Published ahead of print on June 21, 2007, doi:10.1165/rcmb.2006-0395OC
© 2007 American Thoracic Society DOI: 10.1165/rcmb.2006-0395OC Anti-KC Autoantibody:KC Complexes Cause Severe Lung Inflammation in Mice via IgG Receptors1 Department of Biochemistry, University of Texas Health Center, Tyler, Texas; 2 Cardiovascular Research Group, Division of Clinical Sciences (North), Sheffield University, United Kingdom; 3 Section of Pulmonary and Critical Care Medicine, Harborview Medical Center, and 4 Medical Research Service, Seattle VA Medical Center, Seattle, Washington Correspondence and requests for reprints should be addressed to Anna K. Kurdowska, Ph.D., Department of Biochemistry, University of Texas Health Center, 11937 US Highway 271, Tyler, TX 75708-3154. E-mail: anna.kurdowska{at}uthct.edu.
We have shown previously that high concentrations of IL-8 associated with anti-IL-8 autoantibodies (anti–IL-8:IL-8 complexes) are present in lung fluids from patients with the acute respiratory distress syndrome (ARDS), and correlate both with the development and outcome of ARDS. We also detected deposition of these complexes in lung tissues from patients with ARDS but not in control tissues. Moreover, we determined that IgG receptors (Fc  Rs) mediate activity of anti–IL-8:IL-8 complexes. In the current study, we generated anti-KC (KC = chemokine (CXC motif) ligand 1 (CXCL1)) autoantibody:KC immune complexes (KC–functional IL-8) in lungs of mice to develop a mouse model of autoimmune complex–induced lung inflammation. Both wild-type (WT) and -chain–deficient mice that lack receptors for immune complexes (FcRs) were studied. First, the mice were immunized with KC to induce anti-KC autoantibodies. Then, KC was administered intratracheally to generate anti-KC:KC complexes in the lung. Presence of anti-KC:KC complexes was associated with development of severe pulmonary inflammation that was, however, dramatically suppressed in -chain–deficient mice. Second, because sepsis is considered the major risk factor for development of ARDS, we evaluated LPS-treated WT as well as -chain–deficient mice for the presence of anti-KC:KC complexes and pulmonary inflammatory responses. We detected complexes between anti-KC autoantibodies and KC in lung lavages and tissues of mice treated with LPS. Moreover, -chain–deficient mice that lack receptors for immune complexes were protected from LPS-induced pulmonary inflammation. Our results suggest that immune complexes containing autoantibodies contribute to development of lung inflammation in LPS-treated mice.
Key Words: chemokine • autoantibody • immune complex • endotoxin • lung
The acute respiratory distress syndrome (ARDS) affects 150,000 people each year in the United States, and the mortality of severe cases remains greater than 40% despite significant advances in treatment modalities (1, 2). In the lungs, there is an acute inflammatory response with a significant increase in both the total number of neutrophils and the proportion of neutrophils in the alveolar spaces (2). There is considerable evidence linking the number of neutrophils in the alveolar spaces to the severity of disease in most patients with ARDS (3–5). Several studies have demonstrated that high concentrations of interleukin-8 (IL-8, CXCL8), a major neutrophil activator, are present in lung fluids from patients with ARDS (6–12). However, we have found that there is not a consistent relationship between the concentration of IL-8 and either the development or the course of ARDS (13–15). These findings contrast with prior studies, in which IL-8 concentrations were reported to predict the onset and the outcome of ARDS (6, 8–10). Other groups also have not found a relationship between IL-8 and either progression to ARDS or survival once ARDS begins (7, 11, 12). Our studies also show that a portion of the total IL-8 in lung fluids from patients with ARDS is associated with anti–IL-8 autoantibodies (anti–IL-8:IL-8 complexes) (13–15), and that the presence of anti–IL-8:IL-8 complexes in bronchoalveolar (BAL) fluids of patients with ARDS is a prognostic indicator of both the development and the outcome of ARDS (14, 15). Moreover, complexes purified from the lung fluids of patients with acute lung injury (ALI) have the ability to attract and activate human blood neutrophils, and control neutrophil survival (16, 17). Importantly, IgG receptors (Fc RIIa) that interact with immune complexes mediate activity of the anti–IL-8:IL-8 complexes (16, 17). We also demonstrated that the instillation of the purified rabbit anti–IL-8:IL-8 complexes into the lungs of rabbits stimulates an increase in lung fluid concentrations of IL-8 and neutrophils, in contrast to the instillation of a control antibody (18). Finally, we have evaluated lung tissues from patients with lung injury for the presence of anti–IL-8:IL-8 complexes by confocal microscopy. IL-8 co-stained with IgG and immune complex receptors, FcRIIa, in lung tissues from patients with acute respiratory distress syndrome but not in control tissues, suggesting that anti–IL-8:IL-8 complexes are deposited in lungs of patients with ARDS via FcRIIa (19). We were also able to detect anti–IL-8:IL-8 complexes bound to neutrophils present in the alveolar spaces of these patients (our unpublished information).
