To investigate the role of the Lyn kinase in establishing signaling thresholds in hematopoietic cells, a gain-of-function mutation analogous to the Src Y527F-activating mutation was introduced into the Lyn gene. Intriguingly, although Lyn is widely expressed within the hematopoietic system, these mice displayed no propensity toward hematological malignancy. By contrast, analysis of aging cohorts of both loss- and gain-of-function Lyn mutant mice revealed that Lyn(-/-) mice develop splenomegaly, increased numbers of myeloid progenitors, and monocyte/macrophage (M phi) tumors. Biochemical analysis of cells from these mutants revealed that Lyn is essential in establishing ITIM-dependent inhibitory signaling and for activation of specific protein tyrosine phosphatases within myeloid cells. Loss of such inhibitory signaling may predispose mice lacking this putative protooncogene to tumorigenesis.

Genetic ablation of the Lyn tyrosine kinase has revealed unique inhibitory roles in B lymphocyte signaling. We now report the consequences of sustained activation of Lyn in vivo using a targeted gain-of-function mutation (Lynup/up mice). Lynup/up mice have reduced numbers of conventional B lymphocytes, down-regulated surface immunoglobulin M and costimulatory molecules, and elevated numbers of B1a B cells. Lynup/up B cells are characterized by the constitutive phosphorylation of negative regulators of B cell antigen receptor (BCR) signaling including CD22, SHP-1, and SHIP-1, and display attributes of lymphocytes rendered tolerant by constitutive engagement of the antigen receptor. However, exaggerated positive signaling is also apparent as evidenced by the constitutive phosphorylation of Syk and phospholipase Cγ2 in resting Lynup/up B cells. Similarly, Lynup/up B cells show a heightened calcium flux in response to BCR stimulation. Surprisingly, Lynup/up mice develop circulating autoreactive antibodies and lethal autoimmune glomerulonephritis, suggesting that enhanced positive signaling eventually overrides constitutive negative signaling. These studies highlight the difficulty in maintaining tolerance in the face of chronic stimulation and emphasize the pivotal role of Lyn in B cell signaling.

Lyn-deficient mice develop Ab-mediated autoimmune disease resembling systemic lupus erythematosus where hyperactive B cells are major contributors to pathology. In this study, we show that an inflammatory environment is established in Lyn−/− mice that perturbs several immune cell compartments and drives autoimmune disease. Lyn−/− leukocytes, notably B cells, are able to produce IL-6, which facilitates hyperactivation of B and T cells, enhanced myelopoiesis, splenomegaly, and, ultimately, generation of pathogenic autoreactive Abs. Lyn−/− dendritic cells show increased maturation, but this phenotype is independent of autoimmunity as it is reiterated in B cell-deficient Lyn−/− mice. Genetic deletion of IL-6 on a Lyn-deficient background does not alter B cell development, plasma cell accumulation, or dendritic cell hypermaturation, suggesting that these characteristics are intrinsic to the loss of Lyn. However, hyperactivation of B and T cell compartments, extramedullary hematopoiesis, expansion of the myeloid lineage and autoimmune disease are all ameliorated in Lyn−/−IL-6−/− mice. Importantly, our studies show that although Lyn−/− B cells may be autoreactive, it is the IL-6–dependent inflammatory environment they engender that dictates their disease-causing potential. These findings improve our understanding of the mode of action of anti–IL-6 and B cell-directed therapies in autoimmune and inflammatory disease treatment.

