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The Journal of Immunology, 2001, 166: 6861-6868.
Copyright © 2001 by The American Association of Immunologists

Reversing Lipopolysaccharide Toxicity by Ligating the Macrophage Fc{gamma} Receptors1

Jeffrey S. Gerber and David M. Mosser2

Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, PA 19140


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Our laboratory has previously demonstrated that the ligation of phagocytic receptors on macrophages can influence cytokine production. In this study, we examine the cytokine responses to multiple inflammatory stimuli following Fc{gamma}R ligation. Macrophages were stimulated in vitro with LPS, lipoteichoic acid, CD40 ligand, or low molecular mass hyaluronic acid. All of these stimuli were proinflammatory in character, inducing the production of high levels of IL-12, but only modest amounts of IL-10. The coligation of Fc{gamma}R along with these stimuli resulted in an anti-inflammatory profile, abrogating IL-12 production and inducing high levels of IL-10. The modulation of these two cytokines occurred by two independent mechanisms. Whereas the abrogation of IL-12 biosynthesis was a property shared by several macrophage receptors, the induction of IL-10 was specific to the Fc{gamma}R. The biological relevance of these observations was examined in murine models of endotoxemia, in which Fc{gamma}R ligation induced the rapid production of IL-10 and prevented IL-12 synthesis. Mice could be passively immunized with Abs to LPS to reverse inflammatory cytokine production, and the transfer of macrophages whose Fc{gamma}R had been ligated could rescue mice from lethal endotoxemia. Thus, the ligation of the macrophage Fc{gamma}R can be exploited to prevent inappropriate inflammatory cytokine responses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The receptors for the Fc portion of IgG (Fc{gamma}R) allow innate effector cells, such as macrophages, to communicate with the specific, adaptive immune response. The expression of all three classes of Fc{gamma}R (Fc{gamma}RI, Fc{gamma}RII, and Fc{gamma}RIII) on macrophages facilitates the rapid and efficient binding of IgG immune complexes to these cells (1, 2). Although phagocytosis is the most recognized sequella of Fc{gamma}R cross-linking, signaling through Fc{gamma}R may influence other responses such as Ag presentation, Ab-dependent cellular cytotoxicity, and the release of inflammatory mediators (3).

IL-12 is a proinflammatory cytokine produced primarily by APCs. Although transcribed and translated as separate gene products (p40 and p35), the ascribed biologic activity of IL-12 occurs only when the two subunits are joined and secreted as a disulfide-linked heterodimer (p70) (4). IL-12 is essential for the development of an efficient cell-mediated immune response to intracellular pathogens. This occurs primarily through the induction of IFN-{gamma}, leading to macrophage activation and the biasing of Th cells toward a Th1 phenotype (5, 6, 7, 8). Although crucial for host defense to a number of pathogens, the inappropriate overproduction of IL-12 can have adverse affects. The contribution of IL-12 to organ-specific autoimmunity has been documented in both murine and human forms of multiple sclerosis (9, 10) as well as in diabetes, in which macrophages were identified as the source of the pathogenic IL-12 (11). Studies have also implicated IL-12 and IL-12-induced IFN-{gamma} in the morbidity and mortality associated with endotoxemia (12, 13, 14, 15). Therefore, the production of IL-12 must be tightly controlled.

IL-10 is a pleiotropic cytokine produced by monocytes, macrophages, and lymphocytes (16, 17). It was originally identified by its ability to antagonize cellular immunity (18). Among its potent immunosuppressive qualities lies the ability of IL-10 to depress mononuclear cell activation through the prevention of inflammatory mediator production (19, 20, 21, 22) and the down-regulation of Ag-presenting (23) and costimulatory molecule (24) expression in vitro. The important immunomodulatory role of IL-10 in vivo is apparent during septic shock, a pathology that can be triggered by leukocyte activation in response to microbial products (25). Treatment with IL-10 diminishes inflammatory cytokine production and prevents lethality in several animal models of septic shock (26, 27, 28, 29, 30). Conversely, the removal of IL-10 via blocking Ab or gene targeting intensifies proinflammatory cytokine responses and lowers the threshold for induction of endotoxin lethality (31, 32, 33, 34, 35). In human models of endotoxemia, i.v. IL-10 administration reduces inflammatory cytokine responses (36), chemokine production (37), and activation of coagulation and fibrinolysis (38). In human patients suffering from severe Gram-negative septic shock, IL-10 was shown to inhibit the activation of monocytes (39).

