Pathophysiology of human basophils and mast cells in allergic disorders

CLINICAL

IMMUNOI.OGY

AND

Pathophysiology

IMMUNOPA’IHOLOCY

50,

S?+S40 (ll)x9)

of Human Basophils Allergic Disorders’

and Mast Cells in

GIANNI MARONE, VINCENZO CASOLARO, RAFFAELE CIRILLO. CRISTIANA STELLATO, AND ARTURO GENOVESE Department

of Medicine.

Universip

of Naples.

II School

qf Medicine,

80131

Nuples.

Italy

Basophilleukocytes and tissue mast cells are inflammatory cells that are found in virtually all human tissues. They appear to be involved in the pathogenesis of such allergic diseases as allergic rhinitis, bronchial asthma, anaphylaxis, atopic and contact dermatitis, chronic urticaria, and hypersensitivity pneumonitis. By releasing a variety of chemical mediators, they could also play a role in the pathophysiology of a wide range of inflammatory disorders of the joints, and of intestine, lung, coronary, and myocardial diseases. Although these two cell types are similar in several aspects, striking differences have also been observed. Moreover, human mast cells from different anatomical sites and within an individual tissue synthesize different mediators and have different release mechanisms. The recent advent of techniques that yield highly purified basophils and mast cells from diverse tissues will probably lead to major advancements in understanding the biochemical and pharmacological mechanisms that control the release process of these cells. The release of mediators from these cells is also controlled by a series of largely undefined biochemical steps that represent the basis of the concept of basophil and mast cell releasability. Alterations of basophil or mast cell releasability have already been detected in patients with allergic rhinitis, bronchial asthma, atopic dermatitis. and chronic urticaria. Taken together, these findings demonstrate that basophils, mast cells, and their chemical mediators play a pivotal role in several inflammatory disorders. 8 1989 Academic

Press. Inc

INTRODUCTION

Basophil granulocytes and mast cells were first identified by Paul Ehrlich over 100 years ago (1, 2). He called them “Mastzellen” (“well fed cells”) because of the large granules in their cytoplasm (2). Basophils and mast cells are the only cells with high affinity receptors for IgE (3, 4), and they are one of the few cell types to synthesize histamine. Their cytoplasmic granules contain glycosaminoglycans and consequently have metachromatic-staining properties (5-7). In humans these cells synthesize and release upon appropriate stimulation an array of preformed (e.g., histamine) or de n~vo synthesized chemical mediators (e.g., leukotrienes, platelet aggregating factor (PAF), etc.) that possess a wide spectrum of proinflammatory and immunomodulatory effects. Moreover, basophils and mast cells are practically ubiquitous: basophils are circulating cells and mast cells are found in practically all human tissues, hence they are readily available for quick effector function.

’ Presented as part of a symposium entitled “Immunoregulation 1988, Palazzo dei Congressi. Florence, Italy. S24 0090-1229189 $1.50 Copyright All tights

0 1989 by Academic Press. Inc. of reproduction in any form reserved.

