INVITED REVIEW
Role of protein phosphatases in the regulation of human mast cell and basophil function

Matthew J. Peirce1, Michael R. Munday2, and Peter T. Peachell1

1 Section of Molecular Pharmacology and Pharmacogenetics, University of Sheffield, Sheffield S10 2JF; and 2 Department of Pharmaceutical and Biological Chemistry, School of Pharmacy, University of London, London WC1N 1AX, United Kingdom


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INTRODUCTION
PROTEIN SERINE/THREONINE...
PP1 AND PP2A IN...
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Many extracellular stimuli mediate physiological change in target cells by altering the phosphorylation state of proteins. These alterations result from the dynamic interplay of protein kinases, which mediate phosphorylations, and protein phosphatases, which catalyse dephosphorylations. The antigen-mediated aggregation of high-affinity receptors for IgE on mast cells and basophils triggers rapid changes in the phosphorylation of many proteins and culminates in the generation of inflammatory mediators involved in allergic inflammatory diseases such as asthma. Although protein kinases have an established role in this process, less is known about the involvement of protein phosphatases. This imbalance has been redressed in recent years by the availability of phosphatase inhibitors, such as okadaic acid, that facilitate investigations of the role of protein phosphatases in intact cells. Here we review a number of studies in which inhibitors of protein phosphatases have been used to shed light on the potential importance of these enzymes in the regulation of human mast cell and human basophil function.

okadaic acid; calyculin A; cyclosporin; FK506; dephosphorylation; histamine release


    INTRODUCTION
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INTRODUCTION
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THE RECEPTOR WITH HIGH AFFINITY for IgE (Fcepsilon R1) is predominantly expressed on mast cells and basophils (20). The binding of polyvalent antigen to receptor-bound IgE cross-links the IgE receptors and triggers a cascade of intracellular events, culminating in the release of a battery of inflammatory mediators (63, 68, 99). These mediators, which include histamine (83), leukotrienes (65, 83), prostaglandin D2 (PGD2) (55, 65, 83), as well as interleukins (ILs) such as IL-4 (6, 86) and IL-5 (7, 9), are believed to play an important role in acute hypersensitivity responses to antigen challenge. Indeed, as a prominent link between circulating IgE and the release of proinflammatory mediators, mast cells and basophils may contribute to the genesis of allergic diseases such as asthma (99).

Studies using rodent mast cells indicate that antigen-mediated aggregation of Fcepsilon R1 leads rapidly to the phosphorylation, on tyrosine, serine, and threonine residues, of the beta  and gamma  chains of the IgE receptor itself and a number of other proteins (3, 36, 73, 74). These phosphorylations constitute some of the earliest molecular consequences of receptor aggregation and appear to be a prerequisite for the release of inflammatory mediators (74). As a result, the protein kinases responsible for these phosphorylations have been the subject of intense study, and several kinases, such as p56Lyn and p72Syk, now have well-characterized roles in mast cells of rodent origin (27, 36, 48, 82).

Although protein phosphorylations follow receptor aggregation, the disruption of receptor aggregates results in an equally swift dephosphorylation of the receptor and other phosphoproteins (73, 74). These dephosphorylations are mediated by protein serine/threonine phosphatases (PPs) and protein tyrosine phosphatases (PTPs), but, until relatively recently, the importance of these enzymes was largely overlooked. This oversight may in part be due to the view that PPs and PTPs act merely as passive counterbalances to protein kinases. Cell-free experiments suggest that both PPs and PTPs are relatively promiscuous enzymes acting on a wide range of substrates (1, 15, 16). These data in broken cell systems, to some extent, support the view that phosphatases act in a "house-keeping" fashion to undo the work of protein kinases. However, accumulating data challenge this view, and there is a growing awareness that a high degree of enzyme specificity can be achieved in intact cells (15, 30, 33). In many cases this specificity is achieved via the formation of multimeric complexes with "targeting subunits" (29, 45, 46) that serve to target phosphatase activity toward particular substrates or cellular locations. Furthermore, rather than acting in an exclusively inhibitory manner, dephosphorylations and, by extension, phosphatases have been shown to mediate or to regulate a diverse array of intracellular processes in a positive manner (15, 23, 25, 61, 90, 95). Of particular relevance to this review, a substantial body of data now suggests that protein phosphatases may have a role in modulating the signals generated by the IgE receptor and the subsequent production of inflammatory mediators from mast cells and basophils.

