Role of Tyrosine Kinases in Igli-mediated Signal Transduction in Human Lung Mast Cells and Basophils S. E. Lavens, P. T. Peachell, and J. A. Warner Department of Physiology and Pharmacology, University of Southampton, Southampton, and Department of Pharmaceutical Chemistry, School of Pharmacy, London, United Kingdom

Recent evidence suggests that tyrosine kinases play an important role in signal transduction mechanisms utilized by a range of different agonists in many cell types. We have investigated the effectsof four different inhibitors of tyrosine kinases on IgE-dependent histamine release from human lung mast cells and basophils. Genistein inhibited the anti-IgE-induced histamine release from human basophils (at 10 1LM genistein, inhibition = 55 ± 5%, n = 17, P < 0.005) with an IC so of 8 1LM, but was much less effective in the human lung mast cell (at 10 1LM, inhibition = 18 ± 6%, n = 11, P < 0.05). Two inactive analogs of genistein, genistin and diadzein, failed to affect anti-IgE-induced histamine release significantly in either mast cells or basophils. A second inhibitor of tyrosine kinases, tyrphostin 25, inhibited IgE-dependent release from basophils (at 10 1LM, inhibition = 25 ± 7 %, n = 6, P < 0.05) though it was less effective than genistein and failed to affect IgE-induced histamine release from human lung mast cells (at 10 1LM, inhibition = 22 ± 16%, n = 5, P = NS). In contrast, methyl 2,5 dihydroxycinnamate (MDC) failed to inhibit anti-IgE-dependent histamine release in human basophils (at 10 1LM, inhibition = 3 ± 3%, n = 5, P = NS) but proved to be an effective inhibitor of anti-IgE-induced degranulation in human lung mast cells (at 10 1LM, inhibition = 53 ± 16%, n = 5, P < 0.05). Finally, a fourth drug, lavendustin A, failed to inhibit histamine release from either basophils or lung mast cells. The ability of the drugs to affect histamine release in the different cell types did not appear to reflect their reported activity as inhibitors of epidermal growth factor receptor autophosphorylation or their putative sites of action on the enzyme. Genistein did not appear to be inhibiting protein kinase C as it failed to affect the release of histamine in basophils challenged with 10 ng/rnl phorbol myristate acetate (at 10 1LM, inhibition = 7 ± 4 %, n = 5, P = NS). The differences between basophils and mast cells could not simply be ascribed to the different concentrations of anti-IgE used to initiate release as the inhibition caused by 10 1LM genistein in both cell types was independent of the concentration of anti-IgE used and the net release of histamine. The efficacyand potency of genistein and lavendustin A in human basophils were not altered by increasing the pre-incubation period to 2 h. In summary, inhibitors of tyrosine kinases inhibit IgE-dependent histamine release from both human lung mast cells and basophils, though these two cell types exhibit distinct inhibitory profiles, possibly reflecting differences in IgE-dependent signal transduction mechanisms in these two cell types.

We are interested in the signal transduction mechanisms utilized by IgE in human lung mast cells and basophils. Previous evidence has shown that the crosslinking of IgE leads to an increase in intracellular Ca 2 + ([Ca 2 +] i) (1, 2) and an in(Received in originalform March 2. 1992 and in revisedform July 2. 1992) Addresscorrespondence to: 1. A. Warner, Ph.D., Department of Physiology and Pharmacology, University of Southampton, Bassett Crescent East, Southampton S09 3TU, United Kingdom. Abbreviations: intracellular Ca2 +, [Ca2 +]i; dimethyl sulfoxide, DMSO; epidermal growth factor, EGF; formylmethionylleucylphenylalanine, FMLP; methyl 2,5 dihydroxycinnamate, MDC; Pipes-albumin-glucose, PAG; Pipes-albumin-glucose supplemented with CaCl, and MgCh, PAGCM; Pipes supplemented with bovine serum albumin, DNase, Mg2+, Ca2+, and glucose, Pipes+; protein kinase C, PKC; phorbol myristate acetate, PMA. Am. J. Respir. Cell Mol. Bioi. Vol. 7. pp. 637-644, 1992

crease in membrane-associated protein kinase C (PKC) (3). However, the evidence for the involvement of the "canonical" phosphoinositol-specific phospholipase C pathway is far from complete and many questions remain to be answered. The realization that the crosslinking of IgE leads to the synthesis of a range of cytokines (4,5), in addition to the release of short-term mediators such as histamine, suggests that we must consider the regulation of both long-term and shortterm responses. Indeed, recent evidence suggests that the low-affinityIgE receptor may act as a growth factor in some cell types (6, 7). The role of tyrosine kinases in the signal transduction mechanisms of growth factors has been appreciated for a number of years (for review, see reference 8). Receptors for a number of growth factors such as epidermal growth factor

