Although TRALI can be induced by at least two different mechanisms, both of which increase pulmonary vascular permeability, we have confined our study to a mouse model of alloantibody-induced TRALI. Our studies with this model confirm and extend a previously described observation (Looney et al., 2006
): a disorder very similar to human TRALI can be induced by injecting mice with an mAb against MHC class I, with rapid disruption of the alveolar capillary wall, fluid and protein leak into the lungs, a change in breathing mechanics, and a decrease in blood neutrophil count. However, although our experiments use the same mAb and mouse strain as other publications, they reveal a very different pathogenic mechanism than has been proposed. First, we find that almost complete depletion of neutrophils or platelets from BALB/c mice has no effect on the ability of 34-1-2s to induce mTRALI, conclusions which are consistent with our ultrastructural findings that neither cell type is observed in the vicinity of the alveolar capillary wall lesions. We agree with published reports that injection of mice with a rabbit antiplatelet pAb or a high dose of a rat mAb to Ly6C and Ly6G (anti–Gr-1) acutely prevents mTRALI (Looney et al., 2006
). However, injection of either Ab forms immune complexes with platelets or neutrophils that may inhibit mTRALI by consuming complement or binding to inhibitory or stimulatory FcγRs. We hypothesized that this process, rather than loss of platelets or neutrophils, was responsible for mTRALI inhibition. Indeed, formation of mAb complexes with erythrocytes and erythrocyte precursors had the same effect, even though it is unlikely that erythrocyte lineage cells participate directly in mTRALI pathogenesis. Furthermore, repeated injection of rabbit antiplatelet Ab, which maintains thrombocytopenia in the absence of further immune complex production (because of the time required to produce more platelets), does not inhibit mTRALI. Neither does platelet depletion by a nonimmune mechanism (injection of neuraminidase) inhibit mTRALI.
Testing the hypothesis that neutrophils are not required for mTRALI proved more difficult; the incomplete depletion of splenic neutrophils by anti–Gr-1 mAb did not allow this same strategy to be used to deplete blood neutrophils without maintaining immune complex production. Instead, we used a combination of low-dose anti–Gr-1 mAb and the alkylating agent hydroxyurea to fully deplete blood neutrophils and prevent their appearance in the lungs without generating sufficient immune complex to suppress TRALI.
In contrast to the lack of a requirement for either platelets or neutrophils in TRALI pathogenesis, development of this disorder required functional peripheral blood monocytes, as demonstrated by studies that prevented TRALI by these cells with clodronate-containing liposomes (Thepen et al., 1989
; Popovich et al., 1999
; Wang et al., 1999
; Jordan et al., 2003
; Murphy et al., 2004
; Nikolic et al., 2005
; Farley et al., 2006
; Zhao et al., 2006
; Bhatia et al., 2011
) or inactivating and depleting them with gadolinium (Singh and de la Concha-Bermejillo, 1998
; Strait et al., 2002
; Frid et al., 2006
; Thenappan et al., 2011
). The requirement for monocytes is consistent with an essential role in mTRALI for ROIs, which are produced by activated monocytes/macrophages.
Our experiments also differ from published results by questioning the importance of FcγRs in mTRALI pathogenesis. The previously reported conclusion that FcγRs are required for TRALI pathogenesis (Looney et al., 2006
) was based on the observation that mTRALI is not induced by 34-1-2s injection in FcγR-deficient mice, which lack all stimulatory FcRs. Studies with FcRγ-deficient mice have the advantage that this polypeptide is an essential part of all stimulatory FcRs in the mouse, so that FcRγ deficiency simultaneously prevents FcγRI, FcγRIII, FcγRIV, and FcεRI function. However, we found that mTRALI still occurs with only a minor decrease in severity in BALB/c FcRγ-deficient mice and with a greater decrease in severity in FcRγ-deficient mice that have a mixed BALB/c – C57BL/6 genetic background. More importantly, because FcRγ also associates with many other receptors and influences or is required for their signaling, the absence of mTRALI in FcRγ-deficient mice does not necessarily indicate FcγR involvement. The development of mTRALI to nearly the same degree in B10.D2 FcγRI/III double-deficient mice pretreated with a blocking anti-FcγRIV mAb as in untreated WT mice supports the conclusion that stimulatory FcγRs are not essential in mTRALI pathogenesis.
