EARLY STUDIES IN THE CANINE MODEL

TOP ABSTRACT EARLY STUDIES IN THE CANINE FORCE AND VELOCITY CHANGES IN MYOGENIC RESPONSES IN HUMAN INDUCED HYPERRESPONSIVENESS AND POTENTIAL ROLE OF ANTI-IgE PERSPECTIVES REFERENCES

In 1979 Antonissen and coworkers (1) described changes in the contractile properties of airway smooth muscle in a canine model of asthma. Dogs were actively sensitized to a conjugate of dinitrophenol and ovalbumin (DNP-OA) through repeated intraperitoneal injection with aluminium hydroxide as adjuvant, leading to the production of immunoglobin E (IgE) anti-DNP antibodies, and the development of increased airflow resistance, before tracheal smooth muscle (TSM) was removed and studied in vitro. The responses to carbachol and electrical field stimulation were similar in isolated TSM from sensitized animals and from nonsensitized littermate control animals, but some sensitized TSM exhibited spontaneous changes in tension and myogenic responses. Furthermore, the force and velocity characteristics of sensitized TSM were altered, demonstrating a significantly greater shortening velocity and an increased isotonic shortening at any given load after supramaximal electrical field stimulation (Figure 1). This early demonstration of altered airway smooth muscle contractility in a canine model of allergic bronchoconstriction indicated the importance of effector organ responses in asthma and led away from the concept of increased neural activity (2).

View larger version (22K): [in this window] [in a new window]   Figure 1.   Typical force-velocity curves elicited in a paired (littermate) experiment. Linearized transforms prove relationships are hyperbolic; a goodness-of-fit test yielded a correlation coefficient: r2 = 0.95 for both lines. Note the increased velocity of shortening of sensitized TSM, without a significant difference in opening pressure (Po). (Data adapted from Reference 1.)

	FORCE AND VELOCITY CHANGES IN HUMAN AIRWAYS

TOP ABSTRACT EARLY STUDIES IN THE CANINE FORCE AND VELOCITY CHANGES IN MYOGENIC RESPONSES IN HUMAN INDUCED HYPERRESPONSIVENESS AND POTENTIAL ROLE OF ANTI-IgE PERSPECTIVES REFERENCES

The observations of Anthonissen and coworkers (1) were subsequently extended to human airway smooth muscle using a model of passive sensitization. The principle of incubating human airways with serum from atopic individuals has been used in the past by several investigators studying the release of bronchoconstricting mediators, such as slow-reacting substance of anaphylaxis and histamine from lung fragments (3- 7), and also investigating the in vitro responsiveness to histamine and other contractile agonists (8). For our studies (16) serum was prepared from whole blood of individuals demonstrating high total IgE (> 1,000 U/ml) and then frozen until required. Macroscopically normal bronchial tissue was obtained from patients undergoing surgery, mainly for lung cancer. In addition to obtaining tissue only from donors with a negative history of atopy, we also measured serum IgE levels in all tissue donors immediately after surgery to ensure that the tissues had not been sensitized prior to our interventions. After resection, peripheral airways with an internal diameter of 2-5 mm were dissected free of alveolar tissue and cut into rings of 2-4 mm in length. The tissues were then rotated overnight in tubes containing modified Krebs buffer in the absence (nonsensitized control tissue) or presence of sensitizing serum (10% volume in volume). The next morning selected rings were transferred to 10-ml organ baths containing oxygenated modified Krebs buffer at a pH of 7.4 and at 37° C to confirm the sensitization by a contractile response to a polyclonal anti-IgE antibody or to allergen. Another pair of sensitized and sham-sensitized rings were randomly selected for force-velocity studies.

When the maximal velocity of shortening (Vmax) was determined at the maximal slope of the length displacement curve, it was found to be significantly greater for bronchial rings passively sensitized compared with sham-sensitized controls (16). Bronchial smooth muscle from passively sensitized tissues shortened with a higher maximal rate compared with sham-sensitized rings and representative force-velocity curves showed remarkable similarities to the findings after active sensitization published by Antonissen in 1979 (1). The maximal shortening capacity was also approximately 35% greater for bronchial rings from passively sensitized compared with control tissues while the maximal isometric force after electric field stimulation was not affected by passive sensitization, a finding again similar to the studies in the canine model (Figure 2).