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(EGF) have intrinsic tyrosine kinases in their cytoplasmic regions that, when activated, modify tyrosine residues on both the receptor itself and appropriate target proteins, transducing the external signal into the cell interior. Receptors that use this signal transduction mechanism are comparatively large, spanning the membrane and including both extracellular ligand binding domains and intracellular effector enzymes. These tyrosine kinase-dependent signal transduction mechanisms were originally associated with growth factor receptors where they regulated long-term responses such as control of cell replication and protein synthesis. Recently, however, a number of ligands in diverse cell types have been shown to lead to an increase in tyrosine phosphorylation (9, 10) in addition to the activation of other signal transduction mechanisms. It is not yet clear how these tyrosine kinases are integrated with the other signal transduction mechanisms but several alternative mechanisms are possible. The tyrosine kinase could fulfill an intermediary role, linking the occupied receptor to an intracellular effector such as r phospholipase C (11, 12). Alternatively, tyrosine kinases could be activated in parallel with other effectors such as phospholipase C and modulate long-term responses to the stimulus. There is evidence to suggest that tyrosine kinases may fulfill both roles and it is possible that the mechanism will depend on the ligand and the cell type. Recent evidence from rodent mast cells and mast cell lines has shown that the crosslinking of IgE leads to an increase in tyrosine phosphorylation (13-15), suggesting that a tyrosine kinase may be involved in either the activation of phospholipase C or regulation of long-term responses. It therefore seemed appropriate to investigate the effects of a panel of tyrosine kinase inhibitors on the release of histamine from human lung mast cells and basophils.

Materials and Methods Materials Formylmethionylleucylphenylalanine (FMLP), phorbol myristate acetate (PMA) , calcium ionophore A23187, Pipes, and human serum albumin were all purchased from Sigma Chemical Co. (Poole, UK). Dextran T500 was obtained from Pharmacia (Milton Keynes, UK). The tyrosine kinase inhibitors genistein, tyrphostin 25, methyl 2,5 dihydroxycinnamate (MDC), and lavendustin A were purchased from GIBCO BRL (Uxbridge, UK). The two inactive analogs of genistein, genistin and diadzein, were purchased from Scientific Marketing Associates (Barnet, UK). Sheep antihuman IgE was purchased from Serotec (Oxford, UK). In some experiments, we also used goat anti-human IgE, which was a gift from the Immunopharmacology Group at Southampton General Hospital. found no difference between these two sources of anti-IgE. All other reagents that were used were of the highest grade available.

We

Buffers Pipes buffer contained 25 mM Pipes, 110 mM NaCl, and 5 mM KCl adjusted to pH 7.4 with 1 N HCI; Pipes-albuminglucose (PAG)also contained 0.003 % (wt/vol) human serum albumin and 0.1% (wt/vol) glucose; PAGCM was PAG supplemented with 1 mM CaCb and 1 mM MgCl,. For the digestion of human lung tissue, Pipes buffer was sup-

