Gain-of-function Lyn induces anemia: appropriate Lyn activity is essential for normal erythropoiesis and Epo receptor signaling.

Lyn is involved in erythropoietin (Epo)-receptor signaling and erythroid homeostasis. Downstream pathways influenced following Lyn activation and their significance to erythropoiesis remain unclear. To address this, we assessed a gain-of-function Lyn mutation (Lyn(up/up)) on erythropoiesis and Epo receptor signaling. Adult Lyn(up/up) mice were anemic, with dysmorphic red cells (spherocyte-like, acanthocytes) in their circulation, indicative of hemolytic anemia and resembling the human disorder chorea acanthocytosis. Heterozygous Lyn(+/up) mice became increasingly anemic with age, indicating that the mutation was dominant. In an attempt to overcome this anemia, extramedullary erythropoiesis was activated. As the mice aged, the levels of different immature erythroid populations changed, indicating compensatory mechanisms to produce more erythrocytes were dynamic. Changes in Epo signaling were observed in Lyn(+/up) erythroid cell lines and primary CD71(+) Lyn(up/up) erythroblasts, including significant alterations to the phosphorylation of Lyn, the Epo receptor, Janus kinase 2, Signal Transducer and Action of Transcription-5, GRB2-associated-binding protein-2, Akt, and Forkhead box O3. As a consequence of altered Lyn signaling, Lyn(+/up) cells remained viable in the absence of Epo but displayed delayed Epo-induced differentiation. These data demonstrate that Lyn gene dosage and activity are critical for normal erythropoiesis; constitutively active Lyn alters Epo signaling, which in turn produces erythroid defects.

Autoimmune disease in Lyn-deficient mice is dependent on an inflammatory environment established by IL-6.

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.

CTLA4Ig alters the course of autoimmune disease development in Lyn-/- mice.

Lyn-deficient (Lyn(-/-)) mice develop an age-dependent autoimmune disease similar to systemic lupus erythematosus, characterized by the production of IgG anti-nuclear Ab. To determine the extent to which this autoimmune phenotype is driven by T cell costimulation, we generated Lyn(-/-) mice expressing a soluble form of the T cell inhibitory molecule, CTLA4 (CTLA4Ig). Surprisingly, although CTLA4Ig prevented myeloid hyperplasia, splenomegaly and IgG anti-nuclear Ab production in Lyn(-/-) mice, it did not inhibit immune complex deposition and tissue destruction in the kidney. In fact, regardless of CTLA4Ig expression, Lyn(-/-) serum contained elevated titers of IgA anti-nuclear Ab, although generally IgA deposition in the kidney was only revealed in the absence of self-reactive IgG. This demonstrated that activation of autoreactive B cell clones in Lyn(-/-) mice can still occur despite impaired costimulation. Indeed, CTLA4Ig did not alter perturbed Lyn(-/-) B cell development and behavior, and plasma cell frequencies were predominantly unaffected. These results suggest that when self-reactive B cell clones are unimpeded in acquiring T cell help, they secrete pathogenic IgG autoantibodies that trigger the fulminant autoimmunity normally observed in Lyn(-/-) mice. The absence of these IgG immune complexes reveals an IgA-mediated axis of autoimmunity that is not sufficient to cause splenomegaly or extramedullary myelopoiesis, but which mediates destructive glomerulonephritis. These findings have implications for the understanding of the basis of Ab-mediated autoimmune diseases and for their treatment with CTLA4Ig.

Perturbation of the CD4 T cell compartment and expansion of regulatory T cells in autoimmune-prone Lyn-deficient mice.

