Reduced Ag-triggered degranulation in Lyn-deficient mast cells despite suppressed tyrosine phosphorylation of SHIP1
In hematopoietic cells, the lipid phosphatase SHIP1 is a crucial negative regulator of PI3K-mediated processes and compared to wt BMMCs, SHIP1-deficient (-/-) BMMCs showed augmented Ca2+ mobilization, degranulation, PKB activation, and pro-inflammatory cytokine production in response to IgE/Ag stimulation [10, 12, 13]. SHIP1 phosphatase activity was connected to Lyn-mediated Y-P of SHIP1 [5]. Thus, it was not unexpected that Lyn deficiency resulted in enhanced Ag-triggered degranulation, PKB phosphorylation, and cytokine production as well [4–8]. However, Lyn-/- BMMCs have also been reported repeatedly to degranulate less compared to respective wt cells [6, 9, 14], which does not accord with the reported functional interaction between Lyn and SHIP1. So far, the functional connection between Lyn and SHIP1 was not addressed in such Lyn-deficient BMMCs that showed reduced degranulation compared to wt cells.
To learn about the functional interaction of Lyn and SHIP1 in such cells, we used Lyn-/- BMMCs (C57BL/6 x 129/Sv background), which showed severely decreased overall substrate Y-P (Fig. 1a), Ca2+ mobilization (Fig. 1b; Additional file 1: Figure S1), and attenuated degranulation in response to Ag stimulation (Fig. 1c) compared to wt cells. Next, we analyzed if Lyn was controlling SHIP1 Y-P and PKB phosphorylation in these cells as well. Indeed, SHIP1 Y-P was abrogated in Ag-triggered Lyn-/- BMMCs (Fig. 1d). In correlation, Ag-induced phosphorylation of PKB was augmented immensely in Lyn-/- BMMCs compared to wt cells (Fig. 1e). In line with increased PKB phosphorylation/activation, IL-6 and TNF-α production after Ag-mediated FcεRI crosslinking were strongly increased in Lyn-/- BMMCs compared to wt BMMCs (Fig. 1f). In conclusion, reduced Ca2+ mobilization and degranulation did manifest even in the presence of attenuated SHIP1 phosphorylation/activation in Lyn-deficient BMMCs.
Normal differentiation of BMMCs double-deficient for Lyn and SHIP1
Suppressed Y-P of SHIP1 indicated reduced SHIP1 activation in Lyn-/- BMMCs, which would be expected to coincide with augmented PKB phosphorylation, Ca2+ mobilization, degranulation, and pro-inflammatory cytokine production [10, 12, 13]. However, only PKB phosphorylation and cytokine production were enhanced (Fig. 1). Thus, results shown in Fig. 1 suggested that Lyn deficiency concurs with reduced SHIP1 activity with respect to Ag-triggered PKB phosphorylation and cytokine production, but is dominant over the effect of suppressed SHIP1 Y-P/activation with respect to Ca2+ mobilization and degranulation. To follow this up, Lyn- and SHIP1-deficient mice were crossed and mice from the finally resulting F2 generation (wt, Lyn-/-, SHIP1-/-, and Lyn/SHIP1-double-deficient (dko) mice) were used for the differentiation of BMMCs. These cells differentiated comparably as shown by FACS analysis of 5-6 week old cells measuring expression of MC surface markers, FcεRI and Kit (Additional file 2: Figure S2A). Moreover, similar expression of the IL-33 receptor, ST2, and the myeloid cell marker, CD13, were observed (Additional file 2: Figure S2B). Deficiency of Lyn and SHIP1 was verified by Western blotting (Additional file 2: Figure S2C). Compared to wt cells, single- and double-deficient BMMCs proliferated significantly stronger, which is in agreement with a negative role of Lyn and SHIP1 in IL-3-induced proliferation of BMMCs and colony-forming progenitors (Additional file 2: Figure S2D; [15–17]). Moreover, analysis of degranulation in response to ionophore treatment showed no impairment in these cells (data not shown). In conclusion, Lyn and SHIP1 single- and double-deficiencies allowed BMMC differentiation and thus further analysis of these cells.