plemented with bovine serum albumin (1 g/liter), DNase (20 mg/liter), Mg2+ (1 mM), Ca'+ (0.2 mM), and glucose (1 g/ liter) (Pipes + ). Preparation of the Tyrosine Kinase Inhibitors Genistein, genistin, and diadzein were prepared as 100 mM stock solutions in dimethyl sulfoxide (DMSO), tyrphostin 25 as a 50 mM stock solution in DMSO, and lavendustin A and MDC as 10 mM stock solutions in DMSO, and stored at - 20°e. Drugs were diluted to required concentration in PAGCMjust before use. Control experiments confirmed that DMSO at these concentrations « 0.1%) did not affect the release of histamine. Isolation of Human Basophils Mixed leukocytes containing basophils were prepared from the peripheral blood by dextran sedimentation (16). Briefly, 50 ml of blood was mixed with 12.5 ml of 6 % dextran and 5 ml of 0.1 M EDTA, then allowed to sediment for 90 min at room temperature. The upper buffy coat layer was removed, cells were recovered by centrifugation (120 X g, 8 min), washed twice with PAG, then resuspended in PAGCM for use in the histamine release assay. Isolation of Human Lung Mast Cells Briefly, normal human lung tissue from lung resections of patients with carcinoma was stripped of its pleura, finely chopped, and washed to remove contaminating red blood cells and lung macrophages. The tissue was reconstituted in Pipes+ buffer (10 mI/g of tissue) containing collagenase la (25 mg/100 ml) and digested for 1 h at 37°C (17). Cells were separated from remaining tissue by filtration over nylon mesh, washed in Pipes+, and cultured overnight in RPMI 1640 containing 5% fetal calf serum, 1 U/mI penicillin, streptomycin, and gentamycin. Cell Counting Mast cells and basophils were stained with alcian blue (18) and counted in a modified Neubauer hemocytometer. Mediator Release Cells (approximately 2 X 104 per tube) were resuspended in PAGCM and incubated with the drug for 15 min at 3rC before the addition of the relevant stimulus. The final reaction volume was typically 1 ml, Histamine release was allowed to proceed for a further 45 min at 3re. At the end of the experiment, the tubes were centrifuged (250 X g, 3 min) and aliquots of cell-free supernatant were assayed for histamine content by automated fluorometric analysis (19). Basophils were typically challenged with either 1 jl.g/mlrabbit anti-human IgE antibody (Serotec) or a 0.1% (wt/vol) solution of goat anti-IgE, values previously determined to be optimal. Human lung mast cells were challenged with 10fold higher concentrations (10 jl.g/mlSerotec sheep anti-IgE and 1% solution of goat anti-IgE). Calculation of Results The results were based on the mean of duplicate determinations and were expressed as percentage of total histamine, a value that was obtained by lysing the cells in 2 % (wt/vol) perchloric acid. Results were corrected for the spontaneous

Lavens, Peachell, and Warner: Tyrosine Kinases in Human Lung Mast Cells and Basophils

release from unstimulated cells (usually < 5 % during the 45min assay). Each experiment was repeated using cells from at least four different donors and the number of experiments (n) given in the figure legend. Results were compared using ANOVA and Student's paired t test; a value of P < 0.05 was accepted as statistically significant.

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Results Our initial investigations centered on the effects of genistein, a well-known inhibitor of tyrosine kinases (20, 21), on IgEmediated signal transduction in human lung mast cells and basophils. As shown in Figure lA, genistein is an effective inhibitor of anti-IgE-induced histamine release from human basophils with an ICso of 8 I-tM. At 10 I-tM the inhibition was 55 ± 5% (n = 17, P< 0.005), and at 100 I-tM inhibition exceeded 80%. To ensure that this inhibition reflected an interaction with tyrosine kinases, we also assessed the effects of two inactive analogs of genistein, genistin and diadzein. Neither of these agents significantly affected the anti-IgEinduced release of histamine. Genistein alone failed to initiate histamine release at concentrations of up to 100 I-tM (data not shown). We also examined the effect of genistein on A23187induced histamine release and found that there was significant inhibition of histamine release at the highest concentrations tested (30 and 100 I-tM), though the ICso was almost 4-fold greater at 30 I-tM. However, when we examined the two inactive analogs, we found that both genistin (shown in Figure lA) and diadzein (data not shown) were also able to inhibit A23187-induced histamine release significantly at the highest concentrations. Genistein (10 I-tM) also inhibited the anti-IgE-induced release of histamine from lung mast cells (see Figure lB). The drug proved to be much less effective in human lung mast cells and although 10 I-tM genistein resulted in 17 ± 5 % inhibition (n = 11, P < 0.05) there was a great deal of variation in the extent of the response and higher concentrations of genistein (30 to 100 I-tM) failed to significantly inhibit histamine release. The drug also failed to significantly affect histamine release in cells challenged with ll-tg/ml A23187, even at concentrations of 100 I-tM. Both genistin and diadzein were inactive in human lung mast cells challenged with either anti-IgE or A23l87. We followed up these initial observations with genistein by comparing the effects of three other inhibitors of tyrosine kinases on anti-IgE-induced histamine release from both human lung mast cells and basophils (see Figure 2). Tyrphostin 25 inhibited anti-IgE-induced histamine release from human basophils, though it was much less effective than genistein. There was a significant inhibition of histamine release at both 10 I-tM (25 ± 4%, n = 6, P < 0.05) and 100 I-tM (41 ± 18%, n = 6, P < 0.05). Tyrphostin failed to affect histamine release significantly from human lung mast cells; at 10 I-tM, inhibition was 22 ± 16%. In contrast, MDC proved to be an effective inhibitor of anti-Igfi-induced histamine release in human lung mast cells yet failed to affect the release of histamine from human basophils. At 10 I-tM, MDC inhibited anti-IgE-induced histamine release in human lung mast cells by 53 ± 11% (n = 6, P < 0.05). We next examined the effects of MDC on

