Lyn Kinase Controls Basophil GATA-3 Transcription Factor Expression and Induction of Th2 Cell Differentiation

doi:10.1016/j.immuni.2009.02.008

Immunity. Author manuscript; available in PMC 2010 April 17.

Published in final edited form as:

Immunity. 2009 April 17; 30(4): 533–543.

Published online 2009 April 9. doi: 10.1016/j.immuni.2009.02.008

PMCID: PMC2772996

NIHMSID: NIHMS106601

Lyn Kinase Controls Basophil GATA-3 Transcription Factor Expression and Induction of Th2 Cell Differentiation

Nicolas Charles,¹ Wendy T. Watford,² Haydeé L. Ramos,² Lars Hellman,³ Hans C. Oettgen,⁴ Gregorio Gomez,⁵ John J. Ryan,⁵ John J. O’Shea,² and Juan Rivera¹^*

¹Laboratory of Immune Cell Signaling, NIAMS, National Institutes of Health, Bethesda, Maryland 20892, USA

²Molecular Immunology and Inflammation Branch, NIAMS, National Institutes of Health, Bethesda, Maryland 20892, USA

³Department of Cell and Molecular Biology, Uppsala University, SE-751 24 Uppsala, Sweden

⁴Department of Pediatrics, Division of Immunology, Children’s Hospital, Harvard Medical School, Boston, MA 02115

⁵Department of Internal Medicine, Division of Rheumatology, Allergy, and Immunology, and Department of Biology, Virginia Commonwealth University, Richmond, VA 23284.

^*Correspondence: Juan Rivera, NIAMS-NIH, Building 10, Room 13C103, Bethesda, MD. 20892-1930, Tel: 301-496-7592, Fax: 301-480-1580, E-mail: juan_rivera/at/nih.gov

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Abstract

Summary

T_H1/T_H2 balance is key to host defense and its dysregulation has pathophysiological consequences. Basophils are important in T_H2 differentiation. However, the factors controlling the onset and extent of basophil-mediated T_H2 differentiation are unknown. Here, we demonstrate that Lyn kinase dampens basophil GATA-3 expression and the initiation and extent of T_H2 differentiation. Lyn-null mice had a marked basophilia, a constitutive T_H2 skewing that was exacerbated upon in vivo challenge of basophils, produced antibodies to a normally inert antigen, and failed to appropriately respond to a T_H1 pathogen. The T_H2 skewing was dependent on basophils, IgE and IL-4, but was independent of mast cells. Our findings demonstrate that basophil-expressed Lyn kinase exerts regulatory control on T_H2 differentiation and function.

Introduction

T_H1/T_H2 balance leads to an appropriate immune response tailored to the type of infectious pathogen. T_H1 responses, induced by some bacterial or viral infections, are driven by IL-12 and the transcription factors Stat4 and T-bet (Lighvani et al., 2001; Szabo et al., 2000). T_H2 differentiation, which is predominantly associated with infection by parasitic worms, is driven by cytokines like IL-4, IL-5, IL-13, IL-18, and IL-33. There is considerable evidence that thymic stromal lymphopoietin (TSLP) is also required for T_H2-mediated immunity. The transcription factors GATA-3, c-maf and NFATc are known to control T_H2 differentiation (Neurath et al., 2002; Zhu et al., 2006). Impairment of T_H1 or T_H2 responses results in the failure to clear pathogens (Kawakami, 2003) and can also cause an inappropriate response to an otherwise innocuous antigen, resulting in allergies (Capron et al., 2004). Therefore, the differentiation of T cells into their effector subsets is a topic of intensive study with considerable therapeutic implications and much is known about the molecular factors that drive T cell differentiation (Neurath et al., 2002; Zhu et al., 2006). However, beyond the role of dendritic cells, much less is known about the cell types that can cause T cell differentiation, and in particular T_H2 differentiation. Identifying which cell types and what molecules might be responsible for dysregulation of T_H2 responses would provide knowledge that could be beneficial towards controlling these responses.

While basophils had long been considered as redundant “circulating mast cells”, a considerable body of literature has argued for a distinct role of basophils in both humans and in mice (Poorafshar et al., 2000; Schroeder et al., 2001). In mice, only basophils and mast cells are known to constitutively express the high affinity receptor for IgE (FcεRI). When sensitized with allergen-specific IgE and subsequently challenged with allergen both of these cell types are able to degranulate, releasing pro-inflammatory allergic mediators, and neo-synthesize and secrete a wide variety of cytokines (DeLisi and Siraganian, 1979; Segal et al., 1977). Recent mouse studies reveal that basophils are important in promoting allergen-induced T_H2 differentiation and in enhancing humoral memory immune responses (Denzel et al., 2008; Sokol et al., 2008). These cells also have a primary role in IgG-mediated systemic anaphylaxis and in IgE-mediated chronic allergic inflammation (Mukai et al., 2005; Tsujimura et al., 2008). In humans, the basophil has long been associated with allergic inflammation in chronic disease (Schroeder et al., 2001) and both human and mouse basophils are able to produce large amounts of T_H2-promoting cytokines, like IL-4 and TSLP (Poorafshar et al., 2000; Schroeder et al., 2001). However, the mechanism(s) by which basophils may govern the onset and extent of T_H2 responses has not been explored.

The Src family tyrosine kinase Lyn is important in linking FcεRI stimulation with basophil responses (Schroeder et al., 2001). Lyn is expressed in most hematopoietic cells, but not in T cells (Yamanashi et al., 1989). In mice, the absence of Lyn leads to a late life autoimmune phenotype with characteristics of systemic lupus erythmatosus (SLE) (Hibbs et al., 1995; Nishizumi et al., 1995), suggesting that it plays a key role in tolerance. Lyn deficient mice also have high levels of serum immunoglobulins (including autoantibodies) and their B cells are hyperresponsive to IL-4 and CD40 engagement (Hibbs et al., 1995; Janas et al., 1999; Nishizumi et al., 1995). Interestingly, the SLE phenotype is preceded by an atopic allergic-like manifestation in these mice (Janas et al., 1999; Odom et al., 2004).

Because of the allergic-like phenotype of lyn^−/− mice, we sought to identify the cell population and the factor(s) responsible for this phenotype and to determine how the loss of Lyn contributes to increased hypersensitivity. Our findings show that Lyn controls the expression of GATA-3 in basophils governing the onset and extent of T_H2 differentiation.

