Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion-associated mortality in the US. Previously, we established an immune-mediated TRALI mouse model, wherein mice with cognate antigen were challenged with MHC class I mAb. In this study, when mice housed in a rodent, specific pathogen-free barrier room were challenged with MHC I mAb, there was significant protection from TRALI compared with nonbarrier mice. Priming mice with LPS restored lung injury with mAb challenge. Using TLR4-deficient bone marrow chimeras, the priming phenotype was restricted to animals with WT hematopoietic cells, and depletion of either neutrophils or platelets was protective. Both neutrophils and platelets were sequestered in the lungs of mice with TRALI, and retention of platelets was neutrophil dependent. Interestingly, treatment with aspirin prevented lung injury and mortality, but blocking the P selectin or CD11b/CD18 pathways did not. These data suggest a 2-step mechanism of TRALI: priming of hematopoietic cells, followed by vascular deposition of activated neutrophils and platelets that then mediate the severe lung injury. Furthermore, our data offer an explanation for the increased incidence of TRALI in patients with immune priming conditions, and we suggest what we believe to be a novel therapeutic approach.

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At sites of inflammation, ligation of leukocyte integrins is critical for the activation of cellular effector functions required for host defense. However, the signaling pathways linking integrin ligation to cellular responses are poorly understood. Here we show that integrin signaling in neutrophils and macrophages requires adaptors containing immunoreceptor tyrosine-based activation motifs (ITAMs). Neutrophils and macrophages lacking two ITAM-containing adaptor proteins, DAP12 and FcRγ, were defective in integrin-mediated responses. Activation of the tyrosine kinase Syk by integrins required that DAP12 and FcRγ were first phosphorylated by Src family kinases. Retroviral transduction of neutrophils and macrophages with wild-type and mutant Syk or DAP12 demonstrated that the Src homology 2 domains of Syk and the ITAM of DAP12 were required for integrin signaling. Our data show that integrin signaling for the activation of cellular responses in neutrophils and macrophages proceeds by an immunoreceptor-like mechanism.Integrins are transmembrane adhesion receptors that coordinate cellular responses with the extracellular environment. Integrin function is especially important in neutrophils and macrophages, key effector cells that kill or suppress invading microorganisms during the innate immune response. In neutrophils and macrophages, integrin signaling is critical for cellular functions such as firm adhesion, cell spreading, chemotaxis, the production of COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests. Although several molecules required for relaying signals 'downstream' of leukocyte integrins (often called 'outside-in' signaling) have been identified, the initial steps of β 2 integrin signaling remain poorly understood. Src family kinases are involved in an early step of integrin signaling in neutrophils 6 and macrophages 7,8 . Also, the Syk tyrosine kinase is essential for integrin signaling in neutrophils 9 , macrophages 10 and platelets 11 . As Syk is probably involved in a receptor-proximal event during integrin signal transduction, the mechanism of activation of Syk by integrins and its relationship to Src family kinases may be the key to understanding the initiation of integrin signaling. Unfortunately, despite attempts to clarify that issue, the mechanism of activation of Syk by integrins remains poorly understood. HHS Public AccessSyk and the related kinase Zap70 are also essential for signaling downstream of immunoreceptors, such as B cell and T cell receptors and Fc receptors. In contrast to integrin signal transduction, the mechanism of Syk activation initiated by ligation of these immunoreceptors is well characterized. Engagement of immunoreceptors leads to Src family kinase-mediated phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) on receptor-associated transmembrane adaptor proteins 12 . Those adaptors provide docking sites for the tandem Src homology 2 (SH2) domains of the Syk or Zap70 tyrosine kinases, which leads...

Since the first example of conditional gene targeting in mice in 1994, the use of Cre recombinase and loxP flanked sequences has become an invaluable technique to generate tissue and temporal specific gene knockouts. The number of mouse strains expressing floxed-gene sequences, and tissue-specific or temporal-specific Cre-recombinase that have been reported in the literature has grown exponentially. However, increased use of this technology has highlighted several problems that can impact the interpretation of any phenotype observed in these mouse models. In particular, accurate knowledge of the specificity of Cre expression in each strain is critical in order to make conclusions about the role of specific cell types in the phenotypes observed. Cre-mediated deletion specificity and efficiency has been described in many different ways in the literature, making direct comparisons between these Cre strains impossible. Here we report crossing thirteen different myeloid-Cre mouse strains to ROSA-EYFP reporter mice and assaying YFP expression in a variety of naïve unstimulated hematopoietic cells, in parallel. By focusing on myeloid subsets, we directly compare the relative efficiency and specificity of myeloid deletion in these strains under steady-state conditions.

Autoimmunity is traditionally attributed to altered lymphoid cell selection and/or tolerance, whereas the contribution of innate immune cells is less well understood. Autoimmunity is also associated with increased levels of B cell–activating factor of the TNF family (BAFF; also known as B lymphocyte stimulator), a cytokine that promotes survival of self-reactive B cell clones. We describe an important role for myeloid cells in autoimmune disease progression. Using Lyn-deficient mice, we show that overproduction of BAFF by hyperactive myeloid cells contributes to inflammation and autoimmunity in part by acting directly on T cells to induce the release of IFN-γ. Genetic deletion of IFN-γ or reduction of BAFF activity, achieved by either reducing myeloid cell hyperproduction or by treating with an anti-BAFF monoclonal antibody, reduced disease development in lyn−/− mice. The increased production of IFN-γ in lyn−/− mice feeds back on the myeloid cells to further stimulate BAFF release. Expression of BAFF receptor on T cells was required for their full activation and IFN-γ release. Overall, our data suggest that the reciprocal production of BAFF and IFN-γ establishes an inflammatory loop between myeloid cells and T cells that exacerbates autoimmunity in this model. Our findings uncover an important pathological role of BAFF in autoimmune disorders.