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Figure 1. Effect of genistein on anti-IgE-induced histamine release from human basophils and lung mast cells. (A) Human basophils were incubated with genistein (circles) or genistin (triangles) for 15 min at 37°C and challenged with either I JLg/ml anti-lgE (open symbols) or I JLg/ml A23187 (closed symbols). Histamine release was allowed to proceed for 45 min, the supernatant recovered, and histamine measured. Control histamine release was 29 ± 4% for cells challenged with anti-lgE (n ;;. 6 for all points) and 81 ± 13% for cells challenged withA23187 (n = 4). Values are expressed as mean ± SEM. ** P < 0.001; * P < 0.05. (B) Lung mast cells were incubated with genistein (circles) or genistin (triangles) for 15 min at 37°C and challenged with 10 JLg/ml anti-lgE (open symbols) or I JLg/ml A23187 (closed symbols). Histamine release in the absence of the drug was 33 ± 5 % (n = 7) in the lung mast cells challenged with anti-lgE and 72 ± 7% in those challenged with A23187 (n = 9). * P < 0.05. For clarity, standard error bars, which were between 2 and 5 %, have been omitted from all but the lung mast cells treated with genistein and challenged with anti-lgE.

human lung mast cells stimulated with A23l87. At 10 p.M, MDC inhibited histamine release by 30 ± 13%, a value that did not attain statistical significance. In the human basophil, 10 I-tM MDC failed to induce any inhibition of anti-IgEinduced histamine release at all (average inhibition = 3 ± 3%, n = 6, P = NS). The drug was also ineffective against

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Figure 2. The effect of four different tyrosine kinase inhibitors on anti-IgE-induced histamine release from human basophils and lung mast cells. Human basophils (dark bars) and lung mast cells (hatched bars) were incubated with 10 j.!M of the different tyrosine kinase inhibitors for 10 min at 37°C and then challenged with either 1 j.!g/rnl anti-IgE (basophils, control release = 34 ± 3 %) or 10 j.!gf.rnl anti-IgE (lung mast cells, control release = 29 ± 5). ** P < O.Obl; * P < 0.05. Values for 10 j.!M genistein from Figure 1 are included for comparison.

any of the other stimuli used to initiate histamine release from human basophils (data not shown). A fourth drug, lavendustin A, was also tested and failed to affect anti-IgEinduced histamine release from either human basophils (at 10 ttM, inhibition = 12 ± 5 %, n = 6, P = NS) or human lung mast cells (at 10 ttM, inhibition = 5 ± 5 %, n = 6, P = NS). Table 1 compares the effectsof these four drugs on antiIgE-induced histamine release from both lung mast cells and basophils with their reported ability to inhibit other tyrosine kinases such as the EGF receptor or their putative sites of action. It is known that human basophils are more sensitive to inhibitors of PKC than their counterparts in the lung (22), and as genistein has been reported to inhibit PKC (23) we assessed the ability of genistein to inhibit histamine release initiated by the phorbol ester, PMA (see Figure 3). Genistein proved to be ineffective against PMA-induced histamine release, failing to affect histamine release even at the highest concentrations (100 ttM). Neither genistin nor diadzein had any effect on PMA-induced histamine release at concentrations up to 100 ttM. We also examined the effect of genistein on histamine release at concentrations up to 100 JIoM. We also examined the effect of genistein on histamine release in cells challenged with the FMLP. Genistein was ineffective at concentrations below 100 ttM, though there was some inhibition of histamine release at the highest dose. Experiments

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Lavens, Peachell, and Warner: Tyrosine Kinases in Human Lung Mast Cells and Basophils

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Figure 3. Effect of genistein on histamine release from human basophils challenged with FMLP or phorbol ester. Basophils (n = 5) were incubated with either genistein (squares) or genistin (triangles) for 15 min at 37°C and challenged with either I iLM FMLP (open symbols) or 10 ng/ml PMA (closed squares). Control histamine release was 34 ± 5 % in basophils challenged with FMLP and 71 ± 14% in those challenged with PMA. * P < 0.05.

with genistein and diadzein suggest that this inhibition may reflect nonspecific effects of genistein. Human lung mast cells do not degranulate when challenged with either PMA or FMLP and so we were unable to assess the effect of genistein or any of the other inhibitors in these cells. One major difference between anti-IgE-induced histamine release in human mast cells and basophils is the concentration of anti-IgE required to elicit optimum histamine release (24). To ensure that the difference between basophils and mast cells was not simply a reflection of the concentra-

