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
). 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
). 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, FcγRIIa, 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 FcγRIIa (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also able to detect anti–IL-8:IL-8 complexes bound to neutrophils present in the alveolar spaces of these patients (our unpublished information).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, γ-chain–deficient mice lacking stimulatory FcγRs have substantially attenuated pulmonary inflammatory responses, suggesting that the activity of anti-KC:KC complexes is mediated by receptors for IgG (FcγRs). In support of this conclusion, we found that KO mice immunized and treated with KC had amounts of anti-KC:KC complexes in lavage fluids similar to those of WT mice, but virtually no tissue deposition when lung tissues were examined. This finding suggests that the complexes were formed in the lungs of KO mice but were not able to display activity because of lack of relevant receptors in these mice. The observation that phosphorylation of ERK, Akt, and p-38, essential components of FcγR signaling pathway, was significantly decreased in KO mice further strengthens this interpretation.
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
); 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
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
). However, a few reports suggest a more predominant role for FcγRs (39
), and that complement may play a secondary role in immune complex induced inflammation (40
). Indeed, the most recent studies indicate that C5a is only indirectly involved in mediating activity of immune complexes, acting by regulating the expression of FcγRs (40
). In our model, FcγRs are important for the development of lung inflammation, but a role for complement remains possible. Furthermore, KC receptors could also be involved in mediating the proinflammatory activity of anti-KC:KC complexes. Anti–IL-8:IL-8 complexes have the ability to bind to IL-8 receptors on human neutrophils, and both FcγRIIa and IL-8 receptors mediate chemotactic activity of the complexes, with FcγRIIa being a predominant receptor (16
). Other anti-cytokine:cytokine immune complexes can also interact with receptors specific for a cytokine present in the complex (41
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
), ALI/ARDS can occur after peritoneal sepsis. In fact, extrapulmonary ARDS is quite frequent and many cases are due to peritonitis (1
). 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 γ-chain–deficient mice. A substantial portion of IgG present in lung tissue co-localizes with KC in LPS-treated animals, indicating that the deposited immune complexes consist to a large extent of anti-KC:KC complexes. We believe that the presence of anti-KC autoantibody:KC immune complexes in BAL fluids and lung tissues of LPS mice is not a consequence of increased vascular permeability. Overall change in alveolar permeability is rather modest in these mice and occurs more than 8 hours after LPS treatment. On the other hand, deposition of anti-KC:KC complexes is already detectable at 8 hours after intraperitoneal LPS administration, and anti-KC:KC complexes are present in lavage fluid even though permeability still remains unchanged.
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 × 106 cells/ml and 31.1 ± 22.0 × 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., γ-chain–deficient mice lacking stimulatory FcγRs) were protected from LPS-induced lung inflammation. There was virtually no lung tissue deposition of anti-KC:KC complexes (which were, however, present in lavage fluid in quantities similar to those detected in WT/LPS mice). These findings indicate that anti-KC:KC complexes were formed in the lungs of KO mice but did not contribute to the inflammatory response in the lung because of lack of relevant receptors in these mice. The activity of anti-KC:KC complexes is mediated by receptors for IgG (FcγRs) what is evident from studies conducted on KC-treated mice and was confirmed using KO mice treated with intratracheal KC.
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 (FcγRs), and mice express two types of stimulatory FγRs, FcγRI and FcγRIII (27
). Therefore, to study the function of IgG receptors we used mice lacking functional expression of both FcγRI and FcγRIII (γ-chain–deficient mice). Our results demonstrate that anti-KC autoantibodies develop normally in KO mice, and anti-KC autoantibody:KC complexes are present in lavage fluid in quantities similar to those detected in WT mice. This is true for both mouse models characterized in the current study (i.e., LPS- and anti-KC:KC complex–induced lung inflammation). On the other hand, our data indicate that the inflammatory response in the lung, related to formation of anti-KC:KC immune complexes, is substantially downregulated in γ-chain–deficient mice (lacking stimulatory FcγRs). This is consistent with the fact that activity of immune complexes, such as anti-KC:KC complexes, is mediated by FcγRs (28
). Our in vitro
studies showing that anti–IL-8 autoantibody:IL-8 complexes display their pro-inflammatory activity by binding to FcγRs (specifically FcγRIIa) also support this concept (16
). In addition, γ-chain–deficient mice are protected from alveolar inflammation induced by initiating of reverse passive Arthus reaction in the lung (intravenous injection of ovalbumin followed by intratracheal administration of a rabbit antibody against chicken egg albumin to form anti-ovalbumin:ovalbumin complexes in lungs of these animals) (28
). Moreover, no lung tissue deposition of the anti-KC:KC complexes was detected in KO mice in either model (i.e., LPS- or anti-KC:KC complex–induced lung inflammation). These observations are in agreement with data derived from a parallel comparison of BALB/c mice with systemic autoimmunity induced by administration of mercury and γ-chain–deficient mice also treated with mercury (47
). Deposits containing IgG were not present in kidneys and spleens obtained from KO mice, indicating that FcγRs are required for the formation of tissue deposits of immune complexes in autoimmune disease (47
). Similar conclusions can be reached on the basis of our findings—that is, absence of anti-KC:KC immune complexes in lung tissues of KO mice.
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.