Mutation of tyrosine 145 of lymphocyte cytosolic protein 2 protects mice from anaphylaxis and arthritis.

BACKGROUND: Lymphocyte cytosolic protein 2, also known as Src homology 2 domain-containing leukocyte phosphoprotein of 76 kilodaltons (SLP-76), is an essential adaptor molecule in myeloid cells, where it regulates FcepsilonRI-induced mast cell (MC) and FcgammaR- and integrin-induced neutrophil (polymorphonuclear leukocyte [PMN]) functions. SLP-76 contains 3 N-terminal tyrosines at residues 112, 128, and 145 that together are critical for its function. OBJECTIVE: We sought to explore the relative importance of tyrosines 112, 128, and 145 of SLP-76 during MC and PMN activation. METHODS: We examined in vitro MC and PMN functions using cells isolated from knock-in mice harboring phenylalanine substitution mutations at tyrosines 112 and 128 (Y112/128F) or 145 (Y145F). We also examined the effects of these mutations on in vivo MC and PMN activation using models of anaphylaxis, dermal inflammation, and serum-induced arthritis. RESULTS: Mutations at Y112/Y128 and Y145 both interfered with SLP-76 activity; however, Y145F had a greater effect than Y112/128F on most in vitro FcR-induced functions. In vitro functional defects were recapitulated in vivo, where mice expressing Y145F exhibited greater attenuation of MC-dependent passive systemic anaphylaxis and PMN-mediated inflammatory responses. Notably, the Y145F mutation completely protected mice against development of joint-specific inflammation in the MC and PMN-dependent K/B x N model of arthritis. CONCLUSION: Our data indicate that Y145 is the most critical tyrosine supporting SLP-76 function in myeloid cells. Future efforts to dissect how Y145 mediates SLP-76-dependent signaling in MCs and PMNs will increase our understanding of these lineages and provide insights into the treatment of allergy and inflammation.

Cutting edge: rescue of pre-TCR but not mature TCR signaling in mice expressing membrane-targeted SLP-76.

SLP-76 (Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa) organizes signaling from immunoreceptors, including the platelet collagen receptor, the pre-TCR, and the TCR, and is required for T cell development. In this study we examine a mouse in which wild-type SLP-76 is replaced with a mutant constitutively targeted to the cell membrane. Membrane-targeted SLP-76 (MTS) supports ITAM signaling in platelets and from the pre-TCR. Signaling from the mature TCR, however, is defective in MTS thymocytes, resulting in failed T cell differentiation. Defective thymic selection by MTS is not rescued by a SLP-76 mutant whose localization is restricted to the cytosol. Thus, fixed localization of SLP-76 reveals differential requirements for the subcellular localization of signaling complexes downstream of the pre-TCR vs mature TCR.

Extramedullary erythropoiesis in the adult liver requires BMP-4/Smad5-dependent signaling.

OBJECTIVE: In mice, homeostatic erythropoiesis occurs primarily in the bone marrow. However, in response to acute anemia, bone morphogenetic proteins 4 (BMP-4)-dependent stress erythropoiesis occurs in the adult spleen. BMP-4 can also regulate stress erythropoiesis in the fetal liver. In humans, erythropoiesis occurs in the bone marrow. However, in certain pathological conditions, extramedullary erythropoiesis is observed, where it can occur in several organs, including the liver. Given these observations, we propose to investigate whether the BMP-4-dependent stress erythropoiesis pathway can regulate extramedullary erythropoiesis in the livers of splenectomized mice. MATERIALS AND METHODS: Using splenectomized wild-type and flexed-tail (f) mice, which have a defect in BMP-4 signaling, we compared their recovery from phenylhydrazine-induced hemolytic anemia and characterized the expansion of stress burst-forming unit-erythroid in the livers of these mice during the recovery period. RESULTS: Our analysis indicates that in the absence of a spleen, stress erythropoiesis occurs in the murine liver. During the recovery, stress burst-forming unit-erythroid are expanded in the livers of splenectomized mice in response to BMP-4 expressed in the liver. f/f mice, which exhibit a defect in splenic stress erythropoiesis do not compensate for this defect by upregulating liver stress erythropoiesis. Furthermore, splenectomized f/f mice exhibit a defect in liver stress erythropoiesis, which demonstrates a role for the BMP-4-dependent stress erythropoiesis pathway in extramedullary erythropoiesis in the adult liver. CONCLUSIONS: Our data indicate that the BMP-4-dependent stress erythropoiesis pathway regulates extramedullary stress erythropoiesis, which occurs primarily in the murine spleen or in the case of splenectomized mice, in the adult liver.