Production of pro-inflammatory cytokines is controlled by both Lyn and SHIP1
We have shown previously that SHIP1 restricts Ag-triggered production of IL-6 [13]. Moreover, Lyn deficiency resulted in increased IL-6/TNF-α production in response to Ag (Fig. 1; [6]). This suggested that Lyn/SHIP1 double-deficiency should also, or even more, cause augmented production of pro-inflammatory cytokines. To investigate this, wt, Lyn-/-, SHIP1-/-, and dko BMMCs were preloaded with DNP-specific IgE and subsequently stimulated with increasing concentrations of Ag (DNP-HSA). Production/secretion of IL-6 and TNF-α were determined by ELISA. Wt cells showed the typical bell-shaped dose-response pattern with strong suppression of IL-6/TNF-α production starting at 200 ng/ml Ag (Additional file 3: Figure S3A & B). Compared to wt cells, all single- and double-deficient BMMCs secreted markedly more IL-6/TNF-α at all Ag concentrations tested (Fig. 2a & b; Additional file 3: Figure S3A & B). A comparable response pattern was observed when Ag-induced production of Il6 and Tnf mRNA was measured (Fig. 2c & d; Additional file 3: Figure S3C & D). Although pro-inflammatory cytokine mRNA was enhanced in Lyn-/- compared to wt BMMCs in response to optimal Ag concentration in several experiments (Additional file 4: Figure S4), statistical analysis did not yield significance (Fig. 2c & d). Augmented cytokine production correlated with enhanced PI3K-dependent phosphorylation of PKB in Lyn-/- and SHIP1-/- BMMCs (Fig. 1; [13]). Hence, we expected strong PKB phosphorylation in Ag-triggered dko BMMCs as well. Indeed, PKB phosphorylation in dko BMMCs was stronger than in wt and Lyn-/- cells, however, weaker than in SHIP1-/- BMMCs (Fig. 2e). Our results confirmed that Ag-triggered, PI3K-dependent Il6/IL-6 and Tnf/TNF-α production is controlled by Lyn and SHIP1. Moreover, Lyn co-deficiency seemed to weakly attenuate the SHIP1-/- phenotype, suggesting involvement of (a) stimulatory Lyn-dependent pathway(s). Furthermore, the quantitative differences between Lyn-/-, SHIP1-/-, and dko BMMCs indicated that single Lyn deficiency does not coincide with a complete blockade of SHIP1 function.
Lyn deficiency dominates the SHIP1-deficient phenotype with respect to degranulation and preceding signaling events
In comparison to PKB phosphorylation and cytokine production, which were enhanced in both Lyn-/- and SHIP1-/- BMMCs (Fig. 1; [10]), degranulation and Ca2+ mobilization showed opposing trends (Fig. 1; [10]). We used wt, Lyn-/-, SHIP1-/-, and dko BMMCs to challenge the idea whether Lyn deficiency is dominant to a lack of SHIP1 expression with respect to degranulation and preceding signaling processes. IgE-preloaded cells were stimulated with increasing concentrations of Ag and, subsequently, release of the granular enzyme β-hexosaminidase was measured. In response to an optimal concentration of Ag (20 ng/ml DNP-HSA), SHIP1-/- BMMCs, as expected [10], degranulated far stronger than wt cells (Fig. 3a). In both comparisons (wt vs. Lyn-/- as well as SHIP1-/- vs. dko), Lyn-deficiency resulted in significantly reduced degranulation, particularly evident when comparing SHIP1-/- and dko BMMCs (Fig. 3a). In accordance with our initial analysis of wt and Lyn-/- BMMCs (Fig. 1), which showed reduced degranulation in Lyn-/- BMMCs in the absence of SHIP1 Y-P, co-deficiency of Lyn strongly suppressed degranulation of SHIP1-deficient BMMCs. This effect was especially obvious in response to high, supra-optimal Ag concentrations (Additional file 5: Figure S5A).