Our observation that mTRALI can be blocked by pretreating mice with 2.4G2, which activates and then blocks FcγRIIb, FcγRIII, and possibly FcγRIV (Unkeless, 1979
; Clynes et al., 1999
; Strait et al., 2002
; Hirano et al., 2007
), initially seemed to support a role for stimulatory FcγRs in mTRALI pathogenesis. However, our subsequent experiments revealed that 2.4G2 blocks mTRALI in WT and FcγRI/RIII-deficient mice but not in mice deficient in the inhibitory receptor, FcγRIIb. Thus, 2.4G2 inhibits mTRALI by giving a negative signal to cells via FcγRIIb rather than by blocking the stimulatory receptors FcγRI, FcγRIII, or FcγRIV. Inhibition of mTRALI by FcγRIIb activation was confirmed by demonstrating that this disorder could also be suppressed in WT but not FcγRIIb-deficient mice by treatment with an FcγRIIb-specific anti-Ly17.2 mAb.
In contrast to a previous study (Looney et al., 2006
), our observations establish a critical role for complement, particularly C5a, in this disorder. mTRALI fails, in our hands, to develop in mice deficient in C3, C5, or the C5aR, although Looney et al. (2006)
described normal disease development in C5aR-deficient mice. It is possible that differences in animal husbandry and/or bacterial flora account for our different results. Additionally, the use of general anesthesia for anti–H-2d
mAb–treated mice in the study by Looney et al. (2006)
, but not in our experiments, could influence lung physiology and possibly disease development. More importantly, the requirement for C5 in our experiments explains why 34-1-2s injection induces mTRALI in adult male but not female BALB/c mice, which have only ~25% as much plasma C5 as males. In addition, male mice have a form of C5 that is totally lacking in females (Baba et al., 1984
). This quantitative and qualitative gender dimorphism for C5 explains the male/female difference in mTRALI susceptibility, as shown by our experiments in which female mice became susceptible to mTRALI induction when first infused with plasma from WT or C3-deficient male mice but not when infused with plasma from WT females or C5-deficient males. These observations are consistent with the lack of a gender difference in human TRALI susceptibility, inasmuch as there are no known gender differences in human plasma C5 levels.
Although our disease pathogenesis experiments have been restricted to a mouse model of TRALI, the immune complex–related artifacts that we have uncovered are probably not specific to studies of this disorder. In fact, we have previously shown that IgG–platelet complexes caused secondary effects that mistakenly suggested that platelets are essential for IgG-mediated murine anaphylaxis (Strait et al., 2002
). We suspect that immune complex–mediated complications may confuse investigation of several disease models, as can the mistaken assumption that abnormalities in FcRγ-deficient mice necessarily reflect a requirement for IgG or IgE receptors (FcγRI, FcγRIII, FcγRIV, and/or FcεRI).
In addition to establishing critical roles for monocytes and C5a and confirming a role for ROIs in mTRALI pathogenesis, we show that MHC class I must be expressed on non-BM–derived cells (most likely pulmonary vascular endothelium) for mTRALI induction by 34-1-2s. This vascular endothelial location is particularly interesting in view of a report of TRALI induced solely in the transplanted lung of a posttransplant patient by donor plasma that contained Abs reactive with MHC antigens expressed by the lung donor but not by the recipient’s native lung (Dykes et al., 2000
). Collectively with our other observations, this suggests a relatively complex mechanism for mTRALI induction (diagramed in ): Abs binding to MHC class I on pulmonary vascular endothelial cells activate complement with the production of C5a, which is chemotactic for monocytes/macrophages and additionally activates them to release ROIs, which rapidly damage the endothelial cells. It is additionally possible that complement activation also directly damages endothelial cells (although not enough to induce TRALI in the absence of macrophages or ROIs), that the small size of pulmonary capillaries relative to systemic capillaries facilitates macrophage binding and activation, and that the relatively high oxygen tension in the lung makes pulmonary capillaries more susceptible than systemic capillaries to oxidative damage.
Our data provide further information about mTRALI pathogenesis by evaluating the relative ability of different IgG2a anti–H-2d
mAbs to induce mTRALI. Surprisingly, only 34-1-2s, by itself, induced mTRALI out of eight mAbs tested. Although 34-1-2s is the only member of our set of anti–H-2d
mAbs that binds to both H-2Dd
(Ozato and Sachs, 1981
; Ozato et al., 1982
; Noun et al., 1996
), it still induced mTRALI in mice that expressed only one of these MHC class I molecules (). Although 34-1-2s binds to MHC class I with higher affinity than some other members of our mAb panel, it was not the only mAb that bound with high affinity. These observations suggest that it is necessary for Ab to bind to MHC class I in sufficient quantity to activate enough complement with sufficient rapidity to attract and activate monocytes/macrophages. Consistent with this possibility, we found that mTRALI was induced when mice were injected with a combination of anti–H-2d
mAbs that, by themselves, did not induce mTRALI. We hypothesize that the combined binding of these mAbs to different H-2d
epitopes creates sufficient IgG density to meet the rapid complement-activating requirements for mTRALI induction. These observations would be consistent with the threshold model suggested by some (Bux and Sachs, 2007
; Vlaar et al., 2009
) and supported by the finding of a correlation between strength of donor anti-HLA Ab and induction of TRALI (Hashimoto et al., 2010
). These findings may also provide some explanation for why there is no simple relationship between anti-MHC Ab titers in human plasma and the likelihood of inducing TRALI when infused into a susceptible recipient. In addition, they suggest that the anti–MHC class I pAbs generated in multiparous women, which should bind to multiple epitopes on an MHC class I molecule, should be more effective than a single mAb at inducing TRALI.
