IgA is predominantly recognized to play an important role in host defense at mucosal sites, where it prevents invasion of pathogens by neutralization. Although it has recently become clear that IgA also mediates other immunological processes, little remains known about the potential of IgA to actively contribute to induction of inflammation, particularly in nonmucosal organs and tissues. In this article, we provide evidence that immune complex formation of serum IgA plays an important role in orchestration of inflammation in response to pathogens at various nonmucosal sites by eliciting proinflammatory cytokines by human macrophages, monocytes, and Kupffer cells. We show that opsonization of bacteria with serum IgA induced cross-talk between FcαRI and different TLRs, leading to cell type–specific amplification of proinflammatory cytokines, such as TNF-α, IL-1β, IL-6, and IL-23. Furthermore, we demonstrate that the increased protein production of cytokines was regulated at the level of gene transcription, which was dependent on activation of kinases Syk and PI3K. Taken together, these data demonstrate that the immunological function of IgA is substantially more extensive than previously considered and suggest that serum IgA–induced inflammation plays an important role in orchestrating host defense by different cell types in nonmucosal tissues, including the liver, skin, and peripheral blood.

Immunoglobulin A is the most prevalent Ab in the human body, making up ∼75% of daily Ab production (1). IgA is particularly highly expressed at mucosal tissues as dimeric molecules, where it is secreted as secretory IgA (SIgA). In addition, IgA is the second most common Ab in serum, where it is primarily expressed as a monomer (2). The prevailing view on the immunological function of IgA is based on SIgA, which binds to and neutralizes pathogens and, thereby, protects mucosal surfaces from infection. However, in recent years, it has become apparent that IgA, particularly the nonsecreted form, as is present in serum (as monomer) and lamina propria (as dimer), has additional immunological functions. Many of these functions are mediated by binding of IgA to FcαRI (or CD89), which recognizes conventional IgA but not SIgA (3).

FcαRI is expressed by a variety of myeloid cells, including neutrophils, macrophages, monocytes, and Kupffer cells (47). Ligation of FcαRI can induce inhibitory and activating responses. Binding of soluble IgA to FcαRI triggers inhibitory signals by blocking the activation of other receptors (8, 9). In contrast, IgA immune complexes, which are formed upon opsonization of pathogens, have been described to induce activating processes. For example, Kupffer cells, which are tissue-resident macrophages of the liver, use FcαRI to phagocytose and, thereby, remove IgA-opsonized pathogens that have invaded the body via the intestine and the portal circulation (7). In addition, FcαRI stimulation of neutrophils, basophils, and monocytes has been shown to enhance processes, such as phagocytosis, Ab-dependent cellular cytotoxicity, reactive oxygen species production, and nuclear extracellular trap formation (3, 10).

Although it is clear that IgA immune complexes mediate immunological processes, such as phagocytosis, generation of reactive oxygen species, and NETosis, it is still largely unclear whether serum IgA also has the potential to actively contribute to initiation of inflammatory responses by FcαRI-expressing immune cells. Key for the initiation and skewing of innate and adaptive immunity is the production of various kinds of pro- and anti-inflammatory cytokines and chemokines by myeloid APCs (11). Cytokine production by these cells is classically known to be induced by pathogen recognition through different families of pattern recognition receptors (PRRs), which include TLRs (12), NOD-like receptors (13), and C-type lectin receptors (14). However, direct stimulation of FcαRI-expressing myeloid cells with serum IgA has not been demonstrated to have substantial effects on cytokine production by these cells.

In this study, we have taken into account that FcαRI-expressing myeloid APCs that engage pathogens opsonized with serum IgA are simultaneously activated via FcαRI and PRRs. In this article, we show that opsonization of bacteria with serum IgA increased proinflammatory cytokine production. Although individual stimulation with serum IgA immune complexes barely induced cytokine production, costimulation of different PRR ligands with IgA immune complexes strongly amplified the production of proinflammatory cytokines by human macrophages, monocytes, and Kupffer cells. Interestingly, FcαRI–TLR cross-talk amplified the production of cytokines, such as TNF-α, IL-1β, IL-6, and IL-23, in a cell type–specific manner. Furthermore, we show that this effect was regulated at the level of gene transcription and was dependent on activation of kinases Syk and PI3K. Combined, these data indicate that immune complex formation of serum IgA induces protective proinflammatory responses by different myeloid immune cells at various nonmucosal tissues in the human body.