The difficulty in obtaining human mast cells in large numbers and high purities has meant that the biochemical analysis of mast cell function has relied, to a large extent, on studies based on rodent mast cells, particularly the rat basophilic leukemia cell line, RBL-2H3 (2). Although these cells certainly represent a very useful model system for the study of mast cell function, well-documented differences exist even between mast cell populations from different species and, moreover, different tissues within the same organism (8, 60, 77, 78). Thus it remains unclear to what degree findings based on transformed rodent mast cells reflect the situation in their normal human counterparts. For these reasons our own studies, and the large part of this review, focuses on the study of phosphatases in the human lung mast cell (HLMC) and human basophil.


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The protein serine/threonine phosphatases (PPs) comprise a family of at least four major enzyme types (15, 18, 88). PP activities are ascribed to one of these four groupings on the basis of several functional parameters. The type 1 PPs preferentially dephosphorylate the beta -subunit of phosphorylase kinase, whereas the type 2 enzymes exhibit a preference for the alpha -subunit of this substrate. The type 2 PPs are further subdivided into types 2A, 2B (also known as calcineurin), and 2C. The catalytic activity of the type 2A enzymes is independent of divalent metal cations, whereas the activities of the type 2B and 2C enzymes are dependent, respectively, on Ca2+ and Mg2+. The type 1 enzymes can also be distinguished from type 2 PPs using two endogenous inhibitor proteins, inhibitor 1 (I-1) and inhibitor 2 (I-2), which act selectively to inhibit the activity of PP1. Functionally homologous endogenous protein inhibitors of PP2A (56) and PP2B (91) have also recently been identified.

Although clear functional and regulatory differences exist between the various PPs, PP1, PP2A, and PP2B show overlapping substrate specificities (15, 18). However, the activity of each catalytic subunit can be modulated in a substrate-specific manner by the formation of distinct complexes with regulatory subunits (45, 67). In addition to regulating the substrate preference of the catalytic subunits (11, 50, 51, 89), the regulatory proteins with which they associate may also target the activities to distinct subcellular compartments (44, 93). Thus PPs are subject to tight regulation in intact cells.

Although the system of classification detailed above accounts for the majority of PPs identified to date, a number of novel PPs have been identified that do not readily fall into this relatively simple scheme (10, 12, 41). PP3, PP4, and PP5, for example, exhibit some functional similarity and significant sequence homology with PP1 and PP2A within the catalytic domains. However, they are sufficiently different to be regarded as representatives of distinct enzyme families.

In addition to endogenous factors that regulate, target, or inhibit PP activities, a number of naturally occurring toxins have been found to be potent and selective inhibitors of PPs. Of these compounds, the most widely used is okadaic acid (OA) (16). A product of dinoflagellate metabolism, OA is the causative agent of diarrhetic shellfish poisoning. OA exhibits a degree of PP selectivity; subnanomolar concentrations (IC50, 0.1-1 nM) of OA are sufficient to abolish PP2A activity, whereas 10- to 100-fold higher concentrations (IC50, 1-10 nM) are required to inhibit PP1. PP2B is inhibited only at high micromolar concentrations (IC50 > 5 µM), and PP2C is insensitive to OA. Sensitivity to OA may be related to the highly homologous catalytic domains shared by PP1, PP2A, and PP2B (see Fig. 1) (24). Indeed, data from the solved crystal structures of PP1 (26, 34) and PP2B (35, 53) suggest that these enzymes may utilize similar mechanisms of catalysis (24). Furthermore, site-directed mutagenesis studies of PP1 have provided persuasive evidence that both protein and toxin inhibitors of PPs interact with catalytic residues common to PP1, PP2A, and PP2B (43). In contrast, PP2C shares very little sequence homology with the other PPs, even within the catalytic domain (24, 88), and this may explain the insensitivity of PP2C to OA (see Fig. 1).


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Fig. 1.   Gross domain structures of common protein serine/threonine phosphatases (PPs). PP1, PP2A, and PP2B share a highly homologous catalytic domain (shaded regions) of ~280 amino acids while the amino and carboxy termini are divergent. The catalytic domain (solid region) of PP2C is ~290 amino acids in length and is unrelated to those found in PP1, PP2A, and PP2B. PP2B is a Ca2+/calmodulin-dependent PP, and the site of calmodulin binding is shown (hatched region). Approximate molecular masses are indicated for each enzyme. [Adapted from Cohen (18) and Tong et al. (94).]