ultimately lead to splenomegaly and myeloid neoplasia. In this study, we demonstrate that loss of Lyn results in a stem/ progenitor cell-intrinsic defect leading to an age-dependent increase in myeloid, erythroid, and primitive hematopoietic progenitor numbers that is independent of autoimmune disease. Despite possessing increased numbers of erythroid progenitors, and a more robust expansion of these cells following phenylhydrazine challenge, Lyn-deficient mice are more severely affected by the chemotherapeutic drug 5-fluorouracil, revealing a greater proportion of cycling progenitors. We also show that mice lacking SHIP-1 have defects in the erythroid and myeloid compartments similar to those in mice lacking Lyn or SHP-1, suggesting an intimate relationship between Lyn, SHP-1, and SHIP-1 in regulating hematopoiesis. IntroductionThe production and lineage commitment of hematopoietic cells is governed by the actions of a multitude of cytokines, hormones, and growth factors that bind to cell surface receptors activating signal transduction cascades that ultimately regulate the expression of genes that control cell fate and effector function. 1 Signal propagation in these cells is actively counterbalanced by several families of inhibitory gene products including protein tyrosine phosphatases, 2 phosphatidyl-inositol phosphatases, 3 the suppressors of cytokine signaling (SOCS) proteins, 4 and receptors bearing immunoreceptor tyrosine-based inhibitory motifs (ITIMs). 5 The central role played by tyrosine phosphorylation is exemplified by mutations in particular genes that lead to deregulation of hematopoiesis. For example, mutational activation of either the Abl 6,7 or Janus tyrosine kinases [8][9][10] leads to leukemogenesis. Loss of appropriate negative regulation of signaling may also have catastrophic consequences. For example, loss-of-function mutations within the inhibitory phosphatase Src homology 2 (SH2)-containing phosphatase-1 (SHP-1) [11][12][13] in motheaten and motheaten viable mice (Me v ), or disruption of the murine SH2-domain containing 5Ј-inositol phosphatase (SHIP-1) 14,15 gene, lead to severe perturbations in hematopoiesis with myeloid cell consolidation of the lungs of deficient mice leading to premature death. [14][15][16] Thus, the appropriate balance of positive and negative elements of signal transduction is essential for maintaining normal hematopoietic cell self-renewal, differentiation, and immune cell function.Although clearly involved in initiating tyrosine-phosphorylation cascades following hematopoietic cell stimulation, 17 Lyn has emerged as a critical enzyme responsible for establishing signaling thresholds in B cells, 18-21 myelomonocytic cells, 22,23 and mast cells. [24][25][26][27][28] Indeed, loss of Lyn kinase leads to defects in activation of inhibitory phosphatases that likely underlies the hypersensitivity of deficient cells to immunoreceptor and cytokine stimulation. 20,22,29,30 In the case of B cells and mast cells, Lyn deficiency is associated with impaired activation of ...

The etiology of asthma, a chronic inflammatory disorder of the airways, remains obscure, although T cells appear to be central disease mediators. Lyn tyrosine kinase has been implicated as both a facilitator and inhibitor of signaling pathways that play a role in allergic inflammation, although its role in asthma is unclear because Lyn is not expressed in T cells. We show in the present study that Lyn−/− mice develop a severe, persistent inflammatory asthma-like syndrome with lung eosinophilia, mast cell hyperdegranulation, intensified bronchospasm, hyper IgE, and Th2-polarizing dendritic cells. Dendritic cells from Lyn−/− mice have a more immature phenotype, exhibit defective inhibitory signaling pathways, produce less IL-12, and can transfer disease when adoptively transferred into wild-type recipients. Our results show that Lyn regulates the intensity and duration of multiple asthmatic traits and indicate that Lyn is an important negative regulator of Th2 immune responses.

To assess the combined role of G-CSF, GM-CSF, and M-CSF in myeloid cell production, mice deficient in all three myeloid CSFs were generated (G−/−GM−/−M−/− mice). G−/−GM−/−M−/− mice share characteristics found in mice lacking individual cytokines: they are toothless and osteopetrotic and furthermore acquire alveolar proteinosis that is more severe than that found in either GM−/− or G−/−GM−/− mice. G−/−GM−/−M−/− mice have a significantly reduced lifespan, which is prolonged by antibiotic administration, suggesting compromised ability to control bacterial infection. G−/−GM−/−M−/− mice have circulating neutrophils and monocytes, albeit at significantly reduced numbers compared with wild-type mice, but surprisingly, have more circulating monocytes than M−/− mice and more circulating neutrophils than G−/−GM−/− mice. Due to severe osteopetrosis, G−/−GM−/−M−/− mice show diminished numbers of myeloid cells, myeloid progenitors, and B lymphocytes in the bone marrow, but have significantly enhanced compensatory splenic hemopoiesis. Although G−/−GM−/−M−/− mice have a profound deficiency of myeloid cells in the resting peritoneal cavity, the animals mount a moderate cellular response in a model of sterile peritonitis. These data establish that in the absence of G-CSF, GM-CSF, and M-CSF, additional growth factor(s) can stimulate myelopoiesis and acute inflammatory responses.

