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The Journal of Immunology, 2005, 174: 3041-3050.
Copyright © 2005 by The American Association of Immunologists

Macrophages Induce the Inflammatory Response in the Pulmonary Arthus Reaction through G{alpha}i2 Activation That Controls C5aR and Fc Receptor Cooperation1

Julia Skokowa2,3,*, Syed R. Ali3,*, Olga Felda*,{ddagger}, Varsha Kumar*, Stephanie Konrad*, Nelli Shushakova*, Reinhold E. Schmidt*, Roland P. Piekorz{ddagger}, Bernd Nürnberg{ddagger}, Karsten Spicher§, Lutz Birnbaumer, Jörg Zwirner||, Jill W. C. Claassens#, Josef S. Verbeek#, Nico van Rooijen**, Jörg Köhl4,{dagger} and J. Engelbert Gessner5

* Department of Clinical Immunology and {dagger} Institute of Medical Microbiology, Medical School Hannover, Hannover, Germany; {ddagger} Department of Biochemistry and Molecular Biology II, Heinrich Heine University, Dusseldorf, Germany; § Institute of Pharmacology, Berlin Free University, Berlin, Germany; Laboratory of Signal Transduction, National Institute of Environmental and Health Sciences, National Institutes of Health, Department of Health and Human Sciences, Research Triangle Park, NC 27709; || Department of Immunology, Georg August University, Gottingen, Germany; # Department of Genetics, Leiden University, Leiden, The Netherlands; and ** Department of Molecular Cell Biology, Vrije University, Amsterdam, The Netherlands


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Complement and Fc{gamma}R effector pathways are central triggers of immune inflammation; however, the exact mechanisms for their cooperation with effector cells and their nature remain elusive. In this study we show that in the lung Arthus reaction, the initial contact between immune complexes and alveolar macrophages (AM) results in plasma complement-independent C5a production that causes decreased levels of inhibitory Fc{gamma}RIIB, increased levels of activating Fc{gamma}RIII, and highly induced Fc{gamma}R-mediated TNF-{alpha} and CXCR2 ligand production. Blockade of C5aR completely reversed such changes. Strikingly, studies of pertussis toxin inhibition show the essential role of Gi-type G protein signaling in C5aR-mediated control of the regulatory Fc{gamma}R system in vitro, and analysis of the various C5aR-, Fc{gamma}R-, and Gi-deficient mice verifies the importance of G{alpha}i2-associated C5aR and the Fc{gamma}RIII-Fc{gamma}RIIB receptor pair in lung inflammation in vivo. Moreover, adoptive transfer experiments of C5aR- and Fc{gamma}RIII-positive cells into C5aR- and Fc{gamma}RIII-deficient mice establish AM as responsible effector cells. AM lacking either C5aR or Fc{gamma}RIII do not possess any such inducibility of immune complex disease, whereas reconstitution with Fc{gamma}RIIB-negative AM results in an enhanced pathology. These data suggest that AM function as a cellular link of C5a production and C5aR activation that uses a G{alpha}i2-dependent signal for modulating the two opposing Fc{gamma}R, Fc{gamma}RIIB and Fc{gamma}RIII, in the initiation of the inflammatory cascade in the lung Arthus reaction.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The mechanisms by which the immune system controls effector responses to pathogenic Abs and immune complexes (IC)6 are of central importance for understanding how immunologic diseases develop. The initial response to IC challenge is local inflammation, which provokes perivascular edema, hemorrhage, vascular permeability, and leukocyte extravasation. Based on initial work in the classical model of IC inflammation, the Arthus reaction, it has long been understood that formation of IC propagates tissue damage primarily by complement activation via the classical pathway. This view, however, has been changed during the last years by the advent of gene knockout technology in mice. It is now clear that, dependent on the property of the target tissue, the alternative complement pathway also plays a crucial role (1), and that late complement components, C5a and C5aR, are essential effectors in the majority of complement-mediated inflammatory reactions (2, 3, 4). IgG FcR (Fc{gamma}R) are the other well-established key effectors that regulate inflammatory responses by the balance of activating Fc{gamma}RIII and inhibitory Fc{gamma}RIIB signals (5). Activating Fc{gamma}RI and Fc{gamma}RIII are expressed in association with dimers of the signal-transducing FcR{gamma} chain, which contains an ITAM sequence in its cytoplasmic tail (6). Fc{gamma}RIII and Fc{gamma}RIIB bind IC with comparable affinity and specificity, and coupling of Fc{gamma}RIII with Fc{gamma}RIIB, which has an ITIM motif in the cytoplasmic domain, results in tyrosine phosphorylation of Fc{gamma}RIIB ITIM and subsequent inhibition of the Fc{gamma}RIII ITAM-triggered activation signal (7). In vivo, Fc{gamma}RIIB-deficient mice exhibit enhanced susceptibility in models of inflammatory autoimmune disease (8), whereas Fc{gamma}RIII-deficient mice show diminished disease phenotypes (4, 9, 10). Recently published data for Fc{gamma}RI-deficient mice re-emphasize the requirement of Fc{gamma}RI for immune defenses against bacterial infection and, in concert with Fc{gamma}RIII, for the development of autoimmune hemolytic anemia (11).

Despite increased knowledge of how complement and Fc{gamma}R each contribute to various immunologic diseases, the critical role of their possible interaction has only recently been addressed (12). In skin Arthus reactions, the presence of a C3b-independent, C5a-generating pathway can overrule impaired humoral complement activation in C3-deficient mice (13), whereas C5aR modulates Fc{gamma}R in alveolitis and peritonitis (14, 15). These findings indicate that the convergence of complement and Fc{gamma}R effector pathways at the level of C5a:C5aR is of central importance for disease pathogenesis. However, the underlying cellular mechanisms have not yet been fully defined. In this study we show, through the use of adoptive cell transfer systems in conjunction with genetic deletion and pharmacological inhibition studies, that macrophages are the primary effectors that induce the lung Arthus reaction by sequential activation steps and provide novel insights into the specific requirements of the receptor systems involved, C5aR and Fc{gamma}RIIB/Fc{gamma}RIII. Our findings provide direct evidence for a refined model of the inflammatory cascade with local Gi-dependent C5aR-Fc{gamma}R cross talk as the key regulatory event.


