American Journal of Pathology, Vol. 138, No. 3, March 1991 Copyright © American Association of Pathologists
Contrasting Roles for Tumor Necrosis Factor in the Pathogeneses of IgA and IgG Immune Complex Lung Injury Jeffrey S. Warren, Peter A. Barton, and Michael L. Jones From the Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan
Recent studies suggest that development of acute gamma G immunoglobulin (IgG) immune complex lung injury is partially dependent on a tumor necrosis factor (TNF)-dependent mechanism of neutrophil (PMN) recruitment. The authors have sought to further define the role of intrapulmonary TNF in IgG alveolitis and to examine its role in IgA immune complex alveolitis, a neutrophil-independent model of acute lung injury. IgG immune complex lung injury resulted in a marked rise in intrapulmonary TNF activity accompanied by progressive pulmonary PMN accumulation. Intratracheal instillation of neutralizing concentrations of anti-TNF markedly reduced PMN influx measured at 4 hours but had no effect on PMN recruitment quantitated at 2 hours. IgA immune complex deposition resulted in acute lung injury accompanied by increased numbers of intrapulmonary mononuclear phagocytes but few neutrophils. Lung lavage fluids obtained from IgA immune complex-injured rats contained both neutrophil and monocyte chemotactic activities, albeit at twofold to fourfold lower concentrations than observed in IgG-mediated alveolitis. In contrast to IgG
complex-mediated alveolitis, lung lavagefluidsfrom IgA-injured rats contained no TNF activity. Intratracheal administration of anti-TNF antibodies had no effect on the development of IgA lung injury as assessed by morphology and measurements of vascular permeability. In vitro exposure of isolated alveolar macrophages to preformed IgG immune complexes resulted in dose-dependent TNF secretion, while exposure to IgA complexes resulted in very low levels of TNF secretion These data suggest that TNF-mediated pulmonary neutrophil recruitment (in IgG lung injury) is manifest chiefly in the late phase (approximately 4 hours) of developing alveolitis. The virtual absence of intrapulmonary TNF activity in evolving
IgA immune complex alveolitis may in part account for the limited PMN recruitment observed in this model. (Am J Pathol 1991, 138:581-590)
Full development of acute IgG immune complex alveolitis in the rat is dependent on neutrophils and an intact complement system.1 Identically induced gamma A immunoglobulin (IgA) immune complex lung injury also requires complement, but develops fully in the absence of neutrophils.23 In vivo and in vitro studies suggest that alveolar macrophages or recruited monocytes are the chief effector cells in IgA lung injury.45 Preformed IgG immune complexes activate isolated rat neutrophils and alveolar macrophages as measured by release of lysosomal enzymes (in the case of neutrophils) and production of superoxide anion (02-) and hydrogen peroxide (H202).46 In contrast, preformed IgA immune complexes trigger 02- and H202 release by macrophages but induce a negligible response by neutrophils.4 These observations help to distinguish these models of alveolitis, but given that complement is involved in both models, it is not clear why IgG alveolitis is attended by a massive influx of neutrophils while IgA alveolitis is not. Recent in vivo studies suggest that alveolar macrophage-derived tumor necrosis factor (TNF) participates in the pathogenesis of acute IgG immune complex alveolitis through a mechanism involving neutrophil recruitment.7 The mechanisms by which TNF mediates neutrophil recruitment have not been clearly defined in this model, but several processes may be operative. Tumor necrosis factor, as well as other inflammatory mediators such as leukotriene B4 and C5a, can rapidly (in minutes) trigger the increased expression of adhesionpromoting molecules (CD11/CD18; Mac-1/ LFA-1/p`150,95) on neutrophils.8 Several cytokines, including TNF, also induce the upregulation or de Supported by National Institutes of Health grant HL-40526. Accepted for publication October 16, 1990. Address reprint requests to Jeffrey S. Warren, MD, Department of Pathology, Box 0602, University of Michigan Medical School, 1301 Catherne St., Ann Arbor, MI 48109-0602.
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expression of leukocyte adhesion molecules on endothelial cells.9-13 It has been suggested that cytokine-activated endothelium directs and amplifies leukocyte recruitment into evolving inflammatory foci. Endothelial leukocyte adhesion molecule (ELAM)-1 binds neutrophils, is expressed on the surface of TNF-stimulated endothelial cells, and peaks 4 to 6 hours after cytokine exposure.912 Recent in vitro studies indicate that ELAM-1 may also play a role in monocyte-endothelial adhesion.14 This observation emphasizes that simple one-to-one relationships between given adhesion molecules and specific types of leukocytes do not exist. Furthermore antibodies directed against ELAM-1 do not completely block neutrophil or monocyte adhesion, suggesting that additional adhesive mechanisms are operative.14 There is in vitro evidence that other molecules may also be important in very early leukocyte-endothelial adhesion events (eg, GMP-140, iC3b, PAF). 1-18 These varied mechanisms of leukocyte-endothelial adhesion may all contribute to different degrees, depending on the time point examined. Although not as extensively studied, physiologic variables (eg, shear stress) and anatomic factors (site of vascular bed) also may influence leukocyte recruitment.19 The observation that TNF is required for full pulmonary polymorphonuclear neutrophil (PMN) recruitment in IgGinduced alveolitis led us to further define its role in this model and to examine its potential role in the pathogenesis of acute IgA immune complex alveolitis. IgAmediated immune complex alveolitis is induced in a manner analogous to IgG lung injury, but provides an interesting contrast because it is mediated by monocytes or macrophages.2' The present study provides data that suggest that TNF-mediated PMN influx is manifested in the later phase of developing acute IgG immune complex alveolitis. Although a variety of events are necessary for full neutrophil recruitment into an acute inflammatory focus, the virtual absence of intrapulmonary TNF elaboration in evolving IgA immune complex alveolitis may in part explain why there is limited PMN recruitment in this model.
novo
Materials and Methods
Antibodies, Antigens, and Preparation of Immune Complexes Rabbit IgG rich in antibody directed against bovine serum albumin (BSA) was used to induce lung injury and to form precipitating polyclonal IgG-BSA immune complexes. Antibody content and production of immune
complexes have been previously described in detail.1 IgG was purchased from Organon Teknika, West Chester, PA. Stock IgG anti-BSA and BSA (low endotoxin; ICN Biomedicals, Costa Mesa, CA) preparations contained less than 0.02 ng/ml and 0.012 ng/ml of endotoxin activity, respectively, as estimated using the Limulus amebocyte lysate (lAL) assay (E-toxate; Sigma Chemical Co., St. Louis, MO). Affinity-purified monoclonal IgA (MOPC 315) directed against dinitrophenol-conjugated BSA (DNP-BSA) was purchased from Sigma Chemical Co. Dinitrophenolconjugated BSA was prepared according to the method of Eisen.20 The resulting antigen preparation contained an average of 50 DNP groups per BSA molecule. Antigen-antibody equivalence for IgG and IgA immune complexes (wt/wt per unit volume) was determined by quantitative immunoprecipitation as previously detailed.4 IgG anti-TNF alpha (provided by Drs. Steven L. Kunkel and Daniel G. Remick, University of Michigan) was produced by immunization of rabbits with recombinant murine TNF, affinity purified using protein A sepharose, and characterized for specificity against rat TNF as previously described.7 The antibody preparation used in these experiments blocks rat TNF in a dose-dependent manner. In vitro, 35 jig of anti-TNF reduces rat TNF activity (125 units/ml) to less than 20 units/ml, whereas 50 ,ug of anti-TNF reduces TNF activity to less than 5 units/ ml.7 The quantity of anti-TNF (36 ,ug/rat) required to completely neutralize BAL fluid TNF activity (in vivo) was determined empirically.