Several features of OA underlie its particular utility in the study of PPs. The differential sensitivity of PP1 and PP2A to OA has proved extremely useful in classifying unknown PP activities and can be exploited to determine the relative contributions of particular PPs in mixed PP preparations such as crude cell extracts (17). Furthermore, OA is cell permeant (39, 40) and appears to attenuate PPs in a specific manner (e.g., OA appears to be without effect on protein kinases) (16). This has allowed the study of PPs to be extended to intact cells (40). Such studies are further facilitated by the availability of structural analogs of OA that exhibit distinct potencies as PP inhibitors while retaining the ability to enter intact cells (71, 92). These analogs enable studies in which the effects of several different but highly related molecules, with varying abilities to inhibit PP activity, are compared in parallel.


    PP1 AND PP2A IN MAST CELLS AND BASOPHILS
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The Effect of PP Inhibitors on Mediator Release

Data from our own and other laboratories demonstrate that the IgE-dependent release of histamine from HLMC (75, 79, 80), basophils (5, 80, 81), and rat peritoneal mast cells (28) is attenuated by OA. However, the inhibitory effect of OA is not restricted to the release of histamine. The de novo synthesis of PGD2 and sulfidopeptide leukotrienes (sLT) from HLMC and basophils is also effectively inhibited by OA at concentrations similar to those found to be effective against histamine release (see Table 1). Furthermore, OA also blocks the IgE-dependent generation of the proinflammatory cytokine, IL-4, from human basophils (see Table 1) (81). Studies with structural analogs of OA, okadaol and norokadaone, indicate that the order of activity (OA > okadaol >> okadaone) for the inhibition of mediator release from HLMC and basophils follows the reported activity of these compounds as inhibitors of isolated PPs (71, 92) and the extent of inhibition of PP activity when broken cell extracts of HLMC (79) and basophils (81) are treated with these inhibitors.

                              
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Table 1.   Effects of OA and analogs of OA on IgE-dependent mediator release from HLMC and basophils

These data suggest that PP1 and/or PP2A are necessary components or regulators of IgE-dependent signals leading to 1) the exocytosis of a preformed mediator (histamine), 2) the de novo synthesis of membrane-derived mediators (PGD2 and sLT), and 3) the release of a proinflammatory cytokine (IL-4).

The effect of OA is not restricted to IgE-dependent stimuli. Nonimmunological secretagogues are also sensitive to the effects of OA. For example, histamine release induced by the Ca2+ ionophore A-23187 is inhibited by OA to a comparable degree to that seen when IgE-dependent stimuli are used to induce secretion (79, 81). The ionophore induces release by directly translocating Ca2+ into the cell, bypassing many of the events associated with IgE-dependent activation (19, 57). IgE-mediated activation also leads to increases in intracellular Ca2+ in both HLMC and basophils, and these elevations may contribute to exocytosis (62, 64). That OA attenuates both IgE-dependent and ionophore-induced release of histamine from HLMC and basophils suggests that OA may act at targets in signaling pathways common to both stimuli, and these targets may lie downstream of the increases in intracellular Ca2+ that occur following activation with either stimulus. Collectively, these data point to an important role for PPs in regulating pathways leading to the release of mediators from HLMC and basophils.

PP Activity in Extracts of HLMC and Basophils

PPs are believed to be ubiquitous enzymes, and our laboratory has attempted to confirm the presence of PP1 and PP2A, the presumptive targets of OA, in broken cell extracts of HLMC and basophils.

With the use of a substrate (glycogen phosphorylase) against which both PP1 and PP2A are active (17), but under conditions (presence of 1 mM EDTA) that preclude the activity of PP2B and PP2C (which are dependent, respectively, on Ca2+ and Mg2+), it has been possible to detect PP activity in whole cell extracts of both HLMC and basophils. This PP activity could be partially blocked (10-15% inhibition) by a concentration (2 nM) of OA at which PP2A is selectively inhibited, thereby suggesting the presence of PP2A in these extracts. Because a higher (5 µM) concentration of OA, which blocks both PP1 and PP2A activity, further inhibited this PP activity (>= 85% inhibition), these studies also suggested the presence of PP1 in extracts of HLMC and basophils. Studies employing the PP1-selective inhibitor I-2 provided further evidence that PP1 was present in both HLMC and basophils (80). Moreover, the presence of PP2A in extracts of HLMC and basophils was further suggested by the finding that extracts of these cells were capable of dephosphorylating casein, a PP2A-restricted substrate under the assay conditions employed (80).

