Preferential Signaling and Induction of Allergy-promoting Lymphokines Upon Weak Stimulation of the High Affinity IgE Receptor on Mast Cells

Keywords: cytokines, Gab2, IgE, LAT, mast cells

Reagents and Antibodies and Radiolabeling.

Dinitrophenyl (DNP)-specific mouse IgE was produced as described previously (15). Rabbit antibodies to ERK2, JNK1, and p38MAPK were from Santa Cruz Biotechnology, Inc., mouse anti-phospho-ERK2, rabbit anti-phospho-JNK, and rabbit anti-phospho-p38MAPK were from Cell Signaling Technologies. Rabbit anti-phospho-Akt (Ser472/473/474) was from BD Biosciences. Secondary antibodies used for immunoblotting were: sheep anti–mouse IgG-horseradish peroxidase (HRP) and donkey anti–rabbit IgG-HRP (Amersham Biosciences). DNP-human serum albumin (DNP-HSA, Ag), monovalent hapten DNP-lysine (Hap), murine thrombin, and ATP were from Sigma-Aldrich. The MEK1 inhibitor, PD 98059, and the p38MAPK inhibitor, SB202190, were from Calbiochem. Iodination of IgE was as described (16) with the exception that an Iodo-Gen precoated tube (Pierce Chemical Co.) was used. The concentration and bindability of the iodinated IgE was determined as described previously (16).

Bone Marrow Isolation and BMMC Culture.

Animals were maintained and used in accordance with National Institutes of Health (NIH) guidelines. Bone marrow was isolated from 8-wk-old SV129 × C57/bl6 wild-type and gene-disrupted mice as described (17, 18). LAT-null mice were kindly provided by L.E. Samelson (NCI, NIH). Cell responses of the genetically-altered mice were compared with age and sex matched wild type litter mates. BMMCs were grown in RPMI media supplemented with FBS and IL-3 as described (17, 18). Because IL-3 induces cytokine mRNA responses, cells were removed from IL-3 for 4 h before stimulation.

Receptor Occupancy and Hapten Experiments.

BMMCs (1 × 10⁶ cells/ml) were incubated in IL-3 free media containing 0.5 μg of IgE/ml (sensitization) during 4 h at 37°C; conditions that result in 100% receptor occupancy. To achieve varying degrees of receptor occupancy the iodinated-DNP-specific mouse IgE was mixed at varying ratios with unlabeled rat IgE of unknown specificity. The degree of FcɛRI occupancy with DNP-specific IgE was determined by direct calculation of the cell surface bound [¹²⁵I]-DNP–specific IgE after removal of the unbound iodinated-IgE by centrifugation through phtalate oils as described (19). The remaining cells were washed twice by centrifugation (300 g, 10 min) to remove the unbound IgE, stimulated with Ag as described below and the extent of degranulation, cytokine mRNA levels and protein secretion, and MAP kinase activation was determined.

To analyze the effect of hapten on cytokine and chemokine mRNA expression, BMMCs were sensitized as above with saturating amounts of unlabeled DNP-specific IgE and washed twice in Tyrodes/BSA (10 mM HEPES, 130 mM NaCl, 5 mM KCl, 1.4 mM CaCl2, 1 mM MgCl2, and 5.6 mM glucose containing 0.05% BSA) at 37°C. Cells were resuspended in the same buffer at 2 × 10⁶/ml and three aliquots (1 ml) were transferred to tubes containing: (a) 20 μl Tyrodes/BSA, or (b) 20 μl of Hap (2.5 mM) or (c) 20 μl of Hap (2.5 mM) + 30 ng Ag and incubated at 37°C. The remaining cells were also stimulated with 30 ng/ml of Ag at 37°C and 1 ml aliquots were placed in three tubes (one at 37°C, one on ice, and other at 37°C with 10 μl of Hap, 5 mM) at the indicated times. Incubation continued for 60 min and hexosaminidase release was then determined. Cell pellets were used for total RNA isolation.

Lysates, Immunoblots, Akt, MAP Kinase Analysis.

