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Recent evidence suggests that protease release by neutrophils in the bone marrow may contribute to hematopoietic progenitor cell (HPC) mobilization. Matrix metalloproteinase-9 (MMP-9),neutrophil elastase (NE),and cathepsin G (CG) accumulate in the bone marrow during granulocyte colony-stimulating factor (G-CSF) treatment,where they are thought to degrade key substrates including vascular cell adhesion molecule-1 (VCAM-1) and CXCL12. To test this hypothesis,HPC mobilization was characterized in transgenic mice deficient in one or more hematopoietic proteases. Surprisingly,HPC mobilization by G-CSF was normal in MMP-9-deficient mice,NE x CG-deficient mice,or mice lacking dipeptidyl peptidase I,an enzyme required for the functional activation of many hematopoietic serine proteases. Moreover,combined inhibition of neutrophil serine proteases and metalloproteinases had no significant effect on HPC mobilization. VCAM-1 expression on bone marrow stromal cells decreased during G-CSF treatment of wild-type mice but not NE x CG-deficient mice,indicating that VCAM-1 cleavage is not required for efficient HPC mobilization. G-CSF induced a significant decrease in CXCL12 alpha protein expression in the bone marrow of Ne x CG-deficient mice,indicating that these proteases are not required to down-regulate CXCL12 expression. Collectively,these data suggest a complex model in which both protease-dependent and -independent pathways may contribute to HPC mobilization. View Publication

We have generated mice null for IFN-beta and report the diverse consequences of IFN-beta for both the innate and adaptive arms of immunity. Despite no abnormalities in the proportional balance of CD4 and CD8 T cell populations in the peripheral blood,thymus,and spleen of IFN-beta-/- mice,activated lymph node and splenic T lymphocytes exhibit enhanced T cell proliferation and decreased tumor necrosis factor alpha production,relative to IFN-beta+/+ mice. Notably,constitutive and induced expression of tumor necrosis factor alpha is reduced in the spleen and bone marrow (BM) macrophages,respectively,of IFN-beta-/- mice. We also observe an altered splenic architecture in IFN-beta-/- mice and a reduction in resident macrophages. We identify a potential defect in B cell maturation in IFN-beta-/- mice,associated with a decrease in B220+ve/high/CD43-ve BM-derived cells and a reduction in BP-1,IgM,and CD23 expression. Circulating IgM-,Mac-1-,and Gr-1-positive cells are also substantially decreased in IFN-beta-/- mice. The decrease in the numbers of circulating macrophages and granulocytes likely reflects defective maturation of primitive BM hematopoiesis in mice,shown by the reduction of colony-forming units,granulocyte-macrophage. We proceeded to evaluate the in vivo growth of malignant cells in the IFN-beta-/- background and give evidence that Lewis lung carcinoma-specific tumor growth is more aggressive in IFN-beta-/- mice. Taken altogether,our data suggest that,in addition to the direct growth-inhibitory effects on tumor cells,IFN-beta is required during different stages of maturation in the development of the immune system. View Publication

Clinical observations suggest that in chronic myelogenous leukemia (CML),the Philadelphia chromosome (Ph+) clone has a growth advantage over normal hematopoiesis. Patients with CML have high levels of neutrophil elastase,which has recently been shown to antagonize the action of granulocyte-colony-stimulating factor (G-CSF) and other growth factors. We therefore compared the effect of elastase on the growth of normal and CML progenitor cells. In 10-day suspension cultures of normal or CML CD34+ cells supplemented with G-CSF,stem cell factor (SCF),and granulocyte macrophage-colony-stimulating factor (GM-CSF),CML cells had diminished sensitivity to the growth inhibitory effect of elastase. When equal numbers of CML and normal CD34+ cells were cocultured for 10 days,there was no change in the relative proportions of normal and leukemic cells (measured by fluorescence in situ hybridization [FISH] or flow cytometry). However,when elastase was added,CML cells predominated at the end of the culture period (78% vs 22% with 1 microg/mL and 80% vs 20% with 5 microg/mL elastase). CML neutrophils substituted effectively for elastase in suppressing the proliferation of normal CD34+ cells,but this effect was abrogated by serine protease inhibitors. These results suggest that elastase overproduction by the leukemic clone can change the growth environment by digesting growth factors,thereby giving advantage to Ph+ hematopoiesis. View Publication