in Clinical Diseases,‘* May 26-27,

BASOPHILS

GENERAL

AND

PROPERTIES

MAST

CELLS

IN

OF HUMAN

ALLERGIC

DISORDERS

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MAST CELLS AND BASOPHILS

Early studies stressed the similarities between mast cells and basophils, and it was even suggested that they arise from the same progenitor (8). However, more recent evidence indicates there are striking differences between resting or degranulating human mast cells and basophils (9, 10). Basophils, like other granulocytes, differentiate and mature in the bone marrow. They circulate in the blood and are not normally found in connective tissue. In contrast, mast cells are ordinarily present in connective tissue (1 l-13), often adjacent to blood and lymphatic vessels, near or within nerves, and beneath epithelial surfaces, such as those of the respiratory and gastrointestinal systems and the skin. In various mammalian species there appears to be a general inverse quantitative relationship between the number of basophils and mast cells. Rodents, for example, are relatively rich in mast cells, but have few basophils (7). In amphibians as much as 65% of the circulating leukocytes are basophils (14), while both cell types are found in humans. Human basophils isolated from the peripheral blood are round; in tissues and under certain circumstances in vitro, basophils may assume elongated configurations, or may present uropods (U-17). The cytoplasm exhibits a moderate number of granules. The nucleus is lobular, bilobed, or multilobed. Human mast cells and basophils degranulate by different morphological mechanisms (18-20). Mast cell granules swell, lose electron density, and then fuse with other granules before the contents are extruded (20). Stimulation of basophils results in fusion of individual granules with the plasma membrane, without the granules first fusing with each other as seen in mast cells. The degranulation of basophils activated by antigens, CSa, and the lymphokine HRA is similar (21). The ultrastructural events occurring during recovery from IgE-mediated degranulation of human lung mast cells have recently been elucidated (22). A study of the phenotypic membrane profile of human basophils revealed several activation-linked structures on their surface, namely the IL-2 and the IL-3 receptor, the TlO antigen, and the structure p 24 (23). Human mast cells, like basophils, are heterogeneous in shape, appearing in tissues as round, oval, or elongated spindle-shaped cells. The cell surface has many long, thin projections that appear as villous-like profiles. The nucleus is generally round, although occasional cells have nuclei with lobular configurations. Mast cell granules are more heterogeneous in substructural pattern than are basophils. Dvorak identified four basic granule patterns (scroll, crystal, particle, mixed), and described lipid bodies in human lung mast cells (24). Within individual mast cells, granules may have a uniform substructural pattern (e.g., scrolls), or they may contain a mixture of granules of the various substructural types. MAST CELL AND BASOPHIL

HETEROGENEITY

Human lung mast cells are heterogeneous in size (10 to 18 pm in diameter), histamine content (2 to 10 pg/cell), and function as regards their ability to release histamine and prostaglandin DZ (large cells release more histamine and PGD, than

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do small cells) (25). Heterogeneity of histamine-containing bronchoalveolar mast cells has been suggested (26). Mast cell heterogeneity is particularly evident in rodents. There are two forms of rodent mast cells: “typical” or “connective tissue,” and “atypical” or “mucosal” (27-29). Apart from differing in location, these two cell types also differ in ultrastructure, response to mast cell stimuli, and the effect of antiallergic compounds (27-30). Human lung and intestinal mast cells do not seem to possess a similar heterogeneity; indeed they are similar in histamine content, staining characteristics, ultrastructure. response to anti-IgE, and pharmacologic agents (31, 32).

Schwartz and colleagues (33, 34) identified two types of mature human mast cells differing in neutral protease composition. The tryptase-positive, chymasenegative type (T mast cells) is the predominant, but not exclusive type in normal bowel mucosa and lung. The tryptase-positive, chymase-positive (TC mast cells) is the predominant, but not exclusive type in human skin and bowel submucosa (33). Interestingly, in humans with immunodeficiency diseases affecting T lymphocytes, there is a selective depletion of the T, but not TC mast cells in sections of small bowel (35). Although there is some evidence that two human basophil populations of different density behave differently (36). it is not sufficient to support the existence of subpopulations of these cells. MAST CELLS AND BASOPHILS

IN ALLERGIC

DISORDERS

The relevance of mast cells and their chemical mediators in the pathophysiology of bronchial asthma was first recognized 75 years ago (37), and now multiple histologic, physiologic, biochemical, and pharmacologic evidence indicates that mast cells, basophils, and the products of their activation play a significant role in the pathogenesis of several allergic disorders (Table 1). Basophils have been observed in nasal secretions of allergic patients, and their presence was closely related to mediator release and clinical symptoms (40-42). Kimura et al. (51) reported an increase in the number of basophils in the sputum

DISEASES

Disease Anaphylaxis Allergic rhinitis Asthma Atopic dermatitis Urticaria

INVOLVING

TABLE

1

MAST

CELLS

AND

BASOPHILS

Evidence Multiple physiologic, pharmacologic Multiple physiologic, pharmacologic Multiple physiologic, pharmacologic Multiple physiologic, pharmacologic Multiple physiologic, pharmacologic

References

histologic, biochemical,

(38, 39)

histologic, biochemical,

(40-43)

histologic, biochemical,

(44-46)

histologic, biochemical,

(47. 48)

histologic, biochemical,

(49, 50)

