Immobilized IgG Immune Complex Induces Secretion of Tumor Necrosis Factor-a by Porcine Alveolar Macrophages Joon W. Kim, William G. Wierda, and Yoon B. Kim Department of Microbiology and Immunology, University of Health Sciences/The Chicago Medical School, North Chicago, Illinois

Tumor necrosis factor-a (TNF-a) is an important inflammatory mediator produced by activated monocytes and macrophages. We have previously shown that porcine alveolar macrophages (PAM) mediate bystander cytotoxicity through hydrogen peroxide production following activation with immobilized IgG immune complex (IIC) (J. Immunol. 1983; 131:1438-1442). In this report, we have investigated whether lIC induces TNF-a secretion by PAM. Isolated PAM from Minnesota miniature swine were cultured for 18 h with and without recombinant human interferon-y (rhIFN-')'). Cultured PAM were then incubated with lIC or IgG immune complex in suspension (SIC). The supernatants generated were assessed for cytotoxic activity using a TNF-a-sensitive WEHI-164 cell line. Anti-recombinant human TNF-a (rhTNF-a) monoclonal antibody neutralized the observed cytotoxicity of lIC-activated PAM supernatant completely, indicating that this cytotoxicity is mediated by TNF-a. lIC induced TNF-a secretion by PAM after 3 h of incubation, reaching a plateau from 6 to 12 h and decreasing thereafter. TNF-a release was enhanced by pretreatment of PAM with rhIFN-')'. SIC did not induce significant levels of TNF-a secretion by PAM; however, SIC with cytochalasin B-pretreated PAM induced equivalent levels of TNF-a secretion as lICactivated PAM. We conclude that lIC or SIC with cytochalasin B pretreatment, both of which prevent internalization of IgG immune complex-bound Fe receptor (FcR), provide a signal for PAM to generate TNF-a through FcR modulation. This suggests that in vivo, deposited (immobilized) IgG immune complexes-bound fcR may be a stimulus for activation of PAM to generate TNF-a rather than circulating (mobilized) immune complexes, which may contribute to the pathogenesis of diffuse interstitial fibrosis of the lung, especially in idiopathic pulmonary fibrosis.

Diffuse interstitial fibrosis of the lung is characterized by chronic inflammation associated with the destruction of lung architecture and progressive development of fibrosis (1, 2). Although the exact pathogenesis is still not established, it is hypothesized that immune responses within the lung may play an important role in the development and progression of disease. Alveolar macrophages are the most predominant and ubiquitous cells in the alveoli than other inflammatory cells, and their contribution to lung injury and inflammation processes has become an important area of research. Alveo(Received in original form October 1, 1990 and in revised form February 5, 1991) Address correspondence to: Yoon Berm Kim, M.D., Ph.D., Professor and Chairman, Department of Microbiology and Immunology, University of Health Sciences/The Chicago Medical School, 3333 Green Bay Road, North Chicago, IL 60064. Abbreviations: bronchoalveolar lavage, BAL; bovine serum albumin, BSA; balanced salt solution, BSS; chicken red blood cells, CRBC; Fc receptor, FcR; interferon-v, IFN--y; immobilized form of IgG immune complexes, I1C; idiopathic pulmonary fibrosis, IPF; lipopolysaccharide, LPS; pulmonary alveolar macrophage(s), PAM; recombinant human, rh; suspended form of IgG immune complexes, SIC; tumor necrosis factor-a, TNF-a. Am. J. Respir. Cell Mol. BioI. Vol. 5. pp. 249-255, 1991

lar macrophages are thought to playa critical role in normal wound healing and pathologic tissue inflammatory changes through their ability to release a variety of potent inflammatory and fibrogenic mediators after activation (2, 3). Alveolar macrophages can be activated by a variety of stimuli, such as immune complexes, other phagocytic stimuli, and soluble factors including cytokines (4). Especially in idiopathic pulmonary fibrosis (IPF), immune complexes have been found in bronchoalveolar lavage (BAL) fluid, in the peripheral blood circulation, and in the alveolar walls of patients with IPF (5-7). Even though it is relatively clear that alveolar macrophages have the capacity to initiate and promote pulmonary fibrosis, the mechanism of its activation is still unclear. It appears that immune complexes may contribute to local matrix turnover as an initiating stimuli for the activation of alveolar macrophages. However, there is no direct evidence that stimulation of alveolar macrophages by tissue deposited immune complexes or soluble circulating immune complexes is related to fibrogenesis of the lung. In recent years, a variety of macrophage-derived cytokines have been demonstrated in a number of animal models of lung injury and are thought to be involved in the pathogenesis of pulmonary fibrosis (reviewed in reference 8). How-