These studies led us to hypothesize that anti–IL-8:IL-8 complexes may contribute to the severity of the alveolar inflammation in ARDS, and that IgG receptors mediate activity of the complexes in vivo. Therefore, we have developed a mouse model to evaluate the contribution of anti-KC autoantibody:KC complexes generated in situ to lung inflammation and injury (autoimmune complex–induced lung inflammation). Although mice do not express IL-8, murine chemokine (CXC motif) ligand 1 (CXCL1) (KC) is functionally related to human IL-8 (20). We also investigated whether LPS-induced lung inflammation generates anti-KC:KC complexes in mice. Finally, we examined inflammatory responses in lungs of
Animal Models of Lung Inflammation All studies involving animals were approved by the Institutional Animal Care and Use Committee of the UT Health Center and the Veterans Affairs Puget Sound Health Care Systems, the University of Washington, and conform to the NIH guidelines.
BALB/c mice (Taconic, Germantown, NY) were immunized with murine KC (Peprotech Inc., Rocky Hill, NJ) conjugated to an adjuvant, purified protein derivative of tuberculin (PPD; State Serum Institute, Copenhagen, Denmark), or endotoxin-free saline–PPD mixture essentially as previously described (21, 22). Briefly, KC (2 µg) or saline was injected intraperitoneally on Days 0, 7, and 14. Because intraperitoneal immunization induces mainly a systemic antibody response, we used an intranasal administration of KC to induce an intrapulmonary immune response (22). All mice were immunized both intraperitoneally and intranasally on Day 14 (at the time of the last injection). Intranasal immunizations were performed under halothane anesthesia, and the mice received 8 µg of KC in 40 µl of endotoxin-free saline or saline alone. Blood (< 50 µl) for test samples was obtained via tail nip at 1 week after the last injection (on Day 21), and the enzyme-linked immunosorbent assay (ELISA) (for detecting anti-KC autoantibodies) was performed as routinely done in our laboratory (see next paragraph) (13). Autoantibodies against KC were detected only in plasma of KC-immunized mice. Once the presence of autoantibodies was confirmed, KC (4 µg) was administered intratracheally to generate anti-KC:KC complexes in lungs (KC-immunized/KC group). Additional KC-immunized mice received saline instead of KC (KC-immunized/Saline group). Furthermore, mice immunized with saline received either KC (Saline-immunized/KC group) or saline (Saline-immunized/Saline group). In the additional series of experiments, Fourteen hours after intratracheal administration of saline or KC, mice were killed with Beuthanasia (5 µl/g, intraperitoneally; Schering-Plough Animal Health Corp., Kenilworth, NJ) and the lungs lavaged five times with 1 ml of sterile saline. One of the lobes was also fixed for histology, and a second one snap frozen in liquid nitrogen for measurement of myeloperoxidase (MPO).
Other WT BALB/c mice or In addition, lung tissue sections from mice treated with intratracheal LPS were evaluated for the presence of anti-KC autoantibody:KC complexes, and histologic changes indicative of lung injury. C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME) received a dose of LPS at 1.5 µl/g (1 mg/ml of E. coli 0111:B4; List Biologicals, Campbell, CA) under isoflurane anesthesia. Six hours after intratracheal instillation of LPS or saline (control mice), the animals were killed with an intraperitoneal injection of pentobarbital (120 µg/g) and exsanguinated by intracardiac puncture. Then, the lungs were removed and fixed with 4% paraformaldehyde at 15 cm H2O.