Results

Basophils express lyn kinase and lyn-deficiency causes peripheral basophilia

Murine basophils are poorly granulated FcεRl⁺ Kit⁻ CD11b⁺ CD49b⁺ cells (Mukai et al., 2005) with a polymorphic nucleus, and are known to express mMCP-8 as a protease specific to this cell lineage (Poorafshar et al., 2000) ( Figure S1A–E ). Characterization of basophils from the blood, bone marrow and peritoneum revealed some differences in expression of cell surface markers (i.e., higher expression of CD49b in blood and bone marrow basophils), granule proteases (almost no expression of protease transcripts in peritoneal basophils) and differences in the profile of Src family kinase transcripts (see Table S1 for RT-PCR primers used). Despite the absence of mMCP-8 expression by peritoneal basophils, their phenotypic features suggested a basophil-like cell and all the defined basophil populations expressed transcripts and protein for Lyn kinase ( Figure S1D, E ).

Analysis of wild type littermates (WT), lyn heterozygote (lyn^+/−) or lyn null (lyn^−/−) mice, revealed an increased proportion of basophils in the blood of lyn^−/− mice (Figure 1A) and in the peritoneal lavage (PL) (Figure 1B), but normal levels were found in the bone marrow (BM) (Figure 1C) when compared to WT and lyn^+/− mice. The absolute numbers of blood basophils increased by approximately 50% (Figure 1D). The modest amount of Lyn (~ 30% of normal) found in the lyn^+/− cells ( Figure S2A, B ) was sufficient to reverse the peripheral basophilia (Figure 1A–C). As previously described (Hibbs et al., 1995; Nishizumi et al., 1995; Odom et al., 2004), IgE levels were significantly increased in lyn^−/− mice compared to WT mice. This was reversed (and dampened) by the reduced amounts of Lyn expressed in lyn^+/− mice ( Figure S2C ). Collectively, the data suggested a dominant role of Lyn as a negative regulator of basophil growth in vivo and as having both a positive and negative role in IgE production. In mast cells, Lyn was described to have a positive or negative role depending on the genetic background of the mice from which cells are derived (Yamashita et al., 2007). In contrast, the basophilia observed in the absence of Lyn was independent of the genetic background (Figure 1E) demonstrating a dominant role for Lyn in this phenotype.

Figure 1

lyn^−/− mice develop peripheral basophilia

Lyn is a negative regulator of basophil proliferation

The absence of basophilia in the bone marrow of lyn^−/− mice suggested that Lyn was likely involved in basophil proliferation rather than their differentiation. Thus, we monitored the appearance and proliferation of basophils from BM and PL cultures derived from WT or lyn^−/− mice. Culturmg of these cells revealed a large FcεRl⁺ c-Kit⁻ CD11b⁺ CD49b⁺ cell population that fit the criteria for basophils (Figure 2A and Figure S1 ). The identity of sorted BM-derived cells as basophils was confirmed by mMCP-8 expression both at the mRNA and the protein levels ( Figure S1C and data not shown). To directly determine if differences existed in the proliferation of basophils derived from lyn^−/− vs. WT mice, we performed CFSE staining of BM-derived basophils and found an enhanced proliferation in the absence of Lyn (Figure 2B). Both BM-derived and PL-derived basophils were more abundant in lyn^−/− than in WT cultures (Figure 2C, D). However, the disappearance of basophils from both types of cultures showed the same kinetics. The findings demonstrate that the absence of Lyn enhances basophil proliferation and not their survival.

Figure 2

Lyn-deficiency enhances basophil proliferation

Lyn-deficiency enhances basophil IL-4 production but suppresses degranulation

In mast cells, Lyn-deficiency enhances the secretion of various cytokines including IL-4, IL-6, and TNF-α (Hernandez-Hansen et al., 2005; Xiao et al., 2005; Yamashita et al., 2007). Thus, we investigated if Lyn-deficiency in basophils caused dysregulation of cytokine responses. Analysis of cytokine production in BM-derived basophils showed that lyn^−/− basophils were more potent producers of IL-4 and IL-6, but not of TNF-α, than their WT counterparts (Figure 3A, B and Figure S3A, B ).

Figure 3

The production of IL-4 is enhanced in lyn^−/− bone marrow-derived cultured basophils and in blood basophils from lyn^−/− mice

These experiments suggested that blood basophils obtained directly from lyn^−/− mice should produce higher levels of IL-4 relative to their WT counterparts. Using an ex vivo stimulation protocol with anti-IgE ( Figure S4A and Experimental Procedures), we tested this hypothesis and found that lyn^−/− blood basophils produced approximately 3-fold greater levels of IL-4 (Figure 3C). Normalization to the absolute number of basophils present in the blood showed that lyn^−/− basophils produced more IL-4 on a per cell basis (Figure 3D). While T cells do not express Lyn kinase (Yamanashi et al., 1989), they have been described as an important source of IL-4. Anti-CD3/anti-CD28 treatment of naïve T cells from blood induced low levels of IL-4 production, relative to the basophil-derived IL-4, and no significant differences were seen between T cells from WT or lyn^−/− mice in this acute challenge ( Figure S3C, D ). Thus, lyn^−/− basophils have an enhanced ability to produce IL-4.

Since mast cells are not present in the blood ( Figure S4C ), we measured the total amount of histamine in the blood (in both cells and plasma) of IgE-sensitized WT and lyn^−/− mice (prior to ex vivo challenge), which should be primarily derived from basophils. An increased total blood histamine content was observed in lyn^−/− mice, which correlated with their basophilia ( Figure S4D ). However, when normalized to absolute numbers demonstrated an equal amount of histamine per basophil ( Figure S4E ). Unexpectedly, ex vivo anti-IgE stimulation of IgE-sensitized lyn^−/− basophils did not cause significant histamine release, on a per cell basis, when compared to WT basophils ( Figure S4F ). Thus, while the total blood (cells and plasma) content of histamine is elevated in lyn^−/− mice, the ability of blood basophils to release histamine in response to FcεRI stimulation is considerably reduced in the absence of Lyn.

