Signal transduction: Mechanisms in basophils
Authors: Donald MacGlashan
Journal: Journal of Allergy and Clinical Immunology (1994)

In an effort to develop new therapies for blunting the allergic reaction, investigators have attempted to understand the process by which the aggregation of cell surface IgE and its receptor is translated into the secretion of mediators from basophils and mast cells. The number of candidate reactions and pathways involved in this transmembrane signaling is increasing yearly and despite an enormous wealth of information about the process, it is not yet clear what some of the more obvious candidate second messengers do or how the various pathways fit together. With this explosion of information, it is sometimes useful to gain insight by noting how natural regulation of the process occurs. Those studying human basophil histamine release cannot help but notice that 15% to 20% of donors studied have basophils that are essentially unresponsive to IgE-mediated stimulation. It is not known whether this "phenotype" is related to atopy, but it is nevertheless an intriguing problem for those studying signal transduction. With the usual method of expressing histamine release from 0 to 100%, the population distribution is necessarily constrained to fit between these two extremes, and thus these so-called nonreleasers appear to represent a special subset of the population.1Nguyen KL Gillis S MacGlashan Jr, DW A comparative study of releasing and nonreleasing human basophils: nonreleasing basophils lack an early component of the signal transduction pathway that follows IgE cross-linking.J ALLERGY CLIN IMMUNOL. 1990; 85: 1020-1029Abstract Full Text PDF PubMed Scopus (91) Google Scholar Whether this is true is still not clear, but until other measures of the degree of activation are easily obtainable, the distribution will appear bimodal and therefore give the impression that nonreleasers are a special subset of population. We have been slowly accumulating a picture of what differs about these cells in hopes that a common attribute applies to each donor with nonreleasing basophils and that such an attribute will shed some light on how nature chooses to regulate the overall cascade of events leading to mediator release. One observation that has only slowly come to light is that these cells do not secrete any of the three general classes of mediators that basophils can now be shown to release: granule contents such as histamine, newly synthesized arachidonic acid metabolites such as leukotriene C4,1Nguyen KL Gillis S MacGlashan Jr, DW A comparative study of releasing and nonreleasing human basophils: nonreleasing basophils lack an early component of the signal transduction pathway that follows IgE cross-linking.J ALLERGY CLIN IMMUNOL. 1990; 85: 1020-1029Abstract Full Text PDF PubMed Scopus (91) Google Scholar and newly synthesized proteins such as interleukin (IL)-4.2MacGlashan Jr, DW White JM Huang SK Ono SJ Schroeder J Lichtenstein LM Secretion of interleukin-4 from human basophils: the relationship between IL-4 mRNA and protein in resting and stimulated basophils.J Immunol. 1994; 152: 3006-3016PubMed Google Scholar Because evidence is accumulating that secretion of each of these mediators uses some distinct signal transduction pathways, the difference between releasing and nonreleasing cells must occur early in signal transduction. There are also two simple explanations that have been eliminated experimentally. The first is that the basophils of these donors release histamine and leukotriene C4 in response to several other stimuli that operate independently of IgE and its receptor. Therefore the basophils of nonreleasers possess the machinery for secretion.1Nguyen KL Gillis S MacGlashan Jr, DW A comparative study of releasing and nonreleasing human basophils: nonreleasing basophils lack an early component of the signal transduction pathway that follows IgE cross-linking.J ALLERGY CLIN IMMUNOL. 1990; 85: 1020-1029Abstract Full Text PDF PubMed Scopus (91) Google Scholar This observation has other implications that will be discussed later. The second trivial explanation, that these basophils do not have sufficient cell surface IgE, has also been eliminated as a possibility. Like the normal population, basophils from nonreleasers have a median cell surface IgE antibody density of ≈100,000.1Nguyen KL Gillis S MacGlashan Jr, DW A comparative study of releasing and nonreleasing human basophils: nonreleasing basophils lack an early component of the signal transduction pathway that follows IgE cross-linking.J ALLERGY CLIN IMMUNOL. 