Recently, we demonstrated that receptor ligation can influence macrophage cytokine production in vitro (40, 41). We showed that IL-12 production can be abrogated by ligating any one of several macrophage receptors (40). There was a surprising lack of receptor specificity to this effect, because the ligation of the Fc{gamma}, the complement, or the scavenger receptors was capable of diminishing IL-12 production. We also showed that the specific ligation of the Fc{gamma}R could increase the production of IL-10 in response to LPS (41). Because macrophages are in position to effect both innate and adaptive immune responses, we now further characterize macrophage cytokine production following Fc{gamma}R ligation. We examine the specificity and the kinetics of cytokine production and examine the extent to which the in vitro observations that we have made could be extended to in vivo applications.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice and macrophages

Six- to 8-wk-old female C57BL/6 and C57BL/6/IL-10-/- mice were purchased from Taconic Farms (Germantown, NY). C57BL/6J-RAG1-/- breeding pairs were purchased from The Jackson Laboratory (Bar Harbor, ME). All mice were housed in the Temple University School of Medicine Animal Facility. Bone marrow-derived macrophages (BMM{phi})3 were established, as previously described (40). Briefly, femurs were flushed with cation-free Dulbecco’s PBS containing 200 U/ml penicillin G and 200 µg/ml streptomycin. Cells were grown in DMEM containing 20% L929 cell-conditioned medium (as a source of M-CSF), 10% heat-inactivated FCS, 2 mM L-glutamine, 100 U/ml penicillin G, and 100 µg/ml streptomycin. Cells were incubated at 37°C in 5% CO2 for 7–10 days until uniform monolayers of macrophages were established. Twelve hours before use, cells were removed from the original plastic petri dishes using 5 mM EDTA and put in tissue culture-treated plates (Nunc, Naperville, IL) in DMEM containing 10% heat-inactivated FCS, 2 mM L-glutamine, 100 U/ml penicillin G, and 100 µg/ml streptomycin (complete medium) with or without 200 U/ml murine rIFN-{gamma}.

Reagents

IgG-opsonized erythrocytes (E-IgG) were generated by combining sheep erythrocytes (SRBC; Lampire, Pipersville, PA) with rabbit anti-SRBC IgG (Cappel, Durham, NC) at nonagglutinating titers. This suspension was gently rotated for 30 min at room temperature. E-IgG were then washed with HBSS (Life Technologies, Gaithersburg, MD) and resuspended at 2 x 108 cells/ml in HBSS. Complement-opsonized erythrocytes (E-C3b/i) were generated by incubating SRBC with rabbit anti-SRBC IgM (Cappel, Durham, NC) at nonagglutinating titers in the presence of 5% C5-deficient mouse serum for 40 min at room temperature. E-C3b/i were washed twice with HBSS and resuspended at 2 x 108 cells/ml in HBSS. Lipoteichoic acid (LTA) from Staphylococcus aureus, LPS (Escherichia coli 0127:B8, 0128:B12, and K 235), and cytochalasin D were purchased from Sigma (St. Louis, MO). An agonistic Ab to CD40 was prepared from hybridoma supernatants of FGK115 cells. Low molecular mass hyaluronic acid (LMW-HA; ICN, Aurora, OH) was treated with polymyxin B-coated beads (Sigma) before use. IgG-LPS was generated by incubating 10 µg/ml E. coli 0128:B12 LPS with rabbit anti-LPS polyclonal antiserum (Calbiochem-Novabiochem, San Diego, CA) at a 1/1 dilution for 15 min at 4°C. This dilution was selected as the lowest amount of IgG that maximally stimulated macrophage IL-10 production in vitro. Latex microspheres (2 µm) were purchased from Duke Scientific (Palo Alto, CA). Recombinant murine IL-10 and IFN-{gamma} were purchased from R&D Systems (Minneapolis, MN).

Macrophage stimulation and receptor ligation

For in vitro assays, stimuli were added to BMM{phi} at the following concentrations, unless otherwise specified: LPS at 10 ng/ml, LTA at 10 µg/ml, CD40 agonist at 20 µg/ml, IgG-LPS at 100 ng/ml, LMW-HA at 250 µg/ml, murine rIL-10 at 2 ng/ml. For receptor ligation/phagocytosis, E-IgG were added at a ratio of 10 erythrocytes per macrophage. Latex microspheres were added at a ratio of 50 microspheres per macrophage. For phagocytosis studies, cells were incubated with 10 µg/ml cytochalasin D. Unless otherwise specified, inflammatory stimulation and Fc{gamma}R ligation were initiated simultaneously.