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tion of anti-IgE used, we assessed the ability of 10 tLM genistein to inhibit the histamine release initiated by a range of different concentrations of anti-IgE in both human lung mast cells and basophils (see Figure 4). Histamine release from basophils challenged with the highest concentration of anti-IgE, 10 tLg/ml, was 16 ± 4% and in the presence of 10 tLM genistein this decreased to 7 ± 4 %, a 65 ± 12 % inhibition of histamine release. Although control histamine release was greater in basophils challenged with 1 tLg/ml anti-IgE (31 ± 6 %), the effect of 10 tLM genistein was similar, reducing histamine release to 13 ± 4 %, with an average inhibition of 62 ± 8 %. There was no difference between the efficacy of the drug at different concentrations ofanti-IgE (P > 0.05), suggesting that the differences noted in Figure 1 reflect a more fundamental difference between lung mast cells and basophils than simply the concentration of anti-IgE used to initiate histamine release. In a similar manner, the effect of 10 tLM genistein on the release of histamine from human lung mast cells was unaffected by the concentration of antiIgE used to elicit release. Toexclude the possibility that the differences we observed reflected the differential uptake of the drugs into the cells and to ensure that our standard pre-incubation period of 15 min was optimal, we assessed the effects of increasing the preincubation time on the ability of lavendustin A and genistein to inhibit histamine release from human basophils. A preincubation period of up to 2 h with 10 tLM lavendustin A failed to inhibit anti-IgE-induced release in human basophils (see Figure 5). Increasing the incubation period to up to 2 h also failed to increase the inhibition noted with 10 tLM genistein, suggesting that the drug was rapidly reaching its target within the cell.

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pathways used by many different agonists (9, 10). Though initially described as the signal transduction mechanism utilized by growth factor receptors, it has now become apparent that tyrosine kinases can activate a range of intracellular effectors including phospholipase C (11, 12), phosphatidylinositol 3-kinase (25), and microtubule associated protein 2 kinase (26), and are widely involved in a range of different signal transduction mechanisms. The earliest tyrosine kinases to be characterized were an intrinsic part of growth factor receptors and the IgE receptor has no such intrinsic kinase (27). However, it has now also become apparent that tyrosine kinases can be located on distinct proteins associated with different receptors (28). This means that it is no longer possible to exclude tyrosine kinase-dependent signal transduction simply on the basis of the receptor sequence. Although in some cell types tyrosine kinases appear to be an integral link between the receptor and an intracellular effector, in other cells the increase in tyrosine phosphorylation peaks 30 min after the addition of agonist, well after the initial responses are complete (10). This suggests that tyrosine kinases may be activated in parallel with other pathways and that two or more pathways may modulate distinct areas of the cell response. Our understanding of IgE- mediated signal transduction in human cells is still far from complete. Although the crosslinking ofIgE in rodent mast cells results in the mobilization of Ca and the activation of PKC, both characteristic of the inositol phosphate pathway, there is also evidence to suggest that other signal transduction pathways are involved (29, 30). Recently, interest has focused on the activation of tyrosine kinases after the crosslinking of IgE on the surface of rodent mast cell lines (13-15). The phosphorylation of tyrosine residues occurs rapidly, preceding the increase in [Ca2+]" a finding that is consistent with the hypothesis that the activa-

tion of a tyrosine kinase is one of the earliest events in the IgE-dependent cascade in these cell lines (15). However, it is well known that there are clear differences between mast cells from rodents and humans and it is imperative that these investigations are extended to include human cells. The difficulties of obtaining sufficient quantities of highly purified human cells for detailed biochemical studies are generally recognized, and many investigations have used pharmacologic agents to disrupt specific signaling events. The pitfalls of a purely pharmacologic characterization of signal transduction are well known, and a number of supposedly specific inhibitors have later proved to have a wide range of different actions that invalidate many of the early conclusions. We have attempted to circumvent these problems by examining four different putative inhibitors of tyrosine kinases in two different cell types and comparing their effects on both receptor-mediated stimuli and nonphysiologic activation. Our initial investigations with genistein were consistent with tyrosine kinases having a role in IgE-mediated signal transduction in human basophils though, interestingly, the drug was much less effective in human lung mast cells. These initial differences between the lung mast cell and the basophil were reinforced when we extended these studies to characterize three other inhibitors of tyrosine kinases. We found that tyrphostin 25 was able to inhibit histamine release from the basophil, though it was much less effective than genistein and failed to inhibit histamine release in lung mast cells. This observation was in keeping with the reported ability of tyrphostin 25 to inhibit autophosphorylation of the EGF receptor in isolated membranes of A431 cells. However, this correlation did not persist when we characterized two ostensibly more potent inhibitors, lavendustin A and MDC. Whereas lavendustin A was totally inactive in either basophils or lung mast cells, MDC proved to be a potent inhibitor of histamine release in human lung mast cells yet failed to affect the basophil. The high-affinity IgE receptor associates with different tyrosine kinases in the rat basophilic leukemia cell line and the PT-18 mouse mast cell line (31), and our data may well be interpreted as suggesting that different tyrosine kinases are operating in human mast cells and basophils. The observation that high doses (100 p.M) of genistein and related drugs were also able to inhibit histamine release induced by the calcium ionophore A23187 and FMLP suggests that the specificity of the drug may be compromised at these higher concentrations and care must be taken in interpreting these results. These data would suggest that at high doses genistein and its analogs are capable of modifying events downstream of the mobilization of [Ca 2 +], in addition to possibly acting earlier in the signal transduction cascade. One of the two substrates of tyrosine kinases identified in the RBL cells appears to be associated with the cytoskeleton (15) and late exocytotic events, suggesting that tyrosine kinases may well act at more than one site to initiate histamine release. We hope to address the issue of where these agents act, particularly in relation to the mobilization of [Ca2+]" in both mast cells and basophils in future investigations. The differences between lung mast cells and basophils are well known and include the amount of anti-IgE needed to initiate histamine release (24). Although basophils in our hands