Regulatory T cells (Tregs) are a subset of T lymphocytes that are responsible for suppressing the function of other immune cells, and preventing potentially harmful autoimmune responses. Studies in autoimmune-prone mice and human autoimmune diseases have shown reduced Treg number or function as a causative factor for the apparent loss of tolerance that contributes to disease. We have found that Lyn-deficient mice, which develop high titers of autoantibodies with age, have a perturbed Treg compartment. Contrary to what has been observed in some strains of autoimmune-prone mice, aged Lyn-deficient mice have increased numbers of Tregs. This expansion occurs in the presence of elevated serum IL-2 and diminished TGF-beta. Despite expansion of the Treg compartment, Lyn-deficient mice succumb at approximately 1 year of age due to immune complex-mediated glomerulonephritis. We have shown that Lyn is not expressed in Tregs or indeed in any T cell subset, suggesting that the expansion and apparent functional deficiency in Tregs in Lyn-deficient mice is due to extrinsic factors rather than an intrinsic Treg defect. Indeed, using an in vivo colitis model, we have shown that Lyn-deficient Tregs can suppress inflammation. These results suggest that Tregs are expanding in Lyn-deficient mice in an effort to control the autoimmune disease but are simply overwhelmed by the disease process. This study highlights the role of the inflammatory setting in autoimmune disease and its consideration when contemplating the use of Tregs as an autoimmune therapy.

Sustained activation of Lyn tyrosine kinase in vivo leads to autoimmunity.

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 (Lyn(up/up) mice). Lyn(up/up) mice have reduced numbers of conventional B lymphocytes, down-regulated surface immunoglobulin M and costimulatory molecules, and elevated numbers of B1a B cells. Lyn(up/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 Cgamma2 in resting Lyn(up/up) B cells. Similarly, Lyn(up/up) B cells show a heightened calcium flux in response to BCR stimulation. Surprisingly, Lyn(up/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.

Dok-related protein negatively regulates T cell development via its RasGTPase-activating protein and Nck docking sites.

Downstream of kinase (Dok)-related protein (DokR, also known as p56(dok)/FRIP/Dok-R) is implicated in cytokine and immunoreceptor signaling in myeloid and T cells. Tyrosine phosphorylation induces DokR to bind the signal relay molecules, RasGTPase-activating protein (RasGAP) and Nck. Here, we have examined the function of DokR during hematopoietic development and the requirement for RasGAP and Nck binding sites in its biological function. Retroviral-mediated expression of DokR in bone marrow cells dramatically inhibited their capacity to form colonies in vitro in response to the cytokines macrophage colony-stimulating factor and stem cell factor, whereas responses to interleukin-3 and granulocyte macrophage colony-stimulating factor were only weakly affected. When introduced into lethally irradiated mice, hematopoietic cells expressing DokR showed a drastically reduced capacity to repopulate lymphoid tissues. Most notably, DokR dramatically reduced repopulation of the thymus, in part by reducing the number of T cell precursors seeding in the thymus, but equally, through inhibiting the transition of CD4(-)CD8(-) to CD4(+)CD8(+) T cells. Consequently, the number of mature peripheral T cells was markedly reduced. In contrast, a minimal effect on B cell and myeloid lineage development was observed. Importantly, functional RasGAP and Nck binding sites were found to be essential for the biological effects of DokR in vitro and in vivo.

Imbalanced gp130-dependent signaling in macrophages alters macrophage colony-stimulating factor responsiveness via regulation of c-fms expression.

