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The Journal of Immunology, 2001, 166: 5193-5200.
Copyright © 2001 by The American Association of Immunologists

Fc{gamma}RIII-Mediated Production of TNF-{alpha} Induces Immune Complex Alveolitis Independently of CXC Chemokine Generation1

Nelli Chouchakova*, Julia Skokowa*, Ulrich Baumann*, Thomas Tschernig{dagger}, Karel M. H. Philippens{dagger}, Bernhard Nieswandt{ddagger}, Reinhold E. Schmidt* and J. Engelbert Gessner2,*

Departments of * Clinical Immunology and {dagger} Functional Anatomy, Medical School Hannover, Hannover, Germany; and {ddagger} Department of Molecular Oncology, General Surgery, Witten/Herdecke University, Wuppertal, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We recently demonstrated a codominant role of C5aR and Fc{gamma}RIII in the initiation of IgG immune complex-mediated inflammation in mice. In this study, we investigated the relative contribution of Fc{gamma}RIII in the generation of several cytokines during experimental hypersensitivity pneumonitis/alveolitis in vivo. Induction of immune complex-alveolitis in C57BL/6 mice resulted in strong accumulation of neutrophils into the lung and enhanced chemotactic activity within bronchoalveolar lavage fluid accompanied by an increased production of the proinflammatory cytokines TNF-{alpha} and IL-1{beta} as well as the ELR-CXC chemokines macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant (KC). Fc{gamma}RIII-deficient C57BL/6 mice (Fc{gamma}RIII-/-) showed a marked reduction of the inflammatory response due to decreased production of TNF-{alpha}, IL-1{beta}, and MIP-2. Results obtained in C57BL/6 mice either lacking the TNF-{alpha} class I receptor (TNF-{alpha}RI-/-) or treated with neutralizing anti-TNF-{alpha} mAb demonstrated an essential contribution of TNF-{alpha} for mediating IL-1{beta} release, neutrophil influx, and hemorrhage. Surprisingly, MIP-2 and KC chemokine levels remained largely unaffected in TNF-{alpha}RI-/- mice or after functional inhibition of TNF-{alpha}. These data suggest that in immune complex alveolitis, the activation of Fc{gamma}RIII may induce divergent downstream effector pathways with TNF-{alpha} acting independently of CXC chemokines to trigger the inflammatory response in C57BL/6 mice.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Elevated levels of immune complexes (IC)3 are frequently observed in a variety of inflammatory diseases, such as systemic lupus erythematosus, rheumatoid arthritis, Goodpasture’s syndrome, and hypersensitivity pneumonitis/alveolitis, indicating that IC-mediated cellular and tissue injury might play a primary role in their pathogenesis (1, 2, 3, 4). Although it had been suspected for a long time that these processes are mediated mainly by complement activation, the mechanisms by which IC formation triggers inflammation are still not fully understood. Results of experimental type III hypersensitivity responses in the classical Arthus reaction support the possibility that IC diseases may be mediated by FcR-dependent mechanisms (5, 6, 7, 8). Comparison of recent data obtained with various gene knockout mice strongly suggests that activation of the complement system, attributed to C5a and its interaction with C5aR, and of Fc receptors for IgG (Fc{gamma}R) are both required to initiate the process of inflammation (Refs. 9, 10, 11, 12 ; reviewed in Ref. 13).

The degree of dependence on complement and Fc{gamma}R appears to be tissue specific. In the case of Fc{gamma}R, Fc{gamma}RI plays a critical role in IC peritonitis, while Fc{gamma}RIII is more important in the cutaneous Arthus reaction and IC inflammation of the lung (14, 15). The codominant role of Fc{gamma}RIII and C5aR has been demonstrated for the initiation and full expression of the inflammatory response in IgG IC alveolitis (15). In addition, enhanced development of IC inflammation of skin and lung has been found in mice lacking the inhibitory Fc{gamma}RII (11, 16). Accordingly, Fc{gamma}RII-/- mice show Fc{gamma}RIII hyperactivation of effector cells, including alveolar macrophages (AM) and skin mast cells, leading to increased secretion of secondary mediators, such as TNF-{alpha} and chemotactic cytokines (11, 17, 18, 19).

The requirement of TNF-{alpha}, IL-1{beta}, and chemokines released by activated AM is best defined for the rat system (20, 21, 22). Although not directly chemotactic, TNF-{alpha} and IL-1{beta} can induce leukocyte accumulation at extravascular sites by stimulating the expression of adhesion molecules (ICAM-1, P- and E-selectins) on the surface of leukocytes and endothelial cells (23, 24). Moreover, TNF-{alpha} in combination with C5a are suspected to function as autocrine activators of CXC and CC chemokine production by AM, resulting in the recruitment of other inflammatory cells, predominantly neutrophils, to the lung interstitium and alveoli (25). The ensuing release of lysosomal enzymes, oxygen radicals, and vasoactive substances ultimately causes vascular tissue damage, edema, and hemorrhage (24).