For preparation of cell lysates, IgE-sensitized BMMC (7 × 10⁶) were stimulated for various times or with different concentrations of Ag in a final volume of 250 μl. The reaction was stopped by the addition of 250 μl of boiling SDS sample buffer. For MAP kinase experiments using hapten competition, 100 ng/ml of DNP-HSA was added at t = 0, hapten (50 μM final) was added at the indicated times and incubated for 10 min at 37°C before stopping the reaction with SDS sample buffer. Immunoblotting conditions were previously described (20, 21). Akt or MAP kinase phosphorylation was determined by phosphospecific antibodies. Relative quantitation of immunoblots was performed by densitometry. To determine the effect of MAP kinase inhibitors, PD98058 (10 μM, MEK1 and thus ERK2 selective) or SB202190 (5 μM, p38MAPK selective) was added to 5 × 10⁶ cells/ml for 15 min at 37°C before Ag stimulation (100 ng/ml) for 1 h at 37°C. Cells were centrifuged and the pellet was used to isolate total RNA.

Hexosaminidase, Chemotaxis, and Cytokine Assays.

Degranulation was measured by assaying β-hexosaminidase release as described (18). To measure chemotaxis, mast cells were loaded with different amounts of [¹²⁵I]odinated-DNP-specific IgE as described above. Thirty million cells were resuspended in 1 ml of Tyrodes/BSA buffer at 37°C and stimulated with Ag (100 ng/ml) or with an equal volume of buffer for controls. Cells were centrifuged and supernatants collected after 1, 2, or 4 h of stimulation. Chemotaxis was measured by Boyden's blind well chamber technique using 48-well microchambers (Neuroprobe) essentially as described (22). The number of cells migrating through the filter was determined microscopically. To measure lymphokine secretion, BMMCs (5.0 × 10⁷ cells) were incubated with IgE as described above to achieve known receptor occupancies. Cells were stimulated with Ag (10 or 100 ng/ml) at 37°C for varying times (1–24 h) and centrifuged at 4°C for collection of the supernatant. Lymphokine concentration was determined by ELISA (Biosource International) as previously described (23).

RNA Extraction, RT-PCR, and Ribonuclease Protection Assay.

Pellets from stimulated cells (2 × 10⁶ per sample) were solubilized in 1 ml of TRI-Reagent (Molecular Research Center) for 15 min at room temperature. Low abundance mRNA was measured by RT-PCR. First strand cDNA synthesis was with the Life Technologies SuperScript first-strand synthesis system. Primers for amplification of IL-2, IL-3, IL-4, IL-10, and IFN-γ were obtained from CLONTECH Laboratories, Inc., and PCR conditions were: 96°C for 1 min; 2 cycles of 96°C for 1 min and 60°C for 4 min; 28 cycles (IL-4) or 35 cycles (IL-2, IL-3, IL-10, IFN-γ) of 94°C for 1 min, 60°C for 2.5 min, and 72°C for 4 min, additional extension at 72°C for 10 min; and holding at 4°C. Cytokine mRNA detection was linear. Fragments were separated on 2% TBE-agarose gels and a negative exposure was used for densitometric quantitation.

For ribonuclease protection assay (RPA), either commercially available multiprobe template sets or two custom-made templates were used (BD Biosciences). Cytokine genes included were TNF-α, IL-13, M-CSF, LIF, and IL-6. Chemokine genes were MIP1β, MIP1α, MIP-2, LIF, and MCP-1. L32 and GAPDH were control genes. Probe synthesis was with RiboQuant™ In vitro Transcription Kit (BD Biosciences) and 100 μCi of [α-³³P]-UTP (ICN Biomedicals), following the provided protocol. Hybridization used 10 μg of total RNA and 10⁶ cpm of probe in 13 μl of hybridization buffer at 56°C overnight. RNaseI digestion and subsequent steps were per manufacturer's instruction. Precipitation of protected fragments was in the presence of 1.5 μg of carrier, glycoblue (Ambion). Fragment resolution was on a denaturing 6% polyacrylamide gel, which was autoradiographed and developed in Kodak Biomax Transcreen-LE using Kodak BioMax MS (Eastman Kodak Co.) film. Quantitation of the autoradiograph was by densitometry using ImageQuant^® software version 3.3 from Molecular Dynamics. Data was normalized to the control genes L32 or GAPDH.

Mast Cell Cytokine mRNA Profiles Are Similar in Response to Different Stimuli.