Animal Models of Immune Complex Alveolitis Male Long-Evans pathogen-free rats (300 to 400 g; Charles River Breeding laboratories, Inc., Wilmington, MA) were used for all studies. Intraperitoneal injections of ketamine (2.5 to 5.0 mg/i 00 g body weight) and sodium pentobarbital (5 mg/i 00 g body weight) were given for sedation and anesthesia. IgG and IgA immune complex lung injuries were induced as previously described.1' 2 Antibody solution (250 ,ug IgG anti-BSA or 400 ,ug IgA anti-DNP-BSA) or mixtures of antibody and anti-TNF or nonspecific rabbit IgG, were instilled into the lungs through a tracheal cannula. In all cases, a final volume of 300 ,ul was instilled into the lungs. Antigen (BSA, 10 mg; or DNP-BSA, 3.3 mg) was injected intravenously. Rats were killed at the indicated times and lung injury quantitated, bronchoalveolar lavage (BAL) fluid harvested, or lung myeloperoxidase (MPO) activity determined. At time of death, anesthetized rats are exsanguinated through inferior vena caval section before removal of lungs, thus
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resulting in negligible contamination of lungs with blood. Pulmonary injury was quantitated by permeability measurements. Permeability indices were calculated by comparing the leakage of 1251-labeled bovine gamma globulin from the circulation into the lung to the 1251-labeled colloid remaining in 1 ml of blood as previously described.1 Intrapulmonary TNF neutralization was carried out as previously described.7
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Morphometric Analysis of Pulmonary Neutrophil Recruitment Lung samples (1 mm3) were excised from the peripheral aspect of whole lungs that had been fixed in 4% glutaraldehyde under constant pressure inflation (25 cm H20). Samples were washed in 0.1 mol/l (molar) cacodylate buffer (pH 7.3), embedded in 1 -,-thick plastic sections, and stained with toluidine blue. Use of 1-mm3 samples from the peripheral aspects of inflated lung results in sections that contain no large bronchial structures. ('Large bronchial structures' are defined as muscular airways lined by respiratory epithelium.) Plastic embedding (1 ,u thick) allows very high morphologic resolution, thus allowing virtually all alveolar and alveolar septal cells to be easily identified. Morphometric analysis of PMN recruitment was carried out by a pathologist (J. S. Warren) who was blinded to sample origin. For each condition, five lung samples were examined. In each sample, 45 to 60 randomly selected 40x microscopic fields (HPF; highpower field) were analyzed.
Whole Lung Myeloperoxidase Activity as Quantitative Measure of Neutrophil Recruitment To assess the use of tissue MPO activity as a measure of PMN influx, known concentrations of glycogen-elicited PMN were added to normal rat lungs, the tissue homogenized and extracted, and standard curves produced as previously described21 (see Figure 1 for standard curve). Whole lungs were homogenized with a Polytron homogenizer (Tekmar Co., Cincinnati, OH) (4 x 10 seconds at a setting of 4) using 6 ml of homogenization buffer on ice. The homogenization buffer (50 mmol/l [millimolar] phosphate, pH 6.0) contained 0.5% hexadecyltrimethyl ammonium bromide (Sigma) and 5 mmol/l ethylenediaminetetra-acetic acid (EDTA). Homogenized samples were then sonicated (3 x 10 seconds at a setting of 5) and centrifuged (3000g; 30 minutes) at 4°C. Myeloperoxidase activity in supernatants was assayed by measuring the change in A460 resulting from decomposition of H202 in
0
1 o6
1
07
1 o8
Neutrophils Added Figure 1. Relationship between number ofaddedglycogen-elicited neutrophils and tissue myeloperoxidase activity. Supernatants (25 Pd and 50 pl, respectively)from homogenized lungs were assayed as described in Materials and Methods. Change in A460 was monitored for at least 3 minutes to ensure lineari. 7he data are expressed as means ± standard deviations (n = 4 for each point except 0 added neutropbils, wbere n = 2).
the presence of o-dianisidine.22 Multiple aliquots of lung extract were tested to assure assay linearity.21
Collection of BAL Fluid and Isolation of Alveolar Cells Alveolar macrophages were recovered from normal rats using a previously described.6 Lung lavages obtained from normal rats contained greater than 94% alveolar macrophages. Lung lavage contents for TNF measurements and cell counts were collected using 5 ml of 37°C, serum-free RPMI 1640. At least 90% of the administered fluid was always recovered, centrifuged (400g; 7 minutes) to separate cells, and stored at - 200C before analysis. There was no systematic difference in BAL fluid recovery between control and experimental groups of rats.
TNF Assays Tumor necrosis factor activity was measured using a modification of the procedure of Ruff and Gifford.23 Briefly, equal volumes of log2 dilutions (final volume, 0.1 ml) of sample-containing media or serum were made in 96-well microtiter plates (Costar, Cambridge, MA) containing target LM cells (mouse fibroblast cell line derived from NCTC clone 929; American Type Culture Collection) (5 x 1 04 cells per 0.1 ml) in the presence of actinomycin D (final concentration, 1 ,ug/ml; Sigma). In a separate set of wells, a standard consisting of serially diluted recom-
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binant human TNF (Cetus Immune Laboratories, Palo Alto, CA) was added. The cells were incubated at 370C for 18 hours, the supernatants discarded, and the remaining viable adherent cells washed with PBS and then stained by adding crystal violet (0.2% in 2% ethanol) for 5 minutes. Microtiter plates were rinsed three times with PBS and allowed to air dry. The absorbance of each well was read at 620 nm with a MicroELISA Autoreader (BioTek Instruments, Winooski, VT). Units of TNF are defined as the reciprocal of the dilution at which 50% cytolysis occurs. Assay specificity was conferred by the ability of anti-TNF to neutralize sample TNF activity.
here to polystyrene culture dishes (2 x 106/35 mm dish), washed, and incubated for 4 hours in serum-free RPMI 1640 containing preformed immune complexes (IgA or IgG). Culture supernatants were then aspirated and assayed for TNF activity. Negative controls included equivalent concentrations of antibody alone (IgA or IgG), antigen alone (DNP-BSA or BSA), or medium alone. All values were normalized for adherent cell number. The proportions of alveolar macrophages that remained adherent after the wash ranged from 0.53 to 0.74. In no experiment was there more than 14% plate-to-plate variation.
Neutrophil and Monocyte Isolation
Results
Neutrophils were obtained from glycogen-induced rat peritoneal exudates 4 hours after the intraperitoneal injection of 1.0% glycogen in sterile saline. Peritoneal cells (more than 85% neutrophils) were harvested as described elsewhere.6 Human peripheral blood monocytes (80% to 85%) were isolated by centrifugation through Ficoll-Hypaque (Sigma) and Sepracell-MN (Sepratech, Oklahoma City, OK) as described by Vissers et al.24
Neutrophil and Monocyte Chemotaxis Assays Neutrophil and monocyte chemotaxis assays were carried out in 48-well micro chemotaxis chambers as descnbed by Falk et al.25 Cell suspensions (2.25 x 105 cells/well) were added to the top well of the chamber and permitted to migrate through 1 0-, polycarbonate membranes (3-,u porosity) toward the sample-bearing bottom chamber. After a 4-hour incubation (3TC, 5% CO2, humidified), membranes were removed, nonmigrating cells wiped off, and filters fixed for 10 minutes in absolute methanol. Filters were air dried and stained for 30 minutes in 2% toluidine blue. The number of cells migrating through the membrane were counted in three random, 1 0-mm grids at 400x, with the mean ± standard error of the mean (SEM) calculated for triplicate samples. Results are expressed as normalized values representing the percent of maximum f-Met-Leu-Phe (10-7 or 10-8 mol/l; as indicated) (Sigma) positive control, minus negative controls (buffer alone).