Mice lacking granulocyte colony-stimulating factor (G-CSF) are neutropenic with reduced hematopoietic progenitors in the bone marrow and spleen, whereas those lacking granulocyte-macrophage colony-stimulating factor (GM-CSF) have impaired pulmonary homeostasis and increased splenic hematopoietic progenitors, but unimpaired steady-state hematopoiesis. These contrasting phenotypes establish unique roles for these factors in vivo, but do not exclude the existence of additional redundant functions. To investigate this issue, we generated animals lacking both G-CSF and GM-CSF. In the process of characterizing the phenotype of these animals, we further analyzed G-CSF– and GM-CSF–deficient mice, expanding the recognized spectrum of defects in both. G-CSF–deficient animals have a marked predisposition to spontaneous infections, a reduced long-term survival, and a high incidence of reactive type AA amyloidosis. GM-CSF–deficient mice have a modest impairment of reproductive capacity, a propensity to develop lung and soft-tissue infections, and a similarly reduced survival as in G-CSF–deficient animals. The phenotype of mice lacking both G-CSF and GM-CSF was additive to the features of the constituent genotypes, with three novel additional features: a greater degree of neutropenia among newborn mice than in those lacking G-CSF alone, an increased neonatal mortality rate, and a dominant influence of the lack of G-CSF on splenic hematopoiesis resulting in significantly reduced numbers of splenic progenitors. In contrast to newborn animals, adult mice lacking both G-CSF and GM-CSF exhibited similar neutrophil levels as G-CSF–deficient animals. These findings demonstrate that the additional lack of GM-CSF in G-CSF–deficient animals further impairs steady-state granulopoiesis in vivo selectively during the early postnatal period, expand the recognized roles of both G-CSF and GM-CSF in vivo, and emphasize the utility of studying multiply deficient mouse strains in the investigation of functional redundancy.

Granulocyte colony-stimulating factor (G-CSF) is a glycoprotein believed to play an important role in regulating granulopoiesis both at steady state and during an “emergency” situation. Generation of G-CSF and G-CSF receptor–deficient mice by gene targeting has demonstrated unequivocally the importance of G-CSF in the regulation of baseline granulopoiesis. This study attempted to define the physiologic role of G-CSF during an emergency situation by challenging a cohort of wild-type and G-CSF–deficient mice with Candida albicans. Interestingly, after infection, G-CSF–deficient mice developed an absolute neutrophilia that was observed both in blood and bone marrow. In addition, 3 days after Candida infection increased numbers of granulocyte-macrophage (GM) and macrophage (M) progenitors were observed in the bone marrow of G-CSF–deficient mice. Of the cytokines surveyed, interleukin (IL)-6 levels in serum were elevated; interestingly, levels of IL-6 were higher and more sustained in G-CSF–deficient mice infected with C albicans than similarly infected wild-type mice. Despite the higher levels of serum IL-6, this cytokine is dispensable for the observed neutrophilia because candida-infected IL-6–deficient mice, or mice simultaneously deficient in G-CSF and IL-6, developed neutrophilia. Similarly, mice lacking both G-CSF and GM-CSF developed absolute neutrophilia and had elevated numbers of GM and M progenitors in the bone marrow; thus, G-CSF and GM-CSF are dispensable for promoting the emergency response to candidal infection.