The present study was undertaken to investigate the relationship among Fc{gamma}RIII, early response cytokines, TNF-{alpha} and IL-1{beta}, and members of the glutamic acid-leucine-arginine motif (ELR)-CXC subfamily of chemokines, macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant (KC) (26, 27), in the initiation of IgG IC alveolitis in mice. IC formation in the lung of C57BL/6 mice incited an acute inflammatory response highlighted by neutrophil infiltration, reaching a maximum within 8 h, while Fc{gamma}RIII-deficient mice were largely, although not completely, protected. The kinetic data indicated a cytokine dependency by which, upon Fc{gamma}RIII activation, a proximal induction of TNF-{alpha} led to production of the more distal IL-1{beta}. Interestingly, the in vivo blockade of TNF-{alpha}, although effective in attenuating polymorphonuclear leukocyte (PMN) influx, did not result in significantly reduced production of the ERL-CXC chemokines, MIP-2 and KC. Moreover, the comparison of mice lacking Fc{gamma}RIII (Fc{gamma}RIII-/-) and mice lacking TNF-{alpha} receptor class I (TNF-{alpha}RI-/-) mice showed the requirement of Fc{gamma}RIII, but not TNF-{alpha} receptor type I, to induce chemokine production. These results demonstrate the important role of the Fc{gamma}RIII effector system in the release of both inflammatory and chemotactic cytokines and underscore the critical contribution of TNF-{alpha} as one essential downstream mediator in triggering the inflammatory response in the lung of C57BL/6 mice.


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

Fc{gamma}RIII-deficient mice were generated as previously described (9). They were bred for eight generations onto C57BL/6 mice under pathogen-free conditions in the animal facility of Hannover Medical School. The homozygous Fc{gamma}RIII-/- were selected, and wild-type (WT) Fc{gamma}RIII+/+ C57BL/6 littermates were used for all comparisons. C57BL/6 mice homozygous for TNF-{alpha}RI-/- (28) were obtained from The Jackson Laboratory (Bar Harbor, ME). All of these mice were male and were used at 8–12 wk of age. Experiments were conducted in accordance to the regulations of the local authorities.

Experimental IC alveolitis

To induce experimental IC alveolitis in the lung of mice, the trachea was cannulated, and 150 µg of rabbit IgG anti-OVA Ab (Sigma, Munich, Germany) was applied as described previously (15). Immediately thereafter, 20 mg/kg of OVA Ag was given i.v. In some experiments mice received rat anti-mouse mAb V1q directed against TNF-{alpha} to block TNF-{alpha} activity (29). Hereby, a saturating dose of 100 µg of V1q was given i.v. prior to application of anti-OVA IgG. Mice were killed at various time points (ranging from 0 to 72 h) after initiation of IC alveolitis, and bronchoalveolar lavage (BAL) fluids were assayed for PMN accumulation, hemorrhage, chemotactic activity, and production of TNF-{alpha}, IL-1{beta}, MIP-2, and KC. Lung tissues obtained after lavage were processed for histological examination and stained with hematoxylin and eosin according to conventional procedures.

BAL and quantitation of hemorrhage and PMN accumulation in bronchoalveolar space

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 as described previously (15). The volume of collected BAL fluid (BALF) was measured in each sample, and total cell count was assessed with a hemocytometer (Neubauer Zählkammer, Gehrden, Germany). The amount of erythrocytes represented the degree of hemorrhage. For quantitation of PMN accumulation, differential cell counts were performed on cytospins (10 min, 55 x g) stained with May-Grünwald/Giemsa using 300 µl of BALFs.

Determination of chemotactic activity in the BALF

C57BL/6 WT mouse bone marrow cells were suspended at 7.5 x 106 cells/ml RPMI 1640 medium and 0.5% BSA (Sigma). Neutrophil number was routinely determined with a FACSCalibur (Becton Dickinson, Mountain View, CA) for 1 min at 12 µl/min with gating on forward and side scatter to be in the range of 64–68% of total cells. In pilot experiments, staining with FITC-labeled Gr1 (PharMingen, San Diego, CA) had also been used to phenotype PMN, resulting in the same range of 62–70% Gr1-positive cells. One hundred microliters of the bone marrow cell suspension was placed into the insert of a 6.5-mm diameter, 3-µm pore size polycarbonate Transwell chemotaxis chamber (Costar Corning, Corning, NY), and the bottom well was filled with 600 µl of RPMI 1640/0.5% BSA (the medium control) or the same medium supplemented with an optimal concentration of 50 ng of recombinant human C5a (Sigma), which served as an internal positive control, with BALF diluted 1:2 in RPMI 1640/1% BSA, or, where indicated, with 1:2 diluted BALF supplemented with anti-MIP-2, anti-KC polyclonal Abs (R&D Systems, Wiesbaden, Germany) at a final concentration of 5 µg/ml. Inserts were combined to the lower chambers and incubated at 37°C in 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 for 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 percentage of total PMN loaded into the upper chamber. Under these conditions, the medium control results in a mean of >2% cell migration, while the rhC5a-positive control gives reproducible results in the range of 20–25% PMN migration.