We initially tested the hypothesis that differential expression of mast cell cytokine mRNA could occur as a consequence of the stimulus used to activate the cell. Cells were stimulated with IgE/Ag, thrombin, and ATP as the receptors (FcɛRI, proteinase-activated receptor-1 (PAR-1), and purinergic receptors, respectively) responding to each stimulus utilize a different functional coupling mechanism; namely, nonreceptor tyrosine kinases (24), the G_i subclass (25), and the G_q subclass (26) of heterotrimeric GTP binding proteins, respectively. As shown in Fig. 1 , all three stimuli were capable of inducing degranulation. Additionally, all three stimuli induced the same cytokine mRNA profile (Table I). However, ATP showed a tendency to induce a stronger degranulation and cytokine response than IgE/Ag whereas thrombin was only able to induce between 20 to 33% of the IgE/Ag or ATP responses. Notable exceptions were found in IL-2 and the chemokines, MIP-1β, MIP-1α, M-CSF, and MCP-1, where thrombin stimulation elicited ∼50% of the response obtained with IgE/Ag. These initial experiments provided evidence of the potential for mast cells to express similar cytokine profiles to diverse stimuli, but also suggested that differential sensitivity of certain genes to a stimulus could influence the cytokine profile.

Selective IgE-mediated Induction of Mast Cell Cytokine mRNA Occurs in Response to Varying Concentrations of Ag.

To test whether the strength of a stimulus affected the cytokine mRNA profile we focused solely on IgE/Ag-mediated responses because this system was readily amenable to manipulation of the number of receptor aggregates by varying the concentration of Ag or the amount of IgE-occupied FcɛRI. As shown in Fig. 2 A, synthesis of IFN-γ, TNF-α, IL-2, IL-3, IL-4, IL-6, IL-10, IL-13, M-CSF, LIF, MIP-1α, MIP-1β, MIP-2, and MCP-1 was detected by RPA or by RT-PCR. Quantitation of the net response to varying concentrations of Ag revealed that the mRNA acummulation of MIP-1α, MIP-1β, MCP-1, M-CSF, IL-2, and IL-4 reached a maximum at significantly lower concentrations of Ag than IL-3, IL-6, IL-10, LIF, MIP-2, TNF-α, and IFN-γ (Fig. 2, B and C). Quantitative analysis of the effective concentration of Ag required for 50% of the maximal response (EC₅₀), summarized in Table II, showed that genes, like MIP-1α and β, were extremely sensitive to small amounts of Ag whereas others, like IL-3 and IL-10, required almost 10-fold higher concentrations to reach their EC₅₀. Interestingly, some chemokine and cytokine mRNA responses reached their EC₅₀ at concentrations of Ag that elicited minimal degranulation (Fig. 1). This suggested that aggregation of small numbers of receptors was sufficient to induce increased mRNA levels of most tested chemokines and some cytokines in the absence of substantial degranulation.

The Level of Receptor Occupancy with IgE/Ag Causes Differential Expression of Cytokines and Chemokines.

It has been suggested that atopic individuals may differ in their symptoms because the amounts of allergen-specific IgE produced, and thus receptor occupancy, may differ (27, 28). To test this hypothesis cells were loaded with [¹²⁵I]-labeled DNP-specific mouse IgE at varying ratios in order to obtain a known occupancy of receptors. Fig. 3 A shows that degranulation is relatively linear with increasing receptor occupancy, thus, half maximal degranulation is reached when ∼50% of the receptors are occupied. In contrast, ∼50% of the maximal mRNA accumulation for MCP-1, M-CSF, and IL-4 was observed with ∼10% of the receptors occupied by IgE/Ag (Fig. 3, B and C). 50% of the maximal mRNA response for MIP-1α, MIP-1β, and MIP-2, required between 30–40% IgE receptor occupancy. IL-2, IL-6, IL-13, TNF-α, and LIF required greater than 40% IgE receptor occupancy and IL-10 showed a half-maximal response only when receptor occupancy was greater than 60%.