ELISA Assay for Detecting Anti-KC Autoantibodies
Cell Counts and Cytology in BAL Fluid
Measurement of Albumin Concentration and Total Protein Concentration
MPO Activity
Detection of Anti-KC:KC Complexes by ELISA
Determination of Cytokine Levels
Lung Tissue MPO
Confocal Microscopy To evaluate activation of signaling proteins, deparaffinized tissue sections (5 µm) were incubated with blocking buffer (PBS containing 10% normal porcine plasma and 0.5% BSA) overnight at 4°C. After blocking, tissue sections were washed with PBS and incubated with anti-phospho ERK, anti-phospho Akt (Ser 473), or anti-phospho p-38 antibody (Cell Signaling, Beverly, MA) followed by FITC-conjugated secondary antibody (DAKO). Both types of antibodies: primary and secondary were diluted in PBS containing 1% normal porcine serum and 0.5% BSA. Control stainings were performed using only secondary antibodies. After washing with PBS, coverslips were mounted on tissue sections with Permount (Fisher), and evaluated as described above. In some experiments neutrophils were stained with biotinylated rat anti-mouse Ly-6G (Gr-1; eBioscience) and Texas Red–conjugated streptavidin, and epithelial cells were stained with rabbit anti-human surfactant protein B (Chemicon, Temecula, CA) and anti-rabbit FITC antibodies (DAKO).
Pulmonary Histopathology and Histochemistry In addition, immunohistochemistry (IHC) for neutrophils was performed with anti-mouse Ly-6G mAb (BD/Pharmingen, San Jose, CA) in paraffin-embedded tissue. On the day of the experiment, slide-mounted tissue sections were deparaffinized, rinsed twice with PBS for 3 minutes, and rehydrated in a series of graded ethanol. Antigen retrieval was performed using 0.05% pronase, the tissue sections washed in PBS, and then blocked with normal goat serum (Jackson Immuno Research Labs, West Grove, PA). The tissue sections were then incubated overnight with rat anti-mouse Ly-6G/C mAb or isotype-matched control IgG. This was followed by biotinylated goat anti-rat IgG antibody for 2 hours at room temperature. Endogenous peroxidases were blocked with 3% H2O2 in H2O and then the tissue sections were rinsed twice with PBS and incubated with the Vector Laboratories "Elite" ABC-HP kit in a moist chamber for 90 minutes at room temperature. After two rinses with PBS, the sections were incubated with diaminobenzidine substrate (Sigma) for 12 minutes in the dark at room temperature. The slides were counterstained with 1% methyl green for 6 minutes. Digital pictures of the slides were acquired using Nikon DXM1200F camera attached to Nikon Eclipse 80 microscope.
Statistical Analysis
Murine Model of Autoimmune Complex Induced Lung Inflammation To examine the direct contribution of the anti–IL-8:IL-8 complexes to lung inflammation we generated analogous complexes in mice. Although mice do not express IL-8, KC is closely related in function to human IL-8 (20). Thus, female BALB/c mice were immunized with KC or saline. Mice immunized with KC developed plasma autoantibodies to KC, whereas no autoantibodies were found in the plasma of mice inoculated with saline (data not shown). Next, we administered KC intratracheally to form anti-KC:KC immune complexes in the lung (KC-immunized/KC mice). Additional KC-immunized animals received saline (KC-immunized/Saline mice). Importantly, mice assigned to these two groups had similar amounts of plasma anti-KC autoantibodies. The corresponding changes in absorbance expressed as median values with 25 to 75 percentile were: 0.21 (0.16–0.37) and 0.17 (0.14–0.56) for KC-immunized/Saline and KC-immunized/KC, respectively (P > 0.05). KC or saline was also instilled to saline-immunized mice that did not have circulating autoantibodies against KC (Saline-immunized/KC and Saline-immunized/Saline mice, respectively). Fourteen hours after the intratracheal instillation of saline or KC, the lungs were lavaged, and the concentrations of anti-KC:KC complexes were measured in BAL fluids. Anti-KC:KC complexes (at measurable levels [i.e., change in absorbance > 0.1]) were detected only in mice that were immunized with KC and treated with KC (KC-immunized/KC), but not in mice immunized with KC that received saline (KC-immunized/Saline) or mice immunized with saline and treated with intratracheal saline or KC (Saline-immunized/Saline and Saline-immunized/KC, respectively) (Table 1). Mice with anti-KC:KC immune complexes in lungs (KC-immunized/KC) had significantly more (P < 0.05) MPO (indicator of transalveolar neutrophil migration as well as neutrophil activation) in BAL fluid than other groups of mice (Table 1). Similarly, the histologic evaluation of lung tissues revealed significant changes only in KC-immunized/KC mice (P < 0.05, versus other groups of mice) (Table 1).