Monocytes for macrophage differentiation were isolated from buffy coats (Sanquin Blood Supply) by density gradient centrifugation using Lymphoprep (Nycomed) and Percoll (Pharmacia). Macrophages were differentiated by culturing monocytes for 6 d in IMDM (Lonza) containing 5% FBS (Biowest) and 86 μg/ml gentamicin (Life Technologies), supplemented with 20 ng/ml GM-CSF (Invitrogen). At day 2 or 3, half of the medium was replaced with new medium containing cytokines.

For direct stimulation, monocytes were isolated from PBMCs using MACS isolation with CD14 MicroBeads (Miltenyi Biotec).

Human Kupffer cells were obtained from Thermo Fisher Scientific. Cells were thawed according to the manufacturer’s instructions.

Macrophages were harvested at day 6 by removing medium, washing the cells with PBS, and adding TrypLE Select (Invitrogen). Cells (30,000–50,000 per well) were stimulated with 10 μg/ml Pam3CSK4 (Pam3; InvivoGen), 100 ng/ml LPS (from Escherichia coli o111:B4; Sigma-Aldrich), 20 μg/ml polyinosinic-polycytidylic acid (Sigma-Aldrich), 10 μg/ml MDP (InvivoGen), and complexed IgA (c-IgA).

Stimulation with bacteria was performed in X-VIVO 15 culture medium (Lonza) using Pseudomonas aeruginosa PA01 (0.1 bacteria per cell) and Staphylococcus aureus 42D (10 bacteria per cell).

For c-IgA stimulation, 96-well high-affinity MaxiSorp plates (Nunc) were coated with 4 μg/ml serum IgA (MP Biomedicals) or 4 μg/ml dimeric IgA (Nordic-MUbio) diluted in PBS overnight at room temperature, followed by blocking with PBS containing 10% FBS for 1 h at 37°C.

For silencing of Syk, macrophages were harvested at day 3 using TrypLE Select, microporated in the presence of 250 nM Syk SMARTpool siRNA or control small interfering RNA (both from Dharmacon), and cultured for three additional days in the presence of GM-CSF.

Cells were stimulated for 24 h, and supernatant was stored at −20°C until analysis by ELISA. Cytokine levels were measured for TNF-α (eBioscience), IL-1β, IL-6, IL-23 (U-CyTech Biosciences), and IL-10 (BD Pharmingen).

FcαRI expression was determined using 5 μg/ml anti-FcαRI–PE Ab (CD89, MIP8a; Abcam), and fluorescence was measured on a FACSCanto II (BD Biosciences). FcαRI was blocked using 20 μg/ml anti-FcαRI (MIP8a; Abcam) and incubated for 30 min on ice in the presence or absence of FcR Blocking Reagent (Miltenyi Biotec), after which medium and stimuli were added to a final concentration of 5 μg/ml.

Kinases were inhibited by preincubating macrophages with the inhibitor for 30 min at 37°C. The following inhibitors were used: 0.5 μM R406 (Syk), 12.5 μM LY294002 (PI3K; both from Selleckchem), and 100 nM wortmannin (PI3K; InvivoGen).

To determine mRNA levels, cells were lysed at the indicated time points, and mRNA extraction was performed using an RNeasy Mini Kit (QIAGEN), and cDNA synthesis was performed using a RevertAid H Minus First Strand cDNA Synthesis Kit (Fermentas). Quantitative real time PCR was performed using a StepOnePlus Real-Time PCR System, TaqMan Master Mix, and TaqMan primers (all from Thermo Fisher Scientific).

Upon penetrating the body’s barriers, pathogens, such as bacteria, are rapidly opsonized by serum IgA. To determine whether IgA opsonization affects the orchestration of immune responses, we assessed the effect of opsonization of P. aeruginosa, a Gram-negative bacterium. Opsonization of bacteria with serum IgA was verified as previously described (15). The effect of IgA opsonization was assessed by coculturing monocyte-derived (GM-CSF) macrophages with live IgA–opsonized or nonopsonized bacteria for 24 h, after which supernatant was harvested and analyzed for cytokine levels. Interestingly, IgA opsonization of P. aeruginosa strongly increased the production of proinflammatory cytokines TNF-α, IL-1β, IL-6, and IL-23 (Fig. 1). A similar effect, although less pronounced, was observed after stimulation with the Gram-positive bacterium S. aureus (Supplemental Fig. 1). These results indicate that opsonization of bacteria with serum IgA enhances proinflammatory cytokine production by human macrophages.