Early and inappropriate onset of T_H2 responses in lyn^−/− mice

To explore the effect of Lyn-deficiency on the induction of T_H2 responses, a protease allergen (papain) challenge model that was previously demonstrated to elicit a basophil- specific induction of T_H2 responses (Sokol et al., 2008), was used. Given that lyn^−/− basophils produced more IL-4 than their WT counterparts, we hypothesized an altered response of lyn^−/− mice in this particular model. Indeed, lyn^−/− mice showed an early onset of the T_H2 responses with detectable amounts of papain-specific IgE production as early as day 7 post-immunization (Figure 4A and Figure S5B ). While the maximal papain-specific IgE production was lower than for WT mice, this was likely due to the high levels of total IgE produced in lyn^−/− mice, which exceeded the stimulated IgE production of WT mice by almost 3 fold ( Figure S5A ). Dysregulation of T_H2 responses was also supported by the detection of TSLP only in the serum of lyn^−/− mice immunized with papain, which correlated with the early onset of papain-specific IgE production (Figure 4B). Moreover, production of IgE to human serum albumin (HSA) was observed in lyn^−/− mice whereas HSA was previously described to be inert for IgE production in WT mice (Sokol et al., 2008), as confirmed herein (Figure 4C and Figure S5A ). Since the levels of IL-4 in the serum of papain immunized mice could not be measured, we generated mice deficient for both IL-4 and Lyn kinase (IL-4^−/−lyn^−/−) to assess the role of this cytokine in the context of Lyn-deficiency. Peripheral basophilia was still observed in these mice (Figure 4D). After papain immunization of IL-4^−/−lyn^−/− mice, a delayed onset and a poor papain-specific IgE production was observed (Figure 4E). TSLP was not detected in the serum (data not shown) and, relative to WT mice, total IgE production was severely impaired ( Figure S5C ). To exclude the potential involvement of mast cell IL-4 production in the onset of papain-specific IgE production, we papain-immunized mast cell-deficient mice (W^Sh/W^Sh) mice and found that they produced normal levels of papain-specific IgE, albeit with a modest delay in onset relative to their WT littermates ( Figure S5D ). Thus, Lyn-deficiency leads to an inappropriate and early onset of T_H2 responses.

Figure 4

Lyn deficiency causes an early and inappropriate onset of IL-4 dependent T_H2 responses

Constitutive T_H2 skewing and the exacerbated T_H2 responses of lyn^−/− mice is IgE and basophil-mediated

We next explored whether the basophilia in lyn^−/− mice was associated with T_H2 differentiation in the absence of an exogenous challenge. Splenocytes were isolated from unchallenged WT and lyn^−/− mice and CD4⁺ T cells were analyzed for intracellular IL-4 and IFN-γ. This revealed a 2 to 5 fold increase in IL-4 producing CD4⁺ T cells in lyn^−/− versus WT mice (Figure 5A, B). After a 4 hr stimulation with PMA/Ionomycin, the fraction of IL-4 producing CD4⁺ T cells was markedly enhanced (Figure 5A, B) and an increase in IFN-γ-producing CD4⁺ T cells was also observed, suggesting a general hyper-reactivity. Importantly, however, no IFN-γ production was observed in the absence of this non-specific stimulus (Figure 5A and Figure S6A ). Moreover, the T_H2 skewing was strictly dependent on IgE, since IgE^−/− lyn^−/− mice failed to show a spontaneous T_H2 differentiation (Figure 5C), although these mice developed peripheral basophilia to a similar extent as lyn^−/− mice ( Figure S6B and Figure 1A). IFN-γ production by CD4⁺ T cells after PMA/Ionomycin stimulation of IgE^−/− lyn^−/− splenocytes was also observed ( Figure S6C ). These findings demonstrate the requirement for IgE in the constitutive T_H2 skewing of CD4⁺ T cells from lyn^−/− mice, and further reveal the enhanced T_H2 potential of T cells derived from lyn^−/− mice relative to WT mice.

Figure 5

T_H2 skewing of CD4⁺ T cells is driven by in vivo activation of basophils, is exacerbated by Lyn-deficiency, depends on IgE and basophils, but not on mast cells

To directly address whether basophils were key in the T_H2 skewing of lyn^−/− mice, we depleted basophils from these mice using a modified protocol from that previously described (Sokol et al., 2008) (see Experimental Procedures). Depletion in the blood and bone marrow reached at least 90% six days post-first injection ( Figure S7A and data not shown). The almost complete absence of basophils led to a rescue of the constitutive T_H2 skewing in lyn^−/− mice (Figure 5D), demonstrating that this phenotype was strictly dependent on basophils. To further test the involvement of basophils and Lyn in T_H2 differentiation, we generated mast cell- and Lyn- double deficient mice (W^Sh/W^Sh lyn^−/−) in order to selectively stimulate only basophils in vivo via FcεRI. Without mast cells, IgE antibody-dependent anaphylaxis does not occur nor do basophils contribute significantly to this response (Martin et al., 1989; Tsujimura et al., 2008; Zhou et al., 2007). W^Sh/W^Sh lyn^−/− mice developed a peripheral basophilia like lyn^−/− mice ( Figure S6B ) and showed the spontaneous T_H2-skewing of CD4⁺ T cells when compared to W^Sh/W^Sh mice ( Figure S6D ), as well as general hyper-activity of T cells upon PMA/ionomycin restimulation ( Figure S6D, E ). A systemic injection (i.v.) of rat anti-mouse IgE in the control W^Sh/W^Sh led to increased CD4⁺ (IL-4⁺) T cells (Figure 5E, F) similar to the constitutive skewing in unchallenged lyn^−/− mice (Figure 5A, B). Challenge of W^Sh/W^Sh lyn^−/− mice in the same manner caused a marked T_H2 skewing (Figure 5E, F) that was 3–4 fold higher than observed in unchallenged lyn^−/− mice (Figure 5A, B). Hence, in vivo stimulation of basophils selectively initiates T_H2 differentiation in the mouse. PMA/ionomycin stimulation of CD4⁺ T cells from both W^Sh/W^Sh and W^Sh/W^Sh -lyn^−/− showed even a greater enhancement of T_H2 responses (IL-4 production) proportional to the presence or absence of Lyn (Figure 5E, F), as described above for WT and lyn^−/− T cells. This was not limited to IL-4 producing CD4⁺ T cells but was also seen for IL-13 production ( Figure S8 ). Similarly, T_H1 responses were also increased after this non-specific stimulation in all genotypes, in a basophil-independent manner ( Figure S6F , Figure S7C & D ). Thus, FcεRI-stimulation of basophils leads to selective T_H2 differentiation and the extent of this response is tightly controlled by Lyn kinase.