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from asthmatic patients before the onset of an asthmatic attack, and the number of basophils was related to the severity of the relapse. Histamine-containing cells have been found in the lumen of human bronchi (52), and two morphological types of mast cells have been identified in bronchoalveolar lavage (BAL) fluid, the number of BAL mast cells recovered from asthmatic subjects being greater than in controls (46). Moreover, the histamine level is increased in asthmatic BAL (53, 54), and the response to anti-IgE of bronchoalveolar mast cells in asthma is enhanced (54, 55). Patients who died as a consequence of anaphylaxis contained an increased number of lung mast cells (45), while the bronchial walls of patients who died of asthma yielded fewer mast cells compared to patients who died from other causes (44). It would be interesting to study the reasons for these apparently contrasting findings. IgE- and non-IgE-mediated releasability of basophils is altered in asthmatic patients and in other subjects with allergic conditions when compared to controls (56-60).

The role of mast cells, basophils, and their chemical mediators in the pathogenesis of allergic disorders is also supported by pharmacological evidence. All drugs active in the prevention/treatment of these disorders (i.e., methylxanthines, corticosteroids, B-adrenergic drugs, cromolyn, etc.) are inhibitors of in viva or in vitro release of histamine and other mediators from mast cells or basophils (10). Mast Cells and Basophils

in Injlammatory

Disorders

A series of data suggest that mast cells and basophils participate in the pathogenesis of a variety of inflammatory disorders. For example, basophils are prominent in delayed hypersensitivity reactions such as atopic dermatitis, contact dermatitis reactions, and delayed “Jones-Mote” reactions in man (61-63). Basophils, like other granulocytes, probably migrate into areas of active inflammation in response to chemotactic factors released from mast and other cells, and thus prolong the intlammatory reaction. Histologic and biochemical studies indicate that mast cells and products of their activation play a role in the pathogenesis of such inflammatory conditions as rheumatoid arthritis (64-66), inflammatory bowel disease (3 1, 67), progressive systemic sclerosis (68-70), pulmonary fibrosis (71, 72), sarcoidosis (71, 73), and coronary artery disease (74-79). Chemical

Mediators

Synthesized

by Mast Cells and Basophils

The basophil has, on average, I pg of histamine/cell (80). Mast cells purified from human lung, intestine, and skin contain approximately 4 pg of histamine/cell, although lung mast cells show a marked heterogeneity (25). Human basophils and lung mast cells on the average generate approximately 60 ng/106 cells of leukotriene C, (LTC,) (31, 81, 82). Skin mast cells generate a small amount of LTC, (83). Purified lung mast cell and basophil preparations synthesize only LTC,. However, metabolism to leukotriene D, (LTD,) and leukotriene E, (LTE,) occurs in preparations of peripheral mononuclear cells and suspensions of lung or gut cells (31, 32).

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PGD, is the main cyclooxygenase metabolite synthesized by lung. intestinal. and skin mast cells (31, 83-U). Thus, the difference found in rodents, where the mucosal mast cell generates mainly leukotrienes and the connective tissue cell prostaglandins, does not occur in humans (86). It has yet to be established whether or not basophils synthesize cyclooxygenase metabolites. Lung mast cells, but not basophils, rapidly generate several varieties of PAF (87, 88). As in the human polymorphonuclear cell. practically none of the PAF generated is released. Enzymes with TAME-esterase activity have been described in human lung mast cells and basophils (89, 90). In addition, mast cells contain an enzyme resembling tryptase that generates bradykinin from kininogen (91). The major enzyme from human mast cells is trypsin like (90). Human lung mast cells synthesize both heparin and chondroitin sulfate E (92, 93). Basophils also contain major basic protein (94) and Charcot-Leyden crystal protein (95), whereas their presence has been excluded in lung mast cells (96). The eosinophil major basic protein, found in man at the site of allergic inflammation (97, 98), stimulates the release of histamine from human basophils (99). In conclusion, there are marked differences between the array of mediators synthesized by human basophils and those synthesized by mast cells. Therefore, it is likely that these cells play distinct roles in the different aspects of inflammatory reactions. Stimuli