FIGURE 1.

Opsonization of bacteria with serum IgA enhances proinflammatory cytokine production by human macrophages. Macrophages were stimulated with P. aeruginosa (multiplicity of infection = 0.1), which were nonopsonized or opsonized with 5 mg/ml serum IgA for 1 h and washed. Experiments were performed in triplicate. After 24 h, cytokine levels were analyzed using ELISA (mean + SEM). Data are representative of three experiments with different donors.

FIGURE 1.

Opsonization of bacteria with serum IgA enhances proinflammatory cytokine production by human macrophages. Macrophages were stimulated with P. aeruginosa (multiplicity of infection = 0.1), which were nonopsonized or opsonized with 5 mg/ml serum IgA for 1 h and washed. Experiments were performed in triplicate. After 24 h, cytokine levels were analyzed using ELISA (mean + SEM). Data are representative of three experiments with different donors.

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The main receptor for recognizing IgA immune complexes, as formed upon opsonization of bacteria, is FcαRI (16), which is expressed by several human myeloid APCs. Macrophages showed relatively low FcαRI expression, whereas primary monocytes and Kupffer cells expressed relatively high levels of FcαRI (Fig. 2A).

FIGURE 2.

Synergy between IgA immune complexes and TLR ligands amplifies cytokine production by human macrophages, monocytes, and Kupffer cells. (A) FcαRI expression was analyzed on unstimulated macrophages, monocytes, and Kupffer cells by flow cytometry, compared with background fluorescence. (B and D) Cells were stimulated with Pam3, IgA, or both. After 24 h, supernatants were analyzed using ELISA (mean + SEM of triplicate, representative example). (C) Cells were stimulated with Pam3 alone or combined with c-IgA. Each pair of dots represents one donor. *p < 0.05, ***p < 0.001, Mann–Whitney U test.

FIGURE 2.

Synergy between IgA immune complexes and TLR ligands amplifies cytokine production by human macrophages, monocytes, and Kupffer cells. (A) FcαRI expression was analyzed on unstimulated macrophages, monocytes, and Kupffer cells by flow cytometry, compared with background fluorescence. (B and D) Cells were stimulated with Pam3, IgA, or both. After 24 h, supernatants were analyzed using ELISA (mean + SEM of triplicate, representative example). (C) Cells were stimulated with Pam3 alone or combined with c-IgA. Each pair of dots represents one donor. *p < 0.05, ***p < 0.001, Mann–Whitney U test.

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To assess the effect of serum IgA immune complexes on cytokine production, macrophages, monocytes, and Kupffer cells were stimulated with c-IgA, generated by coating serum IgA to a high-affinity plate. c-IgA stimulation alone induced little production of the key proinflammatory cytokine TNF-α (Fig. 2B). Similarly, the three cell types moderately increased TNF-α production after stimulation with the TLR2 ligand Pam3 (Fig. 2B). Strikingly, however, combined stimulation with Pam3 and c-IgA, as would occur upon exposure to IgA-opsonized pathogens, led to a strong upregulation of TNF-α production by all three cell types (representative example in Fig. 2B; multiple donors in Fig. 2C). Dimeric IgA, which is also present in (relatively low concentrations) serum (17), amplified TNF-α production in a similar manner as monomeric IgA (Supplemental Fig. 2).

In addition to TNF-α, we assessed the production of other pro- and anti-inflammatory cytokines. In macrophages, we observed a strong increase in the production of proinflammatory cytokines IL-1β and IL-23, whereas IL-6 production was only moderately affected (Fig. 2D). Interestingly, in macrophages, the anti-inflammatory cytokine IL-10 was also upregulated after costimulation (Fig. 2D). In monocytes, costimulation with Pam3 and c-IgA synergistically increased IL-1β and IL-6 production but did not induce detectable levels of IL-23. In contrast to macrophages, the production of anti-inflammatory IL-10 was strongly downregulated in monocytes after costimulation with Pam3 and c-IgA (Fig. 2D). Costimulation with c-IgA also amplified IL-1β and IL-6 production by Kupffer cells (Fig. 2D), but it did not induce detectable protein levels of IL-10 and IL-23.