Although these studies served to identify the presence of PP1 and PP2A in both HLMC and basophils, a clear difference existed between the two cell types. A combination of I-2 (inhibitor of PP1), at a concentration (20 nM) thought to inhibit PP1 maximally, with a low (2 nM) concentration of OA (inhibitor of PP2A) attenuated glycogen phosphorylase PP activity to a similar degree as a high (5 µM) concentration of OA (inhibits both PP1 and PP2A) in HLMC extracts. These data suggest that the majority of the glycogen phosphorylase PP activity in HLMC is PP1 and PP2A. However, in extracts of basophils, a combination of I-2 with a low concentration of OA was substantially less effective at inhibiting PP activity than a high concentration of OA alone (see Table 2). These data suggest that basophils contain PP species that are sensitive to micromolar concentrations of OA but refractory to I-2. Thus, in contrast to HLMC, in which PP1 and PP2A appear to contribute the large majority of the total PP activity, a substantial proportion of the glycogen phosphorylase PP activity in human basophils may be due to PPs other than PP1 and PP2A. The nature of the non-PP1/PP2A activity in basophils, at this time, remains unknown.

                              
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Table 2.   Assessment of PP content of HLMC and basophils using PP inhibitors

These same studies indicate that, on a per cell basis, the amount of PP1 (taken as the amount of PP activity sensitive to I-2) in HLMC is approximately sixfold greater than that in basophils, and the PP2A activity (identified using the PP2A-restricted substrate, casein) is threefold higher in HLMC than in basophils (80). Thus the ratio of PP1 to PP2A activity is twofold higher in HLMC than in basophils.

The apparent preeminence of PP1 in HLMC relative to basophils may underlie the observation in mediator release experiments that both OA, and an alternative PP inhibitor calyculin A (Cal A), were approximately threefold more potent as inhibitors of histamine release in HLMC than in basophils (see Table 3) (75, 79, 81). Furthermore, Cal A, which is equipotent with OA as an inhibitor of PP2A but 10- to 100-fold more potent than OA against PP1, proved to be ~10-fold more potent than OA as an inhibitor of histamine release in both HLMC and basophils (see Table 3). Because Cal A, the more potent inhibitor of PP1, is also the more potent inhibitor of histamine release, it is possible that PP1 is the more important PP in the context of mediator release from both HLMC and basophils. However, the concentrations of both OA and Cal A used to attenuate histamine release in these studies were several orders of magnitude greater than those at which the selective inhibition of PPs occurs (17, 47), and the concentrations of OA and Cal A achieved intracellularly are unknown. This makes it difficult to ascribe the attenuation of mediator release by PP inhibitors in HLMC and basophils to the inhibition of one PP or another.

                              
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Table 3.   IC50 values for the inhibition of histamine release and PP activity

Although distinct roles for PP1 and PP2A in HLMC and basophils remain to be directly demonstrated, the current data highlight differences in the profile of PP activities resident in the two cell types. One of many future challenges will be to develop an understanding of how these differences affect the responses of HLMC and basophils.


    PP2B AND PP2C IN MAST CELLS AND BASOPHILS
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PP1 AND PP2A IN...
PP2B AND PP2C IN...
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PP2B

Defining the role of the Ca2+-dependent PP, PP2B (also known as calcineurin), in cell function has been facilitated by the immunosuppressant compounds, cyclosporin A and FK506. These agents are believed to form heterodimeric complexes with distinct binding proteins, cyclophilin and FK506 binding protein (FKBP), respectively. These complexes act directly to inhibit the activity of PP2B (32, 59, 87). Because the blockade of PP2B-mediated dephosphorylations by cyclosporin or FK506 profoundly inhibits many T cell responses that follow engagement of the T cell receptor, PP2B has been established as a pivotal enzyme in T cell signaling (14).