Determination of TNF-{alpha}, IL-1{beta}, MIP-2, and KC concentrations in the BALF

The concentrations of TNF-{alpha}, IL-1{beta}, MIP-2, and KC in BALFs were measured in duplicate in appropriately diluted samples with respective TNF-{alpha}-, IL-1{beta}-, MIP-2-, or KC-specific ELISA kits (R&D Systems) according to the manufacturer’s instructions. The detection limits of the assays were 5.1 pg/ml (TNF-{alpha}), 3 pg/ml (IL-1{beta}), 1.5 pg/ml (MIP-2), and 2 pg/ml (KC).

Statistical analysis

To analyze differences in mean values the two-sided unpaired Student’s t test was used; p < 0.05 was considered significant, and p < 0.001 was considered highly significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Kinetics of neutrophil influx in experimental IgG IC-induced alveolitis

The inflammatory response in IC-triggered alveolitis was determined by first analyzing the kinetics of PMN influx into the lung tissue and bronchoalveolar space of C57BL/6 mice. Weak signs of interstitial (Fig. 1Go) and alveolar (data not shown) PMN infiltration were evident at 4–72 h in anti-OVA Ab-injected control mice not receiving the OVA Ag. In contrast, histopathological examination of IgG OVA/anti-OVA IgG IC-treated mice revealed an accumulation of PMN in the lung interstitium at 4 h, reaching a maximum between 8 and 24 h, with a subsequent decline after 72 h, as assessed by conventional hematoxylin/eosin staining (Fig. 1Go). Low numbers of PMN were present in BALF at time 0 and did not increase significantly within the first 2 h after IC challenge. However, a dramatic increase in alveolar PMN influx occurred at 3–4 h, reaching the highest value of >95% neutrophils after 8 h, corresponding to 4.2 ± 0.5 x 106 PMN/mouse (n = 6; Fig. 2GoA). The acute response was transient, and the level of PMN had returned almost to baseline by 72 h postinjection (Fig. 2GoA). This drop in PMN was accompanied by an ~7-fold increase in numbers of AM from 67.6 ± 12.3 x 103 AM/mouse at 8 h to 481.3 ± 73.2 x 103 AM/mouse at 72 h (Fig. 2GoA). Accumulation of apoptotic bodies within AM at 48 and 72 h suggested an active, AM-mediated process in the clearance of PMN from lung alveoli (data not shown).



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  		FIGURE 1. Kinetics of interstitial neutrophil influx in IgG IC alveolitis. Representative hematoxylin- and eosin-stained sections of lavaged and paraffin-embedded lung tissues from C57BL/6 WT mice at 2–72 h after the injection of anti-OVA IgG (Ab) or OVA/anti-OVA IgG IC. Original magnification, x100; inset magnification, x400).

 


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  		FIGURE 2. Kinetics of cellular influx (A) and cytokine production (B) in IgG IC alveolitis. The induction of the inflammatory response in the lung of C57BL/6 WT mice was performed by challenge with 150 µg of anti-OVA Ab intratracheally and then with OVA Ag. Mice were killed at the indicated times (after 1, 2, 3, 4, 8, 24, 48, and 72 h), and the accumulation of AM and PMN in alveoli (A) was assessed by differential cell counts in Giemsa stains of individual BALFs. The production of TNF-{alpha} and IL-1{beta} (B) was determined by specific ELISA of pooled BAL samples. The results in A are expressed as the mean ± SEM (n = 5–8 mice for each time point; in some cases, error bars were so small as to be invisible); in B data are the mean of duplicates.