Taken together, these data demonstrate that complexed serum IgA induces proinflammatory cytokine production through synergy with TLR ligands, resulting in cell type–specific cytokine profiles by human macrophages, monocytes, and Kupffer cells.

Although FcαRI is considered to be the main IgA receptor in humans, other IgA receptors could play a role in the observed amplification of cytokine responses (16). To assess whether FcαRI is responsible for the amplification, we blocked FcαRI using a specific mAb. Notably, blocking of FcαRI almost completely inhibited the upregulation of cytokine production in macrophages, monocytes, and Kupffer cells (Fig. 3). To exclude the potential involvement of FcγRs that could be activated by the anti-FcαRI Ab, macrophages were incubated with FcγR-blocking reagent, which did not affect the inhibition caused by blocking of FcαRI (Supplemental Fig. 3).

FIGURE 3.

Amplification of cytokine production by IgA immune complexes is mediated by FcαRI. Prior to stimulation, cells were incubated with a blocking Ab against FcαRI and stimulated with Pam3 alone or Pam3 and c-IgA. After 24 h, supernatants were analyzed using ELISA (mean + SEM of triplicate). Representative examples of at least three experiments (macrophages and monocytes) or one experiment (Kupffer cells) are shown.

FIGURE 3.

Amplification of cytokine production by IgA immune complexes is mediated by FcαRI. Prior to stimulation, cells were incubated with a blocking Ab against FcαRI and stimulated with Pam3 alone or Pam3 and c-IgA. After 24 h, supernatants were analyzed using ELISA (mean + SEM of triplicate). Representative examples of at least three experiments (macrophages and monocytes) or one experiment (Kupffer cells) are shown.

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These data demonstrate that FcαRI is the main receptor responsible for the induced cytokine production by c-IgA in these three cell types.

Myeloid APCs detect invading pathogens using different PRRs. To determine whether FcαRI cross-talk is restricted to TLR2 or also occurs with other PRRs, we stimulated cells with c-IgA in combination with different PRR ligands. Notably, costimulation with c-IgA also increased TNF-α production induced by the TLR3 ligand, polyinosinic-polycytidylic acid, the TLR4 ligand, LPS, and the TLR5 ligand, flagellin, in macrophages (Fig. 4A) and monocytes (Fig. 4B). Furthermore, simultaneous stimulation of c-IgA also amplified TNF-α production by the NOD2 ligand, MDP (Fig. 4A, 4B), indicating that FcαRI cross-talk is not restricted to TLRs.

FIGURE 4.

FcαRI synergizes with different PRRs. Macrophages (A) and monocytes (B) were stimulated with different PRR ligands, alone or in combination with c-IgA. Stimulated receptors were TLR3 (polyinosinic-polycytidylic acid), TLR4 (LPS), TLR5 (flagellin), and NOD2 (MDP). After 24 h, supernatants were analyzed using ELISA (mean + SEM of triplicate). Data are representative of at least three experiments with different donors.

FIGURE 4.

FcαRI synergizes with different PRRs. Macrophages (A) and monocytes (B) were stimulated with different PRR ligands, alone or in combination with c-IgA. Stimulated receptors were TLR3 (polyinosinic-polycytidylic acid), TLR4 (LPS), TLR5 (flagellin), and NOD2 (MDP). After 24 h, supernatants were analyzed using ELISA (mean + SEM of triplicate). Data are representative of at least three experiments with different donors.

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Taken together, these data indicate that FcαRI is able to synergize with different families of PRRs to amplify cytokine responses by myeloid cells.

Next, we set out to investigate the effect of FcαRI–TLR cross-talk in greater detail. First, we determined cytokine expression over a longer time period. FcαRI–TLR cross-talk amplified TNF-α and IL-1β release by 6 h and continued to do so until ≥48 h (Fig. 5A). In contrast, IL-6 and IL-23 release was not amplified at 6 h, but it did show clear amplification at 24 and 48 h (Fig. 5A). These data indicate that FcαRI–TLR cross-talk modulates immune responses by myeloid cells over a wide timeframe (≥6 h to 2 d).