Although mTRALI could be induced by the simultaneous injection of anti–H-2d mAbs that were individually incapable of inducing mTRALI, mTRALI induction by 34-1-2s could be prevented by first injecting mice with an anti–H-2d mAb that was incapable of inducing mTRALI (). Although we have not yet identified the mechanism responsible for mTRALI prevention by the initial injection of a nonpathogenic mAb (it could involve complement consumption, MHC class I antigen modulation, FcγRIIb activation, FcγRIII-dependent macrophage desensitization, an adaptive response by pulmonary vascular endothelium, or a combination of these), this phenomenon suggests approaches that might be used to reduce the risk of TRALI development in susceptible individuals.
Finally, it is necessary to point out two limitations of our study. First, clinical epidemiology and animal model studies suggest that TRALI can be induced by more than one mechanism, with the common feature being induction of a sufficient increase in pulmonary vascular permeability and fluid leak into the lung parenchyma to compromise breathing and gas exchange (Silliman et al., 1997
; Kopko et al., 2003
; Looney et al., 2006
; Gajic et al., 2007
; Chapman et al., 2009
; Fung and Silliman, 2009
). Epidemiologic features that favor an Ab-mediated pathogenesis include higher incidence of TRALI after transfusion of blood from multiparous female than male donors, by the considerably decreased incidence of TRALI in the USA and UK after reduction in the use of female donors for plasma (Chapman et al., 2009
; Eder et al., 2010
; Stafford-Smith et al., 2010
), and the high frequency of anti–MHC class I, anti–MHC class II, and antileukocyte Abs that react with recipient cells in TRALI-associated donor blood (Popovsky and Moore, 1985
; Kopko et al., 2003
; Kopko, 2004
; Toy et al., 2004
; Silliman et al., 2005
; Bux and Sachs, 2007
). Ab-mediated pathogenesis is also supported by individual case reports, including one in which different units of blood from the same multiparous donor caused TRALI in multiple recipients (Kopko et al., 2002
). In contrast, other studies have shown a relationship between the period of blood storage and the likelihood of TRALI development, with increased storage causing the production of lipid mediators that can increase pulmonary vascular permeability (Silliman et al., 1998
). This model is compatible with the development of TRALI in the absence of detectable alloantibodies in transfused blood and the development of TRALI after transfusion of an individual’s own stored blood. In our view, both the Ab- and the vasoactive mediator–dependent mechanisms are likely to contribute to human disease, and disease may be most likely to develop when both etiologies are present.
Second, although we restricted most of our experiments to mice that initially had no lung or vascular abnormalities, TRALI is most likely to develop in individuals who have preexisting inflammatory conditions. These conditions can activate macrophages, making them more likely to secrete large amounts of ROIs and cause production of vasoactive mediators and cytokines that may act synergistically with ROIs (Gilliss and Looney, 2011
). These conditions may also promote direct Ab-induced, complement-mediated endothelial cell damage that can rapidly increase vascular leak to levels that result in noncardiogenic pulmonary edema (Gilliss and Looney, 2011
). Consistent with this, inoculation of mice with LPS has been shown to considerably increase sensitivity to induction of mTRALI by anti–H-2d
and other mAbs that react with leukocytes (; Khan et al., 2006
; Looney et al., 2009
). Interestingly and relevant to our model, sepsis and associated LPS have also been shown to up-regulate the expression of C5aRs in lung cells (Riedemann et al., 2002
), macrophages, and endothelial cells (Hunt et al., 2005
). Also consistent with this observation are reports that mice kept in an especially clean environment lack mTRALI susceptibility, which is restored by treatment with LPS or housing in an environment where they are exposed to a broad bacterial spectrum (Lögdberg et al., 2009
; Looney et al., 2009
). We agree with suggestions already in the literature that human TRALI is most likely to occur when the effects of preexisting inflammation are exacerbated by infusion of vascular endothelial cell– and/or leukocyte-reactive Abs and vasoactive mediators to simultaneously cause multiple insults to vascular integrity (Shaz et al., 2011