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

The generation of Fc{gamma}RI-, Fc{gamma}RIII-, and G{alpha}i2-deficient mice and their phenotypic characterization has been described in detail (11, 16, 17, 18). G{alpha}i3-deficient mice have been generated as described by Jiang et al. (19), and detailed analysis of this strain will be published elsewhere (our manuscript in preparation). Fc{gamma}RIIB- and C5aR-deficient mice were provided by T. Takai (Department of Experimental Immunology and CREST Program of Japan Science and Technology Agency, Institute of Development, Aging, and Cancer, Tohoku University, Sendai, Japan) (20) and U. Höpken (Department of Tumor Genetics and Immunogenetics, Berlin, Germany) (21). All gene-deleted mice were backcrossed to C57BL/6 mice for four to eight generations and maintained under dry-barrier conditions. CD45.1+ congenic C57BL/6 mice (B6.SJL-PtrcaPep3b/BoyJ) were purchased from The Jackson Laboratory. C57BL/6 control mice were obtained from Charles River. All these mice were used at 8–14 wk of age. Animal experiments were conducted in accordance with current laws in combination with the regulations of the local authorities.

mAb and FACS analysis

The following Abs were used: anti-CD45.1 (clone A20), anti-CD45.2 (clone 104), anti-Fc{gamma}RII/III (clone 2.4G2; all from BD Pharmingen), anti-F4/80 (Serotec), anti-Fc{gamma}RIIB/Ly17.2 (clone K9.361) (22), and anti-C5aR (clone 1/36) (23). F4/80, CD45.1, and CD45.2 Ags on bronchoalveolar lavage (BAL)-alveolar macrophage (AM) cells (2 x 104) were analyzed by flow cytometry using a FACScan flow cytometer (BD Biosciences). Expression of Fc{gamma}RIIB was analyzed by staining with the anti-Fc{gamma}RIIB mAb Ly17.2 conjugated to FITC. To analyze Fc{gamma}RIII expression, BAL-AM cells were first treated with saturating doses of unlabeled anti-Fc{gamma}RIIB Ly17.2 mAb, followed by PE-2.4G2 anti-Fc{gamma}RII/III mAb staining, as previously described (14, 22).

Experimental induction of the lung Arthus reaction

Mice were anesthetized with ketamine and xylazine, the trachea was cannulated, and 150 µg of protein G-purified, rabbit anti-OVA IgG Ab (Sigma-Aldrich) was applied. Immediately thereafter, 20 mg/kg OVA Ag was given i.v.; Ab control animals received PBS instead of OVA Ag. In AM depletion experiments, mice intratracheally (i.t.) received liposome-encapsulated dichloromethylene diphosphonate (Cl2MDP; at a concentration of 5 mg/ml; Lipo-clod) as previously described (Cl2MDP was a gift of Roche Diagnostics) (24). Initial studies indicated a single dose of 100 µl and the application route used to be most effective for selective depletion of AM within 1–3 days, lasting for at least 5–6 days with renewing AM first detectable on day 7 and full repopulation after 18 days. Intratracheal delivery of PBS-containing liposomes (Lipo-PBS) did not show any depletion effects and served as the control. In additional blocking experiments, mice received either 1/36 mAb directed against C5aR to block C5aR activity, pertussis toxin (PTX) to inhibit Gi function, or soluble FcR fragments (sFcR), consisting of the unglycosylated extracellular part of human Fc{gamma}RIIb (25), to inhibit cellular activation of Fc{gamma}R. Doses of 20 µg of 1/36 mAb, 800 ng of PTX, and 50 µg of sFcR were given i.t. before the application of anti-OVA IgG. Mice were killed at various time points (ranging from 2–24 h) after initiation of the Arthus reaction, and lung tissues obtained after lavage were assayed for interstitial polymorphonuclear leukocyte (PMN) accumulation by myeloperoxidase (MPO) activity as previously described (12).

BAL and quantitation of hemorrhage, PMN accumulation, mediator release in the bronchoalveolar compartment, and Fc{gamma}R and C5aR expression on BAL-AM cells

Pulmonary vasculature was gently flushed with PBS with a catheter positioned in the root pulmonary artery. Lungs were lavaged with PBS (1 ml, five times at 4°C) after cannulation of the trachea. The volume of the collected BAL fluid (BALF) was measured in each sample, and total cell count was assessed with a hemocytometer (Neubauer Zählkammer). The amount of erythrocytes represented the degree of hemorrhage. For quantitation of PMN accumulation, differential cell counts were performed on cytospins (10 min at 55 x g) stained with May-Grünwald/Giemsa using 300 µl of BALF. The concentrations of MIP-2, TNF-{alpha}, and IL-1{beta} in BALF were measured in duplicate in appropriately diluted samples with respective MIP-2-, TNF-{alpha}-, and IL-1{beta}-specific ELISA kits (R&D Systems) according to the manufacturer’s instructions. For Fc{gamma}R and C5aR expression analysis, total RNA was prepared from BAL-AM cells. Fc{gamma}R and C5aR mRNA levels normalized to tubulin were quantitated by TaqMan real-time RT-PCR using published Fc{gamma}R/C5aR-specific primers and probes (14, 26).

Determination of C5a-dependent chemotactic activity in BALF and AM cell supernatants

Bone marrow cells (containing 64–68% PMN) from C57BL/6 and C5aR-null mice were suspended at 7.5 x 106 cells/ml RPMI 1640 medium and 0.5% BSA. One hundred microliters of the bone marrow cell suspension (treated, or not, with anti-C5aR 1/36 mAb and PTX) was placed into the insert of a Transwell chemotaxis chamber, and the bottom well was filled with 600 µl of RPMI 1640/0.5% BSA, the same medium supplemented with 5 x 10–9 M rhC5a, appropriately diluted MH-S AM cell culture supernatants obtained at different times after IC activation, or BALF diluted 1/2 in RPMI 1640/1% BSA. BALF were obtained from Lipo-clod-treated and untreated C57BL/6 mice at 2 and 4 h after OVA:anti-OVA IC inflammation. BALF from mice receiving Ab, but not OVA Ag, served as controls. Inserts were combined to the lower chambers and incubated at 37°C and 6% CO2 for 2 h. After the incubation, 50 µl of 70 mM EDTA solution was added to the lower chambers to release adherent cells from the lower surface of the membrane and from the bottom of the well. Plates were further incubated 30 min at 4°C, inserts were removed, and the transmigrated neutrophils were vigorously suspended and counted with a FACSCalibur for 1 min at 60 µl/min with gating on forward and side scatter. Migration of PMN from the insert to the bottom well was quantitated as a percentage of the total PMN loaded into the upper chamber.