Lyn-deficient basophils have an IgE and PI3K–dependent increase in GATA-3 expression and IL-4 production

To address this issue of what factors might control the potent T_H2 skewing ability of lyn^−/− basophils, we investigated if the loss of Lyn led to changes in key differentiation factors that are pivotal for T_H2 differentiation and IL-4 production (Zhu et al., 2006). Assessment of the mRNA levels of various T_H2 transcription factors in lyn^−/− basophils showed a significant increase in the levels of GATA-3 and a trend towards increased c- maf (data not shown). Because increases in GATA-3 expression can induce IL-4 production (Zhu et al., 2006), we explored the expression of GATA-3 (protein) in spleen basophils from WT or lyn^−/− mice. GATA-3 expression was enhanced in Lyn-deficient basophils (Figure 6A and Figure S9B ). This was also true for BM-derived cultured Lyn-deficient basophils (Figure 6B) and FcεRI engagement led to a further enhancement of GATA-3 expression relative to WT cells. This mirrored the constitutive and enhanced T_H2 differentiation observed in lyn^−/− mice (Figure 5A & B). In T cells, Fyn kinase appears to be required for GATA-3 expression and IL-4 production as well as for T_H2 differentiation (Cannons et al., 2004; Davidson et al., 2004). Thus, we investigated the effect of Fyn-deficiency on basophil GATA-3 expression and on T_H2 differentiation. Fyn-deficient basophils showed a modest reduction in GATA-3 expression relative to WT basophils, and a marked reduction relative to lyn^−/− basophils (Figure 6C and Figure S9A & B ). FcεRI engagement of bone marrow-derived Fyn-deficient basophils failed to induce the production of GATA-3 (Figure 6D). Fyn^−/− mice had no basophilia and FcεRI-induced basophil IL-4 production was modestly decreased ( Figures S9C & D ). Consistent with these findings, fyn^−/− mice were not constitutively T_H2 skewed, however, T_H2 differentiation elicited by PMA/ionomycin appeared to be relatively normal ( Figure S9E ). Thus, the increased expression of GATA-3 in lyn^−/− basophils is consistent with their increased production of IL-4 and the observed increase in T_H2 differentiation. In mast cells, Lyn has been shown to dampen Fyn-dependent signals (Odom et al., 2004). Moreover, Fyn is essential for phosphatidylinositol 3-OH kinase (PI3K) activity (Gomez et al., 2005; Parravicini et al., 2002). Thus, the constitutive and FcεRI-enhanced GATA3 expression in lyn^−/− basophils might be due to increased PI3K activity. Inhibition of PI3K activity by treatment of basophils with LY294002 effectively blocked GATA3 expression prior to and after FcεRI engagement (Figure 6E). Concurrently, IL-4 production was also blocked by LY294002 in WT and lyn^−/− basophils (Figure 6F).

Figure 6

GATA-3 expression is increased in lyn^−/− basophils, decreased in fyn^−/− basophils and depends on PI3K activity

GATA3 expression in lyn^−/− basophils could be due to a developmental abnormality, or be related or unrelated to the T_H2 skewing phenotype. Therefore, we analyzed GATA3 expression in spleen basophils from IgE^−/− lyn^−/− mice, which showed a basosphilia but no T_H2 skewing (Figure 5C & Figure S8 ). GATA3 expression in basophils from IgE^−/− lyn^−/− mice was equivalent to that of WT cells ( Figure S10A ). However, these cells were induced to express GATA3 by ex vivo FcεRI stimulation ( Figure S10B ) to levels similar to that of lyn−/− basophils (Figure 6B). Importantly, these conditions also led to enhanced IL-4 production relative to WT basophils ( Figure S10C ). Thus, the findings show that the enhanced GATA3 expression and IL-4 production seen in lyn^−/− mice requires IgE, demonstrating a functional rather than a developmental alteration in basophil homeostasis.

T_H2 skewing impairs the response of lyn^−/− mice to a T_H1 challenge

To assess the pathophysiological consequences of T_H2 skewing, WT and lyn^−/− mice were infected with the T_H1 pathogen Toxoplasma gondii, a well described model requiring a strong T_H1 response to reduce the cyst burden in the brain as well as for survival, both aspects depending on IFN-γ production (Suzuki et al., 1989; Suzuki et al., 1988). Thus, this model would allow us to determine whether T_H1 responses were normal in lyn^−/− mice. Seven days following T. gondii infection, we analyzed the production of IFN-γ in WT and lyn^−/− mice and found an almost 50% inhibition of its production in the absence of Lyn (Figure 7A). At 4 weeks post infection, mice were euthanized and the cyst brain burden was determined. As shown in Figure 7B, a significantly higher number of cysts were present in the brain of lyn^−/− versus WT mice. These findings clearly demonstrate that lyn^−/− mice fail to generate an appropriate T_H1 response to T. gondii.

Figure 7

lyn^−/− mice show impaired production of IFN-γ and fail to mount an appropriate host response to Toxoplasma gondii infection

Discussion

For many years the initiation of T cell subset differentiation was thought to occur primarily through antigen-driven maturation of dendritic cells into specific phenotypes that promote T cell subset differentiation (Reschner et al., 2008). However, mouse studies have recently revealed a key role for the basophil in T_H2 differentiation (Sokol et al., 2008) and humoral memory responses (Denzel et al., 2008). Thus, basophils appear to be key regulatory cells and dysregulation of their function could lead to abnormal immune responses. Our studies on lyn^−/− mice demonstrate an increased proliferative response of Lyn-deficient basophils in vitro, an in vivo basophilia, an enhanced basophil GATA-3 expression and IgE-dependent T_H2 differentiation in the absence of any exogenous challenge. Basophils from lyn^−/− mice were able to produce increased amounts of IL-4, and challenge of these mice with a protease allergen demonstrated enhanced TSLP levels relative to their WT counterparts. This T_H2-skewing results in an inadequate T_H1 response when lyn^−/− mice are challenged with T. gondii.