1990; 85: 1020-1029Abstract Full Text PDF PubMed Scopus (91) Google Scholar Because we have shown that, on average, less than 5000 molecules are required for a maximal response,3MacGlashan Jr, DW Releasability of human basophils: cellular sensitivity and maximal histamine release are independent variables.J ALLERGY CLIN IMMUNOL. 1993; 91: 605-615Abstract Full Text PDF PubMed Scopus (133) Google Scholar nonreleasing basophils certainly possess a high enough density to initiate a normal reaction. Given these initial observations, we can suggest the following explanations for the nonreleaser "phenotype": 1.Poor release results from a missing or inactive component of the IgE-mediated signal transduction pathway.2.Poor release results from an overactive component of a down-regulatory pathway. For example, an overactive desensitization pathway (the normal auto-regulatory pathway of secretion) could prematurely stop secretion. Alternatively, high levels of cyclic adenosine monophosphate (cAMP) might suppress IgE-mediated release specifically, a result of an inactive or low level of phosphodiesterase.3.Poor release results from a missing extrinsic promoter of basophil function, specifically IgE-mediated functions.4.Poor release results from too much of an extrinsic inhibitor of basophil function, specifically IgE-mediated secretion. The first two possibilities fall under the general category of the donor's basophil phenotype, whereas the second two possibilities depend on causes extrinsic to the basophil. It is also possible than an extrinsic promoter or inhibitor is at normal levels on these donors but that the basophil receptors or their signal transduction pathways for these extrinsic factors are abnormal. An intriguing observation is that treating nonreleasing basophils with anti-IgE antibody causes a shift in the cells' responsiveness to another stimulus. In one series of studies it was found that pretreatment increased the sensitivity of the cells to stimulation with phorbol myristate acetate (PMA) to the same degree as observed in releasing basophils.1Nguyen KL Gillis S MacGlashan Jr, DW A comparative study of releasing and nonreleasing human basophils: nonreleasing basophils lack an early component of the signal transduction pathway that follows IgE cross-linking.J ALLERGY CLIN IMMUNOL. 1990; 85: 1020-1029Abstract Full Text PDF PubMed Scopus (91) Google Scholar In a second study it was found that pretreatment with anti-IgE antibody led to a decrease in the cells' responsiveness to the bacterial peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP).4Knol EF Mul FP Kuijpers TW Verhoeven AJ Roos D Intracellular events in anti-IgE nonreleasing human basophils.J ALLERGY CLIN IMMUNOL. 1992; 90: 92-103Abstract Full Text PDF PubMed Scopus (53) Google Scholar Both of these studies suggested that a partial signal occurred in nonreleasing basophils. The studies with PMA suggested that aggregation of cell surface IgE led to some change in protein kinase C (PKC). We have shown that anti-IgE antibody causes an increase in total cellular PKC activity and a specific increase in membrane-associated PKC activity. Because PMA stimulates basophils by activating PKC, it follows that any increase in PKC induced by anti-IgE would affect the responsiveness of the basophil to PMA. Indeed, when nonreleasing basophils were examined, anti-IgE induced an increase in membrane PKC activity. However, the increase was not as great as that observed in releasing basophils. Furthermore, the shape of the kinetic curve for PKC activity after activation resembled the kinetic curve of PKC activity in releasing basophils challenged in the absence of extracellular calcium. The distinction is that in a releasing basophil, PKC activity is transient, decaying to resting levels 30 minutes after stimulation.5Warner JA MacGlashan Jr, DW Protein kinase C (PKC) changes in human basophils: IgE-mediated activation is accompanied by an increase in total PKC activity.J Immunol. 1989; 142: 1669-1677PubMed Google Scholar In releasing basophils challenged in the absence of extracellular calcium, PKC activity rises to 30% to 50% of the peak in the presence of calcium but remains constant for at least 30 minutes. This is the same profile in nonreleasing basophils challenged in the presence of extracellular calcium. The aforementioned studies suggested that nonreleasing basophils lacked a component of the pathway that normally resulted in changes in intracellular calcium. Before reviewing the observations on the cytosolic Ca++ ([Ca++]i) response in nonreleasing basophils, it is useful to review some of the characteristics of the [Ca++]i response in releasing basophils. Four