IC activation of AM MH-S cells in vitro

Mouse MH-S AM cell line cells (27) were maintained in RPMI 1640 medium containing 10% FCS and supplements. In functional assays, 106 MH-S cells were incubated overnight in six-well culture plates containing 1% FCS/RPMI 1640 medium and activated with 100 µg/ml heat-aggregated IgG IC (mouse IgG1) or 5 x 10–9 M rhC5a (Sigma-Aldrich). In some experiments MH-S cells were also incubated with anti-C5aR mAb 1/36 (100 µg/ml) or PTX (500 ng/ml; for 2 h; Calbiochem) to block C5aR-Gi activity. After various time periods (0–16 h), appropriate dilutions of culture supernatants from untreated and stimulated MH-S cells were examined for production of TNF-{alpha} and MIP-2 by ELISA and C5a-dependent chemotactic activity by Transwell migration assays. Total RNA was prepared and analyzed for Fc{gamma}R mRNA synthesis by TaqMan RT-PCR.

Functional expression analysis of G{alpha}i subunits in AM cells

For detection and identification of PTX-sensitive Gi proteins [32P]ADP-ribosylation in the presence of PTX and immunoblot analysis of cell membranes were performed as previously described (28). In brief, mouse embryonic fibroblasts (MEFs) from wild-type (WT), G{alpha}i2(–/–) and G{alpha}i3(–/–) embryos (our manuscript in preparation), MH-S cells, and Jurkat T cells were harvested and disrupted by three freeze-thaw cycles. Cell membranes were isolated and subjected to [32P]ADP ribosylation in the presence of PTX. Cell membranes (100 µg/lane) and PTX-treated membranes (40 µg/lane) were subjected to urea-containing SDS-PAGE, and Gi proteins were either detected by staining with a G{alpha}common Ab (AS 8) (29) and visualized using the ECL chemiluminescence system (Amersham Biosciences) and a Diana chemiluminescence-Imager (Raytest) or subjected to phosphorimaging using a FLA 5000 Fuji-Imager (Raytest), respectively.

Adoptive cell transfer of manipulated CD45.2+ AM cells in CD45.1+ congenic mice

Selective AM depletion in CD45.1+ congenic C57BL/6 recipient mice was performed by i.t. administration of Lipo-clod for 3 days. Reconstitution of such depleted CD45.1+ congenic mice with CD45.2+ AM was performed by i.t. injection of 5 x 105 CD45.2+ BALF-AM cells from WT C57BL/6 donors (pretreated, or not, with either 20 µg/ml anti-C5aR 1/36 mAb or 500 ng/ml PTX for 2 h) or CD45.2+ B6 mice deficient in Fc{gamma}RI, Fc{gamma}RIIB, Fc{gamma}RIII, or C5aR. The effectiveness of depletion of CD45.1+ AM and subsequent adoptive transfer of the different CD45.2+ AM cells was followed in individual mice by FACS analysis using anti-F4/80, anti-CD45.1, and anti-CD45.2 Abs. After allowing 24 h for reconstitution, the lung Arthus reaction was induced and assessed as described above.

Adoptive cell transfer of CD45.1 AM cells in CD45.2 C5aR–/– and Fc{gamma}RIII–/– mice

AM depletion/reconstitution in CD45.2 C5aR–/– and Fc{gamma}RIII–/– mice was performed by i.t. application of Lipo-clod for 3 days and subsequent transfer of CD45.1 AM, followed in individual mice by FACS analysis using F4/80, CD45.1, and CD45.2 staining. One day after reconstitution, the lung Arthus reaction was induced and assessed as described above.

Statistical analysis

Statistical analysis was performed using the SPSS V. 9.0 statistical package. To analyze differences in mean values between groups, a two-sided unpaired Student’s t test was used.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Lung Arthus response depends on macrophages

Nonredundant and codominant roles for C5a/C5aR components of complement and the regulatory Fc{gamma}R system have been implicated in Arthus reactions triggered by IC of foreign Ags depending on the mouse strain and tissue site of inflammation (30). Among the distinct effector cells involved, mast cells are essential in models of vasculitis and arthritis (31, 32). In contrast, they are less important in alveolitis (13). Because AM are major cells in alveoli, we hypothesized that AM may represent the critical effector cells in lung pathology. In C57BL/6 mice, induction of the lung Arthus reaction by rabbit anti-OVA IgG and OVA Ag results in a robust hemorrhagic response with rapid secretion of cytokines and chemokines, followed by accumulation of PMN in lung tissue, reaching maximal levels of PMN transmigration into alveoli at 8–24 h (33). To assess the role of AM effector cells, we evaluated the effect of their depletion in kinetic studies (at 2–8 h) in mice receiving Cl2MDP-containing liposomes (Lipo-clod) i.t. (24). This treatment resulted in AM depletion within 3 days compared with control mice receiving Lipo-PBS (Fig. 1a). IC-induced signs of inflammation specified by PMN accumulation in lung tissue, PMN influx into alveoli, and hemorrhage were all significantly lower in Lipo-clod-treated mice than in Lipo-PBS control animals (Fig. 1b). Moreover, analysis of BALF from AM-depleted mice revealed markedly reduced levels of MIP-2, TNF-{alpha}, and IL-1{beta} (Fig. 1c).



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  		FIGURE 1. AM depletion by Lipo-clod abrogates the lung Arthus response. a, C57BL/6 mice were locally treated with Cl2MDP-encapsulated liposomes (Lipo-clod) or Lipo-PBS and examined on day 3 by flow cytometry (untreated mice served as the control). CD45.2+ BAL-AM cells were isolated and stained for the macrophage marker F4/80. Histograms are representative of three independent experiments. b and c, On day 3 after Lip-clod and Lipo-PBS treatment, the lung Arthus reaction was induced by local injection of 150 µg of purified anti-OVA Ab, followed by systemic 20 mg/kg OVA Ag (IC). Controls received anti-OVA Ab without OVA Ag (Ab). Mice were killed at the indicated times (2, 4, and 8 h), and lung tissues were collected after lavage and analyzed for MPO activity as a marker of interstitial PMN accumulation (b, upper part). BALFs were examined for alveolar PMN influx (b, middle part) and hemorrhage (b, lower part). The concentrations of MIP-2 (c, upper part), TNF-{alpha} (c, middle part) and IL-1{beta} (c, lower part) in BALF were evaluated by ELISA. Results are expressed as the mean ± SEM (n = 6–12 mice for each group). *, p < 0.05; **, p < 0.001.