View larger version (17K): [in this window] [in a new window]   Figure 2.   Representative force-velocity curves of smooth muscle from a passively sensitized and a paired sham-sensitized human bronchial ring. Sensitized tissue demonstrated significantly greater maximal velocity of shortening (Vmax; y-axis intercept) and greater velocities of shortening at all afterloads compared with paired, sham-sensitized bronchial ring. For these preparations, Vmax was 0.1236 Lo/s for sensitized tissue and 0.0803 Lo/s for control tissue. Mean maximal isometric force development (Po; x-axis intercept) was not different for eight pairs of human bronchial rings studied. (Data adapted from Reference 16.)

	MYOGENIC RESPONSES IN HUMAN AIRWAYS

TOP ABSTRACT EARLY STUDIES IN THE CANINE FORCE AND VELOCITY CHANGES IN MYOGENIC RESPONSES IN HUMAN INDUCED HYPERRESPONSIVENESS AND POTENTIAL ROLE OF ANTI-IgE PERSPECTIVES REFERENCES

Since there was a striking similarity between the changes in force-velocity parameters between actively immunized canine airways and passively sensitized human airways, we subsequently assessed the effects of passive sensitization on human bronchial smooth muscle responses to mechanical stretching in vitro (17). Seventh generation bronchial rings were passively sensitized with sera containing high concentrations of IgE and high titers of specific antibodies against Dermatophagoides farinae and compared with sham-sensitized tissues. The airways were fixed isometrically to measure responses to electrical field stimulation (EFS) and quick stretches (QS). As in the earlier reports of Antonissen and colleagues (1), myogenic responses to QS were normalized to a maximal response to EFS.

Bronchial ring preparations from six patients were randomly assigned to either antigen sensitization or sham-sensitized control tissue. Additional pairs of sensitized and control tissues were challenged with D. farinae after sensitization. Control tissues did not respond to antigen challenge, while the allergen-induced contractions in all sensitized bronchial rings amounted to 148 ± 20% of the EFS response. There was no significant difference in the response to EFS between groups during the initial equilibration periods; the response amounted to 280 ± 40 mg in control tissue and 383 ± 89 mg in sensitized tissues (not significant).

Human bronchial rings demonstrated a myogenic contractile response to QS of 0.25 and 0.50 mm, which was amplitude-dependent. For a quick stretch of 0.25 mm all six bronchial rings exposed to passive sensitization responded with a myogenic contraction of 47.9 ± 10.9% EFS, while only three of six sham-sensitized preparations contracted to 13.5 ± 6.4% EFS. Similar differences were found for a quick stretch of 0.50 mm with six of six sensitized tissues responding with a myogenic contraction with a magnitude of 83 ± 21% EFS, while five of six sham-sensitized preparations demonstrated a response of 38 ± 17% EFS. When the experiments were repeated using human sera with low IgE levels instead of buffer for control purposes, the myogenic responses in these tissues were not different from buffer control tissue and significantly differed from tissues sensitized with sera containing high IgE levels.

To further test the hypothesis that IgE in the serum accounts for the hyperresponsiveness to QS observed in these experiments, the effect of a hapten-specific chimeric IgE, JW8, was assessed (18). Adding the chimeric immunoglobulin to low IgE sera led to an augmented QS response in these tissues, which was significantly different from sham-sensitized airways. The parasympathetic contraction caused by EFS, however, was not affected (Figure 3). We found that about 50% of human airways demonstrate modest contractile responses in response to QS and that with sensitization they demonstrate a myogenic contractile response of greater magnitude than was seen in sham-sensitized tissues. The myogenic contractile response depended on the magnitude of the QS, and sensitized bronchi demonstrated greater myogenic responses than sham-sensitized ring preparations. Furthermore, we were able to show that it was in fact IgE that was responsible for tissue hyperresponsiveness since the presence of serum alone did not augment subsequent myogenic responses: tissues incubated with sera either from atopic donors or with sera from nonatopic donors supplemented with JW8 showed augmented myogenic contractile responses. These data suggest that the presence of increased IgE is necessary for the induction and augmentation of myogenic contractile responses to QS in human bronchial rings in vitro.

