Vivian Lam

Monomeric IgE Stimulates Signaling Pathways in Mast Cells that Lead to Cytokine Production and Cell Survival

Although IgE binding to mast cells is thought to be a passive presensitization step, we demonstrate herein that monomeric IgE (mIgE) in the absence of antigen (Ag) stimulates multiple phosphorylation events in normal murine bone marrow-derived mast cells (BMMCs). While mIgE does not induce degranulation or leukotriene synthesis, it leads to a more potent production of cytokines than IgE + Ag. Moreover, mIgE prevents the apoptosis of cytokine-deprived BMMCs, likely by maintaining Bcl-X(L) levels and producing autocrine-acting cytokines. The addition of Ag does not increase this IgE-induced survival. Since IgE concentrations as low as 0.1 microg/ml enhance BMMC survival, elevated plasma IgE levels in humans with atopic disorders may contribute to the elevated mast cell numbers seen in these individuals.

IgE-Induced Mast Cell Survival Requires the Prolonged Generation of Reactive Oxygen Species

We show in this study that the ability of five different monomeric IgEs to enhance murine bone marrow-derived mast cell (BMMC) survival correlates with their ability to stimulate extracellular calcium (Ca2+) entry. However, whereas IgE+Ag more potently stimulates Ca2+ entry, it does not enhance survival under our conditions. Exploring this further, we found that whereas all five monomeric IgEs stimulate a less robust Ca2+ entry than IgE+Ag initially, they all trigger a more prolonged Ca2+ influx, generation of reactive oxygen species (ROS), and ERK phosphorylation. These prolonged signaling events correlate with their survival-enhancing ability and positively feedback on each other to generate the prosurvival cytokine, IL-3. Interestingly, the prolonged ERK phosphorylation induced by IgE appears to be regulated by a MAPK phosphatase rather than MEK. IgE-induced ROS generation, unlike that triggered by IgE+Ag, is not mediated by 5-lipoxygenase. Moreover, ROS inhibitors, which block both IgE-induced ROS production and Ca2+ influx, convert the prolonged ERK phosphorylation induced by IgE into the abbreviated phosphorylation pattern observed with IgE+Ag and prevent IL-3 generation. In support of the essential role that IgE-induced ROS plays in IgE-enhanced BMMC survival, we found the addition of H2O2 to IgE+Ag-stimulated BMMCs leads to IL-3 secretion.

SHIP Represses Th2 Skewing by Inhibiting IL-4 Production from Basophils

We report that SHIP−/− mice, compared to SHIP+/+ mice, are Th2 skewed with elevated serum IgE and twice as many splenic CD4+ Th2 cells that, when stimulated with anti-CD3, produce more IL-4 and less IFN-γ. Exploring the reason for this Th2 skewing, we found that freshly isolated SHIP−/− splenic and bone marrow basophils are present in elevated numbers and secrete far more IL-4 in response to IL-3 or to FcεRI stimulation than do WT basophils. These SHIP−/− basophils markedly skew wild-type macrophage colony stimulating factor–derived macrophages toward an M2 phenotype, stimulate OT-II CD4+ Th cells to differentiate into Th2 cells, and trigger SHIP+/+ B cells to become IgE-producing cells. All these effects are completely abrogated with neutralizing anti–IL-4 Ab. Exploring the cell signaling pathways responsible for hyperproduction of IL-4 by SHIP−/− basophils, we found that IL-3–induced activation of the PI3K pathway is significantly enhanced and that PI3K inhibitors, especially a p110α inhibitor, dramatically suppresses IL-4 production from these cells. In vivo studies, in which basophils were depleted from mast cell-deficient SHIP+/+ and SHIP−/− mice, confirmed the central role that basophils play in the Th2 skewing of naive SHIP-deficient mice. Taken together, these studies demonstrate that SHIP is a potent negative regulator of IL-4 production from basophils and thus may be a novel therapeutic target for Th1- and Th2-related diseases.