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ever, such a pathway, in which deposited or circulating immune complexes generate bioactive cytokines through their ability to activate alveolar macrophages, has not been verified. Thmor necrosis factor-a (TNF-a) is a mediator of particular interest among macrophage-derived cytokines. TNF-a, also called cachectin, is an activated monocyte/macrophage-derived cytokine initially identified by its ability to cause hemorrhagic necrosis of some tumors in vivo and cytotoxic or cytostatic effects against certain cancer cells in vitro (9, 10). It is now recognized that TNF-a is a pleiotropic cytokine that can mediate a wide variety of biologic effects as an important mediator of inflammation. Included among these diverse bioactivities are enhanced stimulation of fibroblast growth (11), stimulation of collagenase release from several mesenchymal cell types (12), enhanced adhesion of neutrophils to endothelial cells (13), and induction of a number of growth factors, including granulocyte/macrophage colony-stimulating factor (14), platelet-derived growth factor (15), and interleukin-l (16, 17). Recent in vivo study demonstrated the effects of exogeneous TNF-a on the development of acute alveolitis (18). Fc receptors for IgG (IgG FcR) are expressed on the plasma membrane of pulmonary alveolar macrophages (PAM) as well as most cells of the immune system (19) and have been implicated in the regulation of immune reactivity. They have been associated with the release of biologically active mediators, autoimmune disorders, and regulation of immune reactive cells through their interaction with antibody complexed to antigen (20). We have previously shown that porcine PAM mediate bystander cytotoxicity through hydrogen peroxide production following activation with immobilized immune complex (IIC) (21, 22). This in vitro model also suggests modulation of IgG FcR is required for activation of PAM to secrete hydrogen peroxide and induce bystander cytotoxicity. We have examined further whether the secretion of TNF-a, which also constitutes an important mediator of macrophage cytotoxicity, can be induced by IIC through FcR modulation. Therefore, we hypothesized that PAM stimulated by IIC induce TNF-a secretion, and that its mechanism is related to IgG immune complex-bound FcR modulation. To test our hypothesis, we have determined TNF-a secretion from activated PAM by an in vitro IIC model, compared the level of TNF-a secretion from PAM incubated with IIC and immune complex in suspension (SIC), and evaluated the mechanism of IgG FcR modulation as a critical signal to produce TNF-a.

Materials and Methods Animals Male and female swine between 2 mo and 2 yr of age used in this study were obtained from the Minnesota miniature swine herd (23) maintained at our facilities. Preparation of PAM PAM were collected as previously described (24). Briefly, phosphate-buffered saline, pH 7.2, was injected intratracheally into healthy young adult Minnesota miniature swine lung killed by CO 2 anesthesia and exsanguination. The lavage fluid was then removed, and the lavagecells were washed