Moreover, anti-KC autoantibodies by themselves appear not to be inflammatory as evident by lack of neutrophil influx (MPO) and histologic changes in KC-immunized/Saline mice (Table 1). These mice developed plasma autoantibodies against KC due to immunization with KC. However, they did not differ from Saline-immunized/Saline mice in which autoantibodies were not induced when MPO concentrations and histologic indices were compared (Table 1). In addition, MPO level was significantly higher in mice that were immunized with saline and had KC instilled intratracheally (Saline-immunized/KC) than in mice immunized and treated with saline (Saline-immunized/Saline) or in saline-treated mice immunized with KC (KC-immunized/Saline) (P < 0.05) (Table 1). Even though BAL fluid concentration of MPO was increased in Saline-immunized/KC mice, we observed no histologic changes in lung tissues from these mice (Table 1). Our findings agree with previously published information on function of KC in the lung, which is primarily recruitment of neutrophils without causing damage to the lung (25, 26). One of the main objectives of the current study was to develop a model of anti-chemokine:chemokine immune complex–induced lung inflammation and evaluate pulmonary responses due to presence of these complexes. Therefore, in the subsequent parts of the article we focus on more detailed assessment of inflammatory responses in lungs of KC-immunized/KC mice, a group with anti-KC:KC complexes in BAL fluid. Moreover, these mice were compared with KO mice that lack receptors for immune complexes (see following section).
Mice Lacking Functional Expression of Stimulatory Fc One group of KO mice was immunized with saline only and had saline administered intratracheally (KO/Saline-immunized/Saline). These mice did not differ from their WT counterparts (Saline-immunized/Saline mice) (Table 1), indicating that baseline parameters are similar in KO and WT mice. A second group of KO mice was immunized with KC. Importantly, these mice developed similar amounts of circulating anti-KC autoantibodies to other groups of mice immunized with KC (KC-immunized/Saline and KC-immunized/KC, respectively). The corresponding changes in absorbance expressed as median values with 25 to 75 percentile were: 0.15 (0.13–0.18), 0.21 (0.16–0.37), and 0.17 (0.14–0.56) for KO, KC-immunized/Saline, and KC-immunized/KC mice, respectively (P > 0.05). After the presence of autoantibodies was confirmed, KO mice had KC administered intratracheally (KO/KC-immunized/KC mice). The levels of anti-KC:KC complexes in BAL fluid of these mice were similar to those of WT mice (KC-immunized/KC) (P > 0.05) (Table 1). However, the concentration of MPO in BAL fluid was decreased in KO mice (P < 0.05) (Table 1). There were also no appreciable histologic changes detected in KO/KC-immunized/KC mice (Table 1).