that Activate Human

Mast Cells and Basophils

Cross-linking of IgE receptors on the surface of basophils and mast cells by antigen and anti-IgE are the classical means with which to induce mediator release from these cells (100, 101). IgG is also present on the surface of basophils, and cross-linking of these surface molecules results in mediator release (102-104). Several substances can activate these cells, i.e., macrophage, monocyte, and lymphocyte products, such as histamine-releasing activity (HRA) (105-109), IL-3 (1 lo), the Ca2+ ionophore A23187 (11 l-l 13), hyperosmolar stimuli (18, 114), deuterium oxide (57), PAF (115). and platelet-derived products (116-I 18). As previously mentioned, the major basic protein stimulates the release of histamine from human basophils and rat mast cells (98, 99). Anaphylatoxins such as C3a and C5a (119, 120), formyl-methionine-containing peptide analogs of bacterial products (f-met peptide ( 121, 122, 123). and phorbol esters (124) activate only human basophils. Gastrin and morphine activate only human skin mast cells (125). Protein A and intact Staphylococci Cowan I induce histamine release from human basophils (126) by interacting through the “alternative site” of protein A with the F(ab’), portion of IgE on the basophil membrane (104, 127). Interestingly, neither protein A nor intact Staphylococci Cowan I induce histamine release from human lung mast cells (54, 128). Arachidonic acid metabolites such as Shydroperoxyeicosatetraenoic acid (S-HPETE), PGD,. and leukotriene B, activate basophils only under certain conditions (129). Table 2 summarizes most of the agents known to activate human basophils and mast cells.

RASOPHILS

AND MAST CELLS IN ALLERGIC TABLE

ACTIVATORS

OF BASOPHILS

Basophils Antigen Anti-&E Anti-IgG Ca*.+ ionophores (e.g., A23187) Hyperosmolarity C3a. CSa Macrophage and monocyte-derived factors Lymphocyte and platelet-derived factors (e.g. HRA) Formyl-methionine peptide Eosinophil major basic protein lnterleukin 3 (IL-3) Maitotoxin Major basic protein Pepstatin A Platelet activating factor (PAF) Protein A of Staphylococcus aureus Phorbol esters Phosphohpase A Deuterium oxide* 5-Hydroperoxyeicosatetraenoic acid* Prostaglandin DZ* Leukotriene B,*

DISORDERS

S29

2 AND

MAST

CELLS

Mast cells Antigen Anti-IgE Ca’+ ionophores (e.g. A23187) Hyperosmolarity Gastrin (cutaneous mast cells) Macrophage-derived factors Maitotoxin Morphine (cutaneous mast cells)

__Agents not included in the list either do not activate the particular cell type or have not been exhaustively examined in the system. * Requires the presence of cytochalasin B. Note.

BIOCHEMICAL AND PHARMACOLOGICAL CONTROL OF MEDIATOR RELEASE FROM HUMAN MAST CELLS AND BASOPHILS

The bulk of the more recent information reported concerning the biochemical events in mast cell activation has come from studies using purified rat peritoneal mast cells. However, there are striking differences between human basophils and mast cells and rodent mast cells (9, 56, 130, 131) and clearly, results obtained in rodent cannot be extrapolated to humans. The recent availability of purified basophils and mast cells has given an impetus to biochemical studies in these cells. It should be noted, however, that biochemical studies with purified human basophils and mast cells are still in an early stage (132, 133) and much remains to be done before results concerning the role of these cells in inflammation are forthcoming. The presence of calcium in the extracellular medium appears to be essential for IgE-mediated histamine release from mast cells and basophils. Stimulation of purified lung mast cells with anti-IgE induces the uptake of radiolabeled calcium and an increase in free cytoplasmic Ca*+ (133, 134). The increase in free cytoplasmic Ca2 + appears to be correlated with the percentage of histamine release. Not only is the activation of these cells associated with Ca2’ uptake, but their desensitization leads to a loss of the Ca2+ uptake response upon subsequent