An intronic sequence mutated in flexed-tail mice regulates splicing of Smad5.

Recent work has identified a growing body of evidence that subtle changes in noncoding sequences can result in significant pathology. These mutations, which would have been called silent polymorphisms in the past, affect gene transcription and mRNA splicing and lead to drastic changes in gene expression. Previous work from our lab has characterized the murine flexed-tail (f) mutation, which encodes Smad5, a transcription factor that functions downstream of the receptors for bone morphogenetic proteins (BMPs). f/f mice are unable to rapidly respond to acute anemia. Our analysis of these mice led to the development of a new model for stress erythropoiesis, where BMP4 expression in the spleen leads to the Smad5-dependent expansion of a specialized population of stress erythroid progenitors during the recovery from acute anemia. f/f mutant mice exhibit a defect in Smad5 mRNA splicing in the spleen such that the majority of Smad5 transcripts are two misspliced mRNAs. One of these mRNAs encodes a truncated form of Smad5 that inhibits BMP4 signaling when overexpressed. Here we show that a mutation in a poly(T) element in intron 4 causes the splicing defect in f/f mutant mice. This subtle mutation (loss of 1 or 2 Ts in a 16-T element) results in defects in splicing throughout the Smad5 gene. Furthermore, we show that this mutation results in tissue-specific splicing defects, which may explain why f/f mice are viable when Smad5-/- mice are embryonic lethal.

Loss of SLP-76 expression within myeloid cells confers resistance to neutrophil-mediated tissue damage while maintaining effective bacterial killing.

The Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) is an adaptor molecule critical for immunoreceptor and integrin signaling in multiple hemopoietic lineages. We showed previously that SLP-76 is required for neutrophil function in vitro, including integrin-induced adhesion and production of reactive oxygen intermediates, and to a lesser extent, FcgammaR-induced calcium flux and reactive oxygen intermediate production. It has been difficult to determine whether SLP-76 regulates neutrophil responses in vivo, because Slp-76(-/-) mice exhibit marked defects in thymocyte and vascular development, as well as platelet and mast cell function. To circumvent these issues, we generated mice with targeted loss of SLP-76 expression within myeloid cells. Neutrophils obtained from these animals failed to respond to integrin activation in vitro, similar to Slp-76(-/-) cells. Despite these abnormalities, SLP-76-deficient neutrophils migrated normally in vivo in response to Staphylococcus aureus infection and efficiently cleared micro-organisms. Interestingly, SLP-76-deficient neutrophils did not induce a robust inflammatory response in the localized Shwartzman reaction. Collectively, these data suggest that disruption of integrin signaling via loss of SLP-76 expression differentially impairs neutrophil functions in vivo, with preservation of migration and killing of S. aureus but reduction in LPS-induced tissue damage and vascular injury.

BMP4 and Madh5 regulate the erythroid response to acute anemia.

Acute anemia initiates a systemic response that results in the rapid mobilization and differentiation of erythroid progenitors in the adult spleen. The flexed-tail (f) mutant mice exhibit normal steady-state erythropoiesis but are unable to rapidly respond to acute erythropoietic stress. Here, we show that f/f mutant mice have a mutation in Madh5. Our analysis shows that BMP4/Madh5-dependent signaling, regulated by hypoxia, initiates the differentiation and expansion of erythroid progenitors in the spleen. These findings suggest a new model where stress erythroid progenitors, resident in the spleen, are poised to respond to changes in the microenvironment induced by acute anemia.