Furthermore, decreased numbers of neutrophils were present in the BAL fluid of these mice as compared with KC-immunized/KC mice (5.1 ± 2.8 x 106 cells/ml versus 9.8 ± 9.1 x 106 cells/ml) (Figure 1A). KC-immunized/KC mice, on the other hand, had more neutrophils than KC-immunized/Saline mice (9.8 ± 9.1 x 106 cells/ml versus 0.3 ± 0.5 x 106 cells/ml) (Figure 1A). The percentage of red blood cells was also significantly higher (P < 0.05) in the former group of mice compared with KC-immunized/Saline mice and KO/KC-immunized/KC mice (Figure 1B). Further, the total number of red blood cells in the BAL fluid was determined as described in MATERIALS AND METHODS. The KC-immunized/KC mice had substantially more red blood cells (indicator of alveolar hemorrhage) when compared with KC-immunized/Saline and KO/KC-immunized/KC mice (P < 0.05) (31.1 ± 22.0 x 106 cells/ml, 2.4 ± 2.9 x 106 cells/ml, and 10.2 ± 9.1 x 106 cells/ml, respectively). Similarly, the concentrations of albumin (indicator of pulmonary vascular and epithelial permeability) were significantly higher (P < 0.05) in KC-immunized/KC mice than KC-immunized/Saline mice and
To evaluate deposition of anti-KC:KC complexes in the lungs, tissue sections were incubated with FITC-conjugated anti-mouse IgG antibody. As shown in Figure 3A, IgG was easily detectable in specimen from KC-immunized/KC mice (green) indicating the presence of immune complexes associated with the lung tissue (28). The same tissue sections were also incubated with biotinylated antibody against mouse KC followed by Texas Red–conjugated streptavidin. There is substantial staining (red) in tissue from KC-immunized/KC mice (Figure 3A). Merging of "green" and "red" channels resulted in significant co-localization (yellow) in lung tissue from these mice (Figure 3A). These results suggest that a significant portion of IgG is associated with KC forming anti-KC:KC complexes in lung tissue from KC-immunized/KC mice. IgG (green) was not visible in tissue from KC-immunized/Saline mice or KO/KC-immunized/KC mice (Figure 3A). Staining for mouse KC (red) was also negligible, and, moreover, virtually no co-localization (yellow) was observed in these tissues (Figure 3A), indicating the absence of appreciable deposition of anti-KC:KC immune complexes in the lungs of KC-immunized/Saline, and KO/KC-immunized/KC mice (Figure 3A). In agreement with these observations correlation coefficients for co-localization between IgG and KC were 0.88, 0.06, and 0.03 for KC-immunized/KC, KC-immunized/Saline, and KO/KC-immunized/KC mice, respectively.
In addition, since our previous studies show that ERK, Akt, and p-38 pathways are evoked by anti–IL-8:IL-8 complexes in human neutrophils in vitro (16), we evaluated activation of these kinases in lung tissues of WT and KO mice. The phosphorylation of ERK, Akt, and p-38 was significantly increased in mice that had anti-KC:KC complexes in lungs (KC-immunized/KC) compared with KC-immunized/Saline and KO/KC-immunized/KC mice (Figure 3B), suggesting an important role for ERK, Akt, and p-38 pathways in lung inflammation triggered by anti-KC:KC complexes. Further, phosphorylated ERK, Akt, and p-38 were detected in inflammatory cells (neutrophils, epithelial cells, and macrophages) (data not shown). In summary, our data indicate that the inflammatory response in the lung related to formation of anti-KC:KC immune complexes is substantially down-regulated in -chain–deficient mice (lacking stimulatory FcRs).
LPS-Induced Lung Inflammation
Moreover, an increased number of red blood cells was present in the BAL fluid of WT/LPS mice compared with WT/Sal and KO/LPS mice (4.2 ± 3.3 x 106 cells/ml, 0.1 ± 0.1 x 106 cells/ml, and 1.2 ± 2.0 x 106 cells/ml, respectively). However, virtually no neutrophils were detected in the BAL fluid of any group of mice (Figure 4A). These results are consistent with lack of appreciable influx of neutrophils after administration of intraperitoneal LPS. On the other hand, the concentration of MPO in lung tissue was significantly higher (P < 0.05) in WT/LPS mice (Table 2). Our findings agree with previously published observations in mice treated with LPS via the intraperitoneal route (30–32). Virtually no neutrophils are found in BAL fluid; however, in lung tissue, neutrophils adhere to endothelium of small parenchymal vessels, suggesting that effect of intraperitoneally administered LPS is limited to intravascular sequestration of neutrophils (31, 32).