DMSO Represses Inflammatory Cytokine Production from Human Blood Cells and Reduces Autoimmune Arthritis.

Dimethyl sulfoxide (DMSO) is currently used as an alternative treatment for various inflammatory conditions as well as for cancer. Despite its widespread use, there is a paucity of data regarding its safety and efficacy as well as its mechanism of action in human cells. Herein, we demonstrate that DMSO has ex-vivo anti-inflammatory activity using Escherichia coli- (E. coli) and herpes simplex virus-1 (HSV-1)-stimulated whole human blood. Specifically, we found that between 0.5%– 2%, DMSO significantly suppressed the expression of many pro-inflammatory cytokines/chemokines and prostaglandin E2 (PGE2). However, a significant reduction in monocyte viability was also observed at 2% DMSO, suggesting a narrow window of efficacy. Anti-inflammatory concentrations of DMSO suppressed E. coli-induced ERK1/2, p38, JNK and Akt phosphorylation, suggesting DMSO acts on these signaling pathways to suppress inflammatory cytokine/chemokine production. Although DMSO induces the differentiation of B16/F10 melanoma cells in vitro, topical administration of DMSO to mice subcutaneously implanted with B16 melanoma cells was ineffective at reducing tumor growth, DMSO was also found to block mouse macrophages from polarizing to either an M1- or an M2-phenotype, which may contribute to its inability to slow tumor growth. Topical administration of DMSO, however, significantly mitigated K/BxN serum-induced arthritis in mice, and this was associated with reduced levels of pro-inflammatory cytokines in the joints and white blood cell levels in the blood. Thus, while we cannot confirm the efficacy of DMSO as an anti-cancer agent, the use of DMSO in arthritis warrants further investigation to ascertain its therapeutic potential.

The Role of SHIP in the Development and Activation of Mouse Mucosal and Connective Tissue Mast Cells

Although SHIP is a well-established suppressor of IgE plus Ag-induced degranulation and cytokine production in bone marrow-derived mast cells (BMMCs), little is known about its role in connective tissue (CTMCs) or mucosal (MMCs) mast cells. In this study, we compared SHIP’s role in the development as well as the IgE plus Ag and TLR-induced activation of CTMCs, MMCs, and BMMCs and found that SHIP delays the maturation of all three mast cell subsets and, surprisingly, that it is a positive regulator of IgE-induced BMMC survival. We also found that SHIP represses IgE plus Ag-induced degranulation of all three mast cell subsets and that TLR agonists do not trigger their degranulation, whether SHIP is present or not, nor do they enhance IgE plus Ag-induced degranulation. In terms of cytokine production, we found that in MMCs and BMMCs, which are poor producers of TLR-induced cytokines, SHIP is a potent negative regulator of IgE plus Ag-induced IL-6 and TNF-α production. Surprisingly, however, in splenic or peritoneal derived CTMCs, which are poor producers of IgE plus Ag-induced cytokines, SHIP is a potent positive regulator of TLR-induced cytokine production. Lastly, cell signaling and cytokine production studies with and without LY294002, wortmannin, and PI3Kα inhibitor-2, as well as with PI3K p85α−/− BMMCs and CTMCs, are consistent with SHIP positively regulating TLR-induced cytokine production via an adaptor-mediated pathway while negatively regulating IgE plus Ag-induced cytokine production by repressing the PI3K pathway.