In vivo assays

For passive immunization studies, RAG1-/- mice (five per group) were injected with anti-LPS Ab or saline in the peritoneum, followed 2 or 18 h later by 4 µg of LPS (0128:B12) i.v. (tail vein). Mice were bled by retro-orbital puncture at the indicated time intervals, and serum cytokine levels were determined by ELISA. For in vivo reconstitution assays, a total of 1 x 106 BMM{phi} was incubated with 1 x 107 E-IgG in the presence of 5 ng/ml LPS (0127:B8) at 37°C in 1.5-ml Eppendorf tubes for 30 min and then injected i.p. into C57BL/6 mice. Three and one-half hours later, mice were injected i.p. with 300 µg of LPS (K 235). Animals were then monitored at 12-h intervals for 6 days.

Cytokine assays

Cell supernatants or serum were assayed for IL-12 p40, IL-12 p70, TNF-{alpha}, or IL-10 protein by ELISA. Macrophages were added to 24-well plates at 2 x 105 cells/well in 0.5 ml complete medium and stimulated for 8–24 h. Culture supernatants were then collected and centrifuged at 13,000 x g, and the supernatants were stored at -80°C. Ab pairs (IL-12 p40, C15.6 and C17.8; IL-12 p70, 9A5 and C17.8; TNF-{alpha}, G281-2626 and MP6-XT3; IL-10, JES-2A5 and JES-16E3) and recombinant standards were purchased from BD PharMingen (San Diego, CA). To determine cytokine mRNA levels, 1.5 x 106 macrophages were added to six-well plates in 2 ml complete medium. Cells were stimulated for 2.5 h, and total cellular RNA was harvested using TRIzol (Life Technologies), according to manufacturer’s instructions. For RT-PCR assays, RNA was reverse transcribed and amplified, as previously described (40). For RNase protection assays, RNA was prepared and assayed using multiprobe template sets mCK-2b and mCK-3b (BD PharMingen), according to manufacturer’s instructions.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Fc{gamma}R-mediated cytokine modulation is stimulus independent

To determine the extent to which receptor ligation could alter macrophage cytokine production, murine BMM{phi} were stimulated in vitro with the Gram-negative cell wall constituent LPS in the presence or absence of E-IgG to ligate the Fc{gamma}R. Cells stimulated with LPS alone produced high levels of IL-12 and TNF-{alpha}, but only modest amounts of IL-10 (Fig. 1Go). The cross-linking of Fc{gamma}R simultaneously with LPS stimulation changed the cytokine profile of these cells. IL-12 production was essentially abrogated, and IL-10 production was dramatically increased (Fig. 1Go). TNF-{alpha} production was not affected by Fc{gamma}R ligation. These data confirm and extend two previous independent observations (40, 41). This assay was repeated with BMM{phi} that were primed with IFN-{gamma} to induce the production of high levels of IL-12 p70 (Fig. 2Go). Several stimuli in addition to LPS were examined to determine whether they were also able to cooperate with Fc{gamma}R ligation and modulate cytokine production. These stimuli included the Gram-positive cell wall component LTA, the ligation of macrophage CD40 with an agonistic Ab that mimics T cell CD40 ligand ({alpha}CD40), and CD44-mediated stimulation with LMW-HA. All of the stimuli tested were proinflammatory in character and induced relatively high levels of IL-12 (Fig. 2GoA, top graph), but only modest levels of IL-10 (Fig. 2GoA, bottom graph), comparable with stimulation with 10 ng/ml of LPS. The coupling of all of these stimuli with Fc{gamma}R ligation resulted in a decrease in IL-12 production to near-background levels (Fig. 2GoA, top graph), and a dramatic increase in IL-10 production (Fig. 2GoA, bottom graph). LPS contamination of LMW-HA was excluded as a potential source of macrophage stimulation by performing this assay on C3H/Hej mice, which have a mutation in Toll-like receptor 4, making them hyporesponsive to LPS. The response of macrophages from these mice to LMW-HA ± E-IgG was not different from that of wild-type mice (data not shown).



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  		FIGURE 1. Fc{gamma}R ligation on LPS-stimulated macrophages affects the production of IL-12 p40 and IL-10, but not TNF-{alpha}. Unprimed BMM{phi} were incubated with 10 ng/ml LPS and/or E-IgG. Eight hours after stimulation, cell supernatants were harvested and assayed by ELISA for IL-12 p40, IL-10, and TNF-{alpha}. Each bar indicates the mean cytokine concentration of triplicate cultures ± SD. These results are representative of three separate experiments. MED, denotes unstimulated cells cultured in complete medium alone.