 
In line with the kinetic data of macrophage depletion, the roles of Fc{gamma}RIII and C5aR have recently been shown to correlate with mast cell-independent secretion of such mediators in the lung Arthus response (13, 33). IC-induced responses of enhanced vs diminished hemorrhage and alveolar PMN infiltration were consistently observed at 24 h in Fc{gamma}RIIB compared with Fc{gamma}RIII and C5aR mutant mice (14, 33, 34) (data not shown), whereas lack of the high affinity Fc{gamma}RI had no substantial influence on the inflammatory reaction (WT vs Fc{gamma}RI knockout: PMN, 20.54 ± 3.92 x 105 vs 17.96 ± 3.83 x 105; n = 8–16; p > 0.6; RBC, 114.59 ± 21.27 x 106 vs 107.20 ± 44.60 x 106; n = 8–16; p > 0.7). Local inhibition of Fc{gamma}R by sFcR or of C5aR by the neutralizing anti-C5aR Ab 1/36 as well as AM depletion each resulted in markedly suppressed alveolar PMN influx and hemorrhage at 24 h in IC-challenged C57BL/6 mice (Fig. 2a). BAL-AM cells displayed changes in the mRNA levels of Fc{gamma}R, but not C5aR at 2 h after IC challenge (Fig. 2b). These early IC-induced Fc{gamma}R alterations are C5aR-dependent, because anti-C5aR 1/36 mAb-treated mice showed neither induction of Fc{gamma}RIII {alpha}{gamma}-chains nor suppression of Fc{gamma}RIIB (Fig. 2b). Surface Fc{gamma}R expression was up-regulated by IC in the case of Fc{gamma}RIII and down-regulated in the case of Fc{gamma}RIIB, with this opposite regulation being sensitive to C5aR blockade (Fig. 2c). Collectively, these results establish macrophages as primary effectors that trigger the lung Arthus reaction and, consistent with our previous data in C5aR-deficient mice (14), validate the dichotomous effects of C5aR on inhibitory Fc{gamma}RIIB and activating Fc{gamma}RIII in the induction of the inflammatory cascade.



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  		FIGURE 2. C5aR-dependent inverse Fc{gamma}R regulation in vivo. The lung Arthus reaction was induced in C57BL/6 mice pretreated with blocking anti-C5aR 1/36 mAb (1/36), soluble Fc{gamma}R (sFcR), chlodronate liposomes (Lipo-clod), or Lipo-PBS by local injection of anti-OVA Ab, followed by systemic OVA Ag (IC). Controls received anti-OVA Ab without OVA Ag (Ab). Mice were killed at different time points (2, 4, and 24 h), and BALF were counted for PMN (a, left panel) and RBC (a, right panel) as markers for alveolar PMN transmigration and hemorrhage at 24 h. b and c, Expression of C5aR and Fc{gamma}R was assessed in BAL-AM cells from C57BL/6 mice at 2 h after IC challenge. b, mRNA analysis by TaqMan RT-PCR showed significantly increased Fc{gamma}RIII and FcR{gamma} and reduced Fc{gamma}RIIB mRNA levels in response to IC challenge ({blacksquare}; IC), which was inhibited by anti-C5aR 1/36 mAb treatment ({cjs2108}; IC and 1/36). C5aR expression did not differ between the treatment groups. Data are expressed as the mean ± SEM (n = 5–6 mice in each group). *, p < 0.05; **, p < 0.001). c, BAL-AM cells (2 x 104) from the indicated treatment groups were analyzed by flow cytometry for changes in surface expression of Fc{gamma}RIII and Fc{gamma}RIIB. Data are expressed as the mean fluorescence intensity (MFI) ± SEM (n = 5–6 mice in each group). *, p < 0.05. IC-induced Fc{gamma}RIII up-regulation and Fc{gamma}RIIB suppression were diminished in the IC plus 1/36 treatment group. d, C5aR-dependent chemotactic activity was determined by Transwell migration assays of neutrophils (PMN isolated from bone marrow of C57BL/6 and C5aR-null mice) elicited with 300 µl of BALF pools obtained from five mice of the Ab, IC, and IC plus Lipo-clod treatment groups at 2 and 4 h. Results are expressed as the percentage of PMN loaded into the upper chamber that had migrated to the bottom well (mean ± SEM for five individual experiments). *, p < 0.05; **, p < 0.001.

 
Macrophages are needed for early C5a production

Several studies of the role of complement have indicated a greater importance of C5a than C3 in models of arthritis and different settings of the Arthus reaction (4, 21), which may be explained by a C3 bypass mechanism in the generation of C5a at sites of inflammation (13). Analysis of chemotactic activities of BALF from IC-challenged mice, measured on C5aR+ compared with C5aR-null PMN, showed (within 2 h) C5aR-dependent PMN chemotaxis that was completely diminished in Lipo-clod-treated mice, which suggests the requirement for AM cells in early C5a production in the lung (Fig. 2d). AM-depleted mice displayed reduced, but detectable, C5aR-dependent chemotaxis 4 h after IC challenge (Fig. 2d), indicating activation of plasma complement as the expected complementary C5a generation pathway. However, such humoral production of C5a was not sufficient to trigger substantial inflammation in the absence of AM.