Lavens, Peachell, and Warner: Tyrosine Kinases in Human Lung Mast Cells and Basophils

typically need 1 J.Lg/ml sheep anti-human IgE, mast cells require 10- to 30-fold higher concentrations to attain maximal release. The reasons for this difference are not fully understood, mast cells typically have much less IgE on their cell surface, but degranulation can proceed with a relatively small percentage of the IgE molecules on the surface crosslinked (32). Higher concentrations of anti-IgE will result in larger IgE clusters on the cell surface and could affect the signal transduction pathways downstream. However, the effects of genistein were independent of the concentration of antiIgE used to initiate release in both human lung mast cells and basophils, suggesting that the concentration of anti-IgE used to initiate histamine release was not critical in these experiments. Some reports have suggested that genistein inhibits PKC with an IC so of 15 J.LM (21), a value similar to the 8 J.LM found with anti-IgE-induced histamine release. However, genistein failed to inhibit PMA-induced histamine release from human basophils, indicating that the drug was not substantially affecting PKC. This is in direct contrast to other inhibitors of PKC, such as H7 (33) and staurosporine (2), which inhibit PMA-induced histamine release from human basophils. The data with staurosporine are particularly relevant to this investigation as, unlike H7, it inhibits anti-IgEinduced histamine release in human basophils and, like genistein, is much less effective in human lung mast cells (22). Although staurosporine was initially assumed to be specific for PKC, it is now known to affect a number of different kinases including tyrosine kinases (34). However, the inhibition of basophil histamine release with staurosporine is quite distinct from that which we have observed with genistein, suggesting that the two drugs are acting at distinct sites and supporting the contention that genistein is not acting via PKC. The characterization of tyrosine kinases is still in its infancy, with relatively little information available on the consensus phosphorylation sequences and the susceptibility of different kinases to inhibitors. The assumption that the potency of various agents against EGF receptor autophosphorylation predicts their ability to inhibit other tyrosine kinases is likely to prove a distinct oversimplification. The ability of various agents to inhibit different classes of Ser/Thr kinases can vary by several orders of magnitude, and it is likely that the tyrosine kinases will prove to be as diverse. A detailed knowledge of the inhibitory profiles of different tyrosine kinases would obviously help in assigning different kinases to different cell types. The situation is further complicated by the possibility that more than one tyrosine kinase may be involved. In rat basophilic leukemia cells, two different kinases are activated by receptor crosslinking, though only one co-purifies with the receptor, and it may well be that more than one tyrosine kinase is activated in both mast cells and basophils. This complication and the fact that pharmacologic inhibitors are unlikely to ever be totally specific suggest that definitive evidence will only come from the biochemical characterization of the tyrosine kinases involved in both human lung mast cells and basophils. In summary, we report that two putative inhibitors of tyrosine kinases inhibit IgE-induced histamine release in human lung mast cells and basophils, suggesting that tyrosine

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kinases may be involved in IgE-dependent signaling in human cells. Genistein proved to be a potent inhibitor of IgEinduced histamine release from human basophils, though at higher concentrations it also inhibited release initiated by A23187 and FMLP and it failed to affect PMA-induced histamine release, suggesting that it was not acting via PKC. In contrast, MDC inhibited IgE-induced histamine release from human lung mast cells and failed to affect release from human basophils. These results suggest that a tyrosine kinase may be involved in IgE-mediated signal transduction pathways in both cell types and highlight the differences between the blood-borne leukocyte and the tissue-resident mast cell, emphasizing again the diversity of human IgE-bearing cells. Acknowledgments: We would like to thank Mr. Matthew Peirce for invaluable technical assistance during the course of these studies and the members of the Immunopharmacology Group, especially Dr. Tim Hunt, at Southampton General Hospital for making their histamine autoanalyzer available throughout these investigations. This work was supported by the Wellcome Trust, the Wessex Medical School Trust, and the Smith Kline Foundation.