Time Course of Pulmonary PMN Recruitment in lgG Immune Complex-mediated Lung Injury Pulmonary PMN accumulation was quantitated at varying times after initiation of lung injury by measuring whole lung MPO activities. There was a progressive increase in pulmonary PMN accumulation over the course of developing alveolitis (Figure 2). The absolute number of PMNs recruited into the lungs of rats with developing IgG immune complex lung injury cannot be accurately determined based on these data. Examination of hematoxylin and eosin (H&E)-stained whole lung sections and Wright-stained smears of cells retrieved by lung lavage disclosed fewer than 1% lymphocytes and no eosinophils (data not shown). 0.5
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In Vitro TNF Secretion by Isolated Alveolar
Macrophages Rat alveolar macrophages suspended in RPMI 1640 with 10% heat-inactivated fetal calf serum were allowed to ad-
Figure 2. Pulmonary neutrophil recruitment in rats with IgG immune complex alveolitis (0) as a function of time. Negative control rats (0) received equivalent concentrations ofnonspecific rabbit IgG instead of anti-BSA. Neutrophil recruitment was quantitated by measuring whole lung MPO activities.2' The data represent cumulative means + SEM offour experiments. At leastsix animals were used for each data point, except for rats at time 0 and negative control rats, in which three ratspergroup were used.
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Local TNF Mediates IgG Immune Complex-induced Pulmonary PMN Recruitment There was an 18-fold increase in BAL fluid TNF levels after induction of IgG immune complex alveolitis (Figure 3). No detectable rise in serum TNF was observed at either 0, 1, 2, or 4 hours after induction of alveolitis (data not shown). Intrapulmonary TNF blocking experiments were carried out to compare the role of TNF in PMN influx measured 2 and 4 hours after initiation of lung injury. Neutralization of intrapulmonary TNF resulted in marked suppression of pulmonary PMN recruitment measured at 4 hours (Table 1). Intratracheal anti-TNF had no effect on PMN accumulation observed at 2 hours, despite neutralization of BAL TNF activity (Table 1). These data suggest that the mechanism(s) responsible for TNF-mediated PMN recruitment are manifested predominantly during the late phase (approximately 4 hours) of developing acute alveolitis.
Neutrophil and Monocyte Chemotactic Activities in IgG and IgA Immune Complex-mediated Acute Lung Injury Analysis of BAL fluid from rats with IgA immune complex lung injury showed neutrophil and monocyte chemotactic
activities, albeit at lower concentrations than in IgG immune complex-injured rat lungs (Figure 4). The identities of the chemotactic factors elaborated in IgA immune complex lung injury are unknown. It is also unclear whether they are the same as in IgG alveolitis. These data suggest that the dearth of neutrophils recruited into sites of alveolar IgA-DNP/BSA deposition cannot be accounted for by an absence of neutrophil chemotactic activity.
Role of TNF in IgA Immune Complex Lung Injury Intratracheal instillation of IgA anti-DNP-BSA followed by intravenous infusion of DNP-BSA resulted in acute lung injury (Table 2, Figure 5). As noted in previous studies, fewer than 10% of the retrieved BAL lavage inflammatory cells were neutrophils (Table 2). Fewer than 5% of the retrieved cells were lymphocytes and no eosinophils were observed (data not shown). Bronchoalveolar lavage fluids from rats with IgA immune complex-induced acute lung injury contained no detectable TNF activity. Intratracheal instillation of anti-TNF into rats receiving IgA DNPBSA resulted in no suppression of lung injury (Table 2), no difference in the cellular contents of retrieved BAL fluid (Table 2), and no change in morphology compared with positive controls (not shown). These observations suggest that IgA immune complex alveolitis is mediated by a TNF-independent pathway.
TNF Secretion by Alveolar Macrophages Stimulated with IgG and IgA Immune Complexes
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0
1
3
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TIME (hours) Figure 3. Elaboration of intrapulmonary (BAL fl uid) TNF in rats during the development of IgG immune complexr-mediated lung injury (0). Negative control rats (0) received int;ratracbeal nonspecific IgG instead of anti-BSA Rats were killed iat the indicated time points and lungs lavaged with serum-free RPi MI 1640 (5 ml). The data represent the cumulative means SEA, ments in which at least three animals were usedfcr each variable. The TNF values expressed represent total activity retrieved per rat. Tumor necrosis factor activity was standardized a nant human TNF (see Materials and Methods).
Previous studies suggest that resident alveolar macrophages are the chief source of intraalveolar TNF in IgG alveolitis.7 Incubation of alveolar macrophaaes with increasing concentrations of preformed IgG immune complexes resulted in dose-dependent release of TNF (Figure 6). Incubation of macrophages with IgG alone or BSA alone resulted in negligible TNF release, suggesting that intact immune complexes, not a contaminant of the antigen or antibody preparation (eg, endotoxin), is responsible for the cellular response. Incubation of alveolar macrophages with equivalent concentrations of IgA immune
complexes resulted in very little TNF release. Exposure of alveolar macrophages to IgA alone resulted in negligible
±
TNF release. Exposure of macrophages to relevant concentrations antigen alone (DNP-BSA) also resulted in
minimal TNF release. Addition of high concentrations
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r
Table 1. Role of Intrapulmonary TNF in IgG Immune Complex Pulmonary PMNRecruitment Neutrophil influx Lung MPO activity
Total BAL TNFt activity (units)
Intervention* 2 Hours Nonspecific IgG (36 ,ug) (positive control) Anti-TNF (36 p,g)
0.08 ± 0.04
39 ± 6
Noninjured (negative)ll 4 Hours Nonspecific IgG (36 [tg) (positive control) Anti-TNF (36 ,g) Noninjured (negative control)"
2± 2 0± 2 177 ± 21 4± 3 3± 3
PMN/40X HPF
0.10 ± 0.05J 0.01 ± 0.01§
17 ± 4-1 NSt 14 ± 5j 3 ± 1§
0.52 ± 0.06 1 P < 0.05t 0.18 ± 0.05 0.03 ± 0.01
78 ± 5-, P < 0.05t 19 ± 3 4±2
-
NSt
* All rats except noninjured (negative controls) received intratracheal IgG anti-BSA and intravenous BSA. Interventions (anti-TNF or equivalent concentrations of nonspecific IgG) were instituted of the same time that immune complex injury was initiated (time zero). Rats were killed after either 2 or 4 hours as indicated. The data represent the cumulative means of at least 6 rats/group, except for noninjured (negative controls) where 3 rats/group were used. 38 t Data were subjected to one-way analysis of variance. NS, no significant difference at P < 0.05. t TNF activity was standardized against recombinant human TNF (see Methods). § P < 0.05 versus positive control (nonspecific IgG); P < 0.05 versus anti-TNF-treated group. 11 Noninjured (negative control) rats received intravenous BSA (10 mg) and nonspecific intratrancheal IgG (instead of anti-BSA) at time zero.
(r400 ,ug/ml) of DNP-BSA resulted in release of TNF up to 14 units/ml (data not shown). Previous studies indicate that high concentrations of DNP-BSA are cytotoxic (unpublished observations). Incubation of alveolar macrophages with a fixed concentration of preformed IgG immune complexes showed that TNF secretion reaches a plateau by 4 hours (Figure 7). Only small amounts of TNF were released by macrophages exposed to IgG or BSA alone. Negligible TNF was secreted by IgA immune complexes, IgA alone, or DNP-BSA alone. These data suggest that alveolar macrophages release substantial amounts of TNF in response to IgG immune complexes but very little in response to IgA complexes.