The concentration of MPO in lung tissue comprises both MPO residing in neutrophils and MPO released from the cells. The amount of lung tissue MPO is similar after LPS administration over time (32, and data not shown). However, the number of intact neutrophils in lung vasculature declines with time after LPS treatment, and maximum is reached between 0.5 and 2 hours (30). Accordingly, neutrophils were easily detectable in lungs of mice at 1 hour after injection of LPS (Figure 4B). Further, percentage of erythrocytes was significantly higher in WT/LPS mice than WT/Sal or KO/LPS mice after 14 h (P < 0.05) (Figure 4C). Normal mice injected with LPS (WT/LPS) had similar albumin concentrations in lavage as compared with WT/Sal and KO/LPS mice (122.8 ± 43.7 µg/ml, 105.8 ± 57.1 µg/ml, and 94.8 ± 28.0 µg/ml for WT/LPS, WT/Sal, and KO/LPS mice, respectively). Lack of a difference in albumin concentrations between WT/LPS and WT/Sal mice agrees with previously reported finding in mice treated with intraperitoneal LPS (31). However, albumin may sometimes not reflect adequately changes in permeability (33). Therefore, we measured total protein concentrations in lavage samples. We detected very modest increase in protein levels in LPS-treated mice (WT/LPS) as compared with WT/Sal and KO/LPS mice (165.9 ± 20.0 µg/ml, 145.2 ± 58.0 µg/ml, and 129.5 ± 42.2 µg/ml for WT/LPS, WT/Sal, and KO/LPS, respectively) (P < 0.05). Interestingly, there was no change in total protein concentration in BAL fluid from WT/LPS mice at 8 hours (data not shown). These findings indicate that there is only subtle increase in vascular permeability in WT/LPS mice.
In addition, the concentration of IL-1
Influx of inflammatory cells, increase in concentrations of cytokines, and elevated activity of MPO are all characteristics of lung inflammation. Appearance of erythrocytes and plasma proteins in the lung lavage fluid is indicative of lung injury, whereas results of direct histopathologic evaluation of lung tissues are used to determine whether damage to the lungs actually occurred (31, 33). We also examined deposition of anti-KC:KC complexes in lung tissues at 8 and 14 hours. Staining with anti-mouse IgG antibody (green) was detected only in WT mice treated with intraperitoneal LPS (WT/LPS) for 14 hours as depicted in Figure 6A, indicating presence of immune complexes associated with the lung tissue (28). The same tissue sections were also incubated with biotinylated antibody against mouse KC and Texas Red–labeled streptavidin (red) (Figure 6A). IgG co-localized with KC in lung tissues from normal mice treated with intraperitoneal LPS (WT/LPS) for 14 hours (yellow) (Figure 6A), indicating formation of complexes between of IgG and KC. These results, though unexpected, suggest that lung tissue deposition of immune complexes, such as anti-KC:KC complexes, occurs in WT/LPS mice (Figure 6A). IgG (green) was almost undetectable, as was staining for KC (red) in lung tissues from WT/Sal and KO/LPS mice (Figure 6A). Co-localization (yellow) between IgG and KC was also minimal in these tissues (Figure 6A), indicating that virtually no anti-KC:KC complex deposition was present in WT/Sal and KO/LPS mice. We calculated correlation coefficients to validate co-localization data which were 0.74, 0.02, and 0.04 for WT/LPS, WT/Sal, and KO/LPS mice, respectively. Similar results were obtained for mice treated with intraperitoneal LPS or saline for 8 hours (i.e., presence of anti-KC:KC complexes in lung tissues from animals which received LPS) (data not shown).
Intratracheal instillation of LPS induces alveolar influx of neutrophils and substantial change in permeability, and causes severe lung injury (30). We analyzed lung tissues from mice which were given LPS via intratracheal route for the presence of anti-KC:KC complexes, and we were able to detect deposition of these complexes at 6 hours after LPS treatment (Figure 6A); the correlation coefficient was 0.89. On the other hand, no complexes were present in lungs of mice that had saline administered intratracheally (data not shown). We also evaluated the tissues for histologic changes indicative of lung injury and confirmed presence of lung injury in these animals (histologic change values were 1.76 ± 0.10 for mice that received intratracheal LPS and 0.00 ± 0.00 for mice that received intratracheal saline). Finally, we evaluated activation of ERK, Akt, and p-38 in the lungs of WT/LPS, WT/Sal, and KO/LPS mice at 14 hours. We detected significantly increased phosphorylation of ERK, Akt, and p-38 in WT/LPS mice but not in the WT/Sal or KO/LPS mice (Figure 6B). Further, phosphorylated ERK, Akt, and p-38 were detected in inflammatory cells (neutrophils, epithelial cells, and macrophages) (data not shown).