characteristics are worth noting in the context of these studies: 1.Oscillations in [Ca++]i are commonly observed in single cells.2.The kinetics of the [Ca++]i response are generally biphasic in nature.3.There is a variable interval of time between the addition of the stimulus and the first transient change in [Ca++]i.4.The distribution of single cell responses in releasing basophils usually shows few or no cells that have no response to stimulation. This is true at optimal and moderately suboptimal levels of stimulation. The first and third observations are characteristics clearly observed only at the single cell level, whereas the biphasic character of the [Ca++]i kinetic curve (No. 2) can be appreciated in whole population studies. This biphasic character is common in other cell types and has been attributed to the release of internal stores of calcium from endoplasmic reticulum-like structures followed by an influx of calcium from the extracellular medium. In fact, a popular model of the calcium responses raises the possibility that the depletion of the internal stores causes an intracellular messenger to signal some component in the plasma membrane to allow calcium to enter the cell. This "capacitance" model6Putney JW A model for receptor-regulated calcium entry.Cell Calcium. 1986; 7: 1-12Crossref PubMed Scopus (2116) Google Scholar has found some support in recent studies where a small unidentified factor has been found that can cause changes in the plasma membrane's permeability for calcium.7Randriamampita C Tsien RY Emptying of intracellular Ca2+ stores releases a novel small messenger that stimulates Ca2+ influx.Nature. 1993; 364: 809-814Crossref PubMed Scopus (789) Google Scholar In many instances, the human basophil shows clearly delineated phases to the [Ca++]i response, especially when stimulated through IgE receptors. However, it is important to note that the initial transient is only observed when basophils are stimulated with supraoptimal concentrations of anti-IgE or antigen. It appears that to adequately observe this sudden release of internal stores, the rate of receptor aggregation must be rapid to synchronize the generation of inositol 1,4,5-triphosphate (IP3) within the cell and therefore synchronize the release of internal stores of calcium. The time lag mentioned in No. 3 can be best appreciated in single cell [Ca++]i kinetic curves. In these kinetic curves, the addition of stimulus is followed by a quiescent period after which there is usually an abrupt increase in [Ca++]i. In basophils this initial increase is often transient, whereas in lung mast cells the increase is relatively stable. It is certainly true that the IP3 receptor itself has many features that explain this sudden rise in [Ca++]i, and both experimental8Finch EA Turner TJ Goldin SM Calcium as a coagonist of inositol 1,4,5,-trisphosphate-induced calcium release.Science. 1991; 252: 443-446Crossref PubMed Scopus (677) Google Scholar and mathematical models9Keizer J De YGW Two roles of Ca2+ in agonist stimulated Ca2+ oscillations.Biophys J. 1992; 61: 649-660Abstract Full Text PDF PubMed Scopus (65) Google Scholar of IP3–receptor dependent calcium release show this behavior. It is not as clear what components can explain the variable length of the quiescent period and the long period of quiescence that can often follow the addition of the stimulus. We have noted that two factors appear to control the length of the time lag: the concentration of the stimulus, that is, the rate of its induced cross-linking reaction, and the strength of the stimulus generated. The first of these factors is expected; the rate of the crosslinking reaction should be slower with lower antigen concentrations, leading to a slow build-up of second messenger activity in the cell. However, one can also show that the strength of the signal generated determines the length of this time lag. We noted many years ago that bivalent penicillin was a weaker stimulus for histamine release in properly sensitized basophils than multivalent conjugates of penicillin to proteins (e.g., human serum albumin [HSA]).10MacGlashan Jr, DW Mogowski M Lichtenstein LM Studies of antigen binding on human basophils. II. Continued expression of antigen-specific IgE during antigen-induced desensitization.J Immunol. 1983; 130: 2337-2342PubMed Google Scholar, 11MacGlashan Jr, DW Peters SP, Warner J, Lichtenstein LM. Characteristics of human basophil sulfidopeptide leukotriene release: releasability defined as the ability of the basophil to respond to dimeric cross-links.J Immunol. 