Effect of Supernatants from IgA Immune Complex-stimulated Alveolar Macrophages on TNF Secretion by IgG Immune Complex-triggered Alveolar Macrophages One potential explanation for the lack of TNF secretion by IgA immune complex-triggered alveolar macrophages is that such cells release an inhibitory or suppressive factor. To address this possibility, alveolar macrophages were incubated with preformed IgG immune complexes in the presence of increasing proportions of culture supernatant obtained from macrophages that had been incubated with IgA complexes. As shown in Table 3, super-
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Figure 4. A: Neutrophil chemotactic activity in lung lavage fluid obtained 4 hours after initiation of either IgG (0) or IgA (0) immune complex lung injuwy. Lavage fluidsfrom negative control rats (intratracheal antibody alone and intravenous antigen alone) contained less than 20 units of activity. B: Monocyte chemotactic activity in lavagefluidfrom either IgG (0) or IgA (0) lung injuwy. Negative controlfluids contained less than 10 units of activity. The data are normalized to fMLP (10 - 7 mol/lfor neutropb ils and 10 - 8 mol/lfor monocytes).
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Table 2. Role of TNF in IgA Immune Complex Lung Injury* Rat Treatments Lung Injury
BALTNF
BAL Cells Retrieved
(permeability Intratracheal
Intravenous
A. Saline (negative control) B. IgA (negative control) C. IgA + nonspecific IgG (36 Rg) D. IgA + Anti-TNF(36 p.g)
Saline
0.02 ± 0.02
0.03 ± 0.04
Saline
0.04 ± 0.01
0.08 ± 0.02
DNP-BSA
0.25 ± 0.02§
0.07 ± 0.05
E. IgG (250 p.g)
F. IgG (250 ,ug) + Anti-TNF
index)
(units)t
Total 6.6 ± 0.7 x 104 6.9 ± 0.6 x 104 15.1 ± 1.9
PMN (%) 6
AMt
8
90
6
88
(%) 93
x 1i4
DNP-BSA
0.26 ± 0.03§
BSA (10 mg)
0.52 ± 0.08
BSA (10 mg)
0.04 ± 0.03
193
0.23 ± 0.06t
6
5
90
± 27
17.3 ± 2.1 x 104 8.3 ± 0.6
71
21
± 5'
x 106 3.8 ± 0.5
77
19
(36 Rg)
1 06t
* All rats received intratracheal IgA (400 ,ug) and intravenous DNP-BSA (3.3 mg), except where indicated. Rats were killed at 4 hours. The data represent the cumulative totals of at least six rats/group except in group A where three rats were used. All data are expressed as means + SEM. t Total TNF activity in serum-free RPMI 1640 (5 ml) retrieved from rats 4 hours after induction of injury. TNF activity was standardized against recombinant human TNF (see Methods).
t AM = alveolar macrophages. § p < 0.05 versus negative controls A. and B. (one-way analysis of variance).38 P < 0.05 versus matched positive control E (for reduced permeability index, BAL-TNF activity, and number of BAL cells retrieved). (One-way analysis of variance).38
natants from IgA immune complex-treated cells exerted no suppressive effect in IgG-complex-induced TNF release. These data suggest that a soluble inhibitory substance is not responsible for the lack of TNF secretion by IgA-activated macrophages.
Discussion Recent studies suggest that intrapulmonary elaboration of TNF is a necessary step in the complete development of IgG immune complex-induced alveolitis.7 Neutralization of intra-alveolar TNF activity attenuates pulmonary neutrophil recruitment by approximately 60%. The objec-
tives of the present study were to further characterize the role of TNF in the pathogenesis of IgG immune complex alveolitis and to examine its role in a complementary model, IgA immune complex-mediated alveolitis. IgA immune complex-induced lung injury develops rapidly (4 hours), but is mediated by monocytes or macrophages rather than neutrophils. The data suggest that TNFmediated pulmonary neutrophil recruitment (in IgG lung injury) is manifest largely in the late phase of developing alveolitis. The virtual absence of intrapulmonary TNF ac-
E
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5.
IgA
immune
complex lung
injury.
There is intra-
alveolar hemorrhage accompanied by increased numbers of alveolar macrophages. Few neutrophils are present. Toluidine blue stained 1-p.m plastic section; original magnification, x400.
10
20
30 gg Antibody
40
50
Figure 6. In vitro production of TNF by alveolar macrophages incubated with increasing concentrations of preformed IgG-BSA immune complexes (0), IgA-DNP/BSA complexes (I), and negative controls. Negative controls included equivalent concentrations of IgG alone (0), BSA alone (A), IgA alone (U), and DNPBSA alone (*). Antigen and antibody concentrations were selected such that immune complexes were formed at antigen:antihoY equivalence. As described previously,4 antigen:antibody equivalence (concentration:concentration) was 1:5 for IgG-BSA and 2:1 for IgA-DNP/BSA These data represent the cumulative means of three separate experiments where each variable was run in duplicate or triplicate. Tumor necrosis factor activity was standardized against recombinant human TNF (see Materials and Methods).
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. ^
Figure 7. In vitro production of TNF by im-
complex-stimulated alveolar macrophages as a function of time. Exactly as demune
lgG i.c. scribedfor Figure 6, adherent alveolar macro-
E
pbages were incubated with preformed immune complexesfor the indicated periods of time. IgG and IgA immune complexes (formed at antigen:antibody equivalence) contained 50 pg/ml antibody. Negative controls (not shown)
(0 C
U-
z
included equivalent concentrations of either
IgG along, BSA alone, IgA alone, or DNP/BSA alone. For all negative controls (at 0, 1, 2, 4, and 7 bours) TNF concentrations were less than IgA i.c. 11 ± 2 units/ml. These data represent the cumulative mean of two separate experiments in wbich each variable was run in tiplicate. Tumor necrosis factor activity was standardized
TIME (hours)
tivity in IgA-induced alveolitis may in part explain the limited neutrophil recruitment observed in this model. Several mechanisms may be operative in TNFmediated neutrophil recruitment. Exposure of isolated neutrophils to TNF results in a rapid (minutes) increase in adhesiveness.8 Exposure of human umbilical vein endothelial cells (HUVECs) to TNF results in interleukin-8 production-6,27 Interleukin-8, also known as neutrophil activating peptide-1 or neutrophil chemotactic factor, is a very potent and specific neutrophil chemotactic factor.28 More recent studies indicate that TNF-activated fibroblasts and alveolar macrophages also produce IL-8.2930 Tumor necrosis factor reportedly triggers the production of platelet-activating factor by several types of cells indigenous to the lung.17'18 Platelet-activating factor exhibits potent neutrophil chemotactic activity.31,32 In vitro studies using rat leukocytes indicate that TNF-triggered PAF production is rapid.17 Finally TNF, as well as several other
against recombinant human TNF (see Materials and Methods).