 
Kinetics of cytokine release and CXC chemokine production in IC alveolitis

The strong and temporary accumulation of PMN in the lung suggested that a local production of recruitment factors for PMN might be enhanced in response to IC stimulation. We obtained pooled BALFs from IC-treated C57BL/6 mice (n = 6) at each time point and measured the early response cytokines, TNF-{alpha} and IL-1{beta}, and the two functionally related ELR-CXC chemokines, MIP-2 and KC, by specific ELISA. A small, but substantial, increase in TNF-{alpha} up to 100 pg/ml BALF was already seen at 1 h, with a maximal accumulation at 3 h after IC challenge (Fig. 2GoB). IL-1{beta} followed different kinetics, with a smaller amount of 6.2 pg/ml first detectable at 2 h, reaching maximal levels of 10–15 pg/ml after 8–24 h (Fig. 2GoB). However, the kinetics of MIP-2 and KC were very similar to that observed for TNF-{alpha}, with concentrations of about 50 pg/ml BALF at 1 h, followed by increasing amounts at 2 h, reaching their maximal levels of >2 ng/ml BALF at 3 h (Fig. 3GoA). This was followed by a subsequent decline at 4 h, with chemokine concentrations no longer detectable after >=8 h (Fig. 3GoA). In accordance, BALFs obtained at 1 h after IC stimulation did not display any chemotactic activity, whereas BALFs recovered after 2 h contained measurable neutrophil chemotactic activity (6.0 ± 1.5% migration of PMN in vitro; n = 6; Fig. 3GoB). The peak in chemotactic activity of 22.2 ± 4.6% PMN migration (n = 5) at 3 h paralleled the MIP-2 and KC peaks within BALF. In contrast to the results obtained in IC-challenged mice, corresponding BALF of Ab-treated control mice contained significantly lower levels of chemotactic activity and mediators (data not shown). Taken together, the kinetic data demonstrate that IgG IC-triggered lung inflammation results in enhanced production of TNF-{alpha}, IL-1{beta}, MIP-2, and KC in IC-alveolitis.



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  		FIGURE 3. Kinetics of CXC chemokine production in IgG IC alveolitis. The induction of the inflammatory response in the lung of C57BL/6 WT mice was performed as described in the legend to Fig. 2Go. A, The levels of MIP-2 and KC were determined by ELISA in BALF pools collected from five to eight mice for each of the investigated time points after initiation of lung reaction. Data are presented as the mean of duplicates. B, Chemotaxis, assessed by Transwell migration assays of bone marrow-derived neutrophils elicited with 300 µl of BALF pools obtained from five to eight mice, was examined for each time point. Data are presented as the percentage of PMN number loaded into the upper chamber that had migrated to the bottom well. Results are expressed as the mean ± SEM of five individual experiments. In addition, BALFs collected 3 h after challenge with anti-OVA Ab ({square}) or OVA/anti-OVA IC ({blacksquare}) were incubated with 5 µg/ml polyclonal anti-MIP-2 and anti-KC Abs and subsequently used in Transwell migration assays (inset). Results are expressed as the mean ± SEM of four individual experiments. Significance was determined by Student’s t test (**, p < 0.001).

 
MIP-2-dependent chemotactic activity in BALF of IC-challenged mice

To determine which of the two functionally similar chemokines, MIP-2 and KC, are responsible for chemotactic activity, BALFs were examined in the presence of polyclonal Abs directed against MIP-2 and KC at a final concentration of 5 µg/ml. This concentration was 10-fold above a level proven to neutralize the chemotactic properties of mouse recombinant MIP-2 and KC in vitro (data not shown). Over four independent experiments, the anti-MIP-2 Ab consistently reduced the chemotactic activity within BALF of mice recovered at 3 h after OVA/anti-OVA IC challenge down to background levels (from 20.8 ± 1.3 to 12.9 ± 0.6% migrated PMN; p < 0.001), compared with 9.0 ± 1.8% migrated PMN measured in chemotaxis assays using BALF of anti-OVA Ab control mice not receiving OVA Ag (Fig. 3GoB). In contrast, incubations with anti-KC Ab resulted in a slight, but not significant, reduction to 17.6 ± 2.7% migrated PMN (n = 4; p = 0.23; Fig. 3GoB), indicating that MIP-2 might be more potent than KC to trigger PMN migration in vitro. This finding is also consistent with a recent observation that MIP-2 can stimulate PMN recruitment to s.c. tissue and peritoneal cavity in vivo, while KC plays a limited role (30).