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stimulation (133). Fewtrell and others, using digital fluorescence ratio imaging of Fura 2 to measure [Ca”]i in individual cells, demonstrated that the kinetic process of the increase in Ca’ ’ concentration associated with the secretion of mediators is a population event. In fact, the lag time between addition of antigen and onset of the increase of [Ca”li varied considerably between cells (135). Several studies have provided evidence of the interrelationship among Ca’+- metabolism. phosphoinositide breakdown. and protein kinase C activation in the control of mediator release from mast cells and probably basophils (136140). Several groups of investigators have demonstrated that cyclic AMP (CAMP)active drugs prevent mast cells and basophils from releasing mediators (141-143). Activation of rat peritoneal mast cells by anti-IgE or antigen stimulus results in a monophasic rise of CAMP followed by a second rise 3 min after addition of the stimulus (144, 145). The second increase in CAMP does not occur if the cells are preincubated with indomethacin, and it is probably due to stimulation of the mast cell adenylate cyclase by prostaglandins, possibly PGD, (145). In contrast, activation of purified human lung mast cells and basophils with anti-IgE did not affect CAMP levels ( 146). l3-adrenergic agonists (fenoterol, salbutamol, etc.) and CAMP phosphodiesterase inhibitors (theophylline, etc.) are used to treat asthma and one of their effects is to inhibit mediator release by mast cells and basophils (9, 147). However, these drugs also exert direct anti-allergic activity on smooth muscle. Several studies showed that the P-adrenergic agonists, PGE, and PGE,. as well as phosphodiesterase inhibitors and other drugs that increase CAMP inhibited mediator release in mast cells and basophils (143, 148, 149). Similarly, the adenylate cyclase agonist forskolin inhibits the release of histamine and peptide leukotriene C, from human basophils and lung mast cells (82, 150). Such nonsteroidal anti-inflammatory agents as indomethacin and acetylsalicylic acid potentiate antigen-induced histamine release from human basophils (149). This probably occurs because nonsteroidal anti-inflammatory drugs shunt arachidonic acid metabolism from the cyclooxygenase pathway to the lipoxygenase pathways. Support for this hypothesis comes from the observation that the lipoxygenase product, 5-HPETE, enhances histamine release from human basophils (151). Moreover, indomethacin or the addition of exogenous arachidonic acid itself leads to increased levels of LTC, (152). Also here there is a striking difference between human basophils and mast cells. In fact, indomethacin and exogenous arachidonic acid do not potentiate IgE-mediated histamine release from human lung or gut mast cehs. The different pharmacological modulation between the release reaction of basophils and that of mast cells was also found with two important autacoids, histamine and adenosine. In fact, histamine and dimaprit (H2 agonist) inhibit the release process in human basophils (153), whereas they have no effect on human lung mast cells (154). Furthermore, adenosine, a natural nucleoside, dosedependently inhibits histamine release from human basophils (148. 155) whereas it significantly potentiates mediator release from human mast cells (131, 156, 157). Over the last decade we have actively investigated the nature of the regulation by adenosine and modified analogs of mediator release from human basophils and

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AND MAST CELLS IN ALLERGIC

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lung mast cells. We have devoted our attention to this topic for several reasons. First, adenosine is a natural nucleoside circulating in micromolar concentrations in normal plasma (158); second, adenosine is a primary mediator of rat mast cells (159); third, it plays a role in the pathogenesis of some immune deficiencies (156) and autoimmune diseases (160) by interacting with specific membrane receptors present on inflammatory cells (161-164); and fourth, it causes bronchoconstriction in asthmatic patients (165). We found that 5’-N-ethylcarboxyamideadenosine (NECA) > 2-chloradenosine > adenosine > ( - )-N6(R-phenyl-isopropyl)-adenosine {( - )-R-PIA} inhibits in vitro IgE-mediated histamine and LTC4 release from human basophils (155, 156) (Fig. 1). These structural congeners of adenosine and the parent nucleoside inhibit the generation of LTC, more effectively than they do histamine release (156). A number of methylxanthines that are antagonists of cell surface adenosine receptors (161, 162) partially block the inhibition of mediator release caused by NECA and adenosine suggesting that they activate a cell surface adenosine receptor that has properties similar to those of an adenosine A,/Ra receptor (155). More recently, we have extensively evaluated the effects of adenosine and its analogs on mediator release from mast cells purified from human lung parenchyma. Figure 2 shows that micromolar concentrations of adenosine, NECA, and (-)-R-PIA enhance the release of histamine from human lung mast cells challenged with anti-IgE (166). This finding confirms our previous reports (130, 131) and contrasts with reports (167-170) that adenosine inhibits IgE-mediated histamine release from lung mast cells. In an attempt to reproduce the latter findings we used mast cells obtained by mechanical or enzymatic treatment of human lung tissues and different times of preincubation with adenosine. We also used populations of lung cells enriched with 1 to 97% of mast cells. Under all experimental conditions micromolar concentrations of NECA, adenosine, and ( -)-R-PIA enhanced the release of histamine induced by anti-IgE or the Ca2+ ionophore A23187 from lung mast cells. Moreover, micromolar concentrations of adenosine,

FIG. 1. Comparison of the effect of varying concentrations of adenosine and its analogs on antigeninduced secretion during the first stage of histamine release from human basophils. Each point represents the mean 2 SEM obtained from 12 experiments. Control histamine release (mean 2 SEM) was 38.1 5 4.5%. Reprinted with permission from Ref. (155).