View larger version (20K): [in this window] [in a new window]   Figure 3.   Myogenic responses of sensitized and sham-sensitized human bronchial rings. In response to quick stretches (QS) of 0.25 and 0.50 mm, sensitized tissues demonstrated significantly greater myogenic contractile responses than paired, sham-sensitized tissues. All data are expressed as % of electrical field stimulation (EFS). *p < 0.05. (Data adapted from Reference 17.)

	INDUCED HYPERRESPONSIVENESS AND THE ROLE OF IgE

TOP ABSTRACT EARLY STUDIES IN THE CANINE FORCE AND VELOCITY CHANGES IN MYOGENIC RESPONSES IN HUMAN INDUCED HYPERRESPONSIVENESS AND POTENTIAL ROLE OF ANTI-IgE PERSPECTIVES REFERENCES

Because bronchial smooth muscle obtained from individuals who are hyperresponsive to histamine and methacholine in vivo do not necessarily exibit hyperresponsiveness in vitro (19), investigations have examined passive sensitization to assess whether this process will lead to the induction of nonspecific hyperresponsiveness in vitro. In an earlier study, Black and colleagues demonstrated that passive sensitization significantly increased responses to histamine while depressing responses to carbachol (8). They further showed that sensitization increased the involvement of the calcium voltage-dependent channels in KCl-mediated contractions. From these studies, the authors concluded that airway hyperresponsiveness may be associated with altered calcium mobilization in airway smooth muscle. Subsequently, Rosetti and associates (13) assessed the role of protein kinase C (PKC) in passively sensitized isolated human bronchial smooth muscle. The authors demonstrated slowly developing and sustained contractions to the phorbol ester PDB, which was reduced by the PKC inhibitor staurosporine, and also showed that long-term exposure to the phorbol ester led to the downregulation of PKC. Removal of external calcium or the addition of the calcium channel antagonist verapamil reduced PDB-induced contractions. Passive sensitization increased the maximal response to PDB, and this was reversed when tissues were allowed to recover unstimulated for 3 h before PDB application. The authors concluded that PKC activation induces maintained contraction in human isolated airway smooth muscle, which is largely dependent on extracellular calcium, and that passive sensitization alters the PKC-mediated contraction in a way similar to that induced by prolonged stimulation of PKC.

Incubation of airways from nonatopic patients with serum from patients with high IgE levels confers to responsiveness to "specific" (allergen) and hyperresponsiveness to "nonspecific" stimuli such as histamine. We have tested the hypothesis that the level of IgE determines the degree of specific and nonspecific responsiveness (20).

Serum was prepared from whole blood of six individuals, demonstrating high total IgE (> 500 U/ml to > 3,000 U/ml) and a range of levels of specific IgE for five common aeroallergens. Macroscopically normal bronchial tissue was obtained from 22 patients undergoing surgery for lung cancer. Pure IgE was used in the form of JW8 (see above), a chimeric, humanized, hapten-specific IgE containing a human  constant region ligated to the murine V_H region and the original murine L chain, with specificity for the hapten 4-hydroxy-3-iodo-5-nitrophenylacetic acid (NIP) (18). This chimeric IgE antibody can mimic natural allergen-specific IgE reactions in biological systems. In vitro responsiveness to allergen and histamine was evaluated and compared with nonsensitized tissues obtained from the same individuals.