Serum inhibits the immunosuppressive function of myeloid-derived suppressor cells isolated from 4T1 tumor-bearing mice

As more groups investigate the role of myeloid-derived suppressor cells (MDSCs) in promoting the growth of primary tumors and distant tumor metastases, it is imperative to ensure the accurate detection and quantification of MDSC immunosuppression ex vivo. MDSCs are defined by their ability to suppress immune responses. Although different in vitro culture conditions have been used to study MDSCs, the effect of different culture conditions on MDSC immunosuppression is unknown. We therefore isolated MDSCs from the lungs and spleens of 4T1 murine mammary tumor-bearing mice and assayed MDSC-mediated suppression of T cell responses under different culture conditions. We found that 4T1-induced MDSCs effectively suppressed T cell proliferation under serum-free conditions, but not when fetal calf serum (FCS) was present. FCS neither altered the immunosuppressive activities of other myeloid cell types (i.e., peritoneal or tumor-associated macrophages) nor modified the susceptibility of T cells to myeloid cell-mediated suppression, but instead acted directly on 4T1-induced MDSCs to significantly reduce their immunosuppressive function. Importantly, we found that bovine serum albumin was a major contributor to the antagonistic effects of FCS on 4T1-induced MDSC immunosuppression by inhibiting reactive oxygen species production from MDSCs. This work reveals that in vitro culture conditions influence the immunosuppressive properties of MDSCs and highlights the importance of testing different culture conditions on MDSC phenotype to ensure that MDSC immunosuppression is not being masked. These data have important implications for the accurate detection and identification of MDSCs, as well as for determining the influence of MDSC-mediated immunosuppression on primary and metastatic tumor growth.

SHIP represses lung inflammation and inhibits mammary tumor metastasis in BALB/c mice

SH2-containing-inositol-5′-phosphatase (SHIP) is a negative regulator of the phosphatidylinositol-3-kinase pathway in hematopoietic cells and limits the development of leukemias and lymphomas. The potential role of SHIP in solid tumor development and metastasis remains unknown. While SHIP restricts the aberrant development of myeloid cells in C57BL/6 mice, there are conflicting reports regarding the effect of SHIP deletion in BALB/c mice with important consequences for determining the influence of SHIP in different model tumor systems. We generated SHIP−/− BALB/c mice and challenged them with syngeneic non-metastatic 67NR or metastatic 4T1 mammary tumors. We demonstrate that SHIP restricts the development, alternative-activation, and immunosuppressive function of myeloid cells in tumor-free and tumor-bearing BALB/c mice. Tumor-free SHIP−/− BALB/c mice exhibited pulmonary inflammation, myeloid hyperplasia, and M2-polarized macrophages and this phenotype was greatly exacerbated by 4T1, but not 67NR, tumors. 4T1-bearing SHIP−/− mice rapidly lost weight and died from necrohemorrhagic inflammatory pulmonary disease, characterized by massive infiltration of pulmonary macrophages and myeloid-derived suppressor cells that were more M2-polarized and immunosuppressive than wild-type cells. Importantly, while SHIP loss did not affect primary tumor growth, 4T1-bearing SHIP−/− mice had 7.5-fold more metastatic tumor cells in their lungs than wild-type mice, consistent with the influence of immunosuppressive myeloid cells on metastatic growth. Our findings identify the hematopoietic cell-restricted protein SHIP as an intriguing target to influence the development of solid tumor metastases, and support development of SHIP agonists to prevent the accumulation of immunosuppressive myeloid cells and tumor metastases in the lungs to improve treatment of metastatic breast cancer.

The Src Homology 2 Containing Inositol 5′ Phosphatases

Publisher Summary This chapter focuses on the relative structure, expression, and biological roles of Src homology 2 containing inositol 5′-phosphatase (SHIP) 1 and SHIP2 in various cell types, and the therapeutic potential of targeting these two phospholipid phosphatases. The phosphatidylinositol (PI)-3-kinase (PI3K) pathway positively regulates many biological processes, including proliferation, survival, differentiation, activation, and chemotaxis, through the generation of the potent second messenger PI-3,4,5-P3 (PIP3). Consistent with its positive role in promoting proliferation and survival, activation of the PI3K pathway has been shown to be a critical event in tumor development, with many oncogenes (e.g., EGFR, Her2, and K-Ras) stimulating tumor growth by enhancing the PI3K pathway. Studies in skeletal muscle and adipocytes, where SHIP2 is highly expressed, suggest that it is an important negative regulator of insulin signaling. While SHIP1 activators in particular hold a lot of promise therapeutically, there are a number of concerns which they share with PI3K inhibitors.