with balanced salt solution (BSS). This routinely yielded PAM that were 90 to 95 % pure and greater than 95 % viable. Preparation of IgG Immune Complex IIC was prepared using an immobilized chicken red blood cell (CRBC) monolayer fixed with methanol and rabbit IgG anti-CRBC serum (preabsorbed with porcine red blood cells) at a final dilution of 1:200. To prepare an immobilized CRBC, 5 to 10 X 106 fresh CRBC in 1 ml of BSS were allowed to spontaneously adhere to the bottom of a 24-well tissue culture plate at room temperature. After 1 h, excess CRBC were removed by washing the wells 3 times with BSS. The adhering CRBC were then fixed with methanol and washed 2 times. These preparations were stored at -20° C until use. In actual assay, 0.5 ml of 1:100 diluted anti-CRBC serum was incubated in the plate of CRBC monolayer for 30 min at room temperature, and 0.5 ml of suspended PAM (2 x 106/ml) were added. SIC was prepared using 6 X 106 CRBC in suspension and rabbit anti-CRBC serum at a final dilution of 1:200. Preparation of Supernatants from Cultured PAM Isolated PAM were suspended in RPMI-1640 tissue culture medium with 2 mM L-glutamine (GIBCO, Grand Island, NY), supplemented with 10% heat-inactivated fetal bovine serum (HyClone Laboratories, Logan, UT), penicillin 100 U/ml, and streptomycin 100 ILg/ml (GIBCO), hereafter referred to as complete media, at a concentration of 2 x 106 cells/ml. PAM (1 X 106 cells) were then placed into wells of a 24-well tissue culture plate (Corning, Corning, NY) containing IIC or SIC in a final volume of 1 ml. Control wells included PAM incubated alone, with antigen (CRBC) only and with antibody (anti-CRBC) alone. They were incubated at 37 0 C with 5 % CO2 for various incubation periods. At the time of harvest, the supernatants were collected and centrifuged at 1,000 X g for 10 min and stored at -70 0 C until tested. In the priming of PAM wth interferon-v (lFN-')'), PAM (2 X 100/ml) were preincubated with 500 U/ml of recombinant human (rh) IFN-')' (sp act, 3 X 107 U/mg) donated by Genentech (South San Francisco, CA) for 18 h before stimulation with actual IIC and SIC systems. To determine effects ofIgG immune complex-bound FcR modulation, PAM incubated in the SIC system were pretreated with 2 ILg/ml·ofcytochalasin B (Sigma Chemical Co., St. Louis, MO), which prevents phagocytosis and internalization of IgG immune complex-bound FcR (21). Cytotoxicity Assay (TNF-a Bioassay) and Antibody Neutralization Experiments A standard 18-h 51Cr release assay was used to measure cytotoxic activity of the test supernatants from PAM. WEHI164 (fibrosarcoma cell line ofBALB/c origin), which is sensitive to TNF-a (25), was maintained in complete media and labeled with 51Cr. Briefly, 1 X 106 cells in 100 III of complete media were incubated with 200 ILCi of Na251Cr0 4 (New England Nuclear, Boston, MA) at 37° C for 1 h with constant mixing. The cells were then washed 3 times with BSS and resuspended at 1 X 105 cells/ml in complete media. One hundred microliters of each diluted test supernatant were placed in wells of a 96-well, flat-bottom microtiter tissue culture plate (Corning). One hundred microliters of

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251

TABLE 1

Effects of Iltl-stimulated PAM supernatants on cytotoxic activity for WEB/-164 cells and neutralization experiments with anti-rhTNF-a % Specific Lysis (WEHI-I64)t Supernatant*

Exp.2

Exp. 1

Control 1 (medium alone) Control 2 (antigen alone) Control 3 (antibody alone) IIC stimulation IIC stimulation + anti-TNF-a mAb:\:

0.7 ± 1.4 ± 1.3 ± 38.8 ±

0.4 1.7 0.5 39.1

0.4 0.6 0.1 4.1

1.2 ± 0.7

± ± ± ±

Exp.3

0.2 0.3 0.2 3.8

2.1 ± 1.1 1.7 ± 0.8 0.4±0.1 26.1 ± 3.4

0.1 ± 0.3

0.6 ± 0.2

Exp.4

3.5 -0.2 -1.1 29.2

± ± ± ±

Exp.5

0.4 0.6 0.4 1.7

2.5 ± 0.6 1.6 ± 0.8 0.9 ± 0.6 29.8±2.1

ND

ND

Definition of abbreviations: IIC = immobilized form of IgG immune complex; PAM = pulmonary alveolar macrophage(s); anti-rhTNF-a mAb = anti-recombinant human tumor necrosis factor-a monoclonal antibody; ND = not done; CRBC = chicken red blood cells. * PAM (1 x 106 cells/ml) were incubated with complete medium alone (control 1), complete medium containing CRBC alone (control 2), or anti-CRBC alone (control 3), and antibody-coated immobilized CRBC as an actual stimulation system (IIC stimulation). After a 6-h incubation period, PAM supernatants were collected to test for their ability to lyse WEHI-I64 target cells. t 5 1Cr-Iabeled WEHI-I64 cells (l X 1()4/wells) were incubated with 1:4 diluted supernatant and assayed for specific 5 1Cr release after 18 h of incubation at 37° C. Data presented are mean of triplicate determinations of each experiment ± SD. :\: Anti-rhTNF-a mAb at a concentration of 250 U/ml was used to inhibit the cytotoxic activity of test supernatants.