The mechanisms by which interleukin-6 (IL-6) family cytokines, which utilize the common receptor signaling subunit gp130, influence monocyte/macrophage development remain unclear. Here we have utilized macrophages devoid of either gp130-dependent STAT1/3 (gp130(Delta STAT/Delta STAT)) or extracellular signal-regulated kinases 1 and 2 (ERK1/2) mitogen-activated protein (MAP) kinase (gp130(Y757F/Y757F)) activation to assess the individual contribution of each pathway to macrophage formation. While the inhibition by IL-6 of macrophage colony-stimulating factor (M-CSF)-induced colony formation observed in gp130(wt/wt) mice was abolished in gp130(Delta STAT/Delta STAT) mice, inhibition of macrophage colony formation was enhanced in gp130(Y757F/Y757F) mice. In gp130(Delta STAT/Delta STAT) bone marrow-derived macrophages (BMMs), both IL-6- and M-CSF-induced ERK1/2 tyrosine phosphorylation was enhanced. By contrast, tyrosine phosphorylation of ERK1/2 in response to M-CSF was reduced in gp130(Y757F/Y757F) BMMs, and the pattern of ERK1/2 activation in gp130 mutant BMMs correlated with their opposing responsiveness to M-CSF-induced proliferation. When compared to the level of expression in gp130(wt/wt) BMMs, c-fms expression was elevated in gp130(Delta STAT/Delta STAT) BMMs but reduced in gp130(Y757F/Y757F) BMMs. Finally, an ERK1/2 inhibitor suppressed M-CSF-induced BMM proliferation, and this result corresponded to a reduction in c-fms expression. Collectively, these results provide a functional and causal correlation between gp130-dependent ERK MAP kinase signaling and c-fms gene activation, a finding that provides a potential mechanism underlying the inhibition of M-CSF-dependent macrophage development by IL-6 family cytokines in mice.

Hematopoietic abnormalities in mice deficient in gp130-mediated STAT signaling.

OBJECTIVE: Studies on mice lacking the common receptor subunit gp130 reveal that activation of gp130-dependent signaling pathways is essential for normal fetal and adult hematopoiesis. However, the extent to which hematopoiesis is dependent upon activation of a particular gp130 signaling pathway, namely STAT1/3 or SHP2/MAPK, is unknown. This study examined the specific contribution of gp130-mediated STAT1/3 signaling to the regulation of hematopoiesis. MATERIALS AND METHODS: Hematopoiesis was examined at various developmental stages in mice homozygous for a targeted carboxy-terminal truncation mutation in gp130 (gp130(delta)/(delta)) that deletes all STAT1/3 binding sites, thereby abolishing gp130-mediated STAT1/3 activation. RESULTS: Adult gp130(delta)/(delta) mice have increased numbers of immature colony-forming unit spleen progenitor cells in the bone marrow and spleen, elevated numbers of committed myeloid progenitor cells in the spleen and peripheral blood, and leukocytosis. Increased progenitor cell production was observed in gp130(delta)/(delta) fetal livers from 14 days of gestation onward. In contrast, the circulating platelet count was reduced by 30% in gp130(delta)/(delta) mice, without any corresponding decrease in the number of bone marrow and splenic megakaryocytes. In liquid cultures, megakaryocytes from gp130(delta)/(delta) mice are smaller than those from wild-type mice and do not increase in size upon stimulation with interleukin-6 or interleukin-11. Administration of either interleukin-6 or interleukin-11 to gp130(delta)/(delta) mice failed to increase platelet numbers, despite an increase in the production of megakaryocytes. CONCLUSIONS: Collectively, these results reveal that gp130-mediated STAT1/3 activation is required to maintain the normal balance of hematopoietic progenitors during fetal and adult hematopoiesis. Furthermore, they suggest two distinct roles for gp130-mediated STAT1/3 activation in hematopoiesis, one restricting the production of immature hematopoietic progenitor cells and the other promoting the functional maturation of megakaryocytes to produce platelets.

Candida albicans can stimulate stromal cells resulting in enhanced granulopoiesis.