cytokines, induces the expression of ELAM-1 by HUVECs maintained in culture.912 Endothelial cell ELAM-1 expression triggered by TNF is protein synthesisdependent, peaking at 4 to 6 hours. Immunohistochemical staining of inflamed dermal blood vessels suggests that a similar time course may occur in vivo.33 Quantitative analysis of the relative contributions of these mechanisms to in vivo neutrophil recruitment has been hampered by the lack of specific reagents that react with homologous mediator substances in animals. The importance of these mechanisms in tissue injury and neutrophil recruitment may also vary according to anatomic site. For instance, intradermal inoculation of anti-TNF antibodies does not reduce vascular permeability associated with the reversed dermal Arthus reaction in rats (JS Warren; unpublished data). In contrast, TNF may be particularly important in lung injury because of the large reservoir of TNF-producing cells (alveolar macrophages) that are ac-
Table 3. Effect of Supernatants* from IgA Immune Complex-stimulated Alveolar Macrophages on TNF Secretion Immune Comple-x-tiggered AlveolarMacrophages Stimulus
Additivet
TNF (units/mI)t
by IgG P
value
A. IgG immune complexes (50 ,g antibody/ml) None 47 ± 8 B. IgG alone (50 ,ug/ml) None 7± 3
Contrasting Roles for Tumor Necrosis Factor in the Pathogeneses of IgA and IgG Immune Complex Lung Injury Jeffrey S. Warren, Peter A. Barton, and Michael L. Jones From the Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan
Recent studies suggest that development of acute gamma G immunoglobulin (IgG) immune complex lung injury is partially dependent on a tumor necrosis factor (TNF)-dependent mechanism of neutrophil (PMN) recruitment. The authors have sought to further define the role of intrapulmonary TNF in IgG alveolitis and to examine its role in IgA immune complex alveolitis, a neutrophil-independent model of acute lung injury. IgG immune complex lung injury resulted in a marked rise in intrapulmonary TNF activity accompanied by progressive pulmonary PMN accumulation. Intratracheal instillation of neutralizing concentrations of anti-TNF markedly reduced PMN influx measured at 4 hours but had no effect on PMN recruitment quantitated at 2 hours. IgA immune complex deposition resulted in acute lung injury accompanied by increased numbers of intrapulmonary mononuclear phagocytes but few neutrophils. Lung lavage fluids obtained from IgA immune complex-injured rats contained both neutrophil and monocyte chemotactic activities, albeit at twofold to fourfold lower concentrations than observed in IgG-mediated alveolitis. In contrast to IgG
complex-mediated alveolitis, lung lavagefluidsfrom IgA-injured rats contained no TNF activity. Intratracheal administration of anti-TNF antibodies had no effect on the development of IgA lung injury as assessed by morphology and measurements of vascular permeability. In vitro exposure of isolated alveolar macrophages to preformed IgG immune complexes resulted in dose-dependent TNF secretion, while exposure to IgA complexes resulted in very low levels of TNF secretion These data suggest that TNF-mediated pulmonary neutrophil recruitment (in IgG lung injury) is manifest chiefly in the late phase (approximately 4 hours) of developing alveolitis. The virtual absence of intrapulmonary TNF activity in evolving
IgA immune complex alveolitis may in part account for the limited PMN recruitment observed in this model. (Am J Pathol 1991, 138:581-590)
Full development of acute IgG immune complex alveolitis in the rat is dependent on neutrophils and an intact complement system.1 Identically induced gamma A immunoglobulin (IgA) immune complex lung injury also requires complement, but develops fully in the absence of neutrophils.23 In vivo and in vitro studies suggest that alveolar macrophages or recruited monocytes are the chief effector cells in IgA lung injury.45 Preformed IgG immune complexes activate isolated rat neutrophils and alveolar macrophages as measured by release of lysosomal enzymes (in the case of neutrophils) and production of superoxide anion (02-) and hydrogen peroxide (H202).46 In contrast, preformed IgA immune complexes trigger 02- and H202 release by macrophages but induce a negligible response by neutrophils.4 These observations help to distinguish these models of alveolitis, but given that complement is involved in both models, it is not clear why IgG alveolitis is attended by a massive influx of neutrophils while IgA alveolitis is not. Recent in vivo studies suggest that alveolar macrophage-derived tumor necrosis factor (TNF) participates in the pathogenesis of acute IgG immune complex alveolitis through a mechanism involving neutrophil recruitment.7 The mechanisms by which TNF mediates neutrophil recruitment have not been clearly defined in this model, but several processes may be operative. Tumor necrosis factor, as well as other inflammatory mediators such as leukotriene B4 and C5a, can rapidly (in minutes) trigger the increased expression of adhesionpromoting molecules (CD11/CD18; Mac-1/ LFA-1/p`150,95) on neutrophils.8 Several cytokines, including TNF, also induce the upregulation or de Supported by National Institutes of Health grant HL-40526. Accepted for publication October 16, 1990. Address reprint requests to Jeffrey S. Warren, MD, Department of Pathology, Box 0602, University of Michigan Medical School, 1301 Catherne St., Ann Arbor, MI 48109-0602.
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expression of leukocyte adhesion molecules on endothelial cells.9-13 It has been suggested that cytokine-activated endothelium directs and amplifies leukocyte recruitment into evolving inflammatory foci. Endothelial leukocyte adhesion molecule (ELAM)-1 binds neutrophils, is expressed on the surface of TNF-stimulated endothelial cells, and peaks 4 to 6 hours after cytokine exposure.912 Recent in vitro studies indicate that ELAM-1 may also play a role in monocyte-endothelial adhesion.14 This observation emphasizes that simple one-to-one relationships between given adhesion molecules and specific types of leukocytes do not exist. Furthermore antibodies directed against ELAM-1 do not completely block neutrophil or monocyte adhesion, suggesting that additional adhesive mechanisms are operative.14 There is in vitro evidence that other molecules may also be important in very early leukocyte-endothelial adhesion events (eg, GMP-140, iC3b, PAF). 1-18 These varied mechanisms of leukocyte-endothelial adhesion may all contribute to different degrees, depending on the time point examined. Although not as extensively studied, physiologic variables (eg, shear stress) and anatomic factors (site of vascular bed) also may influence leukocyte recruitment.19 The observation that TNF is required for full pulmonary polymorphonuclear neutrophil (PMN) recruitment in IgGinduced alveolitis led us to further define its role in this model and to examine its potential role in the pathogenesis of acute IgA immune complex alveolitis. IgAmediated immune complex alveolitis is induced in a manner analogous to IgG lung injury, but provides an interesting contrast because it is mediated by monocytes or macrophages.2' The present study provides data that suggest that TNF-mediated PMN influx is manifested in the later phase of developing acute IgG immune complex alveolitis. Although a variety of events are necessary for full neutrophil recruitment into an acute inflammatory focus, the virtual absence of intrapulmonary TNF elaboration in evolving IgA immune complex alveolitis may in part explain why there is limited PMN recruitment in this model.
novo
Materials and Methods
Antibodies, Antigens, and Preparation of Immune Complexes Rabbit IgG rich in antibody directed against bovine serum albumin (BSA) was used to induce lung injury and to form precipitating polyclonal IgG-BSA immune complexes. Antibody content and production of immune
complexes have been previously described in detail.1 IgG was purchased from Organon Teknika, West Chester, PA. Stock IgG anti-BSA and BSA (low endotoxin; ICN Biomedicals, Costa Mesa, CA) preparations contained less than 0.02 ng/ml and 0.012 ng/ml of endotoxin activity, respectively, as estimated using the Limulus amebocyte lysate (lAL) assay (E-toxate; Sigma Chemical Co., St. Louis, MO). Affinity-purified monoclonal IgA (MOPC 315) directed against dinitrophenol-conjugated BSA (DNP-BSA) was purchased from Sigma Chemical Co. Dinitrophenolconjugated BSA was prepared according to the method of Eisen.20 The resulting antigen preparation contained an average of 50 DNP groups per BSA molecule. Antigen-antibody equivalence for IgG and IgA immune complexes (wt/wt per unit volume) was determined by quantitative immunoprecipitation as previously detailed.4 IgG anti-TNF alpha (provided by Drs. Steven L. Kunkel and Daniel G. Remick, University of Michigan) was produced by immunization of rabbits with recombinant murine TNF, affinity purified using protein A sepharose, and characterized for specificity against rat TNF as previously described.7 The antibody preparation used in these experiments blocks rat TNF in a dose-dependent manner. In vitro, 35 jig of anti-TNF reduces rat TNF activity (125 units/ml) to less than 20 units/ml, whereas 50 ,ug of anti-TNF reduces TNF activity to less than 5 units/ ml.7 The quantity of anti-TNF (36 ,ug/rat) required to completely neutralize BAL fluid TNF activity (in vivo) was determined empirically.