Store-operated Ca2+ entry is a prerequisite for Ag-triggered degranulation. Compared to wt BMMCs, Lyn-/- BMMCs have been reported to show a suppressed Ca2+ signal ([4–7, 9] and Fig. 1). SHIP1-/- BMMCs, on the other side, exerted markedly enhanced Ca2+ mobilization in response to Ag [10, 12]. This was corroborated in Ag (20 ng/ml DNP-HSA)-stimulated wt, Lyn-/-, and SHIP1-/- BMMCs (Fig. 3b). Interestingly, dko BMMCs showed no immediate Ca2+ flux (representing the time window most crucial for degranulation), as compared to wt and SHIP1-/- BMMCs. A slow and steady increase in intracellular Ca2+ content, however, was observed to reach the level seen in wt cells approximately 2 min after Ag addition (Fig. 3b). In principle, the same response pattern was found after stimulation with high to supra-optimal Ag concentrations (200 and 2000 ng/ml DNP-HSA), however, the delayed and sustained increase in intracellular Ca2+ was faster and reached higher levels (Additional file 5: Figure S5B). These data demonstrated that Lyn co-deficiency is able to dominate the SHIP1-deficient phenotype and that characteristics of the SHIP1-/- phenotype might prevail at higher Ag concentrations.
Next, we checked FcεRI-mediated signaling events preceding Ca2+ mobilization and degranulation. Wt, Lyn-/, SHIP1-/-, and dko BMMCs were stimulated with Ag for 1, 5, and 15 min and overall substrate Y-P was analyzed by anti-P-Tyr immunoblotting. Correlating with the qualitative response patterns for Ca2+ mobilization and degranulation, wt and SHIP1-/- BMMCs exerted strong substrate Y-P, whereas Y-P events were blunted in Lyn-/- and dko BMMCs (Fig. 3c). In order to understand how Lyn deficiency regulated degranulation as well as Ca2+ mobilization, we focused on the transmembrane adapter protein LAT1 as well as PLC-γ1. Both proteins are involved in the induction of Ca2+ mobilization [18] and the accepted model of FcεRI signaling involves Lyn in the phosphorylation of both in an indirect manner: LAT1 via Lyn-mediated Syk activation and PLC-γ1 via Lyn-mediated Btk activation [3]. Most notable with respect to the induction of Ca2+ release was the 1 min time point. Whereas LAT1 phosphorylation was induced in wt and SHIP1-/- BMMCs 1 min after Ag addition, this response was only marginal in Lyn-/- and dko BMMCs (Fig. 3d), fitting to the observed Ca2+ signals (Fig. 3b). Whereas in wt cells P-LAT1 was strongest at 5 min and still visible after 15 min, SHIP1-/- BMMCs showed highest LAT1 Y-P at 1 min and considerably reduced signals at 5 and 15 min. Corresponding differences were found between Lyn-/- and dko BMMCs with dko cells showing weaker P-LAT1 signals than Lyn-/- BMMCs particularly at 5 and 15 min (Fig. 3d). The phospho-specific anti-LAT1 antibody used in this analysis is known to also recognize phosphorylated LAT2 (a.k.a. NTAL) [19]. P-LAT2 followed the pattern described for P-LAT1 (Fig. 3d), suggesting comparable dependence on presence of Lyn and/or SHIP1. With respect to signal initiation, PLC-γ1 Y-P showed a comparable pattern to LAT1 Y-P. Ag-triggered wt and SHIP1-/- BMMCs showed PLC-γ1 Y-P within 1 min, whereas in Lyn-/- and dko BMMCs only marginal signals were detectable (Fig. 3d). Conspicuously, PLC-γ1 Y-P was by far strongest in SHIP1-/- BMMCs, suggesting that PIP3-dependent signaling processes promote PLC-γ1 Y-P, at least in SHIP1-deficient cells [20]. The 160 kDa protein, heavily tyrosine-phosphorylated in SHIP1-/- BMMCs (Fig. 3c), most likely represented PLC-γ1. In conclusion, Lyn co-deficiency dominated the SHIP1-/- phenotype of BMMCs concerning degranulation and preceding signaling events.