Fc{gamma}RIII-dependent neutrophil influx and MIP-2 production in IC-alveolitis

Previous data have demonstrated the importance of Fc{gamma}R together with complement in the Arthus reaction (9, 15). Manifestation of cutaneous and pulmonary IC inflammation is largely determined by the codominant action of Fc{gamma}RIII- and C5aR-mediated pathways. This was defined at 4 h by quantitating changes in microvascular permeability, hemorrhage, and neutrophil accumulation in tissue and exudate of Fc{gamma}RIII-deficient mice treated, or not, with a specific antagonist against C5aR (15). In C57BL/6 WT mice, however, cellular influxes into the lung (Fig. 2Go) and skin (data not shown) reach their maximum after 8 h. Therefore, we examined the role of Fc{gamma}RIII in the secretion of chemotactic factors contributing to the activation and recruitment of PMN at different times during IC alveolitis. PMN infiltration and hemorrhage in alveoli of Fc{gamma}RIII-/- mice were significantly lower than those in WT controls at both early (4 h) and late (8 h) time points after OVA/anti-OVA IgG IC application (Fig. 4Go, A and B). Only very weak signs of inflammation were observed when mice were treated with anti-OVA IgG Ab without OVA Ag injection. At 4 h after IC challenge the chemotactic activity in BALF of Fc{gamma}RIII-/- mice was markedly attenuated (Fig. 4GoC), accompanied by significantly reduced levels of MIP-2 (Fig. 4GoD). This indicates that in IC alveolitis, enhanced generation of CXC chemokines, such as MIP-2, are dependent on upstream Fc{gamma}RIII.



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  		FIGURE 4. Role of Fc{gamma}RIII in pulmonary IC inflammation. Experimental alveolitis (IC) in Fc{gamma}RIII-/- mice ({blacksquare}) and C57BL/6 WT mice ({square}) was performed as described in the legend to Fig. 2Go. Ab controls received anti-OVA Ab intratracheally without i.v. injection of OVA Ag (Ab control). After 4 or 8 h, mice were killed, and alveolar PMN infiltration (A) was assessed by differential cell counts in Giemsa stains of BALF, hemorrhage (B) was measured by total cell counts of erythrocytes present in BALF, chemotactic activity (C) was quantitated by a Transwell migration assay of 300 µl of BALF, and MIP-2 chemokine production (D) was determined by MIP-2-specific ELISA of BALF. Data are presented as the mean ± SEM (n = 6–8 mice for each group). Significance differences between WT and Fc{gamma}RIII-/- mice were determined by Student’s t test (*, p < 0.05; **, p < 0.001).

 
Fc{gamma}RIII-dependent release of TNF-{alpha} and IL-1{beta} in IC alveolitis

It had been shown that IgG IC induce the secretion of TNF-{alpha} and IL-1{beta} from mouse and human macrophages in vitro (31, 32). TNF-{alpha} and IL-1{beta} were enhanced during the initiation of inflammation in knockout mice lacking the inhibitory Fc{gamma}RII (11, 33). This together with our finding that the synthesis of TNF-{alpha} and IL-1{beta} preceded PMN influx in alveolitis (Fig. 2Go) prompted us to assess the importance of the activatory IgG-Fc{gamma}RIII pathway to the secondary release of TNF-{alpha} and IL-1{beta} in experimental alveolitis in vivo. The high amounts of TNF-{alpha} present in BALFs of WT mice 4 h after IC challenge were significantly reduced in Fc{gamma}RIII-/- mice (Fig. 5GoA). IL-1{beta} levels were not significantly affected in Fc{gamma}RIII-/- mice 4 h after IC application, whereas a substantial decrease from 66.0 ± 10.9 to 32.4 ± 6.5 pg/BALF (n = 6–8; p < 0.05) was observed after 8 h compared with that in WT mice (Fig. 5GoB). This indicates that Fc{gamma}RIII contributes to the secretion of both TNF-{alpha} and IL-1{beta} in IC alveolitis.



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  		FIGURE 5. Fc{gamma}RIII-dependent release of TNF-{alpha} (A) and IL-1{beta} (B). Experimental alveolitis (IC) was allowed to proceed for 4 or 8 h in Fc{gamma}RIII-/- mice ({blacksquare}) and C57BL/6 WT mice ({square}). Ab controls received anti-OVA Ab intratracheally without i.v. injection of OVA Ag (Ab control). Levels of TNF-{alpha} and IL-1{beta} were determined by specific ELISA of BALFs of individual mice. Data are presented as the mean ± SEM (n = 6–8 mice for each group). Significance differences between WT and Fc{gamma}RIII-/- mice were determined by Student’s t test (*, p < 0.05).

 
TNF-{alpha}-dependent enhancement of IL-1{beta} synthesis in IC alveolitis

Since TNF-{alpha} was secreted earlier than IL-1{beta} in IC alveolitis, we investigated whether the synthesis of IL-1{beta} depends on TNF-{alpha} by using TNF-{alpha}RI-deficient mice (28). At 8 h after IC application, TNF-{alpha}RI-/- mice showed a strong reduction in PMN infiltration and hemorrhage (Fig. 6Go, A and B), comparable with that observed in Fc{gamma}RIII-deficient mice (Fig. 4Go) or in C57BL/6 mice following blockade of TNF-{alpha} (data not shown). Moreover, in the BALF from TNF-{alpha}RI-/- mice the IL-1{beta} levels were significantly reduced compared with those in their C57BL/6 WT controls (Fig. 6GoC). This suggests that after Fc{gamma}RIII-mediated production of TNF-{alpha}, the subsequent activation of TNF-{alpha}RI contributes to the regulation of IL-1{beta} synthesis in IC alveolitis.