 


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  		FIGURE 2. Fc{gamma}R-mediated cytokine modulation is stimulus independent and selective for IL-12 and IL-10. A, IFN-{gamma}-primed BMM{phi} were incubated with different inflammatory stimuli in the absence () or presence ({blacksquare}) of E-IgG. Inflammatory stimulation and Fc{gamma}R ligation were initiated simultaneously. Twelve hours after stimulation, cell supernatants were harvested and assayed for IL-12 p70 (top graph) and IL-10 (bottom graph) by ELISA. Each bar indicates the mean cytokine concentration of triplicate cultures ± SD. These results are representative of three separate experiments. Stimuli: LPS, 10 ng/ml; LTA, 10 µg/ml; CD40 agonist, 20 µg/ml; LMW-HA, 250 µg/ml; E-IgG:M{phi} ratio, 10:1. MED, denotes unstimulated cells cultured in complete medium alone. B, Modulation of IL-12 and IL-10 mRNA levels by ligating Fc{gamma}R. BMM{phi} were exposed to LPS alone, LPS + E-IgG, or E-IgG alone, and RNA was isolated after 2.5 h of culture. Five micrograms of RNA per condition were then subjected to RNase protection assay using multiprobe templates provided by PharMingen. Results are representative of three separate experiments.

 
The suppression of IL-12 and the increase in IL-10 production following Fc{gamma}R ligation were also evident at the mRNA level (Fig. 2GoB). By RNase protection, RNA levels for a number of cytokine genes, including TNF-{alpha}, IL-1, and IL-6, were increased following stimulation of macrophages with LPS alone. The coupling of stimulation with Fc{gamma}R ligation specifically diminished mRNA levels for IL-12 p35 and p40 and increased IL-10 mRNA. Importantly, the majority of the other cytokines that were analyzed remained relatively unaffected by Fc{gamma}R ligation. Thus, the ligation of Fc{gamma}R does not result in an indiscriminate down-regulation of all macrophage cytokines, but rather a specific down-regulation of IL-12 and an induction of IL-10.

Characterization of cytokine modulation following Fc{gamma}R ligation

To determine the receptor specificity of the effects demonstrated in Fig. 1Go, BMM{phi} were stimulated with LPS, coupled with the ligation of either Fc{gamma}R (E-IgG) or complement receptor (E-C3b/i) ligation (Fig. 3Go). IL-12 levels were substantially diminished following the ligation of either of these two receptor classes, as previously described (40). However, the induction of IL-10 was achieved only after Fc{gamma}R ligation (Fig. 3Go). These results reveal a fundamental difference between the two receptor classes with regard to the regulation of IL-10 biosynthesis, and demonstrate that the induction of IL-10 is specific to the Fc{gamma}R, whereas IL-12 inhibition is shared by a number of macrophage receptors, as previously described (40). Thus, the reciprocal alteration in cytokine production that was observed in Figs. 1Go and 2Go is specific to the macrophage Fc{gamma}R.



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  		FIGURE 3. Complement receptor ligation fails to up-regulate IL-10. IFN-{gamma}-primed BMM{phi} were incubated with 10 ng/ml LPS with or without E-IgG (to ligate Fc{gamma}R) or E-C3b/i (to ligate complement receptors). Twelve hours after stimulation, cell supernatants were harvested and assayed for IL-12 p70 (, left axis) and IL-10 (black bars, right axis) by ELISA. Each bar represents the mean cytokine concentration of triplicate cultures ± SD. Results are representative of three separate experiments.

 
The requirement for phagocytosis was addressed by performing assays on cells treated with cytochalasin D. Cells treated with cytochalasin D failed to internalize E-IgG (data not shown), but they down-regulated IL-12 and up-regulated IL-10 as well as did cells undergoing E-IgG internalization (Fig. 4Go). Latex beads were also added to LPS-stimulated BMM{phi} to determine whether nonreceptor-mediated phagocytosis could induce similar cytokine alterations. Although several latex beads were rapidly engulfed by each macrophage, this did not affect cytokine production in response to LPS; LPS stimulation resulted in high IL-12 and low IL-10, and these levels were not significantly altered by bead phagocytosis (Fig. 4Go). Thus, phagocytosis is neither required nor sufficient to modulate LPS-induced macrophage cytokine production in response to Fc{gamma}R ligation.