In response to FcεRI engagement, LAT1 scaffolds several protein complexes at the plasma membrane, amongst them Grb2/Sos resulting in phosphorylation/activation of the MAPKs ERK1/2. Indeed, early phosphorylation (1 min) of ERK1/2 was reduced in Lyn-/- and dko BMMCs compared to wt and SHIP1-/- cells, respectively, correlating with Y-P of LAT1. A comparable pattern was observed for Ag-triggered phosphorylation of the MAPK p38 (Fig. 3e). Attenuated early, Ag-triggered phosphorylation of ERK1/2 and p38 in Lyn-/- BMMCs was in agreement with previous work by Xiao et al. [4].
Lyn deficiency reveals Ca2+-dependence of Ag-triggered activation of the NFκB pathway
NFκB activation downstream of the FcεRI depends on the adaptor protein Bcl-10 and the paracaspase MALT1. It is crucial for Ag-stimulated MC activation, particularly with respect to pro-inflammatory (Il6 and Tnf) gene transcription [21]. Here, we analyzed the impact of Lyn deficiency on NFκB activation in the context of wt and SHIP1-/- BMMCs. Wt, Lyn-/-, SHIP1-/-, and dko BMMCs were stimulated with Ag and phosphorylation of IKKα/β was analyzed by immunoblotting. Particularly evident was the reduced phosphorylation of IKKβ after 5 min of Ag stimulation in Lyn-/- and dko BMMCs (Fig. 4a). Additionally, phosphorylation as well as subsequent degradation of IκBα were studied. Comparable phosphorylation of IκBα was observed after 5 min in wt and SHIP1-/- BMMCs correlating with degradation/disappearance of IκBα (5 and 15 min) (Fig. 4a). In accordance with previous data, basal IκBα expression in SHIP1-/- BMMCs was stronger than in wt cells [13], and a comparable behavior was observed in dko and Lyn-/- BMMCs. The underlying mechanism, however, is unknown at present. Unexpectedly, IκBα phosphorylation was markedly reduced and degradation retarded in Lyn-/- and dko compared to wt and SHIP1-/- BMMCs, respectively (Fig. 4a), despite augmented production of IL-6 and TNF-α (Fig. 2a & b). Thus, Lyn plays a positive regulatory role for NFκB activation in Ag-triggered wt and SHIP1-/- BMMCs.
Due to the discrepancy between IκBα phosphorylation/degradation and pro-inflammatory cytokine production, we next determined if the observed pattern of IκBα phosphorylation/degradation proceeded to transcription of typical NFκB target genes. The genes for IκBα (Nfkbia) and the deubiquitinating enzyme A20 (Tnfaip3) have been demonstrated to be transcribed in an NFκB-dependent manner [22, 23]. Therefore, production of Nfkbia and Tnfaip3 mRNA in Ag-stimulated (90 min) wt, Lyn-/-, SHIP1-/-, and dko BMMCs were measured using RT-qPCR. Indeed, differences in respective mRNA production in response to an optimal Ag concentration (20 ng/ml) followed the pattern observed for IκBα phosphorylation and degradation (Fig. 4b). Lyn-/- and dko BMMCs produced markedly less Nfkbia and Tnfaip3 mRNA compared to wt and SHIP1-/- BMMCs, respectively, with the most dramatic difference seen between SHIP1-/- and dko cells (Fig. 4b). Though Nfkbia and Tnfaip3 mRNA production was increased in wt vs. Lyn-/- BMMCs in response to 20 ng/ml Ag in several experiments (Additional file 6: Figure S7), combined differences did not yield statistical significance (Fig. 4b). In SHIP1-/- BMMCs, particularly high levels of Nfkbia and Tnfaip3 mRNAs were measured, in line with the strong negative regulation of the NFκB pathway by SHIP1 [13]. In agreement with a negative regulatory role of SHIP1 particularly in response to high doses of Ag [10, 24], dko BMMCs produced higher levels of Nfkbia and Tnfaip3 mRNA compared to wt cells (Additional file 7: Figure S6A).