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  		FIGURE 6. Role of TNF-{alpha}RI in pulmonary IC inflammation. Experimental alveolitis (IC) was performed in TNF-{alpha}RI-/- mice ({blacksquare}) and C57BL/6 WT mice ({square}) as described in the legend to Fig. 2Go. Mice receiving anti-OVA Ab without i.v. injection of OVA Ag served as the control (Ab control). After 8 h mice were killed, and alveolar PMN infiltration (A) was assessed by differential cell counts in Giemsa stains of BALF, hemorrhage (B) was measured by total cell counts of erythrocytes present in BALF, and release of IL-1{beta} (C) was determined by IL-1{beta}-specific ELISA. Data are presented as the mean ± SEM (n = 6–8 mice for each group). Differences between WT mice and TNF-{alpha}RI-/- mice were highly significant for all parameters (**, p < 0.001).

 
TNF-{alpha} induces IC alveolitis independently of CXC chemokine production

To further assess the role of TNF-{alpha}, TNF-{alpha} activity was blocked during the initial phase (within 4 h) by the i.v. injection of 100 µg/mouse of the neutralizing rat anti-mouse TNF-{alpha} mAb V1q just before starting IC-induced alveolitis. As shown in Fig. 7GoA, functional inhibition of TNF-{alpha} in C57BL/6 WT mice resulted in a marked decrease in neutrophil count in alveoli from 11.4 ± 2.1 to 3.3 ± 1.9 PMN x 103/mouse (n = 4–6; p < 0.05) at 2 h, from 351.2 ± 91.2 to 63.1 ± 21.5 PMN x 103/mouse (n = 8–10; p < 0.001) at 3 h, and from 383.3 ± 44.8 to 70.5 ± 13.4 PMN x 103/mouse (n = 8–10; p < 0.001) at 4 h. Moreover, anti-TNF-{alpha} treatment strongly reduced interstitial PMN accumulation, vascular permeability, and hemorrhage at the investigated time points by 50–80% (data not shown). In contrast, anti-TNF-{alpha} mAb did not change the chemotactic activity in BALFs obtained 2–4 h after IC application (Fig. 7GoB). Accordingly, the high levels of CXC chemokines, MIP-2 and KC, present in BALF of IC-challenged C57BL/6 mice were only marginally and insignificantly affected by the inhibition of TNF-{alpha} (Table IGo). Similar results were obtained in TNF-{alpha}RI-/- mice (data not shown). This indicates that in IC alveolitis, TNF-{alpha} and its interaction with TNF-{alpha}RI can mediate the inflammatory response independently of CXC chemokine generation.



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  		FIGURE 7. Inhibition of TNF-{alpha} abrogates PMN influx, but not chemotaxis, in IC alveolitis. The inflammatory response in the lung of C57BL/6 WT mice was induced in the absence (IC, {blacksquare}) or the presence (IC + anti-TNF-{alpha}, {diamondsuit}) of anti-TNF-{alpha} mAb V1q as described in the legend to Fig. 2Go. Mice receiving only anti-OVA Ab alone (Ab, {square}) served as a control. PMN infiltration in the alveolar space (A), assessed by differential cell counts in Giemsa stains of BALFs, and neutrophil chemotaxis (B), determined by Transwell migration assays, were evaluated at 2, 3, and 4 h after initiation of lung reaction. Ab control groups comprised four or five animals per group, and IC treatment groups comprised five to eight animals per group. Data are presented as the mean ± SEM. Differences in IC compared with IC + anti-TNF-{alpha} treatment groups were significant or highly significant for PMN influx, but not chemotaxis (*, p < 0.05 to **, p < 0.001).

 

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  		Table I. Enhanced ERL-CXC chemokine production (MIP-2, KC) occurs independently of TNF-{alpha} in IgG IC alveolitis

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
An important role of complement, in particular C5a, has been shown in acute pulmonary inflammation of IC alveolitis in rats and C5aR-deficient mice (10, 34, 35, 36). Studies in the rat model have elucidated the sequelae of humoral and cellular activation by IC resulting in the release of TNF-{alpha} and IL-1{beta} cytokines (20, 21) and secretion of C5 by alveolar macrophages (35), which further produce components that may assemble to form complement convertases and metalloproteases involved in the cleavage of C5 to C5a (36, 37). Both the release of chemokines and the up-regulation of adhesion molecules seem to depend on the presence of C5a and TNF-{alpha} (23, 35, 36). In addition, functional inhibition of C5a is correlated with strongly reduced BAL levels of TNF-{alpha}, indicating a dependency of these two mediators, at least in rats (35). In contrast, C5aR-deficient mice, while protected against IC-induced lung injury, do not display a significant decrease in intrapulmonary TNF-{alpha} (34).