Heat-aggregated (HA)-IgG1-activation of macrophages: multiple C5a-, C5aR-, Gi-, and Fc{gamma}R-dependent steps

AM cells are constitutively active in converting extracellular C5 into C5a (35), but whether AM directly link IC formation to C5a production in an autonomous manner remains unknown. In this report, kinetic studies using supernatants from HA-IgG1-stimulated MH-S AM cells demonstrated the presence of mediators with chemotactic activity on PMN from Fc{gamma}RIII–/– and WT C57BL/6 mice. This activity was greatly ineffective on PMN from C5aR–/[minus) mice (Fig. 3a), the residual migration of C5aR–/– PMN was found to be MIP-2-dependent (data not shown). These findings indicate that activated AM generate chemotactically active C5a independent of humoral complement activation. In line with this, MH-S AM not only displayed regulated Fc{gamma}R mRNA expression after a 2- to 4-h period of incubation with rhC5a (5 x 10–9 M), but also equally responded to HA-IgG1 exposure, showing the same marked reduction in Fc{gamma}RIIB mRNA and enhanced expression of Fc{gamma}RIII (Fig. 3b). We also analyzed whether this HA-IgG1-induced increase in the ratio of Fc{gamma}RIII to the inhibitory Fc{gamma}RIIB depends on C5aR signaling and is required for activation of AM. Stimulation of Fc{gamma}R by IgG1 aggregates, but not of C5aR by rhC5a alone (at 5 x 10–9 M; data not shown), resulted in increased protein levels of MIP-2 and TNF-{alpha} in supernatants of AM cell cultures (Fig. 3c). Importantly, however, IgG1-induced MIP-2 and TNF-{alpha} secretion was completely inhibited by incubation with 100 µg/ml anti-C5aR 1/36 mAb (Fig. 3c), which was not observed with an isotype control Ab of irrelevant specificity (data not shown). Moreover, treatment of cells with 500 ng/ml PTX for 2 h to functionally inactivate C5aR-associated Gi-type G proteins caused a similar impaired IgG1 activation, as documented by strongly reduced synthesis of MIP-2 and TNF-{alpha} (Fig. 3c). These results together with the observation that treatment with either 1/36 or PTX resulted in blockade of the inverse regulation of Fc{gamma}RIIB and Fc{gamma}RIII (Fig. 3d) suggest a tightly regulated IC responsiveness-encoding pathway in AM that involves autonomous C5a production and activation, and Gi family member-dependent signaling of C5aR, Fc{gamma}R modulation toward the Fc{gamma}RIII activation phenotype, and, in turn, chemokine and cytokine production.



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  		FIGURE 3. The HA-IgG1 activation response of MH-S AM cells involves multiple C5a-, C5aR-, Gi-, and Fc{gamma}R-mediated steps. a, Detection of C5a bioactivity in supernatants of MH-S AM cells (medium control) incubated with heat-aggregated IgG1 for 2–16 h. Chemotaxis was determined by Transwell migration assays of PMN isolated from bone marrow of the indicated mice (mean ± SEM; three individual experiments performed in duplicate). b, Detection of Fc{gamma}R regulation. MH-S cells ({square}; medium control) were triggered with HA-IgG1 ({blacksquare}; +100 µg HA-IgG1) or rhC5a ({cjs2108}; +5 x 10–9 M rhC5a) for 2–4 h and assayed for altered Fc{gamma}RIIB and Fc{gamma}RIII mRNA synthesis by TaqMan RT-PCR. c and d, Gi-dependent C5aR signaling in the control of Fc{gamma}R regulation and mediator release. MH-S cells (medium control) were activated for the indicated times with HA-IgG1 or were incubated with anti-C5aR 1/36 mAb (HA-IgG1 + 1/36), or pretreated for 2 h with PTX (HA-IgG1 + PTX) and analyzed for MIP-2 and TNF-{alpha} production by ELISA (c) and for changes in Fc{gamma}RIIB/Fc{gamma}RIII mRNA normalized to {beta}-tubulin by TaqMan RT-PCR (d). Data are expressed as the mean ± SEM from three independent experiments performed in duplicate. *, p < 0.05; **, p < 0.001.

 
C5aR-associated Gi proteins in the Arthus response: critical importance of G{alpha}i2

Because IC-triggered Fc{gamma}R activation on AM appears to be C5a:C5aR-Gi-dependent in vitro, we assessed the significance of PTX-sensitive Gi protein activity in pulmonary IC inflammation in vivo. IC-challenged C57BL/6 mice that received PTX showed diminished PMN and RBC levels in alveoli (Fig. 4a), which indicated that one or more PTX-sensitive G proteins are required for disease induction. A recent analysis demonstrated only G{alpha}i2 and G{alpha}i3 in lung tissue homogenates (36). G{alpha}i2 and G{alpha}i3 are widely expressed and, with respect to lung, MH-S AM cells coexpress Gi proteins with a stoichiometric excess of G{alpha}i2 over G{alpha}i3, as shown by autoradiograms from PTX-catalyzed [32P]ADP ribosylation and immunoblot analysis (Fig. 4c). Thus, we considered G{alpha}i2 to be particularly important for C5aR-dependent effects in immune inflammation. To address this issue, Gi-knockout mice deficient in either G{alpha}i2 or G{alpha}i3 were analyzed in the Arthus reaction. G{alpha}i2/3 double mutants were not included in these experiments due to their embryonic lethality. As illustrated in Fig. 4d, G{alpha}i3–/– mice (n = 6; p > 0.7 compared with C57BL/6 mice) developed normal signs of inflammation at 24 h, whereas G{alpha}i2–/– mice exhibited a strong defect in hemorrhage and alveolar PMN influx that was even more pronounced in C5aR mutant mice (G{alpha}i2–/– vs C5aR–/–: PMN, 6.21 ± 2.09 x 105 vs 1.25 ± 0.35 x 105; n = 7–8; p = 0.03; RBC, 50.79 ± 16.46 x 106 vs 11.37 ± 0.29 x 106; n = 7–8; p = 0.03). These data suggest that C5aR-associated Gi2 is the functionally dominant G protein in this process. with some compensatory role of Gi3, especially when Gi2 is absent.



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  		FIGURE 4. Analysis of general Gi function and the G{alpha}i2/3 requirements in vitro and in vivo. a, The lung Arthus reaction was induced in C57BL/6 mice (WT) treated with either anti-C5aR 1/36 mAb (+1/36) or the general Gi function inhibitor, PTX (+PTX) by IC challenge (IC). Controls only received anti-OVA Ab (Ab). b, The Gi function dependency of C5aR-mediated chemotaxis was assayed with 50 ng/ml rhC5a using the indicated PMN preparations incubated, or not, with 1/36 or PTX. Data are expressed as the mean ± SEM from two independent experiments performed in triplicate. **, p < 0.001. c, Expression of Gi proteins in mouse AM MH-S cells as visualized by PTX-mediated [32P]ADP ribosylation and immunoblot analysis. Track 1, WT MEFs; track 2, G{alpha}i2(–/–) MEFs; track 3, G{alpha}i3(–/–) MEFs; track 4, MH-S cells; track 5, Jurkat T cells. d, Attenuation of the Arthus response by G{alpha}i2, but not G{alpha}i3, deficiency. After 24 h, IC-challenged mice were killed and analyzed for alveolar PMN accumulation (left panel) and hemorrhage (right panel). Results in a and d are expressed as the mean ± SEM (n = 6–9 mice for each group). *, p < 0.05; **, p < 0.01.