We tested if extracellular secretion of lymphokines could occur under conditions of low receptor occupancy. For most lymphokines, 5 h of stimulation was sufficient to reach maximal secretion. When 10% of FcɛRI was occupied by IgE/Ag MCP-1 was potently secreted, with 2076.5 pg/10⁶cells detected in the medium (Table III). This represented ∼42.9% of the secretion observed at 100% occupancy. IL-6, MIP-1β, MIP-1α, and TNF-α were also secreted at amounts that were 10.9%, 48.8%, 48.0%, and 34.1% of the maximal secretion, respectively (Table III). The secreted lymphokines induced a potent monocyte/macrophage (J774.1 cells) chemotactic response that was similar to that observed at full receptor occupancy (Fig. 3 D). MCP-1 alone, at concentrations similar to those secreted (1,000–4,000 pg/10⁶ cells), caused a comparable chemotatic response. IL-4 and IL-10 were not detected at 5 h after stimulation but small amounts were detected after 24 h of stimulation. Under low receptor occupancy only IL-4 was detected whereas IL-10 was not (Table III). Quantitation of secreted IL-6 and secreted MCP-1 showed that at low receptor occupancy almost a 10-fold difference in absolute amounts of these lymphokines exists whereas this difference is strikingly narrowed (2.3-fold) at high receptor occupancy. With the exception of TNF-α, the results are consistent with a skewed lymphokine response that favors chemokines at low receptor occupancy. FcɛRI-mediated TNF-α mRNA responses required higher Ag concentrations or receptor occupancy (Figs. 2 and 3), however, substantial secretion of TNF-α, upon a weak stimulus, may result as a consequence of its synthesis and storage in preformed secretory granules (8).

IgE/Ag-induced Gene Responses Show Dependence or Early Independence of Receptor Aggregation.

We previously demonstrated that TNF-α, which requires high receptor occupancy for complete induction, was exquisitely sensitive to disaggregation of receptors by a monovalent hapten (29). We reasoned that it was possible that genes that required a larger number of receptor aggregates could be most sensitive to disruption of receptor aggregates and vice versa. As shown in Fig. 4 A, degranulation of mast cells required continued aggregation of receptors for up to 6 min after Ag addition. At 3 min after Ag addition, hapten addition resulted in ∼50% of the normal degranulation response. The inhibitory effect of hapten was mirrored by the addition of ice cold buffer indicating the interruption of an active process.

Analysis of the sensitivity of the cytokine and chemokine mRNA response to hapten revealed three distinct responses: first, mRNA accumulation of MIP-1α, MIP-1β, MCP-1, and M-CSF became rapidly independent (between 1–4 min after Ag stimulation) of continued receptor aggregation (Fig. 4 B). Second, mRNA accumulation of LIF, IL-3, IL-4, IL-6, and IL-10 became independent of continued receptor aggregation between 4 and 8 min after Ag stimulation (Fig. 4 C). Third, mRNA accumulation of IL-2, IL-13, IFN-γ, and TNF-α required more than 15 min to show considerable independence of receptor aggregation (Fig. 4 D). Non-linear regression analysis of the mRNA response of IL-2, IL-13, IFN-γ, and TNF-α showed two distinct groups (IL-2, IFN-γ and IL-13, TNF-α). However, the cytokines in each group showed a poor fit suggesting additional regulatory complexity in their response. With some notable exceptions, like IL-2, most mRNA responses induced at low Ag concentrations or low receptor occupancy became rapidly independent of continued receptor aggregation. Vice versa, those genes requiring high Ag concentrations or high receptor occupancy for induction were more dependent on continued receptor aggregation. Despite the noted exception, this suggests that most genes the respond to low Ag or low receptor occupancy conditions can generate intermediates that rapidly become independent of continued receptor aggregation.

Low Ag or Low Receptor Occupancy Favors Fyn/Gab2-initiated Signals and Activation of p38MAPK.