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  		FIGURE 4. Fc{gamma}R-mediated cytokine modulation is not dependent upon phagocytosis. BMM{phi} were stimulated with 10 ng/ml LPS or LPS + E-IgG in the presence or absence of cytochalasin D (CYTO), or LPS + latex beads. Eight hours after stimulation, cell supernatants were harvested and assayed by ELISA for both IL-12 p40 (, left axis) and IL-10 ({blacksquare}, right axis). Each bar indicates the mean cytokine concentration of triplicate cultures ± SD. Results are representative of three separate experiments.

 
The down-regulation of IL-12 does not depend on IL-10 induction

BMM{phi} from mice genetically deficient in IL-10 (IL-10-/-) were examined to determine whether the down-regulation of IL-12 was dependent on the enhanced production of IL-10. The addition of E-IgG to LPS-stimulated IL-10-/- macrophages resulted in a dramatic down-regulation of IL-12 production (Fig. 5Go, main graph). The extent of down-regulation was similar to that observed in parallel monolayers of wild-type BMM{phi} (Fig. 5Go, inset). Thus, the induction of IL-10 that accompanies macrophage Fc{gamma}R ligation is not required for the ablation of IL-12 biosynthesis. However, the addition of exogenous rIL-10 to macrophages of both genotypes potently inhibited IL-12 production (Fig. 5Go). Taken together, these data indicate that both IL-10 dependent and IL-10 independent mechanisms exist to inhibit macrophage IL-12 production.



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  		FIGURE 5. Down-regulation of IL-12 by Fc{gamma}R is IL-10 independent. IFN-{gamma}-primed BMM{phi} from IL-10-/- (main graph) and +/+ mice (inset) were incubated with 10 ng/ml LPS with or without E-IgG. Some cells received 2 ng/ml murine rIL-10 at the same time as LPS stimulation. Twenty-four hours after stimulation, cell supernatants were harvested and assayed by ELISA for IL-12 p70. Each bar indicates the mean cytokine concentration of triplicate cultures ± SD. Results are representative of three separate experiments.

 
Fc{gamma}R ligation is a potent inducer of IL-10

To determine the extent to which Fc{gamma}R ligation was able to induce IL-10, macrophages were incubated with increasing concentrations of LPS in the presence or absence of E-IgG. LPS is a relatively inefficient inducer of IL-10 from macrophages, and at low doses (below 1 ng) LPS failed to elicit the production of detectable levels of IL-10, even when the assays were performed in the presence of serum, as these were (Fig. 6Go). High doses of LPS consistently induced IL-10 production, but the levels of production rarely exceeded 500 pg/ml in these assays, which used a total of 2 x 105 macrophages per 0.5 ml. However, the combination of LPS and E-IgG was a potent inducer of IL-10. Even low doses of LPS, which alone failed to produce detectable IL-10, synergized with Fc{gamma}R ligation to induce high levels of IL-10. This represents a true synergy, because E-IgG alone also failed to induce detectable IL-10. The IL-10 levels that we routinely measure following Fc{gamma}R ligation of stimulated macrophages (2–10 ng/ml) make this the most potent inducer of macrophage IL-10 that we have observed.



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  		FIGURE 6. Fc{gamma}R ligation is a potent inducer of IL-10 from LPS-stimulated macrophages. BMM{phi} were stimulated with increasing doses of LPS in the absence () or presence ({blacksquare}) of E-IgG. Eight hours after stimulation, cell supernatants were harvested and assayed for IL-10 by ELISA. Each bar indicates the mean cytokine concentration of triplicate cultures ± SD. Results are representative of three separate experiments.

 
Kinetics of cytokine production

We examined the kinetics of LPS-induced IL-12/IL-10 production with and without Fc{gamma}R ligation (Fig. 7Go). Total cytokine accumulation over time was determined (main graphs), as was cytokine production during specific time intervals (insets). Following stimulation by LPS alone (), IL-12 p40 accumulated in a fairly linear fashion over the entire 24-h observation period (Fig. 7Go, top graph). IL-10 production was close to the limit of detection (Fig. 7Go, bottom graph, ). However, following Fc{gamma}R ligation ({blacksquare}), there was a rapid and substantial accumulation of IL-10, which increased linearly for approximately the first 8 h and then began to plateau (Fig. 7Go, bottom main graph). An analysis of IL-10 production during individual 4-h time intervals (insets) revealed that the kinetics of IL-10 production following Fc{gamma}R ligation were quite rapid, with the majority of cytokine being produced within the first 4 h of stimulation. Thus, Fc{gamma}R ligation not only induced the production of large amounts of IL-10, but also caused this cytokine to be produced very rapidly following stimulation.