Animal Models of Immune Complex Alveolitis Male Long-Evans pathogen-free rats (300 to 400 g; Charles River Breeding laboratories, Inc., Wilmington, MA) were used for all studies. Intraperitoneal injections of ketamine (2.5 to 5.0 mg/i 00 g body weight) and sodium pentobarbital (5 mg/i 00 g body weight) were given for sedation and anesthesia. IgG and IgA immune complex lung injuries were induced as previously described.1' 2 Antibody solution (250 ,ug IgG anti-BSA or 400 ,ug IgA anti-DNP-BSA) or mixtures of antibody and anti-TNF or nonspecific rabbit IgG, were instilled into the lungs through a tracheal cannula. In all cases, a final volume of 300 ,ul was instilled into the lungs. Antigen (BSA, 10 mg; or DNP-BSA, 3.3 mg) was injected intravenously. Rats were killed at the indicated times and lung injury quantitated, bronchoalveolar lavage (BAL) fluid harvested, or lung myeloperoxidase (MPO) activity determined. At time of death, anesthetized rats are exsanguinated through inferior vena caval section before removal of lungs, thus
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resulting in negligible contamination of lungs with blood. Pulmonary injury was quantitated by permeability measurements. Permeability indices were calculated by comparing the leakage of 1251-labeled bovine gamma globulin from the circulation into the lung to the 1251-labeled colloid remaining in 1 ml of blood as previously described.1 Intrapulmonary TNF neutralization was carried out as previously described.7
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Morphometric Analysis of Pulmonary Neutrophil Recruitment Lung samples (1 mm3) were excised from the peripheral aspect of whole lungs that had been fixed in 4% glutaraldehyde under constant pressure inflation (25 cm H20). Samples were washed in 0.1 mol/l (molar) cacodylate buffer (pH 7.3), embedded in 1 -,-thick plastic sections, and stained with toluidine blue. Use of 1-mm3 samples from the peripheral aspects of inflated lung results in sections that contain no large bronchial structures. ('Large bronchial structures' are defined as muscular airways lined by respiratory epithelium.) Plastic embedding (1 ,u thick) allows very high morphologic resolution, thus allowing virtually all alveolar and alveolar septal cells to be easily identified. Morphometric analysis of PMN recruitment was carried out by a pathologist (J. S. Warren) who was blinded to sample origin. For each condition, five lung samples were examined. In each sample, 45 to 60 randomly selected 40x microscopic fields (HPF; highpower field) were analyzed.
Whole Lung Myeloperoxidase Activity as Quantitative Measure of Neutrophil Recruitment To assess the use of tissue MPO activity as a measure of PMN influx, known concentrations of glycogen-elicited PMN were added to normal rat lungs, the tissue homogenized and extracted, and standard curves produced as previously described21 (see Figure 1 for standard curve). Whole lungs were homogenized with a Polytron homogenizer (Tekmar Co., Cincinnati, OH) (4 x 10 seconds at a setting of 4) using 6 ml of homogenization buffer on ice. The homogenization buffer (50 mmol/l [millimolar] phosphate, pH 6.0) contained 0.5% hexadecyltrimethyl ammonium bromide (Sigma) and 5 mmol/l ethylenediaminetetra-acetic acid (EDTA). Homogenized samples were then sonicated (3 x 10 seconds at a setting of 5) and centrifuged (3000g; 30 minutes) at 4°C. Myeloperoxidase activity in supernatants was assayed by measuring the change in A460 resulting from decomposition of H202 in
0
1 o6
1
07
1 o8
Neutrophils Added Figure 1. Relationship between number ofaddedglycogen-elicited neutrophils and tissue myeloperoxidase activity. Supernatants (25 Pd and 50 pl, respectively)from homogenized lungs were assayed as described in Materials and Methods. Change in A460 was monitored for at least 3 minutes to ensure lineari. 7he data are expressed as means ± standard deviations (n = 4 for each point except 0 added neutropbils, wbere n = 2).
the presence of o-dianisidine.22 Multiple aliquots of lung extract were tested to assure assay linearity.21
Collection of BAL Fluid and Isolation of Alveolar Cells Alveolar macrophages were recovered from normal rats using a previously described.6 Lung lavages obtained from normal rats contained greater than 94% alveolar macrophages. Lung lavage contents for TNF measurements and cell counts were collected using 5 ml of 37°C, serum-free RPMI 1640. At least 90% of the administered fluid was always recovered, centrifuged (400g; 7 minutes) to separate cells, and stored at - 200C before analysis. There was no systematic difference in BAL fluid recovery between control and experimental groups of rats.
TNF Assays Tumor necrosis factor activity was measured using a modification of the procedure of Ruff and Gifford.23 Briefly, equal volumes of log2 dilutions (final volume, 0.1 ml) of sample-containing media or serum were made in 96-well microtiter plates (Costar, Cambridge, MA) containing target LM cells (mouse fibroblast cell line derived from NCTC clone 929; American Type Culture Collection) (5 x 1 04 cells per 0.1 ml) in the presence of actinomycin D (final concentration, 1 ,ug/ml; Sigma). In a separate set of wells, a standard consisting of serially diluted recom-
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binant human TNF (Cetus Immune Laboratories, Palo Alto, CA) was added. The cells were incubated at 370C for 18 hours, the supernatants discarded, and the remaining viable adherent cells washed with PBS and then stained by adding crystal violet (0.2% in 2% ethanol) for 5 minutes. Microtiter plates were rinsed three times with PBS and allowed to air dry. The absorbance of each well was read at 620 nm with a MicroELISA Autoreader (BioTek Instruments, Winooski, VT). Units of TNF are defined as the reciprocal of the dilution at which 50% cytolysis occurs. Assay specificity was conferred by the ability of anti-TNF to neutralize sample TNF activity.
here to polystyrene culture dishes (2 x 106/35 mm dish), washed, and incubated for 4 hours in serum-free RPMI 1640 containing preformed immune complexes (IgA or IgG). Culture supernatants were then aspirated and assayed for TNF activity. Negative controls included equivalent concentrations of antibody alone (IgA or IgG), antigen alone (DNP-BSA or BSA), or medium alone. All values were normalized for adherent cell number. The proportions of alveolar macrophages that remained adherent after the wash ranged from 0.53 to 0.74. In no experiment was there more than 14% plate-to-plate variation.
Neutrophil and Monocyte Isolation
Results
Neutrophils were obtained from glycogen-induced rat peritoneal exudates 4 hours after the intraperitoneal injection of 1.0% glycogen in sterile saline. Peritoneal cells (more than 85% neutrophils) were harvested as described elsewhere.6 Human peripheral blood monocytes (80% to 85%) were isolated by centrifugation through Ficoll-Hypaque (Sigma) and Sepracell-MN (Sepratech, Oklahoma City, OK) as described by Vissers et al.24
Neutrophil and Monocyte Chemotaxis Assays Neutrophil and monocyte chemotaxis assays were carried out in 48-well micro chemotaxis chambers as descnbed by Falk et al.25 Cell suspensions (2.25 x 105 cells/well) were added to the top well of the chamber and permitted to migrate through 1 0-, polycarbonate membranes (3-,u porosity) toward the sample-bearing bottom chamber. After a 4-hour incubation (3TC, 5% CO2, humidified), membranes were removed, nonmigrating cells wiped off, and filters fixed for 10 minutes in absolute methanol. Filters were air dried and stained for 30 minutes in 2% toluidine blue. The number of cells migrating through the membrane were counted in three random, 1 0-mm grids at 400x, with the mean ± standard error of the mean (SEM) calculated for triplicate samples. Results are expressed as normalized values representing the percent of maximum f-Met-Leu-Phe (10-7 or 10-8 mol/l; as indicated) (Sigma) positive control, minus negative controls (buffer alone).