The importance of PP2B is not restricted to T cells, however. We and others have demonstrated that both cyclosporin and FK506 inhibit the release of inflammatory mediators from HLMC (22, 76, 97) and basophils (13, 21, 76) and that FK506 (IC50, 0.02 µM) is ~100-fold more potent than cyclosporin (IC50, 2-3 µM) in this regard. Because FK506 (IC50, 0.4 nM) is also more potent than cyclosporin (IC50, 7 nM) as an inhibitor of PP2B activity in Jurkat T cells (32, 58, 85), these data suggest that cyclosporin and FK506 inhibit the release of inflammatory mediators from HLMC and basophils by attenuating PP2B activity. Notably, in mouse mucosal mast cells, cyclosporin and FK506 have been shown to attenuate PP2B activity and cytokine production but not histamine release (31). These data indicate not only that different secretory pathways may differ in their requirement for PP2B but that the role of PP2B may differ in distinct mast cell populations.

With the use of a phosphorylated peptide corresponding to a sequence from the regulatory subunit of cAMP-dependent protein kinase, demonstrated previously to be a substrate for PP2B (32), Ca2+-dependent PP activity (characteristic of PP2B) was detected in extracts of HLMC and basophils (76). On a per cell basis, the level of Ca2+-dependent PP activity was sevenfold higher in HLMC than basophils.

In addition to the Ca2+-dependent PP activity, Western blotting studies with the use of an antibody to the B subunit of PP2B also demonstrated the presence of PP2B protein in extracts of both HLMC and basophils. Densitometric analysis of these blots demonstrated that, on a per cell basis, HLMC contained ~10-fold more PP2B protein than basophils (76). Thus the relative levels of PP2B protein and Ca2+-dependent PP activity in HLMC and basophils are in close agreement and indicate that HLMC contain about ninefold more PP2B per cell than basophils. Indeed, on a per cell basis, HLMC contained twofold higher levels of both Ca2+-dependent PP activity and immunoreactive PP2B protein than Jurkat cells, a human T cell line demonstrated by others to contain substantial amounts of PP2B (76). These data suggest that mast cells may contain relatively high levels of PP2B.

Although basophils appear to contain about ninefold lower levels of PP2B protein and PP activity than HLMC, this was not reflected in the IC50 values for cyclosporin and FK506 as inhibitors of histamine release, which were almost identical in the two cell types. These data may suggest that cyclosporin and FK506 attenuate mediator release by a mechanism distinct or additional to the inhibition of PP2B. Alternatively, it is possible that factors other than the amount of PP2B in the cell regulate the ability of cyclosporin and FK506 to inhibit mediator release. For example, the levels of the binding proteins cyclophilin and FKBP have yet to be determined in HLMC and basophils and may have a limiting role in the effects of immunosuppressants on these cells.

Thus HLMC and basophils contain PP2B, whereas HLMC contain ninefold higher levels of this protein. Although the presence of PP2B in these cells provides a mechanism of action for cyclosporin and FK506 in the inhibition of mediator release from these cells, further work is required to substantiate whether this is indeed the case.

PP2C

PP2C shares no homology with other PPs, even within the catalytic domain, and this is reflected in the fact that the PP inhibitors used to characterize the roles of other PPs have no effect on PP2C (24, 88). The lack of pharmacological agents able to modulate PP2C activity has hampered the understanding of its role in regulating cell function. Our laboratory has some preliminary data that suggest the presence of low levels of a Mg2+-dependent PP activity (characteristic of PP2C) in extracts of HLMC and basophils (unpublished observations). However, the role subserved by this activity in regulating HLMC and basophil function remains unknown.


    PTPS IN MAST CELLS AND BASOPHILS
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Accumulating data from the RBL cell system demonstrate the potential importance of several PTPs in the regulation of mast cell function. These enzymes include the transmembrane PTP, CD45 (4, 84), the two Src homology 2 (SH2)-domain-containing PTPs, SHP-1 and SHP-2 (52), as well as a novel membrane-located PTP that appears to be regulated by IgE-receptor aggregation (37, 38). However, very little published data exist describing the role of PTPs in human mast cells or basophils. To our knowledge, the only relevant study investigated the role of CD45 in human basophils using an antibody to CD45 (42). Pretreatment of human basophils with anti-CD45 antibody attenuated the IgE-dependent secretion of histamine. Although the effect of antibody binding on CD45 PTP activity was not assessed in this study, it has been demonstrated subsequently that dimerization of CD45 blocks its PTP activity (66, 100). It is quite possible that the anti-CD45 antibody used in the basophil studies induced oligomerization of CD45, thereby inhibiting its activity. These data therefore suggest that CD45 may be required to observe optimal IgE-mediated histamine release.