In tissues sensitized with serum containing high specific IgE for D. farinae, but low specific IgE for horse and dog allergens, there was a concentration-related contraction to D. farinae, but no significant response to either dog or horse epithelium (Figure 4). Histamine caused a concentration-dependent contraction in both serum-sensitized and nonsensitized preparations, but the potency of histamine was significantly increased in the serum-sensitized tissues. Additionally, the magnitude of maximum contractions were also significantly greater in serum-sensitized tissues compared with control tissues (Figure 5). NIP-BSA, the hapten for JW8, caused concentration-related contractions in JW8-sensitized preparations, but was without effect in nonsensitized tissues. Histamine caused concentration-dependent contractions in both JW8-sensitized and nonsensitized preparations, but neither the potency nor the magnitude of maximum contractions were significantly different in JW8-sensitized tissues when compared with nonsensitized tissues from the same individual.

View larger version (14K): [in this window] [in a new window]   Figure 4.   Contractile concentration-effect curves to (a) D. farinae, (b) dog, and (c) horse allergens in nonsensitized human bronchus (open circles) and human bronchus sensitized (closed circles) with serum containing modest total IgE (636 U/ml) high (FAST 4) D. farinae-specific IgE and low (FAST 0) horse- and dog-specific IgE. Contractions are expressed as milligrams of changes in tension. Data are the mean ± SEM of five experiments, using tissue derived from different individuals. (Data adapted fom Reference 20.)

View larger version (21K): [in this window] [in a new window]   Figure 5.   Contractile concentration-effect curves to (a) D. farinae and (b) histamine, in nonsensitized (closed circle) human bronchus and bronchus sensitized (open circles) with serum containing very high total IgE (> 3,000 U/ml) and high specific (FAST 4) IgE for D. farinae. Contractions are expressed as milligrams of changes in tension. Data are the mean ± SEM of five experiments, using tissue derived from different individuals. (Data adapted from Reference 20.)

Interestingly, there was a significant increase in the potency and magnitude of the maximum contractions to histamine in the serum-sensitized tissue when compared with the nonsensitized and JW8-sensitized tissues. This clearly indicates that incubation of tissues with pure IgE is lacking some component of IgE-rich serum that leads to histamine hyperresponsiveness. The magnitude of the maximum contraction to histamine was 1,003 ± 142 mg in serum-sensitized tissues, which was substantially and significantly greater than in JW8-sensitized (572 ± 174 mg) and nonsensitized (369 ± 60 mg) tissues.

These data suggested that "specific" responsiveness to allergen can be induced in nonatopic human airways by passive sensitization with either allergen-specific IgE present in atopic serum or a chimeric hapten-specific IgE. In addition, the specificity of allergen responses in these tissues is related to the level of allergen-specific IgE in the sensitizing serum. However, the "nonspecific" responsiveness to histamine did not appear to be directly linked to IgE, but to some other factor or factors present in the sensitizing serums, levels of which may be influenced by the amount of total IgE. Therefore, allergen-specific IgE appeared to be the only component of serum required for the induction of allergen responsiveness, but is not sufficient for the induction of the increased responsiveness to histamine in vitro.

Several earlier studies of Souhrada and Souhrada (21, 22) have investigated the mechanisms of passive sensitization of airway smooth muscle from guinea pigs. Bronchial preparations were passively sensitized with sera from animals repeatedly sensitized in vivo. The authors demonstrated changes in membrane properties of airway smooth muscle in vitro. They reasoned that the hypothesis that IgE is relevant for immune sensitization of human airways could be tested either by analyzing the effects of heat inactivation or by removal of IgE on subsequent tissue responsiveness. They showed that if immune serum is heated at 56° C for 2 h, a significant prevention of passive in vitro sensitization-induced changes of airway smooth muscle cells was observed, and they concluded that the specific reaginic immunoglobulins involved were IgG and IgE (22). We speculated from our previous findings that specific response to allergen was primarily mediated by IgE, but we were uncertain whether histamine hypersensitivity was also related to this immunoglobulin.

Therefore, the objective of the next part of our investigation was to deplete IgE from the serum of a D. farinae-sensitized patient with asthma through a novel immunomagnetic separation technique (23). We passively sensitized tissues from several nonatopic donors with either the whole serum or the IgE-depleted serum and examined histamine and allergen responsiveness the next day. Passive sensitization with whole serum resulted in the development of sensitivity to D. farinae together with an increased sensitivity to histamine in vitro; incubation with IgE-depleted serum still produced marked histamine hypersensitivity but did not result in a significant response to allergen (Figures 6 and 7).