 
Disease prevention by selective targeting of macrophage C5aR-Fc{gamma}R axis components

The diminished inflammatory response exhibited by mice treated with PTX or lacking C5aR (Fig. 4) may be explained by impaired C5aR-Gi-dependent modulation of Fc{gamma}R that is found after C5aR blockade in vivo (Fig. 2) and during AM stimulation in vitro (Fig. 3). In addition, the other more traditional function of C5a as PMN-specific chemoattractant was abolished by preincubation with either PTX or anti-C5aR 1/36 mAb in vitro (Fig. 4b). Thus, it is not clear from these experiments which one of the two or the combination of C5a:C5aR-Gi-mediated activities is central for disease pathogenesis. To tackle this question, we performed several adoptive cell transfer experiments using CD45.1/2 congenic mice. As an initial strategy, AM cells were first depleted for 3 days with Lipo-clod in congenic CD45.1+ C57BL/6 recipient mice, which then were reconstituted with BALF-AM cells from CD45.2+ C57BL/6 donor mice, followed by IC challenge on day 4 and evaluation on day 5 (Fig. 5a). The allotypic CD45 marker served as a tool to follow the course of AM depletion and reconstitution (Fig. 5b). IC-challenged CD45.1+ and CD45.2+ C57BL/6 mice showed the expected identical response of alveolar PMN migration and hemorrhage, which was inhibited by AM depletion, but was fully restored through CD45.2+ AM engraftment in AM-depleted CD45.1+ hosts (Fig. 5c). PTX and anti-C5aR 1/36 mAb treatments of CD45.2+ AM cells before transfer resulted in no disease recovery (Fig. 5c). Thus, ex vivo-performed functional inactivation of Gi proteins in AM was sufficient for complete disease prevention. This suggests that the C5aR-Gi-triggered pathway in AM is of much greater importance than the C5aR-Gi-dependent chemotactic response of PMN, which remains intact in this adoptive transfer model.



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  		FIGURE 5. Ex vivo inhibition of macrophage C5aR/Gi sufficient for disease prevention. a, Overview of the experimental design. b, FACS analysis of BAL cells from CD45.1+ C57BL/6 mice after AM depletion and CD45.2+ AM transfer with FITC- and PE-labeled Abs specific for F4/80, CD45.1, and CD45.2 Ags revealed efficient depletion of F4/80+ AM by Lipo-clod (at 0 and 3 days) and stable repopulation of transferred CD45.2+ AM (at 4 and 5 days). c, Lipo-clod-induced AM depletion and induction of the Arthus reaction was performed as described in Fig. 1. CD45.1+ and CD45.2+ B6 mice of the indicated treatment groups were assayed for alveolar PMN infiltration and pulmonary hemorrhage in response to IC challenge (IC). Controls received anti-OVA Ab without OVA Ag (Ab). Data are expressed as the mean ± SEM (n = 5–8 mice for each group). In AM-depleted CD45.1 mice (+ Lipo-clod), the IC reaction was completely restored after AM transfer (+ Lipo-clod, + CD45.2 AM). This restoration of disease was completely abolished by anti-C5aR 1/36 and PTX treatments (n = 4–5; mean ± SEM). **, p < 0.001.

 
In the next set of experiments, AM cells from Fc{gamma}R and C5aR knockout mice were used for reconstitution. Recovery of lung pathology through CD45.2+ AM in CD45.1+ C57BL/6 recipient mice was equally achieved by transfer of Fc{gamma}RI-deficient AM and was strongly enhanced or completely abolished after reconstitution with CD45.2+ AM specifically lacking Fc{gamma}RIIB, or Fc{gamma}RIII and C5aR, respectively (Fig. 6b). Flow cytometric detection of F4/80, CD45.1, and CD45.2 Ags by FACS analysis verified the successful repopulation of CD45.2+, F4/80+ AM from the various mouse mutants throughout all experiments (Fig. 6a). Thus, AM with impaired Gi function (see Fig. 5) or lacking C5aR or Fc{gamma}RIII (Fig. 6) do not longer possess disease-inducing capacity. The results define an absolute requirement for the macrophage C5a:C5aR-Gi-Fc{gamma}RIIB/Fc{gamma}RIII axis in the lung Arthus reaction.



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  		FIGURE 6. Disease recovery by transplanted AM dependent on the C5aR-Fc{gamma}RIIB/Fc{gamma}RIII axis. a, Efficient engraftment of mutant AM in CD45.1+ recipients. FACS analysis of BAL-AM cells of CD45.1+ AM-depleted mice recovered 2 days after transfer of CD45.2+ AM from the indicated mice. Representative histograms of individual mice are shown. b, AM depletion and reconstitution was performed as described in Fig. 4. Twenty-four hours after IC challenge (IC), alveolar PMN influx and hemorrhage were assessed in Lipo-clod-treated CD45.1+ mice (IC + Lipo-clod; {square}) and after reconstitution with CD45.2+ AM cells from the indicated gene-deficient mice ({blacksquare}). Results are expressed as the mean ± SEM (n = 4–5 mice for each group. *, p < 0.05; **, p < 0.001.