Our findings show that Lyn kinase dampens the level of GATA-3 expression in basophils. In T cells, GATA-3 is thought to control T_H2 responses through the induction of T_H2 cytokines, the selective growth of T_H2 cells, and inhibition of T_H1 cell-specific factors (Zhu et al., 2006). Similarly, in lyn^−/− basophils, an increase in IL-4 production and an enhanced proliferation mirrored the increased expression of GATA-3. On the other hand, Fyn kinase appears to be required for GATA-3 expression, IL-4 production, and T_H2 differentiation (Cannons et al., 2004; Davidson et al., 2004). This is consistent with our finding that fyn^−/− basophils showed a modest reduction of GATA-3 under resting conditions, failed to elicit increased GATA-3 expression, and had reduced levels of IL-4 secretion following FcεRI stimulation. In the mast cell, a close kin of the basophil, Lyn kinase has been shown to dampen the activity of Fyn kinase, whose level of expression drives PI3K activity and IL-4 production (Gomez et al., 2005; Hernandez-Hansen et al., 2004; Odom et al., 2004; Parravicini et al., 2002; Yamashita et al., 2007). Basophils also failed to induce FcεRI-mediated expression of GATA3 and to produce IL-4 when PI3K activity was inhibited. These findings argue that the interplay between Lyn and Fyn kinase is a key determinant for the regulation of GATA-3 expression and T_H2 differentiation.

Importantly, Lyn governs immune homeostasis as its loss led to an inappropriate response to an otherwise inert immunogen (HSA) and to the production of HSA-specific IgE. This strong global T_H2 response likely explains the spontaneous (Odom et al., 2004) and exacerbated allergic phenotype of lyn^−/− mice seen upon antigen challenge (Beavitt et al., 2005; Yamashita et al., 2007). Importantly, the spontaneous T_H2 skewing was IgE dependent and lyn^−/− mice are known to develop high serum IgE levels within five weeks of birth (Odom et al., 2004). This suggests the possibility that high IgE levels could drive increased T_H2 responses in the absence of a defined antigen.

Denzel and colleagues (Denzel et al., 2008) showed that basophils amplified both T_H2-associated IgG1 responses and T_H1-associated IgG2a responses, thus raising the possibility that basophils elicited both T_H1 and T_H2 responses. We saw a similar effect upon restimulation of CD4⁺ T cells with PMA/ionomycin, as both IL-4 (or IL-13) and IFN-γ production was observed. In contrast, in the absence of stimulation, lyn^−/− mice showed only IL-4 (or IL-13) producing CD4⁺ T cells and the in vivo FcεRI stimulation of basophils in the context of mast-cell deficiency (W^Sh/W^Sh or in W^Sh/W^Sh-lyn^−/− mice) demonstrated the presence of only CD4⁺ IL-4⁺ (or IL-13⁺) T cells. This T_H2 induction was reversed by basophil depletion in both lyn^−/− and W^Sh/W^Sh -lyn^−/−) mice without changing the T_H1 hyperreactivity after CD4⁺ T cells restimulation in vitro. Thus, the findings argue that basophil activation and Lyn-deficiency contributes selectively to T_H2 induction and that the induction of IFN-γ-producing CD4⁺ T cells is a consequence of restimulation independently of basophils.

In humans, a reduced level of Lyn expression is seen in the basophils from as much as 20% of the population (the so called “non-releaser” basophil phenotype, so named because of poor histamine release) (Kepley et al., 1999; Lavens-Phillips and MacGlashan, 2000; Youssef et al., 2007). Significantly reduced levels of both Lyn and/or Syk kinase protein expression has been associated with this basophil phenotype (Kepley et al., 1999). A recent study (Gilmartin et al., 2008), comparing the inflammatory mediators released from basophils of asthmatic and control subjects, demonstrates a trend towards increased numbers of blood basophils in the asthmatic population and found that IL-4 was the most consistently expressed cytokine in resting basophils for both populations (Gilmartin et al., 2008). Our finding of a basophilia in the lyn^−/− mouse is consistent with this observation, which together with normal IL-4 production alone could account for some T_H2 differentiation. Treatment of basophils from control or asthmatic subjects with IL-3, which regulates basophil function, was shown to increase basophil IL-4 production for both groups (Gilmartin et al., 2008). Moreover, IL-3 treatment of human basophils causes the production of retinoic acid and T_H2 differentiation of naïve human T cells (Spiegl et al., 2008). Thus, a role for basophils and Lyn kinase in modulating T_H2 differentiation in humans seems likely.

An intriguing aspect of this work, that remains to be explored, is whether the observed T_H2 skewing in the absence of Lyn is contributory to the development of the systemic lupus erythematosus (SLE)-like phenotype seen in lyn^−/− mice (Hibbs et al., 1995; Nishizumi et al., 1995). Autoimmune disease has traditionally, although not exclusively, been associated with T_H1/T_H17 responses (Dardalhon et al., 2008). However, it has been demonstrated that B cells from certain populations of SLE patients showed reduced levels of Lyn expression (Flores-Borja et al., 2005) and this was associated with increased ubiquitination of Lyn and heightened B cell proliferation and IL-10 production (Flores-Borja et al., 2005). Regardless, our findings demonstrate that basophils and Lyn kinase are key elements in determining the T_H1/T_H2 balance in vivo. Strategies that increase or decrease Lyn activity in the basophil compartment may be effective in controlling the T_H1/T_H2 balance.

Experimental Procedures

Mice

For the majority of experiments, littermate-matched mice (lyn^+/+, fyn^+/+ referred to as WT, lyn^+/− and lyn^−/−, fyn^−/−) of mixed background (129Sv x C57BL/6 (N6)) were used. As indicated, in some experiments C57BL/6 and 129Sv lyn^+/+ and lyn^−/− mice from Taconic (Germantown, NY) were used. Il-4^+/+ lyn^+/+ and Il-4^−/− lyn^−/− mouse strains were generated as littermates on a mixed background (C57BL/6 × 129Sv (N7)). IgE^+/+ lyn^+/+ and IgE^−/− lyn^−/− mouse strains were generated as littermates on a mixed background (C57BL/6 × 129Sv (N4)). W^Sh/W^Sh-lyn^−/− mice were generated by crossing a W^Sh/W^Sh mouse with a C57BL/6 lyn^−/− mouse and generating all four genotypes needed for these experiments (WT, lyn^−/− , W^Sh/W^Sh and W^Sh/W^Sh -lyn^−/−) as littermates. Mice were maintained and used in accordance with National Institutes of Health (NIH) guidelines and National Institute of Arthritis and Musculoskeletal and Skin Diseases–approved animal study proposal A007-03-01.