5ICr labeled WEHI-164 cells (1 X 1{)4) was then added to each well. Each sample was set up in triplicate. The plates were incubated for 18 h in a 37° C, 5 % CO 2 , humidified incubator. After incubation, 100 I.d of culture supernatant was harvested from the wells and 5ICr release was measured in a Micromedic Plus Series Model 28037 gamma counter. Results are expressed as percent specific WEHI-164 cell lysis based upon the following formula:

% specific lysis =

cpm experimental - cpm spontaneous release (SR) cpm maximum release (MR) cpm spontaneous release (SR)

X 100

alone and maximum release (MR) was cpm from target cells incubated in 2 % Nonidet P40 detergent. In all experiments, the percentage of SR was less than 20 %. To confirm TNF-a as the mediator of cytotoxic activity in this bioassay, an anti-rhTNF-a monoclonal antibody (sp act, 2.7 X 106 U/mg) (Genentech) was used as a neutralizing antibody against cytotoxic activity. Fifty microliters of serially diluted anti-rhTNF-a antibody (1 X 103 VIm!) was incubated with 50 pJ of test supernatant for 1 h at room temperature before adding "Cr-labeled target cells. rhTNF-a (sp act, 4.75 X 107 Vlmg) (Genentech) was tested to evaluate the standard level of cytotoxic activity for WEHI-164 cells.

where spontaneous release (SR) was defined as the cpm released from target cells incubated in complete medium

40

o

31.25

62.5

125

250

Anti-rhTNF-a mAb (units/ml) O.L-__, - - - - - - , - - - - . , - - - - - - , - - - - . , . - - - - - - , - - 1:2

1:4

1:8

1:16

1:32

1:64

Dilution of test supernatant Figure 1. Concentration-dependent cytotoxic activity of immobilized IgG immune complex (IIC)-activated pulmonary alveolar macrophages (PAM) supernatants. Isolated PAM (l x 106 cells) were incubated with IIC or complete media alone (control). After 6 h of incubation, supernatants were harvestedand serially diluted for the standard i8-h 5ICr release assay to measure cytotoxic activity. The results are expressed in percent specific lysis of WEHI164 target cells.

Figure 2. Neutralizing effects of anti-recombinant human tumor necrosis factor-a monoclonal antibody (anti-rhl'Nf-o mAb) on cytotoxic activity of IIC-activated PAM supernatant. Supernatants of PAM (l x 106 cells) incubated in IIC or complete media alone (control) were evaluatedfor cytotoxic activity. Fifty microliters of test supernatants was incubated with 50 JLI of serially diluted antirhTNF-a mAb (1 x 103 U/ml) for 1 h at room temperature before adding 100 JLI of "Cr-Iabeled WEHI-164 target cells (l x lQ4 cells). The standard 18-h 5ICr release assay was used to measure cytotoxicactivity of the test supernatants. The resultsare expressed in percent specific lysis of WEHI-164 target cells.

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3

_ _ _ HC stimulation Controll (medium alone) ____ Control 2 (antigen alone) Control 3 (antibody alone) -0--

o

3

6

9 12 15 18 Time of incubation (Hr)

21

24

Figure 3. Kinetics of 'I'Nf-ce-mediated cytotoxic activity of supernatants from lIC-activated PAM. Isolated PAM (1 x 106 cells) were incubated with complete media alone (control 1), or complete media containing antigen alone (control 2), or containing antibody alone (control 3), or with lIC. After various time periods, supernatants were harvested to measure TNf-o-mediated cytotoxicity.