 
Diminished Arthus response in C5aR–/– and Fc{gamma}RIII–/– mice rescued by normalization of macrophage function

Whether PMN can transmigrate into alveoli is probably dependent on their response to the chemotactic factors expressed in lung tissue. The failure of PMN to migrate to the alveolar space in AM-depleted mice could be attributed to a reduced expression of C5a (Fig. 2d) and CXCR2 ligands (MIP-2; Fig. 1c). The reported IC-tethering function of Fc{gamma}RIII also suggests a role of neutrophil Fc{gamma}RIII for PMN migration (37). However, the finding that defects in C5aR-Gi-Fc{gamma}RIII on local AM cells were severe enough to cause severely impaired recruitment of otherwise C5aR- and Fc{gamma}RIII-positive PMN (Figs. 5 and 6) suggests a minor role for C5a:C5aR and IC:Fc{gamma}RIII signaling on PMN in the Arthus reaction. To further confirm this idea, AM cells from CD45.1 mice were transplanted in WT and C5aR- or Fc{gamma}RIII-deficient CD45.2 mice (Fig. 7a). This approach was used to selectively restore the macrophage C5a:C5aR-Gi-Fc{gamma}RIII axis, but leaving C5aR/Fc{gamma}RIII on PMN impaired. Transfer of C5aR/Fc{gamma}RIII+/+ AM rescued the diminished Arthus reaction in both Fc{gamma}RIII–/– and C5aR–/– mice, as shown by a normalized hemorrhagic response (Fig. 7b). This rescue effect was also observed for PMN recruitment with normal numbers of migrated Fc{gamma}RIII–/– and C5aR–/– PMN detectable in BALF after C5aR/Fc{gamma}RIII+/+ AM reconstitution (Fig. 7b). Consistent with the Fc{gamma}RIII–/– and C5aR–/– AM transfer studies in C5aR/Fc{gamma}RIII+/+ mice (Fig. 6), the scenario of a C5a:C5aR-Gi-Fc{gamma}R-influenced inflammatory lung microenvironment that induces the migration of PMN to alveoli thus appears dependent on the local macrophage, rather than the PMN side.



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  		FIGURE 7. Diminished lung Arthus response in C5aR–/– and Fc{gamma}RIII–/– mice rescued by WT AM transfer. a, Engraftment of CD45.1 WT AM in CD45.2 C5aR–/– or Fc{gamma}RIII–/– mice was followed by FACS analysis of BAL-AM cells of C5aR–/– and Fc{gamma}RIII–/– mice after CD45.2 AM depletion and CD45.1 AM transfer. Representative histograms of individual mice are shown. b, One day after reconstitution, the Arthus response was induced, and alveolar C5aR–/– and Fc{gamma}RIII–/– PMN migration and hemorrhage were assessed. Results are expressed as the mean ± SEM (n = 4–5 mice/group). **, p < 0.001.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The results show that the inflammatory response in the Arthus reaction is initiated by macrophages that use a Gi-dependent signal for the communication of two key players of the immune system, the C5aR and the paired Fc{gamma}RIIB/Fc{gamma}RIII. This conclusion is based in part on the observation that AM-depleted mice resisted alveolar IC inflammation and that restoration of susceptibility after AM transfer required the functional presence of C5aR and Fc{gamma}RIII (but not of Fc{gamma}RI) on these effector cells. Our finding that Fc{gamma}RIIB-deficient AM induce a substantially enhanced inflammatory phenotype in this transfer model supports previous work (34), which indicated that the balance between stimulatory Fc{gamma}RIII and inhibitory Fc{gamma}RIIB and their coordinate expression is critical in the development of inflammatory lung IC disease. Taking into consideration that the inhibitory Fc{gamma}RIIB is more abundantly expressed on AM than Fc{gamma}RIII (14), it is important to strengthen that for an efficient IC activation of macrophages, a change in the Fc{gamma}RIIB to Fc{gamma}RIII expression ratio is required. Agonists such as LPS and IFN-{gamma} trigger inverse regulation of Fc{gamma}R in vitro (26), whereas in IC disease the C5a anaphylatoxin plays a central role (14). Local inhibition of lung C5aR abrogates inflammation by preventing C5a-dependent induction of macrophage Fc{gamma}RIII and suppression of Fc{gamma}RIIB. Our adoptive transfer studies demonstrate that functional loss of Gi-coupled C5aR on AM causes unresponsiveness to IC in the lung Arthus reaction without compensation by the other known C5aR-Gi-dependent pathway, that is, C5a-triggered chemotaxis for migration of PMN to sites of acute inflammation.

Earlier work suggested the coupling of C5aR to PTX-sensitive Gi class proteins and G{alpha}15, a PTX-insensitive Gq class {alpha} subunit (38). Genetic inactivation of G{alpha}15 in mice revealed functional redundancy of C5a-stimulated Gq class signaling (39), indicating that C5aR may function through both Gq proteins, i.e., G{alpha}15, and one or more of the three distinct Gi1/2/3 isoforms. Our in vitro results show that IC-activated MH-S AM cells have the capacity for plasma complement-independent generation of bioactive C5a, and that a PTX-sensitive C5aR signal is critically required for the modulation of Fc{gamma}R and IC:Fc{gamma}R-induced MIP-2 and TNF-{alpha} production. Thus, Gi family members play a central role in the cellular C5aR-Fc{gamma}R cross talk. Furthermore, C5aR- and G{alpha}i2-deficient, but not G{alpha}i3-deficient, mice, display a diminished IC response in vivo. These data suggest that the AM effector cell response is largely determined by initial C5aR-dependent Gi2 activation connected to Gi-mediated changes in Fc{gamma}R expression levels shifting the balance of Fc{gamma}RIIB/Fc{gamma}RIII toward Fc{gamma}RIII activation in immune inflammation.

Although playing a minor role in the Arthus response of the lung (13), mast cells are essential effector cells in hypersensitivity type III (Arthus-like) reactions at several other tissue sites. In skin and synovium, for example, they function as a cellular link between autoantibodies and inflammatory disease of autoimmune vasculitis and arthritis (31, 32). C5aR and Fc{gamma}RIII are known activators of mast cell function (40, 41) as well as critical effectors in the mast cell-dependent K/BxN model of autoimmune arthritis induced by articular formation of glucose-6-phosphate isomerase/anti-glucose-6-phosphate isomerase IC (4). Thus, future work will address the question of whether C5aR/Fc{gamma}R communication is also a major cellular event on mast cells, as has been shown in this study for macrophages.