Antibodies and Reagents

DNP-specific mouse IgE was produced as previously described (Liu et al., 1980). Rat antisera to mouse MCP-8 (mMCP-8) were previously described (Poorafshar et al., 2000). All other antibodies and reagents were from commercial sources and are described in Supplementary Materials and Methods.

Reverse-Transcription and PCR analysis

RNA from sorted cells was extracted by using Trizol® reagent from Invitrogen following the manufacturer’s instructions. First-strand synthesis was done with the SuperScript III first strand kit from Invitrogen using 0.8 µg of RNA per reaction. Primers used for PCR are described in Table S1 .

Cell culture

For bone marrow derived mast cells (BMMC) and basophils cultures, bone marrow (BM) was isolated from 8–12-weeks-old mice by flushing out bone marrow from tibias and femurs with cell culture medium. Cells were cultured in both IL-3 and SCF (20 ng/ml each) as previously described (Parravicini et al., 2002). For culture of peritoneal derived mast cells (PDMC) and basophils see Supplementary Materials and Methods.

Enzyme-linked immunosorbent assays

For measurement of serum IgE levels, a mouse IgE ELISA kit from Bethyl Laboratories was used (Montgomery, TX). Thymic Stromal Lymphopoietin (TSLP), IFN-γ and IL-4 levels were quantified by using Quantikine ELISA kit from R&D Systems (Minneapolis, MN). IL-13 ELISA kit was from eBioscience, Inc (San Diego, CA). Histamine was measured with a competitive enzymatic immunoassay (EIA) kit from Beckman Coulter Immunotech (Miami, FL). All kits were used following the manufacturer’s instructions. For papain or HSA-specific IgE detection, see Supplementary Materials and Methods.

Flow cytometry and cell sorting

Red blood cells were lysed with ACK lysing buffer (BioWhittaker, Walkersville, MD) when needed and the indicated cell surface markers were detected after incubation with CD16/32 blocking antibody 2.4G2 and a 30 min incubation with the appropriate fluorophore-labeled antibody at room temperature. FcεRI was detected using MAR-1 (anti-FcεRIα) antibody. Cells were sorted using a MoFlo^TM cell sorter (Dako Inc.; Carpinteria, CA), and samples at a purity of at least 99.5% were used. All other flow cytometry acquisitions were done with a FACSCalibur (BD Biosciences, San Jose, CA) and Cell Quest software. Analysis was done with Flowjo software (Treestar Inc., Ashland, OR). For intracellular staining, after staining of cell surface markers, cells were fixed with 3.7% paraformaldehyde solution in PBS, permeabilized with permeabilization buffer (PBS, 0.1% BSA, 0.05% saponin) and stained in the same buffer with the indicated anti-cytokine antibody. For CFSE staining, manufacturer’s instructions were followed and a concentration of 10µM of CFSE was used for staining. Then cells were kept over 4 days under normal culture conditions before flow cytometry analysis.

Ex vivo stimulation of blood basophils for IL-4 measurement

Mice of 8–12 weeks of age were used in these experiments. The day before the experiment, mice were sensitized with 100 µg of anti-DNP monoclonal IgE by retro-orbital injection. Blood samples were harvested by cardiac puncture with heparin after animals have been euthanized by CO₂ inhalation. Aliquots of 100 µl of blood were used for each condition. Blood was treated with 20 µg/ml of rat anti-IgE (R35–92) (BD Biosciences) or 20 µg/ml of a rat IgG1 isotype control for 4 hours (IL-4 release) at 37°C (see Figure S4A ). Plasma was harvested and IL-4 measurement realized by ELISA.

Papain or HSA immunization and in vivo basophil stimulation

For papain immunization, the protocol used was previously described (Sokol et al., 2008). Briefly, mice were injected in the left rear foot pad with 50 µg of papain or HSA diluted at 1 mg/ml in PBS. Injections occurred on day 0 and day 14 of the experiment. Mice were bled from the tail vein on a weekly basis and fluid was replenished after each bleed with 200 µl of saline injected subcutaneously into the neck. For in vivo basophil stimulation, age and sex matched W^Sh/W^Sh and W^Sh/W^Sh -lyn^−/− mice were injected with 50 µg per mouse of rat anti-mouse IgE (R35–92) by retro-orbital i.v. injection. Twenty hours later, mice were euthanized and the spleen harvested for T cell analysis.

Ex vivo re-stimulation of spleen T cells and analysis

Spleens from mice (of indicated genotypes) were homogenized and red blood cells lysed in ACK lysing buffer (Biowhittaker). Splenocytes were then resuspended at 3 × 10⁶ cells per ml in RPMI 1640 supplemented with L-glutamine, 10% FBS, 20 mM HEPES, 100 µg/ml streptomycin and 100 U/ml penicillin (all from Invitrogen). Non-specific stimulation was with 40 nM PMA (phorbol 12-myristate 13-acetate; Sigma) and 400 nM ionomycin (Calbiochem) for 4 hours at 37°C / 5% CO₂. For intracellular cytokine analysis, 10 µM monensin (Sigma) was added to the unstimulated and stimulated cells 2 hours prior to the end of the incubation period.

Basophil depletion in vivo

Mice were immunized intravenously three times with 100 µg of anti FcεRIα antibody (clone MAR-1, eBioscience) or isotype control, 144h, 120h and 96h before the day of analysis. Depletion of basophils in the blood and bone marrow was analyzed by flow cytometry.

In vivo challenge with Toxoplasma gondii

Age-matched female WT and lyn^−/− mice were challenged intraperitoneally with 20 cysts of the avirulent ME49 strain of T. gondii. Serum was collected at day 7 post infection, and inflammatory cytokines were measured to monitor the acute immune response in vivo. The number of cysts within the brains of 4-week, chronically infected mice, were also quantified. Briefly, brains were isolated and homogenized by sequential passage through 19- and 21-gauge needles, and cysts were counted microscopically.