Interestingly, the observed pattern of NFκB activation strongly correlated with Ag-induced Ca2+ mobilization and LAT1/PLC-γ1 Y-P in the cell types under investigation (compare to Fig. 3). Therefore, we speculated that Ag-triggered NFκB activation contains a Ca2+-dependent signaling step. Consequently, wt and SHIP1-/- BMMCs were left untreated or stimulated with Ag for 5 min after short-term EDTA- or vehicle treatment and phosphorylation/degradation of IκBα was analyzed. In both cell types, EDTA-mediated depletion of extracellular Ca2+ (and thus abrogation of store-operated Ca2+ entry) resulted in attenuated IκBα phosphorylation/degradation compared to control treatment (Additional file 7: Figure S6B). This pointed to a positive function of store-operated Ca2+ entry upstream of FcεRI-mediated IκBα phosphorylation. In this respect, the Ca2+/calmodulin-dependent phosphatase calcineurin has been demonstrated in T helper cells to contribute to activation of the NFκB pathway [25]. Our further analysis revealed that Ag-triggered IκBα phosphorylation/degradation was significantly attenuated in wt and SHIP1-/- BMMCs pre-incubated with the calcineurin inhibitor cyclosporine A (CsA) (Fig. 4c & d; Additional file 7: Figure S6C & D). Consequently, Ag-induced production of Nfkbia and Tnfaip3 mRNA were significantly suppressed in CsA-pretreated SHIP1-/-BMMCs (Fig. 4e). In conclusion, by governing FcεRI-mediated Ca2+ mobilization, Lyn acts as positive regulator of calcineurin-controlled NFκB activation and transcription of NFκB-dependent genes.
Identification of genes transcribed in a Lyn/Ca2+/calcineurin-dependent manner
To learn more about Lyn/Ca2+/calcineurin-dependent gene transcription, a preliminary Affymetrix transcriptome analysis was carried out comparing Ag-triggered SHIP1-/- and dko BMMCs. Transcription of Tnip3, Il1a, Il1b, Tnfsf9, and Stx11 appeared to be significantly reduced in dko vs. SHIP1-/- BMMCs (data not shown). For verification, wt, Lyn-/-, SHIP1-/-, and dko BMMCs were stimulated with Ag for 90 min and mRNA production was analyzed by RT-qPCR. Indeed, in response to 20 ng/ml Ag, wt and SHIP1-/- BMMCs showed stronger production of Tnip3, Il1a, Il1b, Tnfsf9, and Stx11 mRNAs compared to Lyn-/- and dko BMMCs, respectively, though statistical significance was only reached in SHIP1-/- cells (Fig. 5a; Additional file 8: Figure S8). Again, SHIP1-/- BMMCs produced particularly high levels of the respective mRNAs, which is in line with the dominant control of the NFκB pathway by SHIP1 [13]. In agreement with the calcineurin-mediated regulation of Nfkbia and Tnfaip3 mRNA production described above (Fig. 4e), CsA treatment significantly attenuated transcription of Tnip3, Il1a, Il1b, Tnfsf9, and Stx11 in SHIP1-/- BMMCs (Fig. 5b). Interestingly, in addition to Nfkbia and Tnfaip3, Tnip3, Il1a, and Il1b are also known to be transcribed in an NFκB-dependent fashion [26–28]. These data indicate that a Lyn/Ca2+/calcineurin/NFκB-dependent signaling pathway is functional for the regulation of Ag-triggered transcription of various genes that are most likely involved in pro-inflammatory processes.