Results Activation of PAM by IIC to Induce Cytotoxic Activity for WEHI-164 Cells Experiments were designed to establish whether IIC activate PAM to become cytotoxic against WEHI-164 cells. PAM were incubated in complete medium alone (control 1), in medium containing antigen alone (control 2), in medium containing IgO antibody alone (control 3), or with IIC. After 6 h of incubation, supernatants were collected and tested for their ability to lyse WEHI-164 target cells in an 18-h "Cr release assay. The results shown in Table 1 demonstrate that supernatant from IIC-stimulated PAM mediated a significant level of cytotoxic activity, but supernatant from PAM in the three control systems did not induce cytotoxicity. We examined the level of cytotoxicity at different dilutions of test supernatant to evaluate the concentration-dependent cytotoxicity. The results shown in Figure 1 demonstrate that supernatant-induced cytotoxicity was maximal at a 1:2 dilution and decreased with further dilution. Thus, it shows that the level of cytotoxicity depends on the concentration of lytic mediator in the supernatant. These results indicate that resident PAM are stimulated by IIC and induce secretion of cytotoxic mediator to lyse WEHI-164 cells. Effects of Anti-rhTNF-a on WEHI-164 Target Cell Lysis by IIC-activated PAM The above data show clearly that IIC activate PAM to release soluble cytotoxic factor to mediate lysis ofWEHI-164 target cells. Experiments were carried out to determine whether IIC-activated PAM produce TNF-a as a lytic factor. To address this question directly, we tested the ability of antirhTNF-a to inhibit IIC-activated PAM supernatant to mediate lysis ofWEHI-164 target cells. The cytotoxic activity of test supernatants was determined with different concentrations of anti-rhTNF-a. The results shown in Figure 2 indicate that different dilutions of anti-rhTNF-a caused variable but

concentration-dependent inhibition of supernatant-mediated cytotoxicity ranging from 10 to 100% inhibition. The results presented in Table 1 show that anti-rhTNF-a completely neutralized IIC-activated PAM supernatant-mediated lysis of WEHI-164 target cells. Thus, it confirms that TNF-a is the soluble lytic mediator produced by IIC-activated PAM. A standard curve was generated using varying doses of rhTNF-a (10 to 103 Vlml) for cytotoxicity against a WEHI164 target cell line. From this curve, we have estimated levels of porcine TNF-a to be 530, 557, and 65.9 Vlml for 38.8% (experiment 1), 37.1% (experiment 2), and 26.1% (experiment 3) specific lysis. However, we prefer to present actual percent specific lysis data rather than the estimated Vlml. Kinetics of TNF-a Secretion by IIC-activated PAM The kinetics of TNF-a secretion induced by IIC-activated PAM were studied. The results presented in Figure 3 show that the level of TNF-a activity in the supernatant from IICactivated PAM was increased after a 3-h incubation period, reached a plateau after 6 to 9 h, and decreased after a 12-h incubation period. These results suggest that the decrease in TNF-a activity after 12 h may be partly due to binding to TNF-a receptors of PAM themselves and implicated as an autoregulation mechanism of PAM. Effects of IFN-"( Priming on TNF-a Secretion by PAM with IIC Stimulation Experiments were performed to evaluate the effectsof IFN-"( priming on TNF-a secretion by IIC-stimulated PAM. We have previously shown that rhIFN-"( is as effectivein our porcine system as in the human system (26). The priming effects of rbIFN-"( were concentration dependent and were optimal in the range of 100 to 1,000 Vlml. In this experiment, isolated PAM were cultured overnight (18 h) in the presence or absence of rhIFN-"( at a concentration of 500 Vlml, cultured PAM were washed and stimulated with the IIC system, and supernatants were then tested for TNF-a activity. The results shown in Table 2 demonstrate that supernatants from IFN-"(pretreated PAM with IIC stimulation mediated significantly (P < 0.01, standard Student t test) higher levels of TNF-amediated cytoxicity than did supernatants from IFN-,,(-untreated PAM with IIC stimulation. These results indicate that priming with IFN-"( exerts a permissive influence on resident PAM and augments TNF-a production by IIC-activated PAM. The Role of IgO Immune Complex-bound FcR Modulation on TNF-a Secretion by PAM We investigated whether modulation of IgO immune complex-bound FcR is required for IIC-activated PAM to secrete TNF-a. First, experiments were performed to compare the stimulation effects of IIC and SIC on TNF-a secretion by PAM. As shown in Table 3, there were much higher levels of TNFa-mediated cytotoxicity when PAM were stimulated with IIC than with SIC. These results indicate that immobilization of IgO immune complex is important to induce and secrete TNF-a by resident PAM. Second, to examine the possible differences of stimulatory effects to induce TNF-a secretion by PAM between the