References 1. MacGlashan, D. W., Jr. 1989. Single cell analysis of Ca"' changes in human lung mast cells. I. Graded versus all or nothing elevations following IgE mediated stimulation. J. Cell. Bioi. 109:123-134. 2. Warner, J. A., and D. W. MacGlashan, Jr. 1990. Signal transduction events in human basophils: a comparative study of the role of protein kinase C in basophils activated by anti-IgE antibody and formyl methionyl leucyl phenylalanine. J. Immunol. 145: 1897-1905. 3. Warner, J. A., and D. W. MacGlashan, Jr. 1989. Protein kinase C changes in human basophils: IgE-mediated activation is accompanied by an increase in total PKC activity. J. Immunol. 142: 1669-1677. 4. Plaut, M., J. H. Pierce, C. 1. Watson, J. Hanley-Hyde, R. P. Nordan, and W. E. Paul. 1989. Mast cell lines produce lymphokines in response to crosslinkage of Fc,RI or to calcium ionophore. Nature 339:64-67. 5. Wodnar-Filipowicz, A., C. H. Heusser, and C. Moroni. 1989. Production of the hemapoietic growth factors and interleukin 3 by mast cells in response to IgE receptor mediated activation. Nature 339: 150-152. 6. Mossalayi, M. D., J. C. Lecron, A. H. Dalloul et al. 1990. Soluble CD23 (Fe epsilon RIl) and interleukin I synergistically induce early thymocyte differentiation. J. Exp. Med. 171:959-964. 7. Delespesse, G., U. Suter, D. Mossalayi et al. 1991. The CD23 antigen. Adv. Immunol. 49:149-193. 8. Yalden, Y., andA. Ullrich. 1988. Growth factor receptortyrosine kinases. Annu. Rev. Biochem. 57:443-479. 9. Klausner, R. D., and L. E. Samuelson. 1991. The T cell antigen receptor activation pathways: the tyrosine kinase connection. Cell 64:875-878. 10. Gomez-Cambronero, J., E. Wang, G. Johnson, C.-K. Huang, and R. I. Sha'afi. 1991. Platelet activating factor induces tyrosine phosphorylation in human neutrophils. J. Bioi,' Chem. 266:6240-6246. II. Margolis, B., S. G. Rhee,S. Fleder et al. 1989. EGF induces tyrosine phosphorylation on phospholipase C-Il: a potential mechanism for signalling. Cell 57:1101-1109. 12. Meisenhelder, J., P.-G. Suh, S. G. Rhee, and T. Hunter. 1989. Phospholipase C-y is a substrate for the PDGF and EGF receptor protein tyrosine kinases in vivo and in vitro. Cell 57: 1109-1122. 13. Kawakawi, T., H. Fukamachi, K. Ishizaka, and T. Ishizaka. 1991. Rapid activation of a protein tyrosine kinase after crosslinking of FCfRI on mouse mast cells. FASEB J. 5:AI676. (Abstr.) 14. Liotta, M., D. Holowka, and B. Baird. 1991. Evidence for the importance of tyrosine phosphorylation in IgE receptor mediated cellular degranulation. FASEB J. 5:AI676. (Abstr.) 15. Benhamou, M., J. S. Gutkind, K. C. Robbins, andR. P. Siraganian. 1990. Tyrosine phosphorylation coupled to IgE receptor mediated signal transduction and histamine release. Proc. Natl. Acad. Sci. USA 87:5327-5330. 16. Lichtenstein, L. M., and A. G. Osler. 1964. Studies on the mechanism of immediate hypersensitivity phenomena. XI. Histamine release from human leukocytes by ragweed antigen. J. Exp. Med. 120:507-512. 17. Ali, H., and F. L. Pearce. 1985. Isolation and properties of cardiac and other mast cells from the rat and guinea-pig. Agents Actions 16: 138-140. 18. Gilbert, H. S., and L. Ornstein. 1975. Basophil counting with a new staining method using alcian blue. Blood 46:279-282. 19. Siraganian, R. P. 1974. An automated continuous flow system for the extraction and f1uorimetric determination of histamine. Anal. Biochem. 57:283-287.