Oncogenic Kit mutations are found in somatic gastrointestinal (GI) stromal tumors (GISTs) and mastocytosis. A mouse model for the study of constitutive activation of Kit in oncogenesis has been produced by a knock-in strategy introducing a Kit exon 11-activating mutation into the mouse genome based on a mutation found in a case of human familial GIST syndrome. Heterozygous mutant KitV558Delta/+ mice develop symptoms of disease and eventually die from pathology in the GI tract. Patchy hyperplasia of Kit-positive cells is evident within the myenteric plexus of the entire GI tract. Neoplastic lesions indistinguishable from human GISTs were observed in the cecum of the mutant mice with high penetrance. In addition,mast cell numbers in the dorsal skin were increased. Therefore KitV558Delta/+ mice reproduce human familial GISTs,and they may be used as a model for the study of the role and mechanisms of Kit in neoplasia. Importantly,these results demonstrate that constitutive Kit signaling is critical and sufficient for induction of GIST and hyperplasia of interstitial cells of Cajal. View Publication

There is evidence that neutrophil production is a balance between the proliferative action of granulocyte-colony-stimulating factor (G-CSF) and a negative feedback from mature neutrophils (the chalone). Two neutrophil serine proteases have been implicated in granulopoietic regulation: pro-proteinase 3 inhibits granulocyte macrophage-colony-forming unit (CFU-GM) growth,and elastase mutations cause cyclic and congenital neutropenia. We further studied the action of the neutrophil serine proteases (proteinase 3,elastase,azurocidin,and cathepsin G) on granulopoiesis in vitro. Elastase inhibited CFU-GM in methylcellulose culture. In serum-free suspension cultures of CD34+ cells,elastase completely abrogated the proliferation induced by G-CSF but not that of GM-CSF or stem cell factor (SCF). The blocking effect of elastase was prevented by inhibition of its enzymatic activity with phenylmethylsulfonyl fluoride (PMSF) or heat treatment. When exposed to enzymatically active elastase,G-CSF,but not GM-CSF or SCF,was rapidly cleaved and rendered inactive. These results support a role for neutrophil elastase in providing negative feedback to granulopoiesis by direct antagonism of G-CSF. View Publication

The cullin family of proteins is involved in the ubiquitin-mediated degradation of cell cycle regulators. Relatively little is known about the function of the CUL-4A cullin,but its overexpression in breast cancer suggests CUL-4A might also regulate the cell cycle. In addition,since other cullins are required for normal development,we hypothesized that CUL-4A is involved in regulating cell cycle progression during differentiation. We observed that CUL-4A mRNA and protein levels decline 2.5-fold during the differentiation of PLB-985 myeloid cells into granulocytes. To examine the significance of this observation,we overexpressed CUL-4A in these cells and found that modest (textless 2-fold),enforced expression of CUL-4A attenuates terminal granulocytic differentiation and instead promotes proliferation. This overexpression similarly affects the differentiation of these cells into macrophages. We recently reported that nearly one half of CUL-4A+/- mice are nonviable,and in this report,we show that the viable heterozygous mice,which have reduced CUL-4A expression,have dramatically fewer erythroid and multipotential progenitors than normal controls. Together these results indicate that appropriate CUL-4A expression is essential for embryonic development and for cell cycle regulation during granulocytic differentiation and suggest this gene plays a broader role in hematopoiesis. Since enforced CUL-4A expression does not alter the cell cycle distribution of uninduced cells but dramatically increases the proportion of induced cells that remains in S-phase and reduces the proportion that accumulates in G0/G1,our results show that this CUL-4A regulatory function is interconnected with differentiation,a novel finding for mammalian cullins. View Publication