Kim, Wierda, and Kim: Immobilized Immune Complex Induces TNF-a Secretion by Macrophages

253

TABLE 2

Effects of rhIFN-"'( priming on TNF-a-mediated cytotoxicity by PAM supernatant with IIC stimulation % Specific Lysis (WEHI-I64)t Exp.l

Supernatant*

Exp.2

21.7 ± 0.4 32.2 ± 2.9

PAM without rhIFN-')' PAM with rhIFN-')'

23.6 35.1

± 1.2 ± 1.7

Exp.3

Exp. 4

25.0 ± 0.6 38.9 ± 4.1

29.8 ± 1.6 37.3 ± 1.8

Definition ofabbreviations: rhIFN--y = recombinant human interferon-y; SIC = suspended form of IgG immune complex; for other abbreviations, see Table 1. * Isolated PAM (2 x 106 cells/ml) were incubated with and without rhIFN--y (550 U/ml) at 37° C. After 18 h, PAM were washed 3 times, and 1 x 106 cells were placed in the wells of a 24-well tissue culture plate and incubated with liC and SIC. After a 6-h incubation period, PAM supernatants were collected to test for their ability to lyse WEHI-I64 target cells. t "Cr-Iabeled WEHI-I64 cells (1 x 1()4/wells) were incubated with 1:4 diluted supernatant and assayed for specific 51Cr release after 18 h of incubation at 37° C. Values are shown as mean of triplicate determinations of each experiment ± SD.

two forms of IgG immune complexes, experiments were designed in relation with IgG FcR modulation and prevention of internalization. We have previously demonstrated that the phagocytosis inhibitor cytochalasin B prevents the internalization of CRBC/anti-CRBC immune complexes by PAM, as shown by immunofluorescence assay for the internalization of IgG immune complexes (22). Therefore, experiments were approached by using pretreated PAM with and without the cytochalasin B. The results presented in Figure 4 show that cytochalasin B-pretreated PAM with SIC generated high levels of TNF-a-mediated cytotoxicity similar to IICstimulated PAM. However, when PAM were stimulated with SIC in the absence of cytochalasin B pretreatment, there was some but lower levels of TNF-a-mediated cytotoxicity. Experiments were also designed to evaluate the effects of rhIFN-"'( priming. When IFN-"'(-primed PAM were stimulated in the SIC system in the absence of cytochalasin B, the level of cytotoxicity was not significantly different to that without rhIFN-"'( priming. However, when rhIFN-"'(-primed PAM were stimulated in the presence of cytochalasin B, an enhanced level of cytotoxicity was observed as compared to that without rhIFN-"'( priming. These results indicate that IIC and SIC with cytochalasin B pretreatment, both of which prevent internalization of IgG immune complex-bound FcR, induce and secrete TNF-a by PAM, and that rhIFN-"'( priming of PAM enhances TNF-a production.

mechanism of TNF-a generation is related to IgG immune complex-bound FcR modulation. TNF-a was initially identified because of its in vivo tumor necrotizing activity and its in vitro cytotoxicity to certain tumor cell lines, and it is now recognized that TNF-a does possess several other biologic activities. Overall, TNF-a may playa role in acute, chronic inflammatory and infectious diseases, in nonspecific antineoplastic and antiviral systems, and in induction of pulmonary fibrogenesis because of significant fibroblast growth-enhancing activity (16, 27). Especially in IPF, activated PAM and TNF-a activity seems to be implicated in the development and progression of disease. A role for TNF-a as the mediator of cell-mediated cytotoxicity has been well documented by data from several laboratories showing that antibodies against TNF-a inhibit cell-mediated cytotoxicity reaction (28, 29). In this experiment, a bioassay was done using TNF-a-sensitive WEHI164 target cells to measure TNF-a activity (TNF-a-medi-