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r i

‘-’ *

tiI

FIG. 2. Comparison of the effect of varying concentrations of NECA (diamonds), R-PIA (closed triangles). and adenosine (squares) on histamine release induced by anti-IgE from mast cells isolated from human lung by enzymatic dispersion. Lung mast cells were preincubated with one of the analogs for 10 min prior to challenge with stimulus. Each point represents the mean t SEM obtained from I7 experiments. Control histamine release (mean -+ SEM) was 32 2 4%. All concentrations of each of the analogs produced statistically significant levels of potentiation except that point marked with an asterisk. Reprinted with permission from Ref. (166).

NECA, and (- )-R-PIA potentiated the de nova synthesis of LTC, from immunologically activated human lung mast cells (166). All these results clearly indicate that a natural nucleoside might exert opposite immunomodulatory effects on human basophils and mast cells (155, 166). Calmodulin is an intracellular Ca’+ -binding protein present in human leukocytes (171, 172), and we have examined its role in the control of histamine and LTC, release from human basophils and lung mast cells. A series of putative calmodulin antagonists, such as trifluoperazine (TFP) and compound W-7, at appropriate concentrations, inhibit IgE-mediated release of histamine and de nova synthesis of LTC, from basophils and lung mast cells (173, 174). The chlorinedeficient analog of W-7, compound W-5, and the sulfoxide derivative of TFP, TFP-S, which have a very low affinity for calmodulin (172), have practically no inhibitory effect on mediator release from human basophils and mast cells. Therefore calmodulin appears to play a role in these release processes. Corticosteroids are the most potent anti-inflammatory agents available and it has been shown that they inhibit the release of histamine from immunologically activated rat mast cells and human basophils (175-177). They do not inhibit the release reaction initiated by non-IgE-dependent stimuli such as compound A23187, phorbol diesters, or formyl-methionine peptides (177, 178). It is interesting that, unlike human basophils and rat peritoneal mast cells, mast cells isolated from human lung are unresponsive to steroids (179). Although phospholipase A2 (PLA,) is probably an important enzymatic intermediate in the process by which histamine release takes place (113, 180), prevailing evidence suggests that the steroids do not inhibit basophil or murine mast cell histamine release by inhibiting PLA, (176, 179). THE RELEASABILITY

CONCEPT

The hypothesis that the IgE and IgG antibodies

against various allergens are of

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paramount importance in the pathogenesis of allergic disorders began to be challenged in the seventies. At that time it started to be appreciated that the pathophysiology of allergic disorders was characterized by two other important aspects, namely the hyperresponsiveness of the airway smooth muscle, and probably other target organs, to chemical mediators such as histamine, metacholine, and PGF,, (181, 182). Second, it became clear that intrinsic alterations of effector cells (e.g., basophils and mast cells) are involved in the pathophysiology of allergic disorders. It was found that the percentage of histamine release from basophils of different adult donors is essentially unrelated to the serum concentration of IgE or to the number of IgE antibody molecules per cell (183). In addition, basophils with 5000 IgE molecules on the cell surface responded almost as well to anti-IgE as basophils containing 500,000 molecules (183). These observations indicated that the extent of the response to IgE- and non-IgE-mediated stimuli was influenced by unknown “factor(s)” unrelated to the antigen-antibody interaction. The latter observations represent the basis on which the concept of basophil and mast cell releasability has been built over the last 15 years (184). Although early studies of basophil and mast cell releasability were hampered by the lack of purified cell populations, a number of useful observations were made by the few groups working in this area. For example, it was demonstrated that IgE-mediated releasability is very low in young normal donors and that it increases with donor age, particularly in subjects between 1 and 20 years of age (185, 186). This practical aspect should always be taken into serious account in studies aimed at comparing basophi1 releasability of groups of different age or nonmatched groups of patients and controls. It is also well to remember that cells of one donor could release well with anti-IgE, but poorly with another secretagogue, e.g., f-met peptide or C5a and vice versa. The use of purified basophils and lung mast cells in studies on basophil and mast cell releasability will undoubtedly produce basic information, but at present there are still practical difficulties in obtaining sufftcient numbers of cells to perform the appropriate biochemical studies. Therefore, in an attempt to provide a genetic basis to the concept of basophil releasability we performed a study in monozygotic and dizygotic twins. Our results suggested that both IgE-mediated releasability and the response to the Ca ‘+ ionophore, A23187, are mainly controlled by genetic factors, whereas the response tof-met peptide seems to be influenced by environmental factors (187). In the same study it was found that the parameter of IgE-mediated releasability is independent of the control of serum IgE levels. The latter observation may account for the polymorphic genetic traits of allergic individuals. It should be pointed out that results of basophil releasability cannot be extrapolated to human mast cells. In fact, studies conducted on basophils and mast cells not obtained from the same donor have suggested that there are several differences between human basophils and mast cells obtained from different anatomical sites (188). Comparing basophils and lung mast cells obtained from 52 individual donors we found striking differences between IgE- and non-IgE-mediated releasability of these two cell types (54). In general, an increased basophil releasability has been found in patients with