1986; 136: 2231-2239PubMed Google Scholar In other words, for basophils sensitized with a specific density of BPO-specific IgE, BPO2 would cause far less histamine release than BPO21-HSA. In terms of the density of IgE required for equivalent histamine release, a 5 to 10 times higher density of benzyl penicilloyl (BPO)-specific IgE was required when BPO2 was used as a stimulus rather than BPO21-HSA. It was also shown that BPO2 establishes cross-links very rapidly; thus a comparison of the time lags after stimulation with either BPO2 or BPO21-HSA would not be distinguished by the rate of cross-link formation but by the strength of the signal transduction events generated. In this type of comparison, the time lag after BPO2 was 57 seconds, whereas for BPO21-HSA it was 17 seconds. These observations have direct relevance to the nonreleaser problem and will be used to analyze the next studies to be presented. The final point noted previously suggests that in a normal reaction, all basophils participate to varying degrees. Naturally there is a distribution of responses in a population of basophils, but we have found that this distribution rarely includes many cells that do not show any response to stimulation. Even at moderately suboptimal levels of stimulation, few nonresponding cells are observed. It is only when the response is very weak that an appreciable number of nonresponding cells can be observed. This point will also be useful in analyzing the [Ca++]i response in nonreleasing basophils. The studies of PKC activity in nonreleasing basophils suggested that a low level of PKC activity could be induced by anti-IgE antibody, but that the activity resembled a situation where no extracellular calcium was present. One might infer that in nonreleasing basophils no [Ca++]i signal was generated. It is true that there is a good correlation between the net [Ca++]i response and histamine release and that the y intercept for a linear regression analysis of this correlation is near zero. This suggests that nonreleasing basophils have no [Ca++]i response. However, these correlation data were generated by stimulating basophils with concentrations of anti-IgE antibody that were slightly less than optimal, that is, in a zone of activation where the initial release of calcium internal stores is not very evident in the whole population response and an asynchronous response among individual cells would result in a very low average response. However, a close examination of the single cell responses shows several interesting features. First, approximately 80% of the basophils show a [Ca++]i response, but this response has the characteristics of a weak stimulus (Fig. 1). There are a series of [Ca++]i oscillations that rapidly decrease in magnitude until resting conditions are achieved within 20 to 180 seconds. Indeed, the pattern observed is identical to the pattern observed in releasing basophils challenged in the presence of either acid or La+++ to block calcium influx. The average lag time for the response is three times slower than that observed in releasing basophils challenged with the same concentration of anti-IgE antibody. Finally, although the cells are challenged with an optimal concentration of anti-IgE antibody, approximately 20% of the basophils show no response. These data indicate that an initial response can be generated in nonreleasing basophils but that it is weak. Our first impression of these data led us to speculate that what these nonreleasing basophils lacked was the means to mobilize calcium from the extracellular medium, that is, the real defect was focused on the second phase of the [Ca++]i response rather than the overall response. However, if the data in the correlation plots are considered in the context of the single cell behavior observed for the [Ca++]i response, it is more straightforward to conclude that nonreleasing basophils only poorly generate any early second messengers. These observations do not actually allow us to select one of the possible explanations for the "phenotype" discussed previously. However, the observations place some constraints on the nature of any hypothesis. For example, one could speculate that high levels of intracellular cAMP could result in basophils that have a very poor calcium response. Inducing high levels of cAMP in releasing basophils alters the second phase of the calcium response and, generally, this inhibition only occurs in cells stimulated through the IgE receptor. However, these same studies indicate that very high levels of cAMP would be required to completely inhibit the [Ca++]i response and that cAMP elevations have little effect on the initial transient elevation in [Ca++]i or the time lag for this