CD45 has been shown to be essential for the development and proliferation of lymphocytes, particularly T cells (49, 54, 96, 98). It is believed to activate Src family protein tyrosine kinases such as p56Lck and p59fyn by dephosphorylating a negative regulatory tyrosine residue, thereby disrupting an inhibitory intramolecular SH2 domain/phosphotyrosine interaction that retains Src kinase in an inactive conformation (69, 70, 72). Studies in rodent mast cells strongly implicate the Src family kinase, Lyn, in initiating cellular signals in response to IgE-receptor aggregation (3, 27, 36). On this basis, one might predict a requirement for CD45 in Fcepsilon R1-mediated signals consistent with the data above. However, since the role of Lyn in human mast cells and basophils has yet to be unequivocally demonstrated, this putative role for CD45 in mast cells of human origin remains speculative. Indeed, heterogeneity in the conclusions of studies to investigate the role of CD45 in rodent mast cells of various sources (4, 84) highlight the fact that the requirement for CD45 in Fcepsilon R1-mediated signaling may vary in different mast cell populations and underscore the need to perform further studies in human mast cells and basophils.


    FUTURE DIRECTIONS
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OA and alternative PP inhibitors are effective inhibitors of the IgE-triggered release of mediators from HLMC and basophils. This suggests that PPs may modulate or directly mediate secretory responses in these cells. For this reason, attempts have been made to characterize the PPs present in HLMC and basophils, and some progress has been made in this regard. Although the four major classes of PP (PP1, PP2A, PP2B, and PP2C) have been identified in both cell types, there are clearly substantial differences in the composition of these different PPs between the two cell types. HLMC contain more PP1 (6-fold), PP2A (3-fold), and PP2B (9-fold) than basophils. Conversely, basophils have considerably more unclassified OA-sensitive PP activity. How these differences might contribute to cell function is, presently, unknown, but attempting to dissect the actions of these PPs would constitute challenging areas of endeavor. In this regard, characterization of regulatory and targeting subunits may contribute to an understanding of PP function, substrate specificity, and cellular localization. At this time, little is known about the profile of PTPs in human mast cells and basophils. However, attempts to determine whether PTPs, known to be involved in RBL-2H3 cell function, might also be involved in human cells could constitute a useful first step. As such, the role of PPs (and PTPs) in HLMC and basophils merits further study, given the central importance of these cells in diseases with an allergic basis.


    ACKNOWLEDGEMENTS

We are grateful to Mr. G. Cooper and Mr. A. Thorpe (Cardiothoracic Surgery) and Dr. K. Suvarna (Histopathology) at the Northern General Hospital, Sheffield, UK, and to Mr. N. Saunders and Mr. R. Nair (Cardiothoracic Surgery) and Dr. P. DaCosta (Histopathology) at the General Infirmary, Leeds, UK, for their invaluable help in providing lung tissue specimens.


    FOOTNOTES

This work was supported by the National Asthma Campaign, UK, and in part by the Wellcome Trust, UK.

Address for reprint requests and other correspondence: P. T. Peachell, Section of Molecular Pharmacology and Pharmacogenetics, Univ. of Sheffield, Royal Hallamshire Hospital (Floor L), Glossop Rd., Sheffield S10 2JF, UK (E-mail: p.t.peachell{at}sheffield.ac.uk).


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13.   Cirillo, R., M. Triggiani, L. Siri, A. Ciccarelli, G. R. Pettit, M. Condorelli, and G. Marone. Cyclosporine A rapidly inhibits mediator release from human basophils presumably by interacting with cyclophilin. J. Immunol. 144: 3891-3897, 1990[Abstract/Free Full Text].

14.   Clipstone, N. A., and G. R. Crabtree. Identification of calcineurin as a key signalling enzyme in T-lymphocyte activation. Nature 357: 695-697, 1992[Medline].

15.   Cohen, P. The structure and regulation of protein phosphatases. Annu. Rev. Biochem. 58: 453-508, 1989[Medline].

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