FIGURE 5.

Activation of FcαRI strongly increases sensitivity to TLR ligands. (A) Macrophages were stimulated with c-IgA, Pam3, or both. Supernatants were analyzed after 6, 24, and 48 h. (B and C) Macrophages were stimulated with different concentrations of Pam3 or LPS, alone or combined with c-IgA. After 24 h, supernatants were analyzed using ELISA (mean + SEM of triplicate). Representative examples of three experiments are shown.

FIGURE 5.

Activation of FcαRI strongly increases sensitivity to TLR ligands. (A) Macrophages were stimulated with c-IgA, Pam3, or both. Supernatants were analyzed after 6, 24, and 48 h. (B and C) Macrophages were stimulated with different concentrations of Pam3 or LPS, alone or combined with c-IgA. After 24 h, supernatants were analyzed using ELISA (mean + SEM of triplicate). Representative examples of three experiments are shown.

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Second, we assessed the effect of FcαRI–TLR cross-talk using suboptimal concentrations of TLR ligands, for which we stimulated macrophages with serial-step dilutions of TLR ligands in combination with c-IgA. Notably, FcαRI costimulation promoted TLR-induced proinflammatory cytokine production even when using Pam3 and LPS concentrations that were 1000-fold below optimum (Fig. 5B, 5C, respectively). Moreover, in general, individual stimulation with optimal concentrations of TLR agonists Pam3 (10 μg/ml) and LPS (100 ng/ml) induced far less proinflammatory cytokine production than 1000-fold dilutions of Pam3 and LPS in the presence of FcαRI costimulation (Fig. 5B, 5C). These data demonstrate that FcαRI stimulation strongly increases the sensitivity of human macrophages to TLR ligands.

Regulation of cytokine production can be organized at different levels. To determine whether the modulation of cytokine production by FcαRI is regulated at the level of gene transcription, we analyzed mRNA expression over time using quantitative PCR. In general, individual stimulation of cells with c-IgA had little effect on cytokine mRNA expression by macrophages, monocytes, and Kupffer cells (Fig. 6). Individual stimulation with TLR ligands moderately increased cytokine mRNA expression (Fig. 6). Strikingly, however, combined stimulation of c-IgA and TLR ligands strongly affected cytokine mRNA production by all three cell types (Fig. 6). Transcription of TNF, IL1B, and IL23A (encoding IL-23 subunit IL-23p19) was amplified by c-IgA costimulation in all three cell types in a similar manner. However, analogously to what we observed at the protein level, c-IgA costimulation of cytokine mRNA induction was cell type specific for other cytokines. IL6 transcription was not affected in macrophages and monocytes (Fig. 6A, 6B, respectively), whereas c-IgA costimulation did amplify IL6 transcription in Kupffer cells (Fig. 6C). Remarkably, IL-6 production by monocytes appeared to be the only exception for which amplified protein production (Fig. 2D) was not reflected by enhanced gene transcription (Fig. 6B). IL10 transcription was amplified in macrophages and Kupffer cells (Fig. 6A, 6C) but suppressed in monocytes (Fig. 6B). Furthermore, transcription of IL12B (encoding IL-12p40, a subunit of functional IL-12 and IL-23) was suppressed in macrophages and monocytes but amplified in Kupffer cells (Fig. 6). In contrast, transcription of IL12A (encoding IL-12 subunit IL-12p35) was not induced in any of the cell types after stimulation (data not shown).

FIGURE 6.

FcαRI-TLR cross-talk modulates cytokine production by affecting gene transcription. Macrophages (A), monocytes (B), and Kupffer cells (C) were stimulated with Pam3, c-IgA, or a combination and analyzed for mRNA expression of the indicated genes at the indicated time points using quantitative PCR. Representative examples of at least three (A and B) or two (C) experiments are shown.

FIGURE 6.

FcαRI-TLR cross-talk modulates cytokine production by affecting gene transcription. Macrophages (A), monocytes (B), and Kupffer cells (C) were stimulated with Pam3, c-IgA, or a combination and analyzed for mRNA expression of the indicated genes at the indicated time points using quantitative PCR. Representative examples of at least three (A and B) or two (C) experiments are shown.