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such different allergic disorders as atopic dermatitis, allergic rhinitis, bronchial asthma, and food allergy (54, 56, 189). In contrast, a decreased IgE-mediated releasability of basophils has been found in patients with chronic urticaria (156). It is now evident that altered basophil and mast cell releasability is, together with increased IgE synthesis and hyperresponsiveness of target organs, one of the three hallmarks of allergic diseases and this parameter probably has a genetic basis. It is obvious that further studies are necessary to provide a biochemical basis to basophil and mast cell releasability, in normals and in allergic individuals. CONCLUSIONS

Mast cells and basophils are located at the interface of the individual and his environment (mucosal surfaces) in two areas of sense invasion (around nerves and blood vessels) and at the site of chronic inflammation (synovial tissues and fluids from patients with diverse arthritides). The results of a number of biochemical, morphological, and functional studies indicate that basophils and mast cells together with their chemical mediators participate not only in immediate and delayed allergic reactions, but also in inflammatory disorders of the joints, intestine, interstitial disease of the lung, and coronary and myocardial diseases. The observation that several lymphokines and monokines (IL-3, HRA, IgEbinding factor(s), etc.) activate human basophils to release mediators strongly implicates that these cells might play a major role in causing chronic inflammation in humans. The advent of techniques for the isolation and purification of human basophils and mast cells from lung parenchyma, BAL, skin, intestine, and cardiac tissue has been a major breakthrough in this field. The results obtained using these techniques indicate that basophils and mast cells obtained from human tissues differ in their content of mediators, biochemistry, and sensitivity to pharmacological agents. It is hoped that the availability of purified basophils and mast cell will also permit the characterization of the biochemical basis of the concept of releasability. Although it is evident from this brief review how much progress has been made in this field, work with purified cells isolated from different anatomical sites is still in a very early stage. However, investigators working on this topic are confident that insight into the pathophysiological events produced by these cells will be beneficial to individuals suffering from a variety of inflammatory disorders. ACKNOWLEDGMENTS Studies in the author’s laboratory have been supported in part by grants (85.00491.04, 86.00088.04, and 88.00559.04) from the CNR, the MPI, and “Minister0 della Sanita” (Rome, Italy). G.M. is the recipient of the “Angelo Minich” prize.

REFERENCES 1. Ehrlich, P., Thesis, 1878. 2. Ehrlich, P., Arch. Anal. Physiol. (Leipzig) 166, 1879. 3. Rossi, G., In “Advances in Clinical Immunology. The Role of Chemical Mediators in Pulmonary and Cardiac Diseases” (M. Condorelli, G. Marone, and L. M. Lichtenstein, Eds.), pp. 21-35, OK Medical Press, Florence, 1984. 4. Rossi, G., and Quarto, R., In “Human Inflammatory Disease. Clinical Immunology” (G.

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5.

6. 7. 8. 9.

10. Il. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.

30. 31. 32. 33. 34. 35. 36. 37. 38. 39.

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