocyte subsets, spleens and livers were cut into small pieces and digested for 45 min with each 0.25 mg/ml collagenase D (Roche, Penzberg, Germany) and collagenase VIII (Sigma-Aldrich, Steinheim, Germany), and 15 μg/ml DNAse I (Sigma-Aldrich) in RPMI 1640 medium supplemented with glutamine, gentamicin, 2-ME, and 10% heat-inactivated FCS (complete RPMI 1640 medium). The tissue was gently meshed through a 70-μm cell strainer and the suspension was centrifuged to pellet the cells. Liver cells were resuspended and centrifuged through a 40/70% Percoll gradient (Biochrom, Berlin, Germany) for 30 min at 600 × g. Cells were collected from the interface of the gradient and washed in complete RPMI 1640 medium. Erythrocytes were lysed and cells were washed and stored on ice. After collagen digestion, spleen cells were pelleted and erythrocytes were lysed. Cells were washed and stored on ice. For analysis of surface IL-6Rα and induction of IL-6, spleen cells were purified without collagen digestion.
Induction of cytokines and intracellular cytokine staining
Cells were cultured in a volume of 1 ml complete RPMI 1640 medium. Cells were stimulated for 6 h with 107 heat-killed listeria (HKL)/ml. During the final 4 h of culture, 10 μg/ml brefeldin A (Sigma-Aldrich) was added. Cultured cells were washed and incubated for 5 min with rat serum and anti-CD16/CD32 mAb to block unspecific Ab binding. Subsequently, cells were stained with Abs against surface proteins, and after 30 min on ice, cells were washed with PBS and fixed for 20 min at room temperature with PBS/4% paraformaldehyde. Cells were washed with PBS/0.2% BSA, permeabilized with PBS/0.1% BSA/0.3% saponin, and incubated in this buffer with rat serum and anti-CD16/CD32 mAbs. After 5 min, fluorochrome-conjugated anti–IL-6 mAb and anti–TNF-α mAb was added. After a further 20 min on ice, cells were washed with PBS and fixed with PBS/1% paraformaldehyde.
For the preparation of HKL, bacteria from an overnight culture were washed twice in PBS and inactivated by incubation for 30 min at 80°C.
IL-6 and sIL-6Rα determination
Real-time RT-PCR analysis
The following primers were used: 18S, forward, 5′-CAG GGC CGG TAC AGT GAA AC-3′, reverse, 5′-AGA GGA GCG AGC GAC CAA A-3′; CXCL1, forward, 5′-GCA CCC AAA CCG AAG TCA TAG-3′, reverse, 5′-CAA GGG AGC TTC AGG GTC AA-3′; CXCL2, forward, 5′-CAC TGC GCC CAG ACA GAA-3′, reverse, 5′-CAG GGT CAA GGC AAA CTT TTT G-3′; CCL2, forward, 5′-GGC TCA GCC AGA TGC AGT TAA-3′, reverse, 5′-CCT ACT CAT TGG GAT CAT CTT GCT-3′; IL-6, forward, 5′-TGG GAA ATC GTG GAA ATG AGA-3′, reverse, 5′-AAG TGC ATC ATC GTT GTT CAT ACA-3′, TNF-α, forward, 5′-AAA TGG CCT CCC TCT CAT CAG T-3′, reverse, 5′-GCT TGT ACA AAT TTT GAG AAG-3′; SAA, forward 5′-AGA GGA CAT GAG GAC ACC ATT GCT-3′, reverse, 5′-AGG ACG CTC AGT ATT TGT CAG GCA-3′.
All mice per group were independently analyzed. Statistical analysis was performed with GraphPad Prism 4 (GraphPad Software, La Jolla, CA). Quantifiable data were transformed into logarithmic values. Afterward, a Student t test or one-way ANOVA and the Bonferroni multiple posttest was performed to define differences among differently treated mice. All results are expressed as arithmetic means ± SD (bar graphs) or median (dot plots). Differences were considered significant with p < 0.05.
IL-6 and IL-6Rα expression during early L. monocytogenes infection
IL-6 signals via membrane-bound IL-6Rα and gp130 (classical signaling). Alternatively, IL-6 can form complexes with sIL-6Rα, which subsequently interact with gp130 on the cell surface and thereby allow activation of cells lacking surface expression of IL-6Rα. IL-6Rα can be actively shed from the surface of cells under inflammatory conditions. Thus, the mode of IL-6 signaling could switch from classical to trans-signaling following infection. In a first set of experiments, we therefore determined the concentrations of IL-6 and sIL-6Rα in serum and tissues as well as the surface expression level of IL-6Rα on different leukocyte populations following infection of mice with L. monocytogenes.