This discrepancy between rats and mice might be due to a species-specific requirement of C5a and its receptor C5aR in the up-regulation of TNF-{alpha}, arguing for the involvement of other effectors, such as macrophage Fc{gamma}R. In fact, Fc{gamma}RIII and C5aR have recently been defined as the major receptor pathways acting together to mediate the full expression of inflammation in IC alveolitis in mice (15). Fc{gamma}RIII mediates enhanced TNF-{alpha} production in IC peritonitis, and Fc{gamma}RIII-induced release of TNF-{alpha} is responsible for the pathogenesis of autoantibody-mediated vasculitis (14, 19). In addition, the development of autoimmune pulmonary inflammation in lupus-prone MRL/lpr mice is correlated with increased TNF-{alpha} (38). The present study adds to these observations in highlighting a general requirement of the Fc{gamma}RIII effector system in the release of TNF-{alpha} during the course of IC-induced inflammation in the lung, skin, and peritoneum. Furthermore, our data underscore the critical role of TNF-{alpha} as one essential mediator triggering IC alveolitis in mice (Figs. 6Go and 7Go), which is similar to the findings in rats (20). An identical reduction of neutrophil influx, hemorrhage, and IL-1{beta} synthesis into alveoli is observed in both TNF-{alpha}RI-/- and Fc{gamma}RIII-/- mice ( Figs. 4–6GoGoGo), suggesting a cascade of events with TNF-{alpha} acting downstream of Fc{gamma}RIII. In addition, our results provide in vivo evidence of a regulatory role of TNF-{alpha} and its receptor through enhancement of IL-1{beta} synthesis, a cytokine known to function as a proinflammatory mediator in various models of tissue injury, such as septic shock, rheumatoid arthritis, autoimmune diabetes, inflammatory bowel diseases, and many others (for review, see Ref. 39).

Mast cells expressing Fc{gamma}RIII have been demonstrated as major effector cells of autoantibody- and IC-induced injury in skin vasculitis (6, 19, 40). In the lung the alveolar macrophage is the most prominent cell type in alveoli and is suspected to promote the migration and activation of neutrophils through the production of various mediators (TNF-{alpha}, IL-1{beta}, MIP-2, KC, etc.). Strongly increased concentrations of these mediators are found within the alveolar space very early after IgG IC formation, preceding interstitial and alveolar neutrophil influx ( Figs. 1–3GoGoGo). The in vitro stimulation of mouse alveolar macrophages with heat-aggregated 105-2H mouse anti-mouse IgG1 IC, already shown to activate Fc{gamma}RIII as the sole Fc{gamma}R in vivo (41, 42, 43), results in a >200-fold induction of TNF-{alpha} and MIP-2 levels (unpublished observations). In contrast, however, preliminary experiments using mast cell-deficient KitW/KitW-v mice show high concentrations of these mediators, which are not significantly reduced compared with those in their mast cell sufficient congenic Kit+/+ littermates. Thus, it appears that activated alveolar macrophages, but not mast cells, are the major source of TNF-{alpha} and MIP-2 production during pulmonary IC inflammation in mice.

Chemokines play an important role in selectively recruiting certain subsets of leukocytes to specific sites of inflammation and tissue injury. Our present results in Fc{gamma}RIII-/- mice indicate that Fc{gamma}RIII promotes MIP-2 CXC chemokine production (Fig. 4Go), which appears to be essential for chemotactic activity of BALF in vitro (Fig. 3GoB) and, as recently shown in a rat model, for full recruitment of neutrophils in vivo (22). Preliminary experiments combining Fc{gamma}RIII deficiency with the blockade of C5aR result in further decreased chemokine levels of MIP-2 as well as KC compared with either intervention alone. We have also analyzed the role of alveolar macrophage Fc{gamma}RIII in the up-regulation of a third mouse CXC chemokine, the recently cloned Lungkine/WECHE (44, 45). This novel chemokine is mainly expressed by lung bronchoepithelial cells, as documented by immunohistochemical analyses (44). In accordance, Lungkine/WECHE mRNA is equally detectable in the lung tissue of Fc{gamma}RIII-/- mice and their WT controls, while resting and IgG1/Fc{gamma}RIII-stimulated alveolar macrophages are completely negative (data not shown). Lungkine/WECHE is constitutively secreted into the bronchoalveolar space and can induce, similar to MIP-2 and KC, the in vitro and in vivo migration of neutrophils (44, 45). Blockade of CXC chemokines within the BALF of IC-challenged mice indicates a major contribution of MIP-2 over KC to promote IC-induced neutrophil migration in vitro (Fig. 3GoB). Thus, the release of MIP-2 appears to be more closely linked to upstream Fc{gamma}RIII activation of macrophages in IC alveolitis than that of KC and Lungkine/WECHE.