Our results show that the
Although the pathophysiologic properties of autoantibody-containing immune complexes that relate to human disease (e.g., rheumatoid arthritis, diabetes, and thrombocytopenia) are well recognized (34), little is known about the possible involvement of anti–IL-8:IL-8 complexes in triggering and/or maintaining the inflammatory response in lung injury. Several key observations made by our laboratory support the likelihood that anti–IL-8:IL-8 complexes contribute to the initiation, potentiation, and severity of acute lung injury in humans. First, the presence of elevated concentrations of anti–IL-8:IL-8 complexes in lung fluids is associated with progression to ARDS (14, 15). Second, the absolute level of these complexes in the lungs is associated with mortality (14). In contrast, there is no consistent relationship between the concentration of IL-8 in BAL fluid and the course of the disease in ARDS (13–15). Moreover, we have evaluated lung tissues from patients with lung injury for the presence of anti–IL-8:IL-8 complexes by confocal microscopy. IL-8 co-stained with IgG and immune complex receptors, Fc RIIa, in lung tissues from patients with acute respiratory distress syndrome but not in control tissues, suggesting that anti–IL-8:IL-8 complexes are deposited in lungs of patients with ARDS via FcRIIa (19). We were also able to detect anti–IL-8:IL-8 complexes bound to neutrophils present in the alveolar spaces of these patients (our unpublished information). We have developed a mouse model to evaluate the contribution of anti-KC:KC complexes generated in situ to lung inflammation and injury (autoimmune complex–induced lung inflammation). In this model autoantibodies to KC in plasma and the alveolar compartment are first induced by immunization with KC. Once the animals develop anti-KC autoantibodies, KC is administered intratracheally to generate anti-KC:KC complexes in the lung. In these animals we found increased transalveolar influx of neutrophils, increased permeability, and alveolar hemorrhage, together with histologic evidence of increased infiltration of inflammatory cells, interstitial thickening, and presence of alveolar exudate. All of these findings indicate the presence of severe pulmonary inflammation and alveolar damage.
Moreover, The possibility that autologous immune complexes, like anti–IL-8:IL-8 complexes or anti-KC:KC complexes in mice, may be involved in the pathogenesis of lung inflammation/injury has not been considered before. It is known that the deposition of heterologous immune complexes (reverse passive Arthus reaction) can trigger a localized inflammatory response in different tissues, including the lung (28, 35–37); however, the models of immune complex–induced alveolitis differ substantially from our model. A foreign antigen is given intravenously, and immediately after that an antibody against this antigen (usually rabbit antibody) is administered intratracheally. This leads to local formation of heterologous immune complexes, which then trigger the alveolar inflammatory response. In our model mice are immunized with murine antigen (KC) for several weeks. After autoantibodies develop, the antigen (KC) is administered intratracheally, and autologous immune complexes (anti-KC:KC complexes) form in the lung. We believe that this model mimics very well the situation observed in patients with ARDS who have anti–IL-8 autoantibody:IL-8 complexes in their lung fluids as well as deposited in lung tissue (13–15, 19).