View larger version (18K): [in this window] [in a new window]   Figure 6.   D. farinae-induced contractions of human bronchial smooth muscle sensitized with whole serum from a asthma subject with asthma (open circles), or with IgE-depleted serum from a subject with asthma (open squares) and of nonsensitized tissue (closed squares). Contractions are expressed as absolute changes in milligrams of tension (a). (Data adapted from Reference 23.)

View larger version (19K): [in this window] [in a new window]   Figure 7.   Histamine-induced contractions of human bronchial smooth muscle sensitized with whole serum from a subject with asthma (open circles), or with IgE-depleted serum from a subject with asthma (open squares) and of nonsensitized tissue (closed squares). Contractions are expressed as absolute changes in milligrams of tension (a). The potency of histamine (pEC50) was 5.05 ± 0.23 in nonsensitized tissue; 5.64 ± 0.16 in whole serum- sensitized tissue, and 5.57 ± 0.16 in tissue-sensitized with IgE depleted serum (*p > 0.05). (Data adapted from Reference 23.)

	POTENTIAL ROLE OF ANTI-IgE ANTIBODIES

TOP ABSTRACT EARLY STUDIES IN THE CANINE FORCE AND VELOCITY CHANGES IN MYOGENIC RESPONSES IN HUMAN INDUCED HYPERRESPONSIVENESS AND POTENTIAL ROLE OF ANTI-IgE PERSPECTIVES REFERENCES

Finally, we investigated the effect of a novel mouse IgG_2b non-anaphylactogenic anti-human IgE antibody (17-9) on allergen and histamine responses in passively sensitized human airways in vitro to determine the specific contribution of IgE to the sensitization process and to validate the model of passive sensitization for clinical applications. This antibody binds IgE with high affinity in an isotype-specific way and, owing to its particular epitope specificity, can prevent IgE binding to the Fc RI on basophils and mast cells, thereby blocking IgE-mediated cell responses. In contrast to conventional anti-IgE antibodies, 17-9 does not induce histamine release from IgE-sensitized human basophils and hence is a non-anaphylactogenic anti-IgE antibody (24). The antibody 17-9 has been characterized similarly to other described non-anaphylactogenic antibodies of different clonal origin, showing similar properties with a high affinity for IgE.

Bronchial rings from 20 individuals were sensitized with serum containing high levels of allergen-specific IgE (D. farinae) or with the hapten-specific chimeric humanized IgE (JW8). There was again a concentration-dependent contraction of serum-sensitized bronchial rings to D. farinae (517 ± 188 mg tension at 10 U/ml, n = 8) that was not observed in nonsensitized control tissues, and this response was practically abolished when tissues were sensitized in the presence of 100 µg/ ml anti-IgE antibody 17-9 (54 ± 20 mg). In tissues sensitized with the anti-NIP IgE, JW8, there was a concentration-dependent contraction to the specific antigen NIP-BSA (560 ± 154 mg at 0.3 µg/ml, n = 5) that was not observed in nonsensitized control tissues and that was substantially inhibited when 17-9 was present in the sensitization buffer (124 ± 109 mg) (Figure 8). The inhibition with 17-9 was specific, since pretreatment with a non-IgE-specific IgG_2b antibody did not affect allergen responses. Potency and maximum contractions to histamine in serum-sensitized tissues were significantly elevated compared with nonsensitized control tissues, but this was not affected by the presence of 17-9 during sensitization (Figure 9). From these data we concluded (24) that non-anaphylactogenic anti-human IgE antibodies effectively inhibit allergen responses of human airways in vitro but may not affect other factors inducing hyperresponsiveness.