Information about immune effector cells and their functions may impact our understanding of diseases and the design of therapeutic strategies. Different effector cells may exhibit different responses to pathogenic products. Moreover, different receptor systems communicate in inflammatory responses, as shown in this study for C5aR and the regulatory Fc{gamma}RIIB/Fc{gamma}RIII system. Based on the opposing Fc{gamma}R functions, strategies that result in enhanced inhibitory Fc{gamma}RIIB expression have been proposed as potential new therapeutic approaches for the treatment of autoimmune diseases (8). Therefore, because Fc{gamma}R are targets for Ab-mediated effects, the characterization of factors and pathways that are capable of controlling inhibitory as well as activating Fc{gamma}R is an important issue. Our results identify Gi-dependent C5aR signaling in the control of the expression and activation of Fc{gamma}R on macrophages in vitro. Similar to the in vivo blockade of Fc{gamma}R, genetic deletion of G{alpha}i2 and C5aR have beneficial effects in vivo. Targeting of C5aR with an anti-C5aR Ab prevents IC disease through the maintenance of macrophage Fc{gamma}RIIB at high expression levels and Fc{gamma}RIII at low expression levels. Previous work suggests different mechanisms in the therapeutic action of i.v. Ig (IVIG) in animal models of immune thrombocytopenic purpura and asthma (42, 43). In the immune thrombocytopenic purpura study, it is reported that administration of IVIG protects against disease in a Fc{gamma}RIIB-dependent manner. After treatment with IVIG, macrophages show high levels of Fc{gamma}RIIB, indicating that enhanced signaling of the inhibitory Fc{gamma}RIIB might be essential for the protective effect of IVIG. In the asthma study, the protective function of IVIG was shown to depend on neutralization of C3a and C5a. In light of the apparent regulatory interactions between C5aR and Fc{gamma}R, it will be important next to determine whether engagement of an as yet unidentified IVIG receptor (44) or the function of IVIG as a C5a scavenger (43) is responsible for high Fc{gamma}RIIB levels and, thus, disease protection.

In conclusion, our results imply a revised multimolecular model of the inflammatory cascade (as summarized in Fig. 8). IC deposition represents the initial step that may provoke inflammation by classical and alternative pathways of complement activation, resulting in C3-dependent C5a production (1, 30). Remarkably, macrophages function as an alternative source of C5a, which helps to explain the varying importance of C3 and C5a in IC disease (4, 21, 45). The functional loss of C5aR on macrophages completely abolishes IC inflammation in otherwise C5a-, C5aR-, and Fc{gamma}R-competent mice, whereas systemic defects of C3, although required for humoral C5a production, are partially tolerated (13). These data indicate the importance of local C5a-induced cellular mechanisms in disease pathogenesis and show that the traditional function of C5a as a PMN chemoattractant is less important than previously suspected. Downstream of AM- and plasma complement-derived C5a, macrophages trigger disease via multiple intracellular steps, one of which involves the C5aR-associated G{alpha}i2 subunit. It is the Gi-dependent part of the C5aR signaling machinery that is absolutely essential for a switch in the balance of the inhibitory and activating Fc{gamma}RIIB/Fc{gamma}RIII pair toward Fc{gamma}RIII, the previously predicted main initiator of the inflammatory cascade. These findings thus confirm the relevance of the regulatory Fc{gamma}R system as the primary target of IC-mediated effects and refine our understanding of the initiating mechanism by dissecting two different functions of C5a: chemoattraction of PMN and local control of Fc{gamma}R, which is dependent on C5aR-associated Gi function with Gi2 as a novel molecular target for drug design in the treatment of inflammation and autoimmune diseases (46, 47). Finally, the effectiveness of ex vivo Gi inhibition by PTX in adoptive cell transfer studies establishes this system as a valuable tool for mutation-based identification of the relevant signaling molecules of the cellular C5aR-G{alpha}i2-Fc{gamma}R axis in the context of immune inflammation in vivo.



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  		FIGURE 8. Summarized model of the roles of complement and Fc{gamma}R in the initiation of the inflammatory cascade and of the dominant involvement of the C5aR-Gi-Fc{gamma}R axis on local effector cells. Lanes 1 and 2, As discussed in the text, cellular and humoral pathways of C5a production and Fc{gamma}R engagement on local effector cells are insufficient triggers of the classical Arthus response. Lane 3, The key regulatory event is defined by an early C5aR-Gi-dependent switch-on signal that is prerequisite for Fc{gamma}RIII activation in the synthesis of TNF-{alpha} and CXCR2L and subsequent PMN recruitment. The migration of PMN occurs independently of C5aR and Fc{gamma}RIII on neutrophils. As indicated by the stippled arrow, the previously reported TNF-{alpha}RI-dependent release of IL-1{beta} (33 ) may represent a potential negative feedback loop via TNF-{alpha}/IL-1{beta}-mediated reinduction of the inhibitory Fc{gamma}RIIB (26 ).

 

   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Acknowledgments
 
We thank P. Sondermann, T. Takai, and U. Höpken for providing sFcR-, Fc{gamma}RIIB-, and C5aR-deficient mice, and A. Braun for critically reading the manuscript.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by grants from the Deutsche Forschungsgemeinschaft (to J.E.G. (GE892/8-1) and B.N. (Nu53/6-1)) and Fond der Chemischen Industrie. J.S. and S.R.A. received fellowships from the international M.D./Ph.D. program provided by the Freundesgesellschaft der Medizinische Hochschule Hannover. V.K. received a fellowship from the graduate program (GK705) of the Deutsche Forschungsgemeinschaft. Back

2 Current address: Department of Pediatric Oncology and Hematology, Medical School Hannover, 30625 Hannover, Germany. Back

3 J.S. and S.R.A. contributed equally to this work. Back

4 Current address: Division of Molecular Immunology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229. Back

5 Address correspondence and reprint requests to Dr. J. Engelbert Gessner, Abteilung für Klinische Immunologie, Medizinische Hochschule Hannover, 30625 Hannover, Germany. E-mail address: gessner.johannes{at}mh-hannover.de Back

6 Abbreviations used in this paper: IC, immune complex; AM, alveolar macrophage; AU, arbitrary unit; BAL, bronchoalveolar lavage; BALF, BAL fluid; C5aR, C5a anaphylatoxin receptor; HA, heat-aggregated; i.t., intratracheal; IVIG, i.v. Ig; Lipo-PBS, PBS-containing liposomes; MEF, mouse embryonic fibroblast; MPO, myeloperoxidase; PMN, polymorphonuclear leukocyte; PTX, pertussis toxin; rhC5a, recombinant human C5a; sFcR, soluble FcR; WT, wild type. Back

Received for publication September 2, 2004. Accepted for publication December 20, 2004.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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