Antibody-induced neutrophil activation as a trigger for transfusion-related acute lung injury in an ex vivo rat lung model

Transfusion-related acute lung injury (TRALI) is a serious, life-threatening complication of hemotherapy. In the United States and United Kingdom, TRALI is considered the most common cause of transfusion-related fatalities.^1,2  It typically presents within 6 hours after transfusion as a clinical syndrome characterized by acute respiratory distress, hypoxemia, and a bilateral pulmonary edema on chest x-ray.³  Most investigators have postulated that TRALI is an antibody-mediated event because recipient-specific antileukocyte antibodies of donor origin are frequently associated with the syndrome.^4-7  Leukocyte antibodies of different specificity have been implicated, including, HLA-related and granulocyte-specific antibodies (eg, anti-HNA-2a). These findings have been corroborated with the clinical observation that plasma-rich products are most commonly implicated in TRALI. In the recent report from the British hemovigilance system, 17 of 36 suspected cases of TRALI received fresh frozen plasma (FFP) and 10 received platelets. Antileukocyte antibodies supportive for the diagnosis of TRALI were detected in two thirds of these cases.²  Despite the accumulation of data strongly suggesting an etiologic link between transfused antileukocyte antibodies and TRALI, little information has been gained regarding pathophysiology. In this study we demonstrate that antileukocyte antibodies can induce TRALI in an ex vivo lung model via neutrophil activation, a process that is independent from the presence of complement or leukoagglutinating properties of the antibody.

Antibodies recognizing CD177 (HNA-2a) have been implicated in TRALI.⁷  A monoclonal mouse anti-human IgG antibody (mAb) against CD177, 7D8, was used instead of human anti-HNA-2a in all experiments.⁸  Neutrophils were isolated from whole blood and the size of the HNA-2a⁺ subpopulation was determined by flow cytometry.⁹  Granulocytes were suspended in Krebs-Henseleit buffer (KHB) and added to the perfusion circuit of isolated rat lungs. The rat lung model has been described elsewhere.¹⁰  Briefly, isolated rat lungs were ventilated with a gas mixture of 5.3% CO₂, 21.0% O₂, and 73.7% N₂ and perfused via cannulas in the pulmonary artery and the left ventricle with KHB containing 5% hydroxy-ethyl starch. Pulmonary artery pressure (PAP) and lung weight gain were recorded continuously. Capillary filtration coefficient (K_f,c) and total vascular compliance (TVC) were repeatedly determined from sudden venous pressure elevation maneuvers of 10 cm H₂O for 8 minutes. After an isogravimetric steady-state period of 30 minutes, a first maneuver (t = 0) was performed. Subsequent repetitive maneuvers were performed as indicated (Figure 1). Neutrophils (10⁸) were added to the buffer following the first maneuver and 75 μg mAb 7D8 or control IgG following the second maneuver. In a second set of experiments, fMLP was added to the buffer fluid at time point 120 minutes. Histologic sections from all lungs were stained and examined by light microscopy. Priming activity of anti-HNA-2a and its inhibition by diphenylene iodonium was measured using the BurstTest kit from Becton Dickinson (Heidelberg, Germany) as described previously.^11,12 

Under baseline conditions, PAP, K_f,c data, and lung weight ranged within normal values. Application of neutrophils plus control IgG (Figure 1A-B, open circles) and the addition of neutrophils, IgG, and fMLP (Figure 1C-D, open circles) did not induce any biophysical changes, as did application of control IgG or anti-HNA-2a alone (not shown). In addition, isolated rat lungs responded normally to the application of angiotensin II at time point 200 minutes (data not shown), indicating that normal physiologic responses are maintained throughout the experiment.

In contrast, when neutrophils from individuals expressing 70% or more CD177 and anti-HNA-2a were added to the buffer fluid, a delayed, severe increase in lung vascular permeability was observed. A venous pressure elevation performed after 200 minutes was followed by a virtually 18-fold increase in K_f,c in all lungs (Figure 1A, left panel), accompanied by a dramatic increase in lung weight (Figure 1A, right panel). Vascular tone and TVC values remained unaltered (not shown), indicating that the increase in K_f,c must be due to an increase in conductivity of the lung microvasculature. Notably, this reaction was independent from the presence of complement because it occurred in a plasma-free environment.