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  		FIGURE 7. Kinetics of BMM{phi} cytokine production in response to LPS + Fc{gamma}R ligation. BMM{phi} were stimulated with LPS () or LPS + E-IgG ({blacksquare}). Cell supernatants were collected in either of two ways. In the main graphs, cell supernatants from separate wells of identically treated cells (LPS or LPS + E-IgG) were harvested at different time points, thus representing total cytokine accumulation over time. In the inset graphs, cells were stimulated and the supernatants were harvested and replaced every 4 h. Thus, the inset graphs represent cytokine production during defined time intervals. All supernatants were assayed by ELISA for both IL-12 p40 and IL-10. Each bar indicates the mean cytokine concentration of triplicate cultures ± SD. Results are representative of four separate experiments.

 
In vivo modulation of cytokine production

Two different models were used to examine the effect of Fc{gamma}R ligation on macrophage cytokine production in vivo. In the first series of studies, IgG-opsonized LPS was used as the stimulus instead of E-IgG + LPS. In vitro studies (Fig. 8Go, A and B) confirmed that IgG-LPS specifically induced the production of IL-10 and diminished IL-12 production, at both the protein (Fig. 8GoA) and the mRNA (Fig. 8GoB) level. To determine the physiological significance of this in vitro observation, in vivo studies (Fig. 8GoC) were undertaken, in which mice were passively immunized with Ab to LPS, 2 and 18 h before the i.v. administration of LPS. Serum was collected at various intervals thereafter, and cytokine levels were assayed by ELISA. Mice receiving Ab 2 h before i.v. injection of LPS produced significantly lower levels of IL-12 (Fig. 8GoC, left graph) and higher levels of IL-10 (Fig. 8GoC, right graph) relative to mice receiving LPS alone. The IL-10 that was produced by IgG-LPS was only detected at 2 h poststimulation, confirming the rapidity with which this cytokine was produced in vitro. Mice receiving Ab to LPS at 18 h before stimulation exhibited intermediate levels of IL-12, but retained a high production of IL-10 at 2 h poststimulation (Fig. 8GoC). Thus, the in vivo administration of anti-LPS IgG before LPS stimulation decreases the production of IL-12 and induces IL-10 production.



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  		FIGURE 8. IgG-opsonized LPS modulates IL-12/IL-10 in vitro and in vivo. A, BMM{phi} were stimulated with LPS or LPS opsonized with an IgG Ab to LPS (IgG-LPS) for 24 h, and cell culture supernatants were harvested and assayed by ELISA for IL-12 p40 and IL-10. Each bar indicates the mean cytokine concentration of triplicate cultures ± SD. Results are representative of three separate experiments. B, BMM{phi} were stimulated with LPS or IgG-LPS for 3 h, and mRNA was isolated. Competitive RT-PCR was performed on cDNA that was normalized with the housekeeping gene hypoxanthine phosphoribosyltransferase (top bands). Results are representative of three separate experiments. C, In vivo passive immunization with anti-LPS IgG. RAG1-/- mice were injected i.p. with 200 µl of polyclonal anti-LPS IgG (or saline), followed 2 or 18 h later by i.v. injection of 4 µg LPS. Mice were bled at the indicated times after LPS injection, and serum was assayed by ELISA for IL-12 p40 and IL-10. Cytokine concentrations are expressed as the mean of five mice per group ± SD. These results are representative of two separate experiments, each with five mice per group.

 
The second in vivo model used to test the efficacy of Fc{gamma}R ligation in preventing LPS toxicity used the i.p. administration of lethal doses of LPS (300 µg/ml) to C57BL/6 mice. This dose is well in excess of the LD90 of 50–100 µg/ml. Consequently, all mice receiving this dose of LPS succumbed within 3 days of administration (data not shown). To test our model, 4 h before LPS administration, a total of 1 x 106 macrophages was transferred i.p. into mice. Before transfer, macrophages were exposed to low doses of LPS (5 ng/ml) in the presence or absence of E-IgG to ligate Fc{gamma}R. All of the mice that received macrophages pretreated with LPS alone succumbed to endotoxic shock within 3 days postadministration, whereas all of the mice that received macrophages treated with E-IgG and LPS survived (Fig. 9Go). The mice in the latter group remained relatively well groomed and continued to move about the cage when their counterparts in the previous group were moribund. To demonstrate that this effect was due to IL-10 production, similar experiments were performed with macrophages from IL-10-/- mice. In this group, LPS + E-IgG-treated macrophages failed to protect mice; four of the five animals succumbed to endotoxemia within the first 3 days, and the fifth animal remained viable but moribund until it was euthanized at day 4. Thus, Fc{gamma}R-induced IL-10 is able to prevent lethality induced by endotoxin.