Fyn and Lyn kinases are FcɛRI-proximal Src family kinases that propagate signals through the adapters Gab2 and LAT, respectively (30, 31). Gab2 is required for the activation of PI3K and Akt in mast cells whereas LAT is essential for calcium responses (30, 32). We now tested if low Ag concentrations or low receptor occupancy could induce the phosphorylation of the adapters Gab2 or LAT and other proteins downstream of these adapters. As shown in the upper panel of Fig. 5 A, low Ag concentrations were substantially effective in stimulating the phosphorylation of Fyn, Gab2, and the p85subunit of PI3K. Because the available antibodies to Gab2 are of relatively low affinity, phosphorylated Gab2 was difficult to detect below stimulation with 1 ng/ml of Ag. LAT phosphorylation was detected at 10 ng/ml of Ag. Low receptor occupancy also showed a preference for the Gab2/PI3K pathway as the downstream kinase, Akt, was phosphorylated to ∼80% of that seen at high receptor occupancy (Fig. 5 A, bottom panel). In contrast, ERK2 phosphorylation, which depends primarily on LAT (18), was at best 15% of that seen at high receptor occupancy when films were overexposed. This suggested that the Gab2/PI3K pathway was more sensitive in its response to low Ag or low receptor occupancy. Given our previous finding of a potent MCP-1 response under conditions of low Ag concentrations or low receptor occupancy, this suggested that Gab2 might be more important to MCP-1 mRNA responses than LAT. As shown in Fig. 5 B, Gab2-deficient mast cells were defective in the mRNA response of all the tested chemokines whereas cytokine mRNA responses were less severely affected. Strikingly, LAT-deficient mast cells showed the opposite profile with cytokines being more severely inhibited than chemokines. In Gab2-deficient mast cells, the inhibition of chemokine mRNA responses ranged from 65–90% depending on receptor occupancy. Gab2 deficiency also caused a 10–30% inhibition of cytokine mRNA responses. In contrast, LAT-deficient mast cells showed an inhibition of chemokine mRNA responses that ranged from 15–40% with MCP-1 and LIF mRNA responses being unaffected. Cytokine mRNA responses were more severely affected by LAT-deficiency with inhibition ranging from 40–80%.

To gain a further understanding of the influence of receptor occupancy on gene expression we explored the activation (phosphorylation) of MAP kinases. Fig. 6 A demonstrates that p38MAPK was most sensitive to low receptor occupancy with 50% of the maximal phosphorylation detected at ∼12% receptor occupancy. In contrast, ERK2 required more than 25% and JNK1 more than 40% occupancy to reach 50% of their maximal phosphorylation levels. It should be noted that consistent with greater sensitivity of Gab2 to weak stimulation, a previous report demonstrated impaired p38MAPK activation in Gab2-deficient mast cells (32). We postulated that if p38MAPK activation is required for production of lymphokines that respond under weak stimulation, like MCP-1, its activation should become rapidly independent of continued receptor aggregation (like MCP-1). Fig. 6 B shows that p38MAPK becomes rapidly independent of continued receptor aggregation (1 min). The activation of both JNK and ERK2 showed a greater dependence on continued receptor aggregation with independence being achieved after 3 and 8 min post-Ag stimulation, respectively (graph, Fig. 6 B). Together, with prior results on chemokine responses, these findings demonstrate a relationship between sensitivity to a weak stimulus and rapid independence of continued receptor aggregation. They also provide suggestive evidence for the importance of p38MAPK in the mRNA responses (like MCP-1) induced by low Ag concentration or low receptor occupancy.

MCP-1 and TNF-α Genes Show Preferential Requirement for p38MAPK or ERK2, Respectively.

While it is well understood that multiple pathways cooperate with the activity of MAP kinases in gene expression (33, 34), we tested whether the mRNA accumulation of some cytokines and chemokines showed preferential sensitivity to inhibition of p38MAPK and ERK2 by SB202190 and PD98059, respectively. We chose to analyze the sensitivity of two genes that well-represent mRNA induction by low IgE/Ag receptor occupancy (MCP-1) or high IgE/Ag receptor occupancy (TNF-α; see Fig. 3, B and C). Fig. 7 shows that pretreatment of BMMC with PD98059 inhibited TNF-α mRNA accumulation by 70%, whereas SB 202190 inhibited TNF-α mRNA accumulation by ∼30%. In contrast, SB202190 inhibited MCP-1 mRNA accumulation by almost 80% whereas PD98059 had no effect on the mRNA accumulation of this chemokine. However, another analyzed gene, IL-13, which responded to intermediate Ag concentrations or receptor occupancy, was not as effectively inhibited (∼23–38%) by SB202190 or PD98059. The findings are consistent with induction of p38MAPK and ERK2 at low or high IgE/Ag receptor occupancy as important in MCP-1 and TNF-α mRNA responses, respectively.

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