Next, neutrophils from individuals expressing 30% or less CD177 were administered, followed by the addition of anti-HNA-2a. Under these conditions, we were unable to observe any significant changes of the lung weight at any time point. A minor increase of K_f,c was observed after 200 minutes, but seems negligible when compared to the K_f,c data obtained in other experiments (Figure 1B). Thus, changes in the lung microvasculature depend on the presence of both anti-HNA-2a antibodies and the number of neutrophils expressing the cognate antigen.

In vivo responses of neutrophils are known to be modulated in the presence of bioactive substances, such as fMLP. When anti-HNA-2a neutrophils from individuals expressing CD177 on 70% or more of their cells and fMLP were administered, the increase in lung vascular permeability was dramatically accelerated (Figure 1C). This effect of neutrophil costimulation was also present when neutrophils from individuals expressing CD177 on less than 30% of their cells were administered. Whereas only a minor increase in lung vasculature permeability was observed in the absence of fMLP when anti-HNA-2a was administered (Figure 1B), addition of fMLP led to a virtually 14-fold increase in K_f,c and an 8-fold increase in lung weight (Figure 1D). These data demonstrate that the response of neutrophils to antileukocyte antibodies can be potentiated when neutrophils are stimulated with bioactive substances such as fMLP, a situation that resembles clinical conditions of surgery or infection.¹³ 

The fact that in control experiments fMLP did not induce TRALI (Figure 1C-D) needs to be mentioned, because plastic tubing has been reported to prime neutrophils in certain clinical situations such as hemodialysis.¹³  Although we cannot generally exclude that such nonspecific priming due to contact with plastic tubing partially adds to effects seen in our ex vivo lung model, it seems unlikely that it adds substantially to effects seen herein.

Two major events discussed in the literature that may eventually lead to endothelial damage and, subsequently, induction of TRALI are complement activation and leukocyte agglutination.¹⁴  Anti-HNA-2a antibodies used in our study did not induce leukoagglutination in standard in vitro assays (data not shown). Furthermore, in the rat lung model, complement activation was not a prerequisite for TRALI induction. Our findings indicate that certain antileukocyte antibodies may induce a direct response of the neutrophil once they have bound. Such direct cellular responses have been elegantly demonstrated by Nardi et al,¹⁵  who gave evidence that antiplatelet IgG was able to cause complement-independent platelet fragmentation via the induction of reactive oxygen species (ROS). We conclude from our data that direct antibody-mediated cellular responses contribute to the pathogenesis of immune TRALI. This hypothesis is corroborated by the finding that fMLP was able to accelerate TRALI induction in our model. Furthermore, the mAb 7D8 as well as IgG fractions obtained from patients with anti-HNA-2a induced the production of ROS in CD177⁺ neutrophils in vitro (Figure 2A). Preincubation of neutrophils with the flavin antagonist diphenylene iodonium led to a 64% to 98% reduction in ROSs production (not shown), indicating that radicals may be produced by NAD(P)H-like oxidase systems of the neutrophil rather than by other oxygenases.¹² 

We suggest that binding of antileukocyte antibodies can activate neutrophils, a process that might lead to an oxidative response, cytotoxicity, and subsequent endothelial damage in the lung microvasculature. This process can be potentiated by neutrophil coactivation. Only recently, preliminary evidence has been presented that anti-HNA-3a might also be able to augment the oxidative response of neutrophils to a subsequent stimulus with fMLP,¹⁶  indicating that a direct effect of antileukocyte antibodies is not specific to anti-HNA-2a. Interestingly, it was also demonstrated that nonimmune TRALI can be induced in a rat lung model by the application of lipopolysaccharide (LPS) and plasma or lipids from stored blood,¹⁷  where lipids are thought to activate LPS-primed neutrophils. A common pathway for both immune and nonimmune TRALI will likely emerge in future.

Taken together, we have demonstrated that (1) antileukocyte antibodies can induce TRALI in the presence of the cognate antigen; (2) complement activation is not an absolute requirement for TRALI induction; (3) TRALI can be promoted by neutrophil coactivation; and (4) antileukocyte antibodies can activate neutrophils to produce ROS.

These results may focus our view on the activating capability of transfused antibodies as an important pathomechanism in TRALI and subsequent ROS production as a central process in endothelial damage and capillary leakage.