Basophils (Ba) and mast cells (MC) are important effector cells of inflammatory reactions. Both cell types derive from CD34(+) hematopoietic progenitors. However,little is known about the cell subsets that become committed to and give rise to Ba and/or MC. We have generated a monoclonal antibody (MoAb),97A6,that specifically detects human Ba,MC (lung,skin),and their CD34(+) progenitors. Other mature hematopoietic cells (neutrophils,eosinophils,monocytes,lymphocytes,platelets) did not react with MoAb 97A6,and sorting of 97A6(+) peripheral blood (PB) and bone marrow (BM) cells resulted in an almost pure population (textgreater98%) of Ba. Approximately 1% of CD34(+) BM and PB cells was found to be 97A6(+). Culture of sorted CD34(+)97A6(+) BM cells in semisolid medium containing phytohemagglutinin-stimulated leukocyte supernatant for 16 days (multilineage assay) resulted in the formation of pure Ba colonies (10 of 40),Ba-eosinophil colonies (7 of 40),Ba-macrophage colonies (3 of 40),and multilineage Ba-eosinophil-macrophage and/or neutrophil colonies (12 of 40). In contrast,no Ba could be cultured from CD34(+)97A6(-) cells. Liquid culture of CD34(+) PB cells in the presence of 100 ng/mL interleukin (IL)-3 (Ba progenitor assay) resulted in an increase of 97A6(+) cells,starting from 1% of day-0 cells to almost 70% (basophils) after day 7. Culture of sorted BM CD34(+)97A6(+) cells in the presence of 100 ng/mL stem cell factor (SCF) for 35 days (mast cell progenitor assay) resulted in the growth of MC (textgreater30% on day 35). Anti-IgE-induced IgE receptor cross-linking on Ba for 15 minutes resulted in a 4-fold to 5-fold upregulation of 97A6 antigen expression. These data show that the 97A6-reactive antigen plays a role in basophil activation and is expressed on multipotent CD34(+) progenitors,MC progenitors,Ba progenitors,as well as on mature Ba and tissue MC. The lineage-specificity of MoAb 97A6 suggests that this novel marker may be a useful tool to isolate and analyze Ba/MC and their progenitors. View Publication

RATIONALE: Adults with cystic fibrosis (CF) are at increased risk of developing osteoporosis. During infective exacerbations,increased production of proinflammatory cytokines and markers of bone resorption have been reported. OBJECTIVE: The aim of this study is to investigate the growth and proliferation of potential osteoclast precursor cells before,during,and after intravenous antibiotic treatment of infective exacerbations in patients with CF. METHODS: Hematopoietic precursor cell growth was examined using colony formation assays using Methocult culture medium. Circulating potential osteoclast precursors were identified using four-color flow cytometry by CD14,CD33,CD34,and CD45 expression. RESULTS: At the start of an infective exacerbation increases in hematopoietic precursor colony formation (15.42 colonies/10(5) cells plated,p = 0.025),proliferation (28.5%,p textless 0.001),and the numbers of circulating potential osteoclast precursors (6.5%,p textless 0.001) were seen in comparison with baseline levels. These increases declined after treatment with intravenous antibiotics to a level close to baseline. CONCLUSIONS: The results demonstrate an increase in the production of potential osteoclast precursors in the peripheral blood during CF infective exacerbations. This may result in increased bone resorption and contribute to bone loss in patients with CF. View Publication

BACKGROUND: Basophils are increasingly utilized as indicators of allergic inflammation and as primary allergic effector cells to study signalling pathways. However,until the present,their enrichment has been time consuming,costly and limited to relatively few specialized laboratories. OBJECTIVE: We have therefore devised a reproducible and rapid method for the purification of human basophils from small quantities of peripheral blood within 1.5 h,which does not require the use of specialized equipment such as elutriators. METHODS: Human basophils were obtained from healthy volunteers undergoing venipuncture. Heparinized or K3-ethylenediaminetetraacetic acid blood samples were first subjected to centrifugation in Hetasep,directly followed by negative selection using immunomagnetic beads. Basophil morphology and purity were assessed by May-Grünwald staining of cytospins. IgE-mediated histamine release was analysed spectrofluorometrically and IL-4 and IL-13 production by quantitative RT-PCR. CD203c and CD63 surface expression was measured using flow cytometry before and after activation with anti-IgE. RESULTS: Using this protocol,basophils were enriched close to homogeneity in most cases with a mean purity of 99.34+/-0.88% (range 97-100%,n=18) and a mean recovery of 75.6 (range 39-100%,n=8). Basophil viability following purification was 99.6+/-0.89% using Trypan blue exclusion. The purification procedure gave rise to basophils with normal functional responses to anti-IgE regarding histamine release as well as IL-4 and IL-13 mRNA expression. Moreover,constitutive cell-surface CD203c/CD63 expressions were not elevated before anti-IgE stimulation. CONCLUSION: The rapidity,simplicity and reproducibility of this method will facilitate the employment of basophils in high-output ex vivo studies. View Publication