Infection of mice with L. monocytogenes caused a profound increase in serum levels of IL-6, which reached a maximum at day 2 postinfection (Fig. 1A). Enhanced IL-6 concentrations were also measured in tissue extracts from spleens but not from liver (Fig. 1B, 1C), although both organs are main target sites for listeria replication. A similar pattern was observed for IL-6 mRNA (Fig. 1F). IL-6 mRNA induction was largely confined to the spleen (100- to 1000-fold induction) and only marginally in the liver (<10-fold induction). Most likely as a consequence of the high serum concentration of IL-6, a strong induction of the mRNA for the IL-6 target gene saa followed by enhanced SAA protein concentration was observed in livers of infected mice (Fig. 1E, 1G). In contrast to the strong increase in IL-6, there was only a marginal change in the serum concentrations of sIL-6Rα during the first days of L. monocytogenes infection (Fig. 1D).
In a next set of experiments, we determined the expression of IL-6Rα on innate immune cells following L. monocytogenes infection. As shown before (46, 47), infection of mice with L. monocytogenes caused a rapid accumulation of neutrophils and inflammatory monocytes in spleen and liver (Supplemental Figs. 3, 4). Flow cytometric analysis revealed IL-6Rα surface expression on inflammatory monocytes as well as on CD4+ and CD8+ T cells of naive mice. In contrast, neutrophils and B cells displayed only relatively low mIL-6Rα expression (Figs. 2, 3). Closer examination of CD4+ and CD8+ T cells showed that in particular naive CD4+ T cells (CD44lowCD62L+) were IL-6Rα+. Activated and memory CD4+ T cells (CD44highCD62L− or CD44highCD62L+) contained both IL-6Rα+ and IL-6Rα− populations. Following infection, we observed a significant loss of IL-6Rα surface expression on all cells analyzed (Figs. 2, 3). Loss of IL-6Rα expression was particular strong on CD4+ T cells and included the naive CD44lowCD62L+CD4+ T cell population (Fig. 3). These results indicate that following L. monocytogenes infection, all analyzed cell populations rapidly lose the ability to respond to IL-6 via classical IL-6 signaling and become dependent on IL-6 trans-signaling.
Loss of surface IL-6Rα expression could be due to proteolytic shedding (22). However, it is also possible that the receptor is internalized in response to IL-6 signaling. Finally, IL-6 could interfere with the binding of the anti–IL-6Rα mAb to mIL-6Rα. To distinguish between these processes, IL-6KO mice were infected with L. monocytogenes and the expression of mIL-6Rα was analyzed. On all leukocyte subsets including CD4+ T cells, CD8+ T cells, granulocytes, and inflammatory monocytes, we observed a similar loss of mIL-6Rα expression in wild-type (wt) and IL-6KO mice (Fig. 4 and data shown). Therefore, loss of mIL-6Rα expression is most likely due to shedding and is not a consequence of receptor internalization or epitope masking.
Inflammatory monocytes are not an essential source of IL-6 and sIL-6Rα
Different cell types have been identified as sources of IL-6 during inflammation and infection (1). Because we observed a strong accumulation of granulocytes and inflammatory monocytes, we tested whether these cells were potential sources of IL-6 during early L. monocytogenes infection. Upon stimulation of spleen cells with HKL, a large fraction of inflammatory monocytes were indeed able to produce IL-6 as determined by intracellular cytokine staining (Fig. 5). Frequencies of inflammatory monocytes producing IL-6 in response to HKL increased until day 2 of infection and then declined again. In these cells, IL-6 production was accompanied by a strong expression of TNF-α. Without stimulation, neutrophils showed already low expression levels of IL-6, but expression did not increase after addition of HKL. Frequencies of granulocytes producing IL-6 declined postinfection.