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Combined, these data strongly suggest that c-IgA–induced cytokine modulation is regulated at the level of gene transcription and provide additional evidence that the effect of costimulation with c-IgA is cell type specific.

Next, we set out to identify the key components in FcαRI signaling that are responsible for the increased proinflammatory cytokine production. Several other (activating) FcαRI-mediated effects are induced by the association of FcαRI with FcRγ, leading to activation of kinases Syk and PI3K (18, 19). To determine whether Syk is also involved in FcαRI-induced cytokine production, we blocked this kinase using the small molecule inhibitor R406 (tamatinib). Importantly, inhibition of Syk almost completely blocked FcαRI-induced TNF-α production by human macrophages and monocytes (Fig. 7A, Supplemental Fig. 4A, respectively). To validate these findings, we silenced Syk expression in macrophages, resulting in a 76% mean reduction in SYK mRNA expression. Similarly to the Syk inhibitor R406, silencing of Syk resulted in strong suppression of FcαRI-TLR–induced TNF-α production (Fig. 7B).

FIGURE 7.

FcαRI–TLR cross-talk is dependent on kinases Syk and PI3K. Macrophages were treated with 0.5 μM Syk inhibitor R406 (A), 100 nM PI3K inhibitor wortmannin (C), 12.5 μM PI3K inhibitor LY294002 (D), or a corresponding volume of DMSO and stimulated with Pam3 alone or combined with c-IgA. (B) Macrophages were silenced for Syk using specific small interfering RNA. Control-silenced cells (si-C) and Syk-silenced cells (si-Syk) were stimulated with Pam3 or Pam3 combined with c-IgA. After 24 h, supernatants were analyzed using ELISA (mean + SEM of triplicate). Data are representative of at least three experiments with different donors.

FIGURE 7.

FcαRI–TLR cross-talk is dependent on kinases Syk and PI3K. Macrophages were treated with 0.5 μM Syk inhibitor R406 (A), 100 nM PI3K inhibitor wortmannin (C), 12.5 μM PI3K inhibitor LY294002 (D), or a corresponding volume of DMSO and stimulated with Pam3 alone or combined with c-IgA. (B) Macrophages were silenced for Syk using specific small interfering RNA. Control-silenced cells (si-C) and Syk-silenced cells (si-Syk) were stimulated with Pam3 or Pam3 combined with c-IgA. After 24 h, supernatants were analyzed using ELISA (mean + SEM of triplicate). Data are representative of at least three experiments with different donors.

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To assess PI3K involvement, we used the small molecule inhibitors wortmannin and LY294002, which block all PI3K subtypes. Both inhibitors suppressed FcαRI-induced TNFα production in macrophages (Fig. 7C, 7D). In addition, wortmannin suppressed FcαRI–TLR cross-talk in monocytes (Supplemental Fig. 4B). These data indicate that, similar to several other activating FcαRI functions, FcαRI-induced proinflammatory cytokine production is dependent on kinases Syk and PI3K (for the model, see Fig. 8).

FIGURE 8.

Model for cytokine induction by FcαRI–PRR cross-talk. Due to high levels of IgA directed against numerous Ags, bacteria are rapidly opsonized when they penetrate the body’s barriers, resulting in simultaneous activation of PRRs and FcαRI. Activation of FcαRI amplifies PRR-induced cytokine production by enhancing gene transcription by signaling through kinases Syk and PI3K. This FcαRI–PRR cross-talk results in cell type–specific cytokine production by macrophages, monocytes, and Kupffer cells.

FIGURE 8.

Model for cytokine induction by FcαRI–PRR cross-talk. Due to high levels of IgA directed against numerous Ags, bacteria are rapidly opsonized when they penetrate the body’s barriers, resulting in simultaneous activation of PRRs and FcαRI. Activation of FcαRI amplifies PRR-induced cytokine production by enhancing gene transcription by signaling through kinases Syk and PI3K. This FcαRI–PRR cross-talk results in cell type–specific cytokine production by macrophages, monocytes, and Kupffer cells.