All Trans Retinoic Acid, Transforming Growth Factor β and Prostaglandin E2 in Mouse Plasma Synergize with Basophil-Secreted Interleukin-4 to M2 Polarize Murine Macrophages.

In previous studies we found that macrophages (MФs) from SH2-containing inositol-5′-phosphatase (SHIP) deficient mice are M2 polarized while their wild type (WT) counterparts are M1 polarized and that this difference in MФ phenotype can be recapitulated during in vitro derivation from bone marrow if mouse plasma (MP), but not fetal calf serum, is added to standard M-CSF-containing cultures. In the current study we investigated the mechanism by which MP skews SHIP-/- but not +/+ MФs to an M2 phenotype. Our results suggest that SHIP-/- basophils constitutively secrete higher levels of IL-4 than SHIP+/+ basophils and this higher level of IL-4 is sufficient to skew both SHIP+/+ and SHIP-/- MФs to an M2 phenotype, but only when MP is present to increase the sensitivity of the MФs to this level of IL-4. MP increases the IL-4 sensitivity of both SHIP+/+ and -/- MФs not by increasing cell surface IL-4 or CD36 receptor levels, but by triggering the activation of Erk and Akt and the production of ROS, all of which play a critical role in sensitizing MФs to IL-4-induced M2 skewing. Studies to identify the factor(s) in MP responsible for promoting IL-4-induced M2 skewing suggests that all-trans retinoic acid (ATRA), TGFβ and prostaglandin E2 (PGE2) all play a role. Taken together, these results indicate that basophil-secreted IL-4 plays an essential role in M2 skewing and that ATRA, TGFβ and PGE2 within MP collaborate to dramatically promote M2 skewing by acting directly on MФs to increase their sensitivity to IL-4.

Effect of age on chronic inflammation and responsiveness to bacterial and viral challenges

To identify reliable biomarkers of age-related changes in chronic inflammation and responsiveness to bacterial and viral challenges, we evaluated endogenous and ex vivo stimulated levels of 18 inflammatory markers, using whole blood collected in EDTA and sodium heparin tubes from 41 healthy volunteers, i.e., 11 men + 10 women aged 20–35 and 10 men + 10 women aged 50–77. These studies revealed significant differences in the levels of inflammatory markers when blood was collected in EDTA versus sodium heparin and age related differences in these biomarkers were confirmed with blood collected in EDTA from 120 healthy volunteers in 3 age categories, ie, 20 men + 20 women, aged 20–35, 36–49 and 50–77. Studies with unstimulated blood samples, to measure levels of chronic inflammation, revealed a significant increase with age in IL-12p70, CRP and PGE2, consistent with the concept of “inflammaging”, and a decrease in G-CSF in both men and women. Interestingly, in response to E. coli stimulation, PGE2 levels were markedly reduced in the 50–77 year old cohort while they were increased following Herpes Simplex virus-1 (HSV-1) stimulation, along with IL-8. In addition, unlike E. coli, HSV-1 potently stimulated IFNα production, but levels were dramatically reduced in the older cohort, consistent with a reduced ability to generate an anti-viral response. We also found platelets and CD8+ T cells were reduced with age while CD4+ T cells were significantly increased, resulting in a substantially higher CD4/CD8 ratio in the older cohort. Surprisingly, however, we found that the older cohort exhibited more T cell proliferation and IFNγ production in response to anti-CD3+anti-CD28 stimulation. Importantly, there was considerable person-to-person variation in these inflammatory markers in all age groups, making possible comparisons between a person’s “inflammage” and chronological age. These assays should help to identify individuals at high risk of autoimmune disorders and cancer.