Time Course of Pulmonary PMN Recruitment in lgG Immune Complex-mediated Lung Injury Pulmonary PMN accumulation was quantitated at varying times after initiation of lung injury by measuring whole lung MPO activities. There was a progressive increase in pulmonary PMN accumulation over the course of developing alveolitis (Figure 2). The absolute number of PMNs recruited into the lungs of rats with developing IgG immune complex lung injury cannot be accurately determined based on these data. Examination of hematoxylin and eosin (H&E)-stained whole lung sections and Wright-stained smears of cells retrieved by lung lavage disclosed fewer than 1% lymphocytes and no eosinophils (data not shown). 0.5
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TIME (hours)
In Vitro TNF Secretion by Isolated Alveolar
Macrophages Rat alveolar macrophages suspended in RPMI 1640 with 10% heat-inactivated fetal calf serum were allowed to ad-
Figure 2. Pulmonary neutrophil recruitment in rats with IgG immune complex alveolitis (0) as a function of time. Negative control rats (0) received equivalent concentrations ofnonspecific rabbit IgG instead of anti-BSA. Neutrophil recruitment was quantitated by measuring whole lung MPO activities.2' The data represent cumulative means + SEM offour experiments. At leastsix animals were used for each data point, except for rats at time 0 and negative control rats, in which three ratspergroup were used.
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Local TNF Mediates IgG Immune Complex-induced Pulmonary PMN Recruitment There was an 18-fold increase in BAL fluid TNF levels after induction of IgG immune complex alveolitis (Figure 3). No detectable rise in serum TNF was observed at either 0, 1, 2, or 4 hours after induction of alveolitis (data not shown). Intrapulmonary TNF blocking experiments were carried out to compare the role of TNF in PMN influx measured 2 and 4 hours after initiation of lung injury. Neutralization of intrapulmonary TNF resulted in marked suppression of pulmonary PMN recruitment measured at 4 hours (Table 1). Intratracheal anti-TNF had no effect on PMN accumulation observed at 2 hours, despite neutralization of BAL TNF activity (Table 1). These data suggest that the mechanism(s) responsible for TNF-mediated PMN recruitment are manifested predominantly during the late phase (approximately 4 hours) of developing acute alveolitis.
Neutrophil and Monocyte Chemotactic Activities in IgG and IgA Immune Complex-mediated Acute Lung Injury Analysis of BAL fluid from rats with IgA immune complex lung injury showed neutrophil and monocyte chemotactic
activities, albeit at lower concentrations than in IgG immune complex-injured rat lungs (Figure 4). The identities of the chemotactic factors elaborated in IgA immune complex lung injury are unknown. It is also unclear whether they are the same as in IgG alveolitis. These data suggest that the dearth of neutrophils recruited into sites of alveolar IgA-DNP/BSA deposition cannot be accounted for by an absence of neutrophil chemotactic activity.
Role of TNF in IgA Immune Complex Lung Injury Intratracheal instillation of IgA anti-DNP-BSA followed by intravenous infusion of DNP-BSA resulted in acute lung injury (Table 2, Figure 5). As noted in previous studies, fewer than 10% of the retrieved BAL lavage inflammatory cells were neutrophils (Table 2). Fewer than 5% of the retrieved cells were lymphocytes and no eosinophils were observed (data not shown). Bronchoalveolar lavage fluids from rats with IgA immune complex-induced acute lung injury contained no detectable TNF activity. Intratracheal instillation of anti-TNF into rats receiving IgA DNPBSA resulted in no suppression of lung injury (Table 2), no difference in the cellular contents of retrieved BAL fluid (Table 2), and no change in morphology compared with positive controls (not shown). These observations suggest that IgA immune complex alveolitis is mediated by a TNF-independent pathway.
TNF Secretion by Alveolar Macrophages Stimulated with IgG and IgA Immune Complexes
IL
-
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s
m
II
I
0
1
3
2
4
TIME (hours) Figure 3. Elaboration of intrapulmonary (BAL fl uid) TNF in rats during the development of IgG immune complexr-mediated lung injury (0). Negative control rats (0) received int;ratracbeal nonspecific IgG instead of anti-BSA Rats were killed iat the indicated time points and lungs lavaged with serum-free RPi MI 1640 (5 ml). The data represent the cumulative means SEA, ments in which at least three animals were usedfcr each variable. The TNF values expressed represent total activity retrieved per rat. Tumor necrosis factor activity was standardized a nant human TNF (see Materials and Methods).
Previous studies suggest that resident alveolar macrophages are the chief source of intraalveolar TNF in IgG alveolitis.7 Incubation of alveolar macrophaaes with increasing concentrations of preformed IgG immune complexes resulted in dose-dependent release of TNF (Figure 6). Incubation of macrophages with IgG alone or BSA alone resulted in negligible TNF release, suggesting that intact immune complexes, not a contaminant of the antigen or antibody preparation (eg, endotoxin), is responsible for the cellular response. Incubation of alveolar macrophages with equivalent concentrations of IgA immune
complexes resulted in very little TNF release. Exposure of alveolar macrophages to IgA alone resulted in negligible
±
TNF release. Exposure of macrophages to relevant concentrations antigen alone (DNP-BSA) also resulted in
minimal TNF release. Addition of high concentrations
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r
Table 1. Role of Intrapulmonary TNF in IgG Immune Complex Pulmonary PMNRecruitment Neutrophil influx Lung MPO activity
Total BAL TNFt activity (units)
Intervention* 2 Hours Nonspecific IgG (36 ,ug) (positive control) Anti-TNF (36 p,g)
0.08 ± 0.04
39 ± 6
Noninjured (negative)ll 4 Hours Nonspecific IgG (36 [tg) (positive control) Anti-TNF (36 ,g) Noninjured (negative control)"
2± 2 0± 2 177 ± 21 4± 3 3± 3
PMN/40X HPF
0.10 ± 0.05J 0.01 ± 0.01§
17 ± 4-1 NSt 14 ± 5j 3 ± 1§
0.52 ± 0.06 1 P < 0.05t 0.18 ± 0.05 0.03 ± 0.01
78 ± 5-, P < 0.05t 19 ± 3 4±2
-
NSt
* All rats except noninjured (negative controls) received intratracheal IgG anti-BSA and intravenous BSA. Interventions (anti-TNF or equivalent concentrations of nonspecific IgG) were instituted of the same time that immune complex injury was initiated (time zero). Rats were killed after either 2 or 4 hours as indicated. The data represent the cumulative means of at least 6 rats/group, except for noninjured (negative controls) where 3 rats/group were used. 38 t Data were subjected to one-way analysis of variance. NS, no significant difference at P < 0.05. t TNF activity was standardized against recombinant human TNF (see Methods). § P < 0.05 versus positive control (nonspecific IgG); P < 0.05 versus anti-TNF-treated group. 11 Noninjured (negative control) rats received intravenous BSA (10 mg) and nonspecific intratrancheal IgG (instead of anti-BSA) at time zero.
(r400 ,ug/ml) of DNP-BSA resulted in release of TNF up to 14 units/ml (data not shown). Previous studies indicate that high concentrations of DNP-BSA are cytotoxic (unpublished observations). Incubation of alveolar macrophages with a fixed concentration of preformed IgG immune complexes showed that TNF secretion reaches a plateau by 4 hours (Figure 7). Only small amounts of TNF were released by macrophages exposed to IgG or BSA alone. Negligible TNF was secreted by IgA immune complexes, IgA alone, or DNP-BSA alone. These data suggest that alveolar macrophages release substantial amounts of TNF in response to IgG immune complexes but very little in response to IgA complexes.