Because inflammatory monocytes expressed IL-6Rα on their surface and were able to secrete IL-6 upon stimulation with listeria, we determined the impact of this cell population on serum levels of IL-6 and IL-6Rα during the early phase of L. monocytogenes infection. Serbina and Pamer (48) recently demonstrated that following L. monocytogenes infection, mobilization of inflammatory monocytes from the bone marrow and accumulation of these cells in infected tissues were strictly dependent on the chemokine receptor CCR2. Therefore, wt and CCR2KO mice were infected with L. monocytogenes and analyzed 3 d postinfection. Determination of L. monocytogenes burden in spleen and livers revealed 100-fold higher titers in CCR2KO mice (Fig. 6A). Compared to infected wt mice, infected CCR2KO mice completely failed to recruit inflammatory monocytes to the spleen and had significantly reduced frequencies of these cells in the liver (Fig. 6B). In contrast, CCR2KO mice displayed high frequencies of granulocytes in spleen and liver following infection (data not shown). Determination of IL-6 revealed increased serum IL-6 levels in infected CCR2KO mice when compared with infected wt mice (Fig. 6C). Serum concentrations of sIL-6Rα were reduced in naive and infected CCR2KO mice when compared with wt controls (Fig. 6C), but reduction did not reach a significant level in infected animals (naive, wt 16.73 ± 0.86 ng/ml versus CCR2KO 12.21 ± 1.13 ng/ml, p = 0.018; infected, wt 15.68 ± 1.28 ng/ml versus CCR2KO 12.25 ± 1.57 ng/ml, p = 0.054). In summary, our results indicate that although inflammatory monocytes are fundamental in control of L. monocytogenes they are not an essential source for IL-6 and they only partially contribute to the generation of sIL-6Rα.
Blocking of conventional IL-6 signaling but not IL-6 trans-signaling caused impaired control of L. monocytogenes infection
Our results demonstrated the presence of IL-6 and sIL-6Rα during the early phase of L. monocytogenes infection. Thus, IL-6 could interact with target cells both via classical and trans-signaling. To determine the impact of classical and trans-signaling on the control of L. monocytogenes, mice were treated with a neutralizing anti–IL-6 mAb, which blocks all IL-6 signaling (44), or with a sgp130Fc fusion protein to selectively block IL-6 trans-signaling (26–29). Treatment of mice with a neutralizing anti–IL-6 mAb caused an increase in the listeria burden in the spleen and particularly in the liver of infected mice (Fig. 7A). In contrast, mice treated with sgp130Fc showed listeria titers in both organs, which were comparable to infected control animals. Thus, classical IL-6 signaling but not IL-6 trans-signaling was important for the control of L. monocytogenes infection.
Following infection, we observed pronounced accumulation of inflammatory monocytes in spleen and liver, which was not further affected by the treatment with anti–IL-6 mAb or sgp130Fc (Fig. 7B). At day 3 postinfection, there was still an enlarged neutrophil population in spleens of infected mice. Pretreatment of mice with sgp130Fc but not with anti–IL-6 mAb resulted in a reduction of this neutrophil population. In the liver, neither treatment with anti–IL-6 mAb or with sgp130Fc caused an alteration of neutrophil frequencies.
To determine changes in the degree of inflammation following different treatments, the mRNA levels of SAA, IL-6, TNF-α, and of different chemokines were measured. L. monocytogenes infection caused a strong induction of SAA mRNA in livers of mice, which was reduced by anti–IL-6 mAb but not by sgp130Fc treatment (Fig. 8A). The mRNA for IL-6 was enhanced in spleens of infected mice but was not further affected by anti–IL-6 mAb or sgp130Fc treatment (Fig. 8B). There was limited induction of IL-6 mRNA in the livers of mice. Treatment with anti–IL-6 mAb caused enhanced IL-6 mRNA expression, but the difference did not reach a significant level. The mRNA for TNF-α was increased in spleens and livers of infected mice, but neither anti–IL-6 mAb nor sgp130Fc caused a change in expression (Fig. 8C). We detected enhanced levels of the neutrophil-attracting chemokines CXCL1 and CXCL2 and of the monocyte-attracting chemokine CCL2 (Fig. 8D–F). All three chemokines were more strongly induced in the spleen than in the liver. For all three chemokines, we observed the strongest expression levels in spleens of anti–IL-6 mAb–treated mice, which were significantly higher than the expression levels in spleens of sgp130Fc-treated mice.
IL-6 deficiency but not Tg expression of sgp130Fc resulted in impaired control of L. monocytogenes
To confirm results from studies with anti–IL-6 mAb and sgp130Fc treatment, IL-6KO mice and sgp130FcTg mice were infected with L. monocytogenes. spg130FcTg mice express a gp130Fc fusion protein under the control of the liver-specific phosphoenolpyruvate carboxykinase promoter, resulting in constitutively high serum levels of sgp130Fc protein and selective blockade of IL-6 trans-signaling (24). Deficiency in IL-6 caused enhanced listeria titers in spleen and liver (Fig. 9A). In contrast, Tg expression of sgp130Fc did not impair the control of L. monocytogenes infection. Listeria titers were even slightly lower than titers of infected wt mice. Similar to mice treated with sgp130Fc, sgp130FcTg mice demonstrated reduced accumulation of granuloytes in spleens of infected mice (Fig. 9B). However, sgp130FcTg mice in addition displayed diminished frequencies of inflammatory monocytes in spleen and liver.