Directional migration of leukocytes is an essential step in leukocyte trafficking during inflammatory responses. However,the molecular mechanisms governing directional chemotaxis of leukocytes remain poorly understood. The Slit family of guidance cues has been implicated for inhibition of leuocyte migration. We report that Clara cells in the bronchial epithelium secreted Slit2,whereas eosinophils and neutrophils expressed its cell-surface receptor,Robo1. Compared to neutrophils,eosinophils exhibited a significantly lower level of Slit-Robo GTPase-activating protein 1 (srGAP1),leading to activation of Cdc42,recruitment of PI3K to Robo1,enhancment of eotaxin-induced eosinophil chemotaxis,and exaggeration of allergic airway inflammation. Notably,OVA sensitization elicited a Slit2 gradient at so-called bronchus-alveoli axis,with a higher level of Slit2 in the bronchial epithelium and a lower level in the alveolar tissue. Aerosol administration of rSlit2 accelerated eosinophil infiltration,whereas i.v. administered Slit2 reduced eosinophil deposition. In contrast,Slit2 inactivated Cdc42 and suppressed stromal cell-derived factor-1α-induced chemotaxis of neutrophils for inhibiting endotoxin-induced lung inflammation,which were reversed by blockade of srGAP1 binding to Robo1. These results indicate that the newly identified Slit2 gradient at the bronchus-alveoli axis induces attractive PI3K signaling in eosinophils and repulsive srGAP1 signaling in neutrophils through differential srGAP1 expression during lung inflammation. View Publication

Pathogen induced migration of neutrophils across mucosal epithelial barriers requires epithelial production of the chemotactic lipid mediator,hepoxilin A3 (HXA3). HXA3 is an eicosanoid derived from arachidonic acid. Although eosinophils are also capable of penetrating mucosal surfaces,eosinophilic infiltration occurs mainly during allergic processes whereas neutrophils dominate mucosal infection. Both neutrophils and eosinophils can respond to chemotactic gradients of certain eicosanoids,however,it is not known whether eosinophils respond to pathogen induced lipid mediators such as HXA3. In this study,neutrophils and eosinophils were isolated from human blood and placed on the basolateral side of polarized epithelial monolayers grown on permeable Transwell filters and challenged by various chemotactic gradients of distinct lipid mediators. We observed that both cell populations migrated across epithelial monolayers in response to a leukotriene B4 (LTB4) gradient,whereas only eosinophils migrated toward a prostaglandin D2 (PGD2) gradient. Interestingly,while pathogen induced neutrophil trans-epithelial migration was substantial,pathogen induced eosinophil trans-epithelial migration was not observed. Further,gradients of chemotactic lipids derived from pathogen infected epithelial cells known to be enriched for HXA3 as well as purified HXA3 drove significant numbers of neutrophils across epithelial barriers,whereas eosinophils failed to respond to these gradients. These data suggest that although the eicosanoid HXA3 serves as an important neutrophil chemo-attractant at mucosal surfaces during pathogenic infection,HXA3 does not appear to exhibit chemotactic activity toward eosinophils. ?? 2013 Elsevier Ltd. All rights reserved.

Recently,we reported that human neutrophils produce biologically active amounts of IL-6 when incubated with agonists activating TLR8,a receptor recognizing viral single strand RNA. In this study,we demonstrate that IFNα,a cytokine that modulates the early innate immune responses toward viral and bacterial infections,potently enhances the production of IL-6 in neutrophils stimulated with R848,a TLR8 agonist. We also show that such an effect is not caused by an IFNα-dependent induction of TLR7 and its consequent co-activation with TLR8 in response to R848,but,rather,it is substantially mediated by an increased production and release of endogenous TNFα. The latter cytokine,in an autocrine manner,leads to an augmented synthesis of the IkBζ co-activator and an enhanced recruitment of the C/EBPβ transcription factor to the IL-6 promoter. Moreover,we show that neutrophils from SLE patients with active disease state,hence displaying an IFN-induced gene expression signature,produce increased amounts of both IL-6 and TNFα in response to R848 as compared to healthy donors. Altogether,data uncover novel effects that type I IFN exerts in TLR8-activated neutrophils,which therefore enlarge our knowledge on the various biological actions which type I IFN orchestrates during infectious and autoimmune diseases. View Publication