Close modal

The prevailing concept regarding the immunological function of IgA is that it binds to and neutralizes pathogens to prevent infection at mucosal sites of the body. Although it has become clear more recently that IgA also mediates other immunological functions, its potential to actively contribute to the initiation of inflammatory responses, particularly at nonmucosal sites, is less well known. In this article, we show that formation of serum IgA immune complexes, as occurs upon opsonization of pathogens, strongly promotes proinflammatory cytokine production by human macrophages, monocytes, and Kupffer cells. This amplification of proinflammatory cytokines is induced by cross-talk of FcαRI with various PRRs, which is orchestrated at the level of gene transcription and is dependent on kinases Syk and PI3K (for model see Fig. 8).

Upon infection with a given pathogen, proinflammatory immune responses need to be tailored to the pathogen and the tissue involved. This context-specific immunity strongly depends on the cytokine profile induced by local APCs, which, in turn, is ultimately shaped by the interaction of different pathogen and danger sensing receptors on these cells. Although cytokine production was initially considered to be controlled primarily by cross-talk between different PRRs, it is becoming increasingly clear that other families of receptors can contribute as well. In this article, we show that FcαRI also plays an important role in shaping cytokine responses. In contrast to PRRs, FcαRI-induced cytokine production is not dependent on recognition of pathogens but is induced by detection of IgA immune complexes, thereby indicating that IgA immune complex formation functions as an important danger signal. Notably, FcαRI activation modulated the response of PRRs that are located on the plasma membrane, as well as PRRs expressed in endosomes (e.g., TLR3) and the cytosol (e.g., NOD2). This indicates that FcαRI–PRR cross-talk does not require a physical interaction between the two receptors but results from induction of individual signaling pathways that converge downstream to amplify cytokine responses.

FcαRI costimulation affected the expression of different cytokines in a selective manner. Macrophages, monocytes, and Kupffer cells all showed a very strong upregulation of TNF-α and IL-1β, whereas IL-6 upregulation was less pronounced. Although IL-23 was not detectable at the protein level in monocytes and Kupffer cells, all three cell types showed FcαRI-induced upregulation of IL23A transcription (encoding IL-23p19), the rate-limiting subunit of IL-23 (20). Because IL-1β, IL-6, IL-23, and TNF-α are all associated with promoting Th17 polarization in humans (21, 22), these data suggest that IgA immune complex formation skews Th cell responses toward Th17, which is particularly important for counteracting extracellular pathogens, such as bacteria and fungi (23).

Interestingly, FcαRI-induced cytokine production was partially cell type specific. Although IL-10 production was enhanced by macrophages and Kupffer cells, IL-10 production was strongly suppressed by monocytes. IL-10 is a classic anti-inflammatory cytokine that is often induced by PRRs as a negative-feedback mechanism to prevent overactivation (24). Why FcαRI costimulation suppresses IL-10 production by monocytes is still speculative, but it could be related to the injurious role of IL-10 during sepsis, where it is associated with increased severity and fatality (25). As such, opsonization of bacteria by serum IgA in the blood could provide a mechanism to protect the host by limiting adverse inflammatory effects. From a mechanistic point of view, the differences in cross-talk between these cells is most likely related to cell type–specific expression of receptors (PRR or FcαRI), signaling proteins, and transcription factors, which together results in a unique FcαRI–PRR cytokine profile. This unique FcαRI-PRR cytokine profile stimulation is likely to contribute to the generation of cell type–specific and therefore probably also tissue-specific immune responses to invading pathogens.

Remarkably, we showed that FcαRI costimulation strongly promoted proinflammatory cytokine production by Kupffer cells, which are generally considered to be a tolerogenic population of phagocytes (26, 27). Kupffer cells are present in high numbers in the vasculature of the liver, where they play a crucial role as gatekeeper by clearing portal blood of pathogens that originate from the intestine (26, 27). Importantly, under basal conditions, Kupffer cells display immune nonresponsiveness or tolerance to the continual exposure to pathogen-derived molecules, such as LPS (28, 29). In contrast, upon infection with live pathogens, this tolerogenic response should be converted to an inflammatory response. Our findings suggest that the formation of serum IgA immune complexes in the portal vein, which arise upon opsonization of bacteria that invade the circulation via the intestine, provides an “inflammatory switch” that breaks the tolerance of Kupffer cells to TLR-activating bacterial structures. As a result, FcαRI–TLR cross-talk allows Kupffer cells to discriminate between homeostatic conditions, during which TLR activation drives immune tolerance, and bacterial infections, during which the combination of TLR and FcαRI activation provides protective immunity via the production of cytokines that promote antibacterial Th17 responses.