View larger version (21K): [in this window] [in a new window]   Figure 8.   Contractile concentration-effect curves to allergen and specific antigen in human bronchus sensitized with serum containing high IgE and high (FAST > 3) Dermatophagoides farinae-specific IgE (a, n = 8) or with 10 mg/ml NIP-specific chimeric IgE JW8 (b, n = 5). Responses to D. farinae (a) and hapten-carrier complex NIP-BSA (b) in nonsensitized (open squares) and sensitized tissues (open circles), and tissues sensitized in the presence of 100 µg/ml 17-9 (closed circles). Contractions are expressed as percent histamine maximum and are the mean ± SEM of the indicated numbers of experiments using tissue derived from different individuals. (Data adapted from Reference 24.)

View larger version (27K): [in this window] [in a new window]   Figure 9.   Contractile concentration-effect curves to histamine in nonsensitized (open squares), high-IgE serum-sensitized control tissues (open circles) and tissues sensitized in the presence of 100 µg/ ml 17-9 (closed circles). Contractions are expressed as milligrams of tension changes and represent mean ± SEM from eight experiments using tissue derived from different individuals. (Data adapted from Reference 24.)

	PERSPECTIVES

TOP ABSTRACT EARLY STUDIES IN THE CANINE FORCE AND VELOCITY CHANGES IN MYOGENIC RESPONSES IN HUMAN INDUCED HYPERRESPONSIVENESS AND POTENTIAL ROLE OF ANTI-IgE PERSPECTIVES REFERENCES

Passive sensitization of human airway smooth muscle has been described previously. It is evident from these studies that incubation of airways from nonatopic individuals with serum from atopic patients produces in vitro responsiveness to specific allergen and nonspecific hyperresponsiveness to bronchoconstrictor agents, such as histamine, and also to neuropeptides (3, 8, 9, 11, 12, 14, 25, 26). The nature of the specific responsiveness (the contractile response to allergen) has been extensively studied, and it is widely accepted that cysteinyl leukotrienes, and to a lesser extent histamine, mediate this response in sensitized tissue (3, 4, 7, 25, 27, 28). The nature of the nonspecific hyperresponsiveness induced by passive sensitization of human airways is less well understood. Several studies have investigated potential changes in the smooth muscle (postjunctional factors) that might account for the hyperresponsiveness, including changes in the velocity and magnitude of smooth muscle cell shortening (16), alterations in Ca²⁺ mobilization (8), and changes in PKC-mediated responses (13).

The role of IgE present in the sensitizing serum in the induction of specific hyperresponsiveness and nonspecific hyperresponsiveness is, however, largely uninvestigated. It is not known whether the degree of responsiveness (specific and nonspecific) is in some way related to the concentration of allergen-specific IgE in the sensitizing serum, whether IgE alone is sufficient to confer specific (allergen) and nonspecific (histamine) responsiveness, or whether allergen responsiveness and histamine hyperresponsiveness are interdependent. These questions may have clinical relevance, since Burrows and colleagues (29) demonstrated a close relationship between the levels of circulating IgE in the serum and the frequency of reported asthma in patients both with and without positive skin prick tests. Additionally, nonspecific airway hyperresponsiveness can be demonstrated in a number of other conditions where allergen-specific IgE is not implicated, such as chronic obstructive bronchitis (30), during viral infections (31), and in cigarette smokers (32).

While most in vitro investigations of airway hyperresponsiveness rely on the measurement of isometric stress, it has been demonstrated that tracheal smooth muscle strips that are allowed to shorten auxotonically are more sensitive to bronchoconstrictor agents than those under isometric conditions (33). Our findings in human airways (16), which are generally remarkably similar to those in the canine model (1), demonstrate no difference in isometric maximal stress in response to electrical field stimulation but demonstrate a significant augmentation in Vmax and Lmax of human and canine smooth muscle. Since it is reasonable to assume that human airways constrict auxotonically rather than as a result of isometric force generation, the observed changes in Vmax and Emax after passive sensitization might be associated with an increased narrowing of the airways in vivo (1, 34), and it may be speculated that these parameters indicate directly the airways capacity to narrow in hyperresponsive disease states such as asthma.