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  		FIGURE 9. Reversal of LPS toxicity by Fc{gamma}R ligation. A total of 1 x 106 C57BL/6 or C57BL/6/IL-10-/- BMM{phi} was stimulated in vitro with 5 ng/ml LPS in the presence or absence of 1 x 107 E-IgG. After 30 min, these macrophages were transferred i.p. to C57BL/6 mice. Three and one-half hours later, mice (five per group) were given 300 µg LPS i.p., and their viability was monitored every 12 h. *, The last remaining mouse in this group was euthanized on day 4 for humane reasons.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It is essential that a balance between immune activation and deactivation is maintained to allow the host to mount efficient responses to potential pathogens that are minimally damaging to self. In the present work, we have identified an unexpected way to manipulate this balance by preventing the production of unwanted inflammatory responses. We have examined two cytokines, IL-12 and IL-10, that play diametrically opposed roles in maintaining immune homeostasis. The primary role of IL-12 is to drive the proinflammatory cell-mediated immune response. IL-10, in contrast, antagonizes inflammation. When in harmony, both activating and deactivating immune mediators provide the balance needed to maintain immune homeostasis. However, when they are uncontrolled an inflammatory response may exacerbate the pathology associated with disease. This has been demonstrated in both mice and humans for diseases such as organ-specific autoimmunity (42) and septic shock (26, 27, 28, 29, 30), in which the suppression of inflammatory cytokines by deactivating cytokines such as IL-10 ameliorates pathology and improves outcome.

In previous studies, we had begun to examine cytokine production by stimulated macrophages following the ligation of phagocytic receptors. We (40) and others (43, 44, 45, 46, 47, 48, 49) have demonstrated that the ligation of a variety of macrophage receptors could result in a decrease in IL-12 production in response to bacterial products. We also previously showed that the specific ligation of the Fc{gamma}R could cause an induction of IL-10 in response to LPS (41). Together, these studies suggested that we could reliably and predictably influence the cytokine profile of a macrophage simply by ligating the correct receptor class. In the present study, we extend and characterize these observations, and we test their therapeutic potential.

We studied the effects of Fc{gamma}R ligation on cytokine production using several types of leukocyte stimuli. All of the stimuli tested yielded identical results; Fc{gamma}R ligation prevented the induction of IL-12 and dramatically up-regulated the production of IL-10. This cytokine modulation occurs whether the inflammatory signal was set off by the innate pattern recognition of Gram-negative or Gram-positive bacterial extracts (LPS or LTA through Toll-like receptor) (50), an acquired immunity ligand on the surface of activated T cells (CD40L-CD40) (51), or a component of the extracellular matrix (LMW-HA through CD44) (13). Each of these stimuli induced IL-12 synthesis, and all of them were down-regulated by Fc{gamma}R ligation. The common pathway that allows all of these diverse stimuli to synergize with Fc{gamma}R to induce IL-10 is less obvious, and studies to identify this pathway are currently underway.

We have begun to define the requirements for altered macrophage cytokine production. Studies using cells treated with cytochalasin D or incubated with latex beads demonstrate that phagocytosis is neither necessary nor sufficient for Fc{gamma}R-mediated cytokine modulation. To address the somewhat controversial issue regarding the ability of complement receptors to up-regulate IL-10 (41, 52), we assayed culture supernatants from macrophages stimulated with LPS in the presence of E-C3b/i. In this study, the down-regulation of IL-12 by complement receptor ligation provided the necessary internal control, and indicated that the failure of complement receptors to induce IL-10 was not due to a lack of receptor signaling. Thus, the reciprocal alteration in cytokine production that we identify is specific to the macrophage Fc{gamma}R.