The lung inflammation induced by heterologous immune complexes is complement dependent, and specifically C5aR plays a crucial role in initiating of the alveolar inflammation (36–38). However, a few reports suggest a more predominant role for Fc
Even though certain animal models, such as those involving direct pulmonary insult or dual hit models, are thought to reflect more adequately pathophysiologic changes that are characteristic of full-blown ARDS (26, 31), ALI/ARDS can occur after peritoneal sepsis. In fact, extrapulmonary ARDS is quite frequent and many cases are due to peritonitis (1, 29). Most importantly, we chose the intraperitoneal route of LPS administration to generate a relatively straightforward model of mild lung injury. Increase in alveolar permeability is rather modest and occurs more than 8 hours after LPS treatment. We also found that immune complexes, including anti-KC:KC complexes, are present in lungs of mice treated with LPS, and may contribute to lung inflammation, because inflammatory responses to LPS are diminished in Our results indicate that changes in alveolar permeability occurring in WT/LPS mice are relatively mild. We detected very modest increase in protein levels in LPS-treated mice (WT/LPS) as compared with saline controls and KO mice (WT/Sal and KO/LPS, respectively). Even though percentages of erythrocytes are similar in WT/LPS mice and KC-immunized/KC mice (animals that were immunized with KC and had KC instilled intratracheally), the number of erythrocytes was much smaller in WT/LPS mice compared with KC-immunized/KC mice (4.2 ± 3.3 x 106 cells/ml and 31.1 ± 22.0 x 106 cells/ml, respectively). Importance of anti-KC:KC complexes in LPS-induced lung inflammation was supported by experiments with mice treated with intratracheal LPS. Intratracheal instillation of LPS induces alveolar influx of neutrophils and substantial change in permeability, and causes severe lung injury (30). We analyzed lung tissues from mice that were given LPS via intratracheal route for the presence of anti-KC:KC complexes, and we were able to detect deposition of anti-KC:KC complexes. Our findings support the hypothesis that anti-KC:KC complexes play a role in pathogenesis of lung inflammation and injury. In agreement with this concept, we showed deposition of anti–IL-8:IL-8 complexes in lung tissues from patients with ARDS but not control tissues (19). (IL-8 is functionally related to KC in mice that do not express IL-8 [20].)
Furthermore, KO mice (i.e., Our findings suggest for the first time that there is a possible link between LPS-induced lung inflammation and autoimmune responses. A recent study showing ability of LPS to induce a relapse of autoimmune encephalomyelitis in normal mice supports this hypothesis (42). Further, LPS is capable of inducing autoantibody production in mice (43). In agreement with this finding, we detected production of anti-KC autoantibodies by splenocytes from mice that received LPS, upon stimulation with LPS, but not by splenocytes from saline-treated mice (data not shown). It has been postulated that LPS could stimulate proliferation and differentiation of B cells by bridging the B cell receptor with Toll-like receptors (44). Further, antibody-forming cells are capable of migrating to the lung from the spleen (45). Taking into consideration the presence of cells producing anti-KC autoantibodies in the lungs of mice injected intraperitoneally with LPS, and also the release of KC in response to LPS treatment it is logical that anti-KC:KC complexes will be formed. Our studies demonstrate that anti-KC:KC complexes contribute to lung inflammation and ultimately may cause lung injury. Excessive activation of various signaling pathways by the complexes (e.g., Akt, ERK, p-38) will lead to release of unrestrained amounts of proinflammatory mediators, recruitment, and activation of abundant inflammatory cells, and tissue injury within the lung. The clinical relevance of these events is underscored by the observation that activation of specific signaling proteins, such as Akt, is related to survival in patients with ARDS (46).
Activity of immune complexes is mediated by receptors for IgG (Fc In summary, we showed that anti-chemokine:chemokine immune complexes containing autoantibodies can induce severe lung inflammation in mice. This finding supports our prior observations describing proinflammatory activity of anti–IL-8:IL-8 complexes purified from lung fluids obtained from patients with acute lung injury (16). Our model of chemokine–autoimmune complex–triggered lung inflammation is ideal for studying the function of such complexes in vivo, and can be used to test of new therapeutic interventions. The observation that anti-KC:KC complexes are deposited in lungs of LPS-treated mice, and may contribute to development of lung inflammation in these animals, is novel, and provides a mechanism to understand some of the pathologic features of acute lung injury.
The authors thank Dr. Timothy C. Allen (Department of Pathology) for his help in preparing digital images of lung tissue section stained for presence of neutrophils.
This work was supported in part by a grant HL073245 from the National Institutes of Health (A.K.K). Originally Published in Press as DOI: 10.1165/rcmb.2006-0395OC on June 21, 2007 Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Received in original form October 24, 2006 Accepted in final form May 23, 2007
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Abstract
CLINICAL RELEVANCE
(IL-1
autoantibodies induced by immunisation with homologous IL-1