Intrapulmonary blockade of TNF-{alpha} by the V1q Ab and in TNF-{alpha}RI-/- mice causes a substantial decrease in lung injury similar to that found in Fc{gamma}RIII-/- mice, as assessed by neutrophil influx, hemorrhage, and IL-1{beta} synthesis. In contrast, chemokine generation is markedly reduced in Fc{gamma}RIII-/- mice, while inhibition of TNF-{alpha} results in only a slight decrease in MIP-2 and KC (~20%), which does not reach significance (Table IGo). This finding shows the critical involvement of Fc{gamma}RIII in enhancing chemokine production, whereas TNF-{alpha} plays a more limited role. The minor contribution of TNF-{alpha} in the regulation of MIP-2 and KC production in mice appears to be in contrast to the TNF-{alpha} dependency of CC and CXC chemokine synthesis observed in a rat model of IC alveolitis (36). Whether this is due to species-specific requirements of TNF-{alpha} awaits further investigation. Nevertheless, our data suggest that, at least in mice, the engagement of Fc{gamma}RIII on macrophages triggers two divergent and largely independent pathways, with TNF-{alpha} and MIP-2 as key mediators. Whether Fc{gamma}RIII and C5a/C5aR are equally essential for MIP-2 generation in vivo remains to be investigated. Preliminary experiments using a further blockade of C5aR in Fc{gamma}RIII-/- mice show a stronger inhibition of chemokine production. This might indicate that C5aR can synergistically enhance CXC chemokine generation by alveolar macrophages during Fc{gamma}RIII-induced alveolitis in mice.

In summary, we have used Fc{gamma}RIII- and TNF-{alpha}RI-deficient mice together with a neutralizing anti-TNF-{alpha} mAb to distinguish Fc{gamma}RIII- and TNF-{alpha}-mediated effects. This approach enabled us to demonstrate that TNF-{alpha} acts downstream of Fc{gamma}RIII in the initiation of the inflammatory response in pulmonary IC disease. Furthermore, the comparison between Fc{gamma}RIII-/- and TNF-{alpha}RI-/- mice shows that alveolar macrophage-derived TNF-{alpha} can function as a positive regulator of IL-1{beta} cytokine synthesis. The earlier observations in rats suggested TNF-{alpha} as an autocrine activator to promote CXC and CC chemokine generation (25). We observed a minor role of TNF-{alpha} in the up-regulation of MIP-2 and KC CXC chemokines. In addition, our findings support the concept that Fc{gamma}RIII together with C5aR (10, 15, 34) is a critical receptor triggering acute inflammation through the enhanced production of both inflammatory and chemotactic cytokines. Finally, our data endorse earlier reports (11, 19) suggesting Fc{gamma}RIII and TNF-{alpha} as potential targets in immunotherapy. With respect to Fc{gamma}RIII, current approaches on resolving the IgG interaction of a human Fc{gamma}RIII (46, 47, 48) in combination with engineered mice expressing human instead of mouse Fc{gamma}RIII (49, 50, 51) will allow to establish the significance of Fc{gamma}RIII blockade as a therapeutic modality of IC disease in humans.


   Acknowledgments
 
We thank W. Falk (Regensburg, Germany) for providing the neutralizing anti-TNF-{alpha} mAb V1q. We thank D. Stelte for graphic art work and the members of our laboratory for valuable discussions and comments on the manuscript. Also, we appreciate the collaboration with J. Sjef Verbeek (Leiden, The Netherlands) in the generation of Fc{gamma}RIII-deficient mice.


   Footnotes
 
1 J.S. was supported by a postgraduate grant from the state of Niedersachsen. Transgenic and other research was supported by grants from the Deutsche Forschungsgemeinschaft (to J.E.G.; Ge892/5-1 and Ge892/7-1). Back

2 Address correspondence and reprint requests to Dr. J. Engelbert Gessner, Abteilung für Klinische Immunologie, Medizinische Hochschule Hannover, 30625 Hannover, Germany. Back

3 Abbreviations used in this paper: IC, immune complex; AM, alveolar macrophage; ELR, glutamic acid-leucine-arginine motif; MIP-2, macrophage inflammatory protein-2: KC, cytokine-induced neutrophil chemoattractant; BAL, bronchoalveolar lavage; BALF, BAL fluid; C5aR, receptor for C5a anaphylatoxin; PMN, polymorphonuclear leukocytes; WT, wild type; TNF-{alpha}-RI, TNF-{alpha} receptor class I. Back

Received for publication October 10, 2000. Accepted for publication February 5, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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