644

AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 7 1992

20. Akiyama, T., J. Ishida, A. Nakagawa et al. 1987. Genistein, a specific inhibitor of tyrosine-specific protein kinases. J. Bioi. Chem. 262:55925595. 21. Trevillyan, J. M., Y. Lu, D. A. Huru, C. A. Phillips, andJ. M. Bjorndahl. 1990. Differential inhibition of T cell receptor signal transduction and early activation events by a selective inhibitor of protein tyrosine kinase. J. lmmunol. 145:3223-3230. 22. Massey, W. A., V. L. Cohan, D. W. MacGlashan, Jr., L. M. Lichtenstein, A. Kagey-Sobotka, and J. A. Warner. 1991. Protein kinase C activation modulates IgE-mediated responses in human mast cells from lung and skin. J. Pharmacol. Exp. Ther. 258:824-829. 23. Onoda, T., H. Iinurna, Y. Sasaki et al. 1989. Isolation of a novel protein tyrosine kinase inhibitor, lavendustin A, from Streptomyces grisoslavendus. J. Nat. Prod. 52: 1252-1257. 24. Fox, C. C., A. M. Dvorak, D. W. MacGlashan, Jr., and L. M. Lichtenstein. 1984. Histamine containing cells in human peritoneal fluid. J. Immunol. 132:2177-2179. 25. Gold, M. R., R. Aebersold, W.-F. Chan, and A. L. DeFranco. 1992. Regulation of phosphatidylinositol 3-kinase by membrane immunoglobulin crosslinking in B lymphocytes. FASEB J. 6:AI128. (Abstr.) 26. Casillas, A., C. Hanekom, K. Williams, R. Katz, and A. E. Nel. 1991. Stimulation of B cells via the membrane immunoglobulin receptor or with phorbol myristate 13-acetate induces tyrosine phosphorylation and activation of a 42kDa microtubule associated protein 2 kinase. J. Bioi. Chem. 266: 19088-19094. 27. Blank, U., C. Ra, L. Miller, K. White, H. Metzger, andJ.-P. Kinet. 1989. Complete structure and expression in transfected cells of high affinity IgE receptor. Nature 337:187-189. 28. Weiss, A., G. Koretzky, R. C. Schatzman, and T. Kadlecek. Functional

29. 30. 31. 32. 33.

34.

35. 36. 37.

activation of the T cell antigen receptor induces tyrosine phosphorylation of phospholipase C 1'1. Proc. Natl. Acad. Sci. USA 88:5484-5488. Kennerly, D. A. 1987. Diacylglycerol metabolism in mast cells. Analysis oflipid mediators pathways using molecular species analysis of intermediates. J. Bioi. Chem. 262:16305-16310. Gomperts, B. D. 1990. G,: a GTP binding protein mediating exocytosis. Annu. Rev. Physiol. 52:591-607. Eiseman, E., and 1. B. Bolen. 1992. Engagement of high-affinity IgE receptor activates src protein related tyrosine kinases. Nature 355:78-80. MacGlashan, D. W., Jr., and L. M. Lichtenstein. 1983. Studies of antigen binding on human basophils. I. Antigen binding and functional consequences. J. lmmunol. 130:2330-2334. Thueson, D.O., J. A. Kennedy, C. D. Wright, and M. C. Conroy. 1987. Specific inhibition of phorbol ester and DiC8 induced release by the protein kinase C inhibitors H7 and H9. Biochem. Biophys. Res. Commun. 144:732-740. Nakano, H., E. Kobayashi, I. Takahashi, T. Tamaoki, Y. Kuzun, and H. Iba. 1987. Staurosporine inhibits tyrosine-specific protein kinase activity of rous sarcoma virus transforming protein p-60. J. Antibiot. (Tokyo) 15:706-708. Geahlen, R. L., N. M. Koonchanok, J. L. MacLaughlin, and D. E. Pratt. 1989. Inhibition of protein tyrosine kinase activity by flavenoids and related compounds. J. Nat. Prod. 52:982-986. Yaish, P., A. Gazit, C. Gilon, and A. Levitzki. 1988. Blocking of EGFdependent cell proliferation by EGF receptor kinase inhibitors. Science 242:933-935. Gazit, A., P. Yaish, C. Gilon, and A. Levitzki. 1989. Tyrphostins. I: synthesis and biological activity of protein tyrosine kinase inhibitors. J. Med. Chem. 32:2344-2352.