Discussion The present study demonstrates the in vitro stimulatory effects of IIC on TNF-a generation by PAM and that the

PAM alone

TABLE 3

% Specific Lysis (WEHI-164)

IIC stimulation SIC stimulation

PAM

cyto B

Comparison between the stimulation effects of IIC and SIC system on TNF-a-mediated cytotoxicity by PAM Supernatant*

IFN-yprimed PAM

Exp. 1

Exp.2

Exp.3

21.7 ± 0.4 3.4 ± 0.3

23.6 ± 1.2 7.7 ± 0.9

25.0 ± 0.6 6.0 ± 1.3

Definition of abbreviations: see Table 2. * Supernatants are harvested from 6-h-incubated PAM (1 x 106ml) with IIC and SIC system. Values are shown as mean of triplicate determinations of each experiment ± SD.

IFN-YP?med PAM Cyto B

Pretreatment of PAM

Figure 4. Effect of cytochalasin B-pretreated PAM with SIC stimulation on TNF-a-mediated cytotoxicity. Isolated PAM (2 x 106 cells/ml) were incubated with and without rhIFN-)' (500 U/ml) at 37° C. After 18 h, PAM were washed 3 times and 1 x 106 cells were placed in the wells of a 24-well tissue culture plate and pretreated with cytochalasin B (2 JLg/ml). After 15 min of incubation at 37° C, PAM were washed again, and chicken red blood cells (CRBC) (6 x 106 cells) and rabbit anti-CRBC antibody were added at a final dilution of 1:200. After 6 h of incubation, supernatants were harvested to measure TNf-ce-mediated cytotoxicity.

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ated cytotoxicity) and confirmed by antibody neutralization experiments with anti-rhTNF-a monoclonal antibody. These data also show that rhTNF-a antibody cross-reacts with porcine TNF-a. It has been demonstrated that most of the effects of TNF-a are not species specific, and comparison of the nucleotide sequence indicates greater than 85 % homology between porcine and human TNF-a gene (30). The present study shows clearly that IIC alone could be a significant stimulus for activation of PAM to produce TNF-a. Initial studies demonstrated that macrophages were the main source of TNF-a and that these cells must first be primed or activated before challenging with an inducer such as bacterial endotoxin (lipopolysaccharide, LPS) to generate significant amounts of TNF-a (31). TNF-a was not produced by unstimulated culture. Some data showed that TNF-a was spontaneously produced from PAM of interstitial lung disease such as sarcoidosis, coal miner pneumoconiosis, and asbestosis (32-34); however, little is known of TNF-a production by resident PAM using immune complex as a stimulatory signal in a controlled animal system. Therefore, in this experiment, the results that significant levels of TNF-a activity were produced from IIC-activated PAM in the absence of other "priming" or "activating" agents are an important finding. The present data demonstrate that priming with IFN-'Y augments IIC-induced TNF-a production by PAM. It has been shown that recombinant IFN-y-primed murine macrophage/monocyte produce TNF-a in response to subsequent LPS stimulation (35). We have previously demonstrated that IFN -'Y alone does not activate PAM to produce TNF-a production, but rhlf'N'-y-primed PAM produce significant levels of TNF-a in response to triggering agents such as LPS, lipid A, or monophosphoryllipid A (data prepared for publication). These results suggest that IFN-'Y exerts a permissive influence on PAM to produce TNF-a in response to IIC stimulation as well. That activated alveolar macrophages produce TNF-a is related to the central role played by these cells in the lower respiratory tract and may contribute to the development and progression of pulmonary fibrosis in concert with other macrophage-derived growth factors. The present study demonstrates that when alveolar macrophages are stimulated with IIC they generate TNF-a. However, SIC induce lower levels of TNF-a generation by PAM than IIC. This result seemingly conflicts from those of Warren and colleagues (18) describing that incubation of rat alveolar macrophages with increasing concentrations of preformed IgG immune complexes (bovine serum albumin [BSA] and anti-BSA) resulted in a dose-dependent release of TNF-a. This may be explained by the fact that some BSA and anti-BSA complexes may have spontaneously immobilized as shown in our previous reports (21, 22), that some spontaneous immobilization occurred in SIC, or it may also be due to differences in the physical nature of the antigen (soluble protein versus particulate cell) in the SIC (i.e., BSA-anti-BSA versus CRBC-antiCRBC). It has been demonstrated that soluble circulating IgG immune complexes and tissue deposits of IgG immune complexes were present in IPF (4-6), but there was no correlation between circulating immune complexes and disease activity (36). It appears that the ability of preformed IgG immune complexes to incite an immune inflammatory reaction