Previously, we have reported that although unperturbed granulocyte colony-stimulating factor (GCSF)-deficient (G-CSF-/-) mice are neutropenic, when challenged with Candida albicans, they develop a profound neutrophilia. In an attempt to understand the basis of Candida-induced neutrophilia in G-CSF-deficient mice, we have modified the Dexter bone marrow culture system to produce an in vitro model that mimics emergency granulopoiesis in vivo. In this model, stromal cultures are overlaid with bone marrow cells in the presence or absence of heat-inactivated (HI) Candida. Irrespective of the genotype of mice used as a source of bone marrow-derived stromal cells, stimulation of these cultures with HI Candida led to a significantly greater recovery of cells compared to unstimulated stromal cultures. In addition, there was a marked increase in the number of colony-forming units granulocyte-macrophage (CFU-GM), as well as in the percentage of granulocytes in the population of nonadherent cells recovered from HI Candida-stimulated cultures. The conditioned medium generated from stromal cultures derived from either wild-type or G-CSF-/- mice exposed to HI Candida, when applied to bone marrow cells in a soft agar clonogenic assay stimulated M-, GM-, and G- type colonies. Interleukin-3 (IL-3) and GM-CSF could not be detected in the conditioned medium from either HI Candida stimulated or unstimulated stromal cultures. However, IL-6 was detected in the conditioned media from both wild-type and G-CSF-/- stromal cultures. Addition of anti-IL-6 antibody significantly impaired granulopoiesis in unstimulated and HI Candida-stimulated, wild type, and G-CSF-/- stromal cultures. Conditioned medium generated from G-CSF/IL-6-deficient stromal cells had the capacity to stimulate bone marrow cells to form colonies comprised of granulocytes and macrophages in soft agar clonogenic assay. This study demonstrates that stromal cells can be stimulated with HI Candida and gives an insight into Candida mediated granulopoiesis.

Mice lacking both G-CSF and IL-6 are more susceptible to Candida albicans infection: critical role of neutrophils in defense against Candida albicans.

Neutrophils play an important role in the host's defense against infection with various pathogenic organisms. Granulocyte colony stimulating factor (G-CSF) is regarded as a major regulator of neutrophil production and function. Mice lacking G-CSF or its receptor are neutropenic. IL-6 is another cytokine that has been shown to promote neutrophil production and modulate the function of many types of immune cells. We have analyzed G-CSF/IL-6 double deficient (G-CSF(- / - )/IL-6(- / - )) mice to gain an insight into the possible contribution of IL-6 to the residual granulopoiesis in G-CSF-deficient (G-CSF(- / - )) mice. Furthermore, we have evaluated the ability of G-CSF(- / - )/IL-6(- / - ) mice to combat an experimental infection with Candida albicans. Our data shows that IL-6 plays a role in granulopoiesis during early post natal period but it is dispensable for steady-state granulopoiesis in adult mice. However, adult G-CSF(- / - )/IL-6(- / - ) mice are more susceptible to Candida infection than similarly infected G-CSF(- / - ) mice. Although, the candidacidal function of neutrophils of G-CSF(- / - )/IL-6(- / - ) mice is deficient, the ability to produce IFN-gamma and TNF-alpha in response to Candida infection is not compromised. Similarly, nitric oxide production by peritoneal macrophages from G-CSF(- / - )/IL-6(- / - ) mice in response to Candida is comparable to G-CSF(- / - ) mice.

Mice lacking three myeloid colony-stimulating factors (G-CSF, GM-CSF, and M-CSF) still produce macrophages and granulocytes and mount an inflammatory response in a sterile model of peritonitis.

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.

Lyn-deficient mice develop severe, persistent asthma: Lyn is a critical negative regulator of Th2 immunity.

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.

Perturbed myelo/erythropoiesis in Lyn-deficient mice is similar to that in mice lacking the inhibitory phosphatases SHP-1 and SHIP-1.

The Lyn tyrosine kinase plays essential inhibitory signaling roles within hematopoietic cells by recruiting inhibitory phosphatases such as SH2-domain containing phosphatase-1 (SHP-1), SHP-2, and SH2-domain containing 5'-inositol phosphatase (SHIP-1) to the plasma membrane in response to specific stimuli. Lyn-deficient mice display a collection of hematopoietic defects, including autoimmune disease as a result of autoantibody production, and perturbations in myelopoiesis that 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.