Following L. monocytogenes infection, we measured high serum levels of IL-6 at days 2 and 3 postinfection, which is in agreement with published results (39–41). Inflammatory monocytes are essential for the protective innate immune response against L. monocytogenes (33, 48), and we could demonstrate that these cells produced IL-6 as well as TNF-α following stimulation with HKL. However, despite their fundamental role in L. monocytogenes control and their IL-6 response to listeria Ag, inflammatory monocytes were not an essential source for systemic IL-6. Infection of CCR2KO mice, which failed to mobilize inflammatory monocytes from the bone marrow (48), resulted in even enhanced IL-6 serum concentration when compared with infected control mice. Alternative IL-6 sources could include other myeloid cells such as tissue-resident macrophages, dendritic cells, and granulocytes or T and B cells, but also nonhematopoietic cells such as endothelial or epithelial cells (49). Owing to the more severe infection, some of these cells might respond even stronger in CCR2KO mice.
In naive mice, we detected mIL-6Rα expression on T cells and inflammatory monocytes. In particular, CD4+ T cells expressed high levels of mIL-6Rα. Granulocytes expressed only low mIL-6Rα levels and B cells were largely negative. Naive T cells (CD62LhighCD44low) were uniformly mIL-6Rα+, and effector as well as memory T cells (CD62LlowCD44high or CD62LhighCD44high) contained mIL-6Rα+ and mIL-6Rα− subpopulations. Following L. monocytogenes infection, all analyzed leukocyte subsets lost mIL-6Rα expression. On T cells, reduction of mIL-6Rα was observed on naive as well as preactivated subpopulations. A similar reduction of mIL-6Rα expression was also observed in infected IL-6KO mice, which excludes IL-6–induced receptor internalization or interference of IL-6 with anti-IL6Rα mAb binding to IL-6Rα as a cause for low IL-6Rα surface staining (50). Shedding of mIL-6Rα by ADAM10 and ADAM17 has been identified as a general mechanism of sIL-6Rα generation in mice (51), and it is very likely that shedding is responsible for the loss of IL-6Rα surface expression following L. monocytogenes infection. TCR triggering has been described as a signal for IL-6Rα shedding (52, 53); however, the global loss of IL-6Rα on naive and effector cells argues against a specific TCR signal at this early stage of infection. It is more likely that loss of IL-6Rα expression is due to shedding induced by inflammatory mediators such as proinflammatory cytokines, chemokines, or acute phase proteins (52, 53). Our present work aims at defining these mediators in our infection model.
Despite the profound loss of mIL-6Rα on all analyzed leukocyte subsets, we observed only a marginal increase in serum concentration of sIL-6Rα following L. monocytogenes infection. This result would argue that leukocytes are not a major source of sIL-6Rα or that concentrations of sIL-6Rα are tightly controlled and excessive sIL-6Rα is rapidly eliminated from the circulation. Interestingly, naive and infected CCR2KO mice showed a reduction of sIL-6Rα concentrations. Thus, inflammatory monocytes or other CCR2-dependent cells represent a source for sIL-6Rα already under homeostatic conditions. Recently, it was demonstrated that IL-6Rα deficiency in hepatocytes (AlbCre+Il6rαfl/fl mice) results in a 30% reduction of sIL-6Rα serum levels, and deficiency in granulocytes and macrophages (LysMCre+Il6rαfl/fl mice) results in a 60% reduction of these levels (54). Because lysozyme M is most likely also expressed in inflammatory monocytes, our results are in accordance with this conclusion and add inflammatory monocytes to the list of cells involved in constitutive sIL-6Rα production.