Effect of Supernatants from IgA Immune Complex-stimulated Alveolar Macrophages on TNF Secretion by IgG Immune Complex-triggered Alveolar Macrophages One potential explanation for the lack of TNF secretion by IgA immune complex-triggered alveolar macrophages is that such cells release an inhibitory or suppressive factor. To address this possibility, alveolar macrophages were incubated with preformed IgG immune complexes in the presence of increasing proportions of culture supernatant obtained from macrophages that had been incubated with IgA complexes. As shown in Table 3, super-
B. Monocyte Chemotaxis
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Figure 4. A: Neutrophil chemotactic activity in lung lavage fluid obtained 4 hours after initiation of either IgG (0) or IgA (0) immune complex lung injuwy. Lavage fluidsfrom negative control rats (intratracheal antibody alone and intravenous antigen alone) contained less than 20 units of activity. B: Monocyte chemotactic activity in lavagefluidfrom either IgG (0) or IgA (0) lung injuwy. Negative controlfluids contained less than 10 units of activity. The data are normalized to fMLP (10 - 7 mol/lfor neutropb ils and 10 - 8 mol/lfor monocytes).
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Table 2. Role of TNF in IgA Immune Complex Lung Injury* Rat Treatments Lung Injury
BALTNF
BAL Cells Retrieved
(permeability Intratracheal
Intravenous
A. Saline (negative control) B. IgA (negative control) C. IgA + nonspecific IgG (36 Rg) D. IgA + Anti-TNF(36 p.g)
Saline
0.02 ± 0.02
0.03 ± 0.04
Saline
0.04 ± 0.01
0.08 ± 0.02
DNP-BSA
0.25 ± 0.02§
0.07 ± 0.05
E. IgG (250 p.g)
F. IgG (250 ,ug) + Anti-TNF
index)
(units)t
Total 6.6 ± 0.7 x 104 6.9 ± 0.6 x 104 15.1 ± 1.9
PMN (%) 6
AMt
8
90
6
88
(%) 93
x 1i4
DNP-BSA
0.26 ± 0.03§
BSA (10 mg)
0.52 ± 0.08
BSA (10 mg)
0.04 ± 0.03
193
0.23 ± 0.06t
6
5
90
± 27
17.3 ± 2.1 x 104 8.3 ± 0.6
71
21
± 5'
x 106 3.8 ± 0.5
77
19
(36 Rg)
1 06t
* All rats received intratracheal IgA (400 ,ug) and intravenous DNP-BSA (3.3 mg), except where indicated. Rats were killed at 4 hours. The data represent the cumulative totals of at least six rats/group except in group A where three rats were used. All data are expressed as means + SEM. t Total TNF activity in serum-free RPMI 1640 (5 ml) retrieved from rats 4 hours after induction of injury. TNF activity was standardized against recombinant human TNF (see Methods).
t AM = alveolar macrophages. § p < 0.05 versus negative controls A. and B. (one-way analysis of variance).38 P < 0.05 versus matched positive control E (for reduced permeability index, BAL-TNF activity, and number of BAL cells retrieved). (One-way analysis of variance).38
natants from IgA immune complex-treated cells exerted no suppressive effect in IgG-complex-induced TNF release. These data suggest that a soluble inhibitory substance is not responsible for the lack of TNF secretion by IgA-activated macrophages.
Discussion Recent studies suggest that intrapulmonary elaboration of TNF is a necessary step in the complete development of IgG immune complex-induced alveolitis.7 Neutralization of intra-alveolar TNF activity attenuates pulmonary neutrophil recruitment by approximately 60%. The objec-
tives of the present study were to further characterize the role of TNF in the pathogenesis of IgG immune complex alveolitis and to examine its role in a complementary model, IgA immune complex-mediated alveolitis. IgA immune complex-induced lung injury develops rapidly (4 hours), but is mediated by monocytes or macrophages rather than neutrophils. The data suggest that TNFmediated pulmonary neutrophil recruitment (in IgG lung injury) is manifest largely in the late phase of developing alveolitis. The virtual absence of intrapulmonary TNF ac-
E
IL
z
0
r
Uw Figure
5.
IgA
immune
complex lung
injury.
There is intra-
alveolar hemorrhage accompanied by increased numbers of alveolar macrophages. Few neutrophils are present. Toluidine blue stained 1-p.m plastic section; original magnification, x400.
10
20
30 gg Antibody
40
50
Figure 6. In vitro production of TNF by alveolar macrophages incubated with increasing concentrations of preformed IgG-BSA immune complexes (0), IgA-DNP/BSA complexes (I), and negative controls. Negative controls included equivalent concentrations of IgG alone (0), BSA alone (A), IgA alone (U), and DNPBSA alone (*). Antigen and antibody concentrations were selected such that immune complexes were formed at antigen:antihoY equivalence. As described previously,4 antigen:antibody equivalence (concentration:concentration) was 1:5 for IgG-BSA and 2:1 for IgA-DNP/BSA These data represent the cumulative means of three separate experiments where each variable was run in duplicate or triplicate. Tumor necrosis factor activity was standardized against recombinant human TNF (see Materials and Methods).
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. ^
Figure 7. In vitro production of TNF by im-
complex-stimulated alveolar macrophages as a function of time. Exactly as demune
lgG i.c. scribedfor Figure 6, adherent alveolar macro-
E
pbages were incubated with preformed immune complexesfor the indicated periods of time. IgG and IgA immune complexes (formed at antigen:antibody equivalence) contained 50 pg/ml antibody. Negative controls (not shown)
(0 C
U-
z
included equivalent concentrations of either
IgG along, BSA alone, IgA alone, or DNP/BSA alone. For all negative controls (at 0, 1, 2, 4, and 7 bours) TNF concentrations were less than IgA i.c. 11 ± 2 units/ml. These data represent the cumulative mean of two separate experiments in wbich each variable was run in tiplicate. Tumor necrosis factor activity was standardized
TIME (hours)
tivity in IgA-induced alveolitis may in part explain the limited neutrophil recruitment observed in this model. Several mechanisms may be operative in TNFmediated neutrophil recruitment. Exposure of isolated neutrophils to TNF results in a rapid (minutes) increase in adhesiveness.8 Exposure of human umbilical vein endothelial cells (HUVECs) to TNF results in interleukin-8 production-6,27 Interleukin-8, also known as neutrophil activating peptide-1 or neutrophil chemotactic factor, is a very potent and specific neutrophil chemotactic factor.28 More recent studies indicate that TNF-activated fibroblasts and alveolar macrophages also produce IL-8.2930 Tumor necrosis factor reportedly triggers the production of platelet-activating factor by several types of cells indigenous to the lung.17'18 Platelet-activating factor exhibits potent neutrophil chemotactic activity.31,32 In vitro studies using rat leukocytes indicate that TNF-triggered PAF production is rapid.17 Finally TNF, as well as several other
against recombinant human TNF (see Materials and Methods).
cytokines, induces the expression of ELAM-1 by HUVECs maintained in culture.912 Endothelial cell ELAM-1 expression triggered by TNF is protein synthesisdependent, peaking at 4 to 6 hours. Immunohistochemical staining of inflamed dermal blood vessels suggests that a similar time course may occur in vivo.33 Quantitative analysis of the relative contributions of these mechanisms to in vivo neutrophil recruitment has been hampered by the lack of specific reagents that react with homologous mediator substances in animals. The importance of these mechanisms in tissue injury and neutrophil recruitment may also vary according to anatomic site. For instance, intradermal inoculation of anti-TNF antibodies does not reduce vascular permeability associated with the reversed dermal Arthus reaction in rats (JS Warren; unpublished data). In contrast, TNF may be particularly important in lung injury because of the large reservoir of TNF-producing cells (alveolar macrophages) that are ac-
Table 3. Effect of Supernatants* from IgA Immune Complex-stimulated Alveolar Macrophages on TNF Secretion Immune Comple-x-tiggered AlveolarMacrophages Stimulus
Additivet
TNF (units/mI)t
by IgG P
value
A. IgG immune complexes (50 ,g antibody/ml) None 47 ± 8 B. IgG alone (50 ,ug/ml) None 7± 3