Paradoxically, despite the important immunological functions attributed to IgA, individuals with IgA deficiency are often clinically asymptomatic (30, 31). With regard to the function of SIgA in pathogen neutralization at mucosal surfaces, it is thought that IgM can compensate for the lack of IgA, because IgM can be transported to mucosal surfaces using the same polymeric IgR as IgA (30). Because our data indicate that (serum) IgA also has another important immunological function (i.e., the induction of protective immunity against pathogens at nonmucosal sites), this may suggest that the human immune system also has a compensatory mechanism for IgA-induced cytokine production. It is tempting to speculate about a role for IgG in this regard. IgG is present in serum at even higher concentrations than IgA, and most FcαRI-expressing myeloid APCs also express FcγRs, although the expression profiles do not fully overlap (32). Moreover, FcγR stimulation by IgG-opsonized bacteria is known to promote Th17 responses induced by human dendritic cells (15) and elicits similar cytokine responses by monocytes and macrophages (33).

In addition to the protective role of IgA in host defense against pathogens, Abs of the IgA isotype are increasingly associated with various inflammatory disorders, including rheumatoid arthritis (RA), Sjögren’s syndrome, IgA nephropathy, alcoholic liver cirrhosis, and various skin-blistering diseases (10). Recently, it was shown that IgA autoantibodies, such as rheumatoid factor, enhance proinflammatory cytokine production by human macrophages in an FcαRI-dependent manner (34). Interestingly, immune complex formation by these IgA autoantibodies particularly amplified the production of RA-associated proinflammatory cytokines, such as TNF-α, IL-1β, and IL-6 (34), similar to our observations after FcαRI–TLR costimulation. Because RA is also strongly associated with increased TLR activation, not by recognition of microbial structures but via endogenous danger signals (35), FcαRI–TLR cross-talk is likely to play a role in amplifying inflammation in RA and various other diseases in which IgA immune complex formation occurs and, thereby, may provide a new and currently underexposed factor in the pathogenesis of these diseases.

It has been shown that cross-linking of FcαRI results in activation of the kinases Syk and PI3K (18, 19). In this article, we have identified that cross-talk of FcαRI with TLRs is also dependent on these kinases. This indicates that, similar to the other “activating” functions of FcαRI, FcαRI-induced cytokine production is induced via the FcRγ. The FcRγ signals through an ITAM, which is a common signaling module used by a variety of receptors, including BCRs, TCRs, and other members of the Fc receptor family (36). Interestingly, cross-talk with TLRs has previously been described for FcγRIIa (bearing an ITAM sequence in its cytoplasmic tail) on human dendritic cells (15) and macrophages (37), as well as for FcεRI (which signals through the FcRγ) on human mast cells (38) and basophils (39). The fact that different Fc receptors on different cells all induce a similar effect suggests that the ITAM signaling module uses a general mechanism to adjust TLR-induced cytokine responses, analogous to the previously described collaboration between the ITAM signaling module and JAK–STAT signaling pathways (40).

Taken together, our data indicate that serum IgA plays an important role in orchestrating host defense in nonmucosal tissues by regulating cytokine responses by different myeloid immune cells. Because undesired activation of this proinflammatory mechanism by IgA autoantibodies is likely to contribute to the pathology of chronic inflammatory disorders, such as RA, targeting of FcαRI–TLR cross-talk may be a valuable tool to suppress inflammation in IgA immune complex–associated diseases.

This work was supported by grants from the Dutch Digestive Foundation (Maag Lever Darm Stichting Career Development Grant 2012), the Academic Medical Center (AMC Fellowship 2015), and the Netherlands Organization for Scientific Research (VENI Project 91611012).

The online version of this article contains supplemental material.

Abbreviations used in this article:

c-IgA

complexed IgA

Pam3

Pam3CSK4

PRR

pattern recognition receptor

RA

rheumatoid arthritis

SIgA

secretory IgA.

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D.L.P.B. is a part-time employee of Union Chimique Belge. The other authors have no financial conflicts of interest.

Supplementary data