Differential blockade of global IL-6 signaling by neutralizing mAb and IL-6 trans-signaling by sgp130Fc indicated that classical signaling is sufficient for early control of L. monocytogenes infection. Infection of IL-6KO and sgp130FcTg mice confirmed this observation. Compared to wt mice or mice treated with sgp130Fc proteins, sgp130FcTg mice demonstrated even better control of L. monocytogenes infection. It is currently unclear why sgp130Tg mice show this improved control. The constant Tg expression of sgp130Fc and the constant inhibition of IL-6 trans-signaling could cause alterations in development and cellular composition of the immune system, which might be responsible for the enhanced control of infection. In mice with a global blockade of IL-6, the increase in listeria titers was more pronounced in the liver, and thus IL-6 could be either particularly important for the innate response in the liver (e.g., through the hepatic induction of acute phase proteins) or there might be compensatory mechanisms in the spleen. Listeria infection induced a strong upregulation of the mRNA for several proinflammatory cytokines and chemokines. We observed a slight increase in chemokine mRNA expression following anti–IL-6 mAb treatment; however, it is unclear whether this was due to defective IL-6 signaling or due to a higher bacterial load. In contrast, treatment of mice with sgp130Fc did not change the mRNA expression levels of any of the analyzed factors. Infection with L. monocytogenes induced accumulation of neutrophil granulocytes in spleen and liver. Interestingly, sgp130Fc treatment caused a reduction of neutrophil accumulation in the spleen. Recent studies from Shi et al. (46) and Carr et al. (47) analyzed the role of neutrophils for the control of L. monocytogenes. In those studies, depletion of neutrophils had either no consequences (46) or only affected control of infection when relatively high doses of bacteria where applied (47). Under the latter conditions, depletion of neutrophils mainly impaired control of infection in the liver (47). Thus, it is not surprising that reduction in neutrophil numbers in the spleen was not accompanied by a more severe infection.
It has been demonstrated that IL-6 trans-signaling is responsible for a switch from a granulocyte to a monocyte/macrophage response (55). We observed reduction of monocyte frequencies in sgp130FcTg mice, but there was no change in monocyte frequencies in sgp130Fc- or anti-IL-6–treated animals. Thus, blockade of classical IL-6 signaling or IL-6 trans-signaling does not generally result in a reduced recruitment of monocytes to sites of L. monocytogenes infection. The failure to observe profound changes in the monocyte responses is most likely due to the infection model. Control of L. monocytogenes infection is highly dependent on recruitment of monocytes to the site of infection. Chemokines such as CCR2 ligands have been shown to release monocytes from the bone marrow (48). L. monocytogenes or material from these bacteria can induce production of CCR2 ligands in infected sites but also within the bone marrow, and this increase in local chemokines might be sufficient for releasing monocytes into the circulation (56). Alternatively, CCR2 ligands and chemokines in general appear to be dispensable for the accumulation of monocytes in the infected spleen and liver (48, 57). Thus, monocyte recruitment following L. monocytogenes infection could be largely independent of IL-6 and IL-6–mediated chemokine production at the sites of infection.
In summary, our results indicate that the protective function of IL-6 occurs in the absence of IL-6 trans-signaling, which also implies that protection is mainly due to mIL-6Rα+ cells. The identity of these cells is currently unclear. Although recruitment of inflammatory monocytes is not impaired by IL-6 deficiency, these cells might be less functional without IL-6 stimulation. IL-6 could also operate in the activation and differentiation of NK cells, of unconventional “innate” T cells, or of conventional T cells. Finally, IL-6 might induce protective mechanisms in hepatocytes, for example, via the induction of the acute phase proteins. The response of these different cell types is currently under investigation by our group. Our results also suggest that IL-6 trans-signaling is not essential for protection against certain bacterial infections. Owing to this residual antibacterial immune response, blockade of IL-6 trans-signaling could therefore be of advantage to a global blockade of IL-6 for the treatment of chronic inflammatory diseases.
Dr. Rose-John is an inventor on patents describing the function of sgp130Fc. He is also a shareholder of the CONARIS Research Institute (Kiel, Germany), which is commercially developing sgp130Fc proteins as therapeutics for inflammatory diseases. The other authors have no financial conflicts of interest.
We thank Georg Waetzig for the production of sgp130Fc protein and Lorenz Knackstedt for technical support.
This work was supported by Deutsche Forschungsgemeinschaft Grants SFB841 (to S.R.-J. and H.-W.M.), SFB877 (to S.R.-J. and A.C.), GRK841 (to J.H. and I.Y.), and KFO228 (to D.R.E., O.M.S., and H.-W.M.), and by the Cluster of Excellence “Inflammation at Interfaces” (to S.R.-J. and A.C.).
The online version of this article contains supplemental material.
Abbreviations used in this article:
- heat-killed listeria
- membrane-bound IL-6Rα
- serum amyloid A
- soluble gp130
- soluble gp130 Fc fusion protein
- soluble IL-6Rα
- Received April 10, 2012.
- Accepted October 27, 2012.
- Copyright © 2013 by The American Association of Immunologists, Inc.