in the lung through PAM depends on its physicochemical characteristics. In this respect, we demonstrated that immobilization of IgG immune complexes, not merely IgG immune complexes, has a significant role as a stimulus to generate TNF-a by PAM. This suggests that in vivo, circulating immune complexes may not act as efficiently as the tissue deposited immune complex as a stimulus to generate significant levels of TNF-a. Furthermore, the activity and progression of pulmonary fibrosis, which are possibly related with IgG immune complex in its pathogenesis, may depend on the level of immune complex deposit and on its persistency. Alveolar macrophages from IPF patients have been shown to have occupied IgG FcR and C3b receptors, suggesting the presence of adherent immune complex on their membrane surface (2,37), and recent in vivo studies showed increased TNF-a activity in BAL sample of IgG immune complex-mediated alveolitis (18). These findings strongly support our concept that TIC-stimulated PAM-released TNF-a could contribute to the initiation and progression of the immune inflammatory circuit leading to pulmonary fibrosis. Finally, we investigated the different mechanisms between IIC and SIC stimulation to induce TNF-a production by PAM. Previously we found that IIC-activated PAM generated significant levels of hydrogen peroxide which mediate nonspecific (bystander) cytotoxicity, and that IgG immune complex modulated FcR and prevention of internalization was implicated in this mechanism (22). To evaluate the effects of IgG immune complex-bound FcR modulation to induce TNF-a by PAM, experiments were performed in which PAM were pretreated with the phagocytosis inhibitor cytochalasin B and subsequently stimulated with SIC. The data demonstrate that cytochalasin B-pretreated PAM are activated to generate significant levels of TNf-« by SIC. Previously we demonstrated that cytochalasin B pretreatment as well as immobilization of immune complexes prevents internalization oflgG immune complex-bound FcR (22), thus we conclude that IgG immune complex-bound FcR modulation and prevention of internalization is an important mechanism of signal induction for activation of PAM to produce and secrete TNF-a. The present data also show enhanced production of TNF-a by cytochalasin B-pretreated PAM after rhIFN-'Y priming with subsequent stimulation in SIC. This enhancing effect can be interpreted by the previous observation that IFN-'Y increases FcR on human monocyte and alveolar macrophages (38) and suggested the role ofFcR for activation of PAM. In addition, the observation that IFN-'Y enhances macrophage transcription of TNF-a (39) could be a potential mechanism by which IFN -'Y primed PAM to release increased levels of TNF-a upon subsequent exposure to IIC stimulation. Therefore, the present study strongly supports the concept that IIC or SIC with cytochalasin B pretreatment, both of which prevent the internalization of IgG immune complex-bound FcR, provide a signal for PAM to generate TNF-a through FcR modulation. This suggests that in vivo, deposited (immobilized) IgG immune complexes-bound FcR may be a stimulus for activation of PAM to generate TNF-a rather than circulating (mobilized) immune complexes, which contribute to the pathogenesis of diffuse interstitial fibrosis of the lung, especially in IPF. Acknowledgments: This work was supported in part by Grant HL-36012 from the National Heart, Lung and Blood Institute, National Institutes of Health. The

Kim, Wierda, and Kim: Immobilized Immune Complex Induces TNF-a Secretion by Macrophages

writers wish to acknowledge Genentech, Inc. for their generous supply of rhTNF-a, rhIFN-')', anti-rhTNF-a monoclonal antibody, and anti-rhlf'Nvy monoclonal antibody for this study.

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