What Determines Asthma Phenotype? Is It the Interaction between Allergy and the Smooth Muscle?

JUDITH L. BLACK and PETER R. A. JOHNSON

Department of Pharmacology, University of Sydney, Sydney, Australia

	INTRODUCTION

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Passive sensitization provides a useful model for the examination of the effect of the allergic process on smooth muscle reactivity and growth. This technique involves the incubation of bronchial tissue segments or bronchial smooth muscle cells in culture with serum from patients with allergic asthma or, alternatively, serum from nonallergic nonasthmatic patients. The allergic serum contains a high concentration of total IgE, usually greater than 1,000 IU/ml, and is taken from patients who exhibit a positive skin test response to one or more common allergens, whereas control nonallergic serum has a total IgE concentration of less than 20 IU/ml and is derived from patients who are skin test negative (1). One of the major advantages of this model is that comparisons can then be made between tissues or cells from the one patient, thus minimizing the inevitable variability in between-patient responses so familiar to those who work with human tissue.

	WHAT IS KNOWN

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Passive Sensitization Results in Changes in Human Airway Smooth Muscle Tone, Growth, and Cytokine Production

Initially, the observation was made that passive sensitization results in an increased contractile response to histamine and K⁺ and that this was likely to be associated with increased calcium influx into the airway smooth muscle cell (1). Since then, the findings have been confirmed (2) and extended by numerous other groups who have reported that passive sensitization results in increased contractile responses to tachykinins and cholinergic agonists and decreased relaxation responses to calcium channel antagonists, potassium channel openers, vasoactive intestinal polypeptide, and, inconsistently, -adrenoceptor agonists (3).

However, there are several different types of functional consequences of passive sensitization (Figure 1). First, bronchial ring segments exposed to allergic serum exhibit a contractile response to exogenous allergen, whereas segments from the same patient exposed to nonallergic serum do not. That this response is mediated by IgE and that the site of this reaction is mast cells located within the tissues is now well established. This evidence arises from studies in which chimeric IgE has been substituted for allergic serum (8), serum has been depleted of IgE (7), and anti-IgE has been substituted for allergen (13). Moreover, immunohistochemical analysis of passively sensitized tissue has revealed that there is an increase in IgE-bearing cells in these tissues and that these cells are principally mast cells (14).

View larger version (28K): [in this window] [in a new window]   Figure 1.   Passive sensitization of human bronchial segments results in a contraction in response to antigen, which is due to the presence of IgE in the sensitizing serum, and increased contractile and decreased relaxation responses, which are due to an unknown serum-derived factor(s). Sensitization of human primary airway smooth muscle cells in culture results in increased growth, increased production of extracellular matrix proteins, and increased message for cytokines. The mechanism of these effects in cultured cells remains to be elucidated.

Second, there are the changes in contraction and relaxation responses outlined above. Evidence for the mechanisms underlying these changes is conflicting, but the increase in contractile responses is consistent except for a single study in which differences in protocol may be relevant (15). In addition, the alteration in smooth muscle response is observed whether the changes are recorded as shortening velocity and capacity (4), isometric force (1, 2, 3, 5, 7, 11), or induction of myogenic response to quick stretch (6).

Third, when human airway smooth muscle cells in culture are exposed to allergic serum and challenged with allergen, there is increased protein expression of the oncogenes c-fos and c-jun and cycling of the cells as measured by incorporation of tritiated thymidine (16). Hakonarson and colleagues have reported that passive sensitization of human bronchus markedly increases the expression of interleukin 1 (IL-1) (10) as well as the mRNA for the low-affinity IgE receptor FcRII (9). Moreover, in one study, in response to passive sensitization, human airway smooth muscle cells have been reported to express increased mRNA for both helper T cell type 1 (Th1) and Th2 cytokines, as well as protein for the respective receptors (17). Hirata and colleagues (18) found that allergen challenge of passively sensitized human bronchi produced upregulation of intercellular adhesion molecule (ICAM), vascular cell adhesion molecule (VCAM), and E-selectin on bronchial vessels and this was associated with increased release of tumor necrosis factor  (TNF-), but not IL-1 into the supernatant.

Fourth, passive sensitization produces changes in the extracellular matrix proteins secreted by human airway smooth muscle cells in culture. Exposure to allergic serum produces increased amounts of fibronectin, perlecan, and chondroitin sulfate compared with cells exposed to nonallergic serum (19).

Thus, passively sensitized human airway smooth muscle exhibits properties that are of relevance to the asthmatic airway: increased contraction, decreased relaxation, increased growth, which could contribute to hyperplasia, and participation in the inflammatory/allergic reaction by a response to, as well as elaboration of, important cytokines.

There Are Important Differences in the Properties of Actively Sensitized and Nonsensitized Bronchial Smooth Muscle

The relationship between the sensitization status of the lung tissue and the clinical definition of atopy cannot always be studied directly. In our laboratory tissues from approximately one-third of the patients contract in response to application of allergen. Thus we describe these patients as "actively sensitized" but since we do not have available corresponding data on skin test reactivity, we cannot strictly call them atopic. Nevertheless, we have noticed marked differences in the in vitro characteristics of tissue taken from this subset of patients, compared with those exhibiting no such response to allergen. Supernatants from stimulated neutrophils, when applied to human bronchial segments in vitro, caused a direct contractile response in nonsensitized tissue that was not observed in sensitized tissues (20). In contrast, when these supernatants were applied to tissues subjected to electrical field stimulation (EFS), a potentiation of the contractile response was observed in sensitized but not nonsensitized tissues: an effect mediated prejunctionally on parasympathetic nerve fibers. Exogenous TNF-, however, potentiated the contractile response to EFS only in nonsensitized tissues (21). Johnson and coworkers (22) reported that the mast cell mediator tryptase causes potentiation of the contractile response to histamine in sensitized but not nonsensitized tissues. Moreover, the myosin light chain kinase content of actively sensitized tissues was increased compared with that of nonsensitized patients (23). In addition, in an immunohistochemical study (24) enumerating inflammatory cells in sections of human bronchus taken from sensitized and nonsensitized patients, whose sensitization status was established in vitro, the number of mast cells located in the smooth muscle of sensitized patients was increased compared with that of controls. Thus, there are significant differences in the in vitro properties of human airway tissue that can be directly related to the allergic status of the patient. It is interesting to contemplate the relationship between these findings and those in a study by Robinson and coworkers (25), who tested the hypothesis that immature progenitors, which may have the potential to develop into eosinophils, are present in the bronchial mucosa in asthma. They examined CD34⁺ cell numbers and found that they were increased in the mucosa of both atopic, nonasthmatic, and atopic asthmatic subjects whereas cells positive for both CD34 and IL-5 receptor  (IL-5R) mRNAindicative of IL-5 responsivenesswere present only in subjects with asthma. The idea that differentiation of progenitors could occur in situ at the site of allergic inflammation raises the possibility that local conditions in the lung could play a role in the interaction of the allergic process and smooth muscle reactivity.

	WHAT IS CONFLICTING

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Are the Changes in Contractility, Cell Cycling, and Cytokine Production Due to the High Concentrations of IgE in the Allergic Serum?

Whether the changes in in vitro reactivity outlined above are all due to the presence of high IgE is a matter of debate (26). Hakonarson and colleagues (9) have shown that passive sensitization is associated with upregulation and expression of the low-affinity IgE receptor FcRII and that the application of immune complexes mimicked this response. Others have shown, however, that depletion of the allergic serum of its IgE content (7) resulted in abolition of the contractile response to allergen but retention of the sensitization induced hyperresponsiveness to histamine, and in addition that increased contractile responses to histamine were unaffected by a novel specific anti-IgE antibody, 17-9 (27). Furthermore, when a chimeric humanized IgE was substituted for allergic serum, no increase in response to histamine occurred (8). These results would suggest that some serum-derived factor or factors that are unrelated to IgE are contributing to sensitization-induced hyperresponsiveness. The results of clinical trials examining the efficacy of anti-IgE therapy may shed some light on this conflict.

Are the Changes Induced by Passive Sensitization the Result of the Actions of Cytokines such as IL-1 and/or TNF-?

Again, although there is some evidence of a role for IL-1 in mediating sensitization-induced changes in smooth muscle reactivity (10), Hirata and coworkers found that, whereas TNF- is released on allergen challenge of sensitized tissue, IL-1 was not increased (18). For IL-1 to play the pivotal role that has been suggested, exogenous IL-1 should potentiate histamine-induced contractile responses in human bronchus, and as yet this evidence is not available.

	IMPORTANT QUESTIONS

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• What are the factors present in allergic serum that cause changes in smooth muscle contractile behavior, changes in cell cycling, and changes in cytokine message, protein release, and receptor expression?

This question needs to be answered by the use of careful fractionation of the serum and examination of all of the functional consequences of passive sensitisation. Logistical problems will be encountered, however, if combinations of factors present in separate fractions of the serum are producing all or some of the different cellular responses.

• Does passive sensitization produce changes in smooth muscle properties that represent a step in the spectrum of nonsensitized, actively sensitized, through to sensitized asthmatic smooth muscle?

By studying these different "degrees" of allergic response, can we gain valuable information about the interaction between the smooth muscle and the allergic response? We need to consider not only the interaction between allergic serum-derived factors and the smooth muscle cells but also the influence of resident or recruited inflammatory cell populations. These, although not present in primary cultures of human airway smooth muscle cells, may have produced changes in the muscle cell that alter its subsequent contractile and growth properties.

We need to know whether the increased numbers of mast cells present in the smooth muscle of sensitized patients have the potential to alter smooth muscle properties or if the increase in mast cells is the result of smooth muscle-induced differentiation of airway inflammatory cell progenitors.

It will be important to delineate the differences in the phenotype of muscle cells derived from nonsensitized, actively sensitized, and passively sensitized lungs.

• What are the signal transduction pathways leading to increased contraction, decreased relaxation, and increased cycling in passively sensitized airway smooth muscle?

Although there is an increasing body of evidence to suggest that sensitization-induced changes in contractility of human airway smooth muscle may be associated with alterations in calcium flux (1, 2), this may not necessarily underlie the upregulation of cytokines and the increased cycling of the cells. It is likely that these pathways will be complex and whether there is cross-over between the mechanisms leading to changes in contractility, in growth, and in cytokine production will require further study and specifically in human airway tissue.

	Footnotes

Correspondence and requests for reprints should be addressed to J. L. Black, Ph.D., Department of Pharmacology, University of Sydney, NSW 2006, Australia. E-mail: judblack{at}pharmacol.usyd.edu.au

	References

TOP INTRODUCTION WHAT IS KNOWN WHAT IS CONFLICTING IMPORTANT QUESTIONS REFERENCES

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