Statistical Analysis

For all comparisons between two populations, unpaired two-tailed student t tests were performed. When 3 or more populations were compared, a one way ANOVA test was done first and, if significance was reached (p<0.05), then an unpaired two-tailed student t tests was performed between each compared population, except when indicated. Data are reported as a mean ± standard error of mean (sem), ns.: not significant, *: p<0.05, **: p<0.01, ***: p<0.001. Statistical analysis was performed using Prism software (GraphPad Software, Inc., La Jolla, CA).

Supplementary Material

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Acknowledgements

This work was supported by the intramural research program of the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) of the National Institutes of Health. NC was supported in part by the Fondation pour la Recherche Médicale of France. HCO is supported by NIAID R01-AI054471. We thank Dragana Jankovic (NIAID) and Jinfang Zhu (NIAID) for providing key reagents. We also thank the Laboratory Animal Care and Use Section, the Flow Cytometry Unit, and the Light Imaging Section of NIAMS for support of this work. We are grateful for helpful discussions with Drs. Hajime Karasuyama (Tokyo Medical and Dental University) and Richard L. Stevens (Harvard). The authors have no conflict of interest to declare.

Footnotes

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Supplemental Data

Supplemental Materials and Methods and eight supplemental figures can be found with this article online at http://www.immunity.com/cgi/content/