first increase. Therefore this possibility appears less likely on the basis of data obtained from nonreleasing basophils. We have examined several other hypotheses that focus on the first explanation offered, that is, that poor release results from a missing or inactive component of the IgE-mediated signal transduction cascade. A couple of years ago, Jean-Pierre Kinet and I speculated that nonreleasing basophils lacked the β subunit of the IgE receptor. At that time preliminary data indicated that the α and γ subunits could be expressed in the absence of the β subunit and that the β subunit might be involved in [Ca++]i signaling. It is clear today that αγ receptor combination is expressed in human monocytes without the β subunit so that the absence of a β subunit could occur in nonreleasing basophils. However, we found that equivalent levels of mRNA for the β subunit were expressed in releasing and nonreleasing basophils. Whereas these data do not exclude the possibility that expression of β subunit protein could be decreased in nonreleasers, this seems less likely. However, we are still working on the possibility that the β subunit itself is not the same in nonreleasing cells. The CD45 protein commonly expressed in leukocytes has been implicated in the early signal transduction steps associated with signaling in T cells. Indeed, in the absence of CD45, which is a phosphatase, no early signals are generated in T cells activated through the T-cell receptor. In a similar context, Hook et al.12Hook WA Berenstein EH Zinsser FU Fishler C Siraganian RP Monoclonal antibodies to the leukocyte common antigen (CD45) inhibit IgE-mediated histamine release from human basophils.J Immunol. 1991; 147: 2670-2676PubMed Google Scholar found that certain monoclonal antibodies to CD45 on human basophils could specifically modify the IgE-mediated response. Several hours of preincubation with the HB10 monoclonal antibody would inhibit IgE-mediated release 30% to 80%. In collaboration with Drs. Hook and Siraganian, we have found that this antibody selectively inhibited both phases of the [Ca++]i response in basophils stimulated with anti-IgE antibody but not with FMLP. We then suggested that nonreleasing basophils would express CD45 differently than releasing basophils, although we could not be certain whether expression would be increased or decreased because we did not know the role of CD45 in signal transduction or what the monoclonal antibody HB10 did during pretreatment. However, in calibrated flow cytometric studies, we could find no difference in CD45 expression between releasers and nonreleasers. For some time we have considered the possibility that circulating levels of IL-3 (or some other cytokine) could determine the "releasability" of basophils studied in vitro. An extension of this hypothesis is that nonreleasing basophils result in donors expressing very low levels of circulating IL-3 or that the basophils themselves did not possess the IL-3 receptor. This hypothesis is appealing because IL-3 is such a potent and efficacious up-regulator of basophil function and because many investigators now believe that leukocytes require specific cytokines to maintain function. In other words, a leukocyte is destined to die (experience apoptosis) unless function is maintained by cytokines. This hypothesis has not yet been fully evaluated in basophil studies. However, we have noted that a 24-hour pretreatment of nonreleasing basophils with IL-3 does not markedly alter their responsiveness to IgE-mediated stimulation. In addition, IL-3 is known to up-regulate the response of basophils to all stimuli, and yet nonreleasing basophils respond well to non–IgE dependent stimuli. One might guess that if IL-3 were missing in the circulation of donors with nonreleasing basophils, their basophil response to other stimuli would also be diminished. A more direct study of the IL-3 hypothesis is currently hampered by technical issues, but the data generated thus far are not supportive. In summary, the cause of the nonreleasing basophil "phenotype" has not yet been found. However, we now know that these basophils only poorly generate the second messengers typically occurring in releasing basophils. Both the PKC and [Ca++]i responses are blunted, but the data also suggest that the influx of calcium is particularly blunted in the nonreleasing basophil. Although the data do not rule out a number of possible explanations for the weak signaling in these cells, we currently favor the possibility that a component of signal transduction operating early in the reaction is either inactive or missing in these donors' basophils.