Previous studies have showed that airways of subjects with asthma have increased electrical activity when compared with nonasthmatic volunteers (37). Spontaneous action potentials of airway smooth muscle have also been demonstrated to accompany the induction of myogenic contraction responses to QS in canine airway tissue (32, 38), and it has been shown in the canine model of allergic bronchospasm that the normal quiescent tracheal smooth muscle demonstrates spontaneous contractile activity (39). Compared to the bronchial smooth muscle from sham-sensitized animals smooth muscle strips from sensitized animals demonstrate significant spontaneous electrical activity (40). In the canine model an altered excitation induced by immune sensitization was suggested, and it was argued that it is the basis of the observed allergic bronchospasm in the animal model and indeed the underlying cause of hyperresponsiveness observed in asthmatic patients (38). Our findings of altered force-velocity characteristics of passively sensitized human airways (16) and the findings of Jiang and coworkers (43) who described a twofold increase in actomyosin adenosine triphosphatase activity in the immune-sensitized canine model, with a 30% increase in myosin light chain kinase content and activity (43), suggest that the increased myogenic response observed on quick stretch of human bronchus may be due to increased contraction coupling, perhaps through an alteration of membrane excitability somehow caused by passive sensitization by IgE. Increased sensitivity of the smooth muscle to mechanical stretch could be a consequence of an alteration in potassium channel activity (39). Alternatively, the attachment of the crystallizable fragment (Fc) of IgE to mast cells or smooth muscle membranes could alter calcium flux through voltage-dependent channels, with a concomitant change in the membrane potential (21).

Our data confirm previous reports that passive sensitization of human bronchial smooth muscle leads to increased responsiveness to histamine and allergen. The demonstration of a leftward shift in the concentration-effect curve to histamine, along with an increase in the magnitude of maximal contraction, are novel findings. Additionally, the lack of response to the common allergens dog and horse epithelium in tissues sensitized with serum containing high levels of D. farinae-specific IgE demonstrated for the first time the specificity of the in vitro allergen sensitization process. These observations are in keeping with the concept that exposing airways from non-atopic individuals to serum from atopic donors containing high allergen-specific IgE results in the loading of high-affinity IgE receptors (Fc RI) on tissue mast cells and basophils with the specific IgE (44). Subsequent exposure of these airways to the allergen results in Fc RI crosslinking and cell activation, with the release of bronchoconstrictor mediators such as cysteinyl leukotrienes and histamine (7, 25, 28, 45, 46). The IgE-dependence of this effect has been demonstrated previously by Tunon de Lara and colleagues (14), who showed that IgG was unable to elicit such responses.

Exposing tissues to serum containing approximately equivalent concentrations of allergen-specific IgE, but high versus modest (> 3,000 versus 636 U/ml) total IgE, resulted in approximately equivalent responses to specific allergen. However, an increase in the potency and magnitude of maximal contractions to histamine was observed only in the tissues sensitized with high IgE. This observation suggested that the non-specific hyperresponsiveness to histamine was unrelated to the concentration of specific IgE in the sensitizing serum but seems to be related to the level of total IgE. In tissues sensitized with the chimeric IgE antibody JW8, an increased sensitivity to the specific hapten/carrier complex, NIP-BSA, could be demonstrated without any associated change in nonspecific (histamine) responsiveness. We interpreted these results again as an indication that IgE per se does not lead to nonspecific hyperresponsiveness. These findings were again interpreted as an indication that nonspecific hyperresponsiveness was unrelated to the presence of specific IgE. Interestingly, although there was no significant change in histamine responses in tissues sensitized with low IgE-containing serum compared with nonsensitized tissues, there was a leftward shift in the response to histamine that was absent in tissues sensitized with JW8, suggesting that the development of hyperresponsiveness, although related to total IgE, required the presence of some other serum factor(s).

The results of the IgE depletion studies (23) confirmed the IgE dependence of the allergen response, but, on the other hand, histamine sensitivity was still significantly greater than in nonsensitized tissues, indicating that histamine hypersensitivity occurs in the absence of allergen responsiveness. This clearly demonstrates a distinction between specific responsiveness and nonspecific hypersensitivity, and suggests that the induction of histamine hypersensitivity requires factors other than IgE.