Our data reveal several important practical aspects of Fc{gamma}R-induced cytokine modulation. We demonstrate that there are actually two independent mechanisms for down-regulating IL-12 following Fc{gamma}R ligation. It is clear that signaling through Fc{gamma}R prevents IL-12 production independently of IL-10, as studies using IL-10-/- macrophages show that these cells fail to produce IL-12 following Fc{gamma}R ligation. However, a second mechanism that can also contribute to the decrease in IL-12 production is the induction of IL-10 following Fc{gamma}R ligation. We show that exogenous IL-10 can effectively halt IL-12 production (Fig. 5Go), as previously reported (53). These two mechanisms may cooperate to maximally inhibit the production of IL-12 at inflammatory sites. The first mechanism exerts a direct effect on the cells whose Fc{gamma}R have been cross-linked, whereas the second mechanism may also influence the activation state of surrounding cells. It is undoubtedly this second mechanism that accounts for the data shown in Fig. 9Go, in which a relatively small number of macrophages can rescue mice from LPS lethality.

We also examined the kinetics of cytokine production and observed a surprising acceleration in the biosynthesis of IL-10 following Fc{gamma}R ligation. Previous studies (19) have shown that macrophages stimulated with LPS experience a lag in IL-10 production, generally producing IL-10 only after these cells have made ample amounts of inflammatory cytokines. These kinetics would suggest that the primary role of macrophage-derived IL-10 is to control the overproduction of inflammatory mediators, allowing damage to the pathogen, while limiting damage to the host. However, the coupling of this stimulation with Fc{gamma}R ligation revealed a fundamental change in these kinetics. The majority of IL-10 production occurred during the first 4 h of stimulation. This production is as rapid as any cytokine that we have observed, including TNF-{alpha}, the earliest cytokine present in the endotoxic cascade. Thus, whereas LPS-stimulated macrophages usually secrete IL-10 as part of a negative feedback cycle subsequent to inflammatory cytokine production (i.e., TNF-{alpha}) (19, 54), Fc{gamma}R ligation induces early IL-10 production, positioned for the prevention rather than the down-regulation of inflammation.

Finally, we adapted our model to study murine endotoxemia in vivo. First, mice were passively immunized with Abs to LPS and then given an i.v. bolus of LPS 2 or 18 h later. It should be noted that these initial in vivo studies were done in RAG-/- mice, which, unlike normal mice, lack naturally occurring Abs to LPS. It is for this reason that these mice are hypersusceptible to LPS (55). Mice that were passively immunized with Ab to LPS produced dramatically less IL-12 and more IL-10 than did mice receiving LPS alone. The IL-10 was only detectable at 2 h postinjection, whereas the differences in IL-12 were evident throughout the 24-h observation period. These data may have practical implications, as a prophylactic treatment with Abs to LPS could benefit patients at risk for endotoxemia. Similar approaches have been previously attempted, using IgM Abs (56, 57), which we have shown to be unable to modulate cytokine production in our in vitro system (data not shown). We hypothesize that an IgG Ab to LPS would prove most effective in ameliorating endotoxemia due to both the accelerated clearance of LPS and the reciprocal alteration in cytokine production.

Our second animal model demonstrates the powerful immunosuppressive effects of Fc{gamma}R-induced IL-10. In this study, delivery of Fc{gamma}R-ligated macrophages protected C57BL/6 mice from a lethal dose of LPS. The addition of only one million macrophages whose Fc{gamma}R had been ligated was enough to overcome the inflammatory response mounted by endogenous cells whose receptors were obviously not ligated. Importantly, the administration of Fc{gamma}R-ligated macrophages from IL-10-/- mice failed to rescue the mice. This failure suggests that it is the production of IL-10 that is primarily responsible for preventing endotoxic shock in this model. Thus, although several studies have reported that the ligation of macrophage Fc{gamma}Rs induces inflammatory cytokine production in vitro (3), these in vivo models of murine endotoxemia demonstrate a dominant physiological anti-inflammatory effect of Fc{gamma}R ligation. This effect is mediated primarily by the production of IL-10, accompanied by the abrogation of IL-12 synthesis. These observations establish the biological relevance of Fc{gamma}R-induced cytokine modulation.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant AI46805. J.S.G. was supported by the Temple University School of Medicine M.D./Ph.D. Program. Back

2 Address correspondence and reprint requests to Dr. David M. Mosser, Department of Cell Biology and Molecular Genetics, 1104 Microbiology Building, College Park, MD 20742. E-mail address: dm268{at}umail.umd.edu Back

3 Abbreviations used in this paper: BMM{phi}, bone marrow-derived macrophage; E-C3b/i, complement-opsonized erythrocytes; E-IgG, IgG-opsonized erythrocytes; LMW-HA, low molecular mass hyaluronic acid; LTA, lipoteichoic acid. Back

Received for publication December 20, 2000. Accepted for publication March 23, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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