References

Beavitt SJ, Harder KW, Kemp JM, Jones J, Quilici C, Casagranda F, Lam E, Turner D, Brennan S, Sly PD. Lyn-deficient mice develop severe, persistent asthma: Lyn is a critical negative regulator of Th2 immunity. J. Immunol. 2005;175:1867–1875. [PubMed]
Cannons JL, Yu LJ, Hill B, Mijares LA, Dombroski D, Nichols KE, Antonellis A, Koretzky GA, Gardner K, Schwartzberg PL. SAP regulates T(H)2 differentiation and PKC-theta-mediated activation of NF-κB1. Immunity. 2004;21:693–706. [PubMed]
Capron A, Dombrowicz D, Capron M. Helminth infections and allergic diseases: from the Th2 paradigm to regulatory networks. Clin. Rev. Allergy Immunol. 2004;26:25–34. [PubMed]
Dardalhon V, Korn T, Kuchroo VK, Anderson AC. Role of Th1 and Th17 cells in organ-specific autoimmunity. J. Autoimmun. 2008;31:252–256. [PubMed]
Davidson D, Shi X, Zhang S, Wang H, Nemer M, Ono N, Ohno S, Yanagi Y, Veillette A. Genetic evidence linking SAP, the X-linked lymphoproliferative gene product, to Src-related kinase FynT in T(H)2 cytokine regulation. Immunity. 2004;21:707–717. [PubMed]
DeLisi C, Siraganian RP. Receptor cross-linking and histamine release. I. The quantitative dependence of basophil degranulation on the number of receptor doublets. J. Immunol. 1979;122:2286–2292. [PubMed]
Denzel A, Maus UA, Gomez MR, Moll C, Niedermeier M, Winter C, Maus R, Hollingshead S, Briles DE, Kunz-Schughart LA. Basophils enhance immunological memory responses. Nat. Immunol. 2008;9:733–742. [PubMed]
Flores-Borja F, Kabouridis PS, Jury EC, Isenberg DA, Mageed RA. Decreased Lyn expression and translocation to lipid raft signaling domains in B lymphocytes from patients with systemic lupus erythematosus. Arthritis Rheum. 2005;52:3955–3965. [PubMed]
Gilmartin L, Tarleton CA, Schuyler M, Wilson BS, Oliver JM. A comparison of inflammatory mediators released by basophils of asthmatic and control subjects in response to high-affinity IgE receptor aggregation. Int. Arch. Allergy Immunol. 2008;145:182–192. [PubMed]
Gomez G, Gonzalez-Espinosa C, Odom S, Baez G, Cid ME, Ryan JJ, Rivera J. Impaired FcεRI-dependent gene expression and defective eicosanoid and cytokine production as a consequence of Fyn deficiency in mast cells. J. Immunol. 2005;175:7602–7610. [PubMed]
Hernandez-Hansen V, Bard JD, Tarleton CA, Wilder JA, Lowell CA, Wilson BS, Oliver JM. Increased expression of genes linked to FcεRI Signaling and to cytokine and chemokine production in Lyn-deficient mast cells. J. Immunol. 2005;175:7880–7888. [PubMed]
Hernandez-Hansen V, Smith AJ, Surviladze Z, Chigaev A, Mazel T, Kalesnikoff J, Lowell CA, Krystal G, Sklar LA, Wilson BS, Oliver JM. Dysregulated FcεRI signaling and altered Fyn and SHIP activities in Lyn-deficient mast cells. J. Immunol. 2004;173:100–112. [PubMed]
Hibbs ML, Tarlinton DM, Armes J, Grail D, Hodgson G, Maglitto R, Stacker SA, Dunn AR. Multiple defects in the immune system of Lyn-deficient mice, culminating in autoimmune disease. Cell. 1995;83:301–311. [PubMed]
Janas ML, Hodgkin P, Hibbs M, Tarlinton D. Genetic evidence for Lyn as a negative regulator of IL-4 signaling. J. Immunol. 1999;163:4192–4198. [PubMed]
Kawakami K. Promising immunotherapies with Th1-related cytokines against infectious diseases. J. Infect. Chemother. 2003;9:201–209. [PubMed]
Kepley CL, Youssef L, Andrews RP, Wilson BS, Oliver JM. Syk deficiency in nonreleaser basophils. J. Allergy Clin. Immunol. 1999;104:279–284. [PubMed]
Lavens-Phillips SE, MacGlashan DW., Jr The tyrosine kinases p53/56lyn and p72syk are differentially expressed at the protein level but not at the messenger RNA level in nonreleasing human basophils. Am. J. Respir. Cell Mol. Biol. 2000;23:566–571. [PubMed]
Lighvani AA, Frucht DM, Jankovic D, Yamane H, Aliberti J, Hissong BD, Nguyen BV, Gadina M, Sher A, Paul WE, O’Shea JJ. T-bet is rapidly induced by interferon-γ in lymphoid and myeloid cells. Proc. Natl. Acad. Sci. U S A. 2001;98:15137–15142. [PubMed]
Liu FT, Bohn JW, Ferry EL, Yamamoto H, Molinaro CA, Sherman LA, Klinman NR, Katz DH. Monoclonal dinitrophenyl-specific murine IgE antibody: preparation, isolation, and characterization. J. Immunol. 1980;124:2728–2737. [PubMed]
Martin TR, Galli SJ, Katona IM, Drazen JM. Role of mast cells in anaphylaxis. Evidence for the importance of mast cells in the cardiopulmonary alterations and death induced by anti-IgE in mice. J. Clin. Invest. 1989;83:1375–1383. [PMC free article] [PubMed]
Mukai K, Matsuoka K, Taya C, Suzuki H, Yokozeki H, Nishioka K, Hirokawa K, Etori M, Yamashita M, Kubota T. Basophils play a critical role in the development of IgE-mediated chronic allergic inflammation independently of T cells and mast cells. Immunity. 2005;23:191–202. [PubMed]
Neurath MF, Finotto S, Glimcher LH. The role of Th1/Th2 polarization in mucosal immunity. Nat. Med. 2002;8:567–573. [PubMed]
Nishizumi H, Taniuchi I, Yamanashi Y, Kitamura D, Ilic D, Mori S, Watanabe T, Yamamoto T. Impaired proliferation of peripheral B cells and indication of autoimmune disease in lyn-deficient mice. Immunity. 1995;3:549–560. [PubMed]
Odom S, Gomez G, Kovarova M, Furumoto Y, Ryan JJ, Wright HV, Gonzalez-Espinosa C, Hibbs ML, Harder KW, Rivera J. Negative regulation of immunoglobulin E-dependent allergic responses by Lyn kinase. J. Exp. Med. 2004;199:1491–1502. [PMC free article] [PubMed]
Parravicini V, Gadina M, Kovarova M, Odom S, Gonzalez-Espinosa C, Furumoto Y, Saitoh S, Samelson LE, O’Shea JJ, Rivera J. Fyn kinase initiates complementary signals required for IgE-dependent mast cell degranulation. Nat. Immunol. 2002;3:741–748. [PubMed]
Poorafshar M, Helmby H, Troye-Blomberg M, Hellman L. MMCP-8, the first lineage-specific differentiation marker for mouse basophils. Elevated numbers of potent IL-4-producing and MMCP-8-positive cells in spleens of malaria-infected mice. Eur. J. Immunol. 2000;30:2660–2668. [PubMed]
Reschner A, Hubert P, Delvenne P, Boniver J, Jacobs N. Innate lymphocyte and dendritic cell cross-talk: a key factor in the regulation of the immune response. Clin. Exp. Immunol. 2008;152:219–226. [PubMed]
Schroeder JT, MacGlashan DW, Jr, Lichtenstein LM. Human basophils: mediator release and cytokine production. Adv. Immunol. 2001;77:93–122. [PubMed]
Segal DM, Taurog JD, Metzger H. Dimeric immunoglobulin E serves as a unit signal for mast cell degranulation. Proc. Natl. Acad. Sci. U S A. 1977;74:2993–2997. [PubMed]
Sokol CL, Barton GM, Farr AG, Medzhitov R. A mechanism for the initiation of allergen-induced T helper type 2 responses. Nat. Immunol. 2008;9:310–318. [PubMed]
Spiegl N, Didichenko S, McCaffery P, Langen H, Dahinden CA. Human basophils activated by mast cell-derived IL-3 express retinaldehyde dehydrogenase-II and produce the immunoregulatory mediator retinoic acid. Blood. 2008;112:3762–3771. [PubMed]
Suzuki Y, Conley FK, Remington JS. Importance of endogenous IFN-γ for prevention of toxoplasmic encephalitis in mice. J. Immunol. 1989;143:2045–2050. [PubMed]
Suzuki Y, Orellana MA, Schreiber RD, Remington JS. Interferon-γ: the major mediator of resistance against Toxoplasma gondii. Science. 1988;240:516–518. [PubMed]
Szabo SJ, Kim ST, Costa GL, Zhang X, Fathman CG, Glimcher LH. A novel transcription factor, T-bet, directs Th1 lineage commitment. Cell. 2000;100:655–669. [PubMed]
Tsujimura Y, Obata K, Mukai K, Shindou H, Yoshida M, Nishikado H, Kawano Y, Minegishi Y, Shimizu T, Karasuyama H. Basophils play a pivotal role in immunoglobulin-G-mediated but not immunoglobulin-E-mediated systemic anaphylaxis. Immunity. 2008;28:581–589. [PubMed]
Xiao W, Nishimoto H, Hong H, Kitaura J, Nunomura S, Maeda-Yamamoto M, Kawakami Y, Lowell CA, Ra C, Kawakami T. Positive and negative regulation of mast cell activation by Lyn via the FcεRI. J. Immunol. 2005;175:6885–6892. [PMC free article] [PubMed]
Yamanashi Y, Mori S, Yoshida M, Kishimoto T, Inoue K, Yamamoto T, Toyoshima K. Selective expression of a protein-tyrosine kinase, p56lyn, in hematopoietic cells and association with production of human T-cell lymphotropic virus type I. Proc. Natl. Acad. Sci. U S A. 1989;86:6538–6542. [PubMed]
Yamashita Y, Charles N, Furumoto Y, Odom S, Yamashita T, Gilfillan AM, Constant S, Bower MA, Ryan JJ, Rivera J. Cutting edge: genetic variation influences FcεRI-induced mast cell activation and allergic responses. J. Immunol. 2007;179:740–743. [PubMed]
Youssef LA, Schuyler M, Gilmartin L, Pickett G, Bard JD, Tarleton CA, Archibeque T, Qualls C, Wilson BS, Oliver JM. Histamine release from the basophils of control and asthmatic subjects and a comparison of gene expression between "releaser" and "nonreleaser" basophils. J. Immunol. 2007;178:4584–4594. [PubMed]
Zhou JS, Xing W, Friend DS, Austen KF, Katz HR. Mast cell deficiency in Kit(W-sh) mice does not impair antibody-mediated arthritis. J. Exp. Med. 2007;204:2797–2802. [PMC free article] [PubMed]
Zhu J, Yamane H, Cote-Sierra J, Guo L, Paul WE. GATA-3 promotes Th2 responses through three different mechanisms: induction of Th2 cytokine production, selective growth of Th2 cells and inhibition of Th1 cell-specific factors. Cell Res. 2006;16:3–10. [PubMed]