A number of potential candidates of serum factors exist that may be related to histamine hypersensitivity, including interleukin (IL)-1, tumor necrosis factor alpha (TNF-) and mast cell tryptase (47). In a passive sensitization model in the rabbit, Hakonarson and colleagues (47) have demonstrated both IL-1 and TNF- to be important for sensitization-induced impairment of receptor-coupled relaxation in passively sensitized tracheal smooth muscle, and it has been shown in rats and in normal human subjects that the inhalation of TNF- may increase airway reactivity to exogenous spasmogens (50, 51). TNF- has also been shown to increase responsiveness of nonsensitized human bronchial tissue to EFS by a prejunctional mechanism (52). Renzetti and colleagues (48) have demonstrated a marked inhibition of allergen-induced airway hyperreactivity in sensitized guinea pigs and rats after treatment with the TNF receptor fusion protein Ro 45-2081 (48).

Additionally, elevated levels of TNF- have been demonstrated in the airways and the bronchoalveolar lavage fluid of patients with asthma (53, 54). Thus, the observation that TNF- is released via IgE-dependent activation of mast cells (55) could explain the apparent correlation of hypersensitivity with total IgE levels regardless of allergen-specific IgE levels observed both in vitro (20) and under clinical conditions (29). It is also of interest that TNF- levels are significantly increased in serum in allergic asthma, which is associated with elevated IgE, in contrast to nonallergic asthma (56). Mast cell tryptase has also been shown to potentiate contractile response to histamine in human bronchi taken from individuals demonstrating pre-existing sensitivity to allergen but not in tissues from patients that were nonatopic (49). In light of the reported studies on the range of pro-inflammatory cytokines that may be capable of inducing hypersensitivity, it is conceivable that in the sensitizing serum one or more of these may be present in sera from patients with asthma and those with an atopy and that these cytokines may at least in part be responsible for the observed nonspecific hyperreactivity. At present the nature of the serum factors is not known, but there is now ample evidence to support the hypothesis that hypersensitivity to histamine may occur largely independent of IgE, while the allergen responses are directly related to the circulating level of this immunoglobulin. Since hypersensitivity was previously linked to circulating IgE levels, it is possible that the mechanisms that lead to increase in total IgE are also linked to the generation of these unknown factors.

We demonstrated that the non-anaphylactogenic, monoclonal anti-IgE antibody 17-9 prevents the IgE-dependent passive sensitization of human airways in vitro (24). This was observed in tissues sensitized with either allergen-specific high-IgE titer homologous serum or with a purified hapten-specific chimeric IgE (JW8). This effect is mediated specifically through the interaction of 17-9 with IgE, since a control antibody of the same IgG_2b class directed against an irrelevant antigen did not show any inhibition. While 17-9 was effective when present during sensitization, it failed to inhibit allergen responses when applied to pre-sensitized tissues 60 min prior to allergen stimulation. This suggests that 17-9 blocks IgE in the sensitizing serum/buffer from binding but that, under these experimental conditions, 17-9 is unable to displace bound IgE from its receptors on the tissue.

Similar studies have shown that anti-IgE and a chimeric construct of Fc RI-IgG were able to prevent sensitization of tissue for allergen-induced bronchial tissue contraction (57). Those studies were based on sensitization with atopic serum and do not preclude participation of other serum factors besides IgE in the sensitization process. Our studies with purified IgE extend these investigations and demonstrate the effect of the non-anaphylactogenic anti-IgE antibody 17-9 to be accounted for purely by prevention of IgE sensitization of tissues. In contrast to its effects on allergen responses, 17-9 had no effect on responses to histamine in any tissues and did not prevent the induction of histamine hyperresponsiveness in tissues sensitized with IgE-containing serum. Non-anaphylactogenic antibodies clearly can prevent tissue sensitization by IgE and thus are highly attractive therapeutic principles. However, two possible limitations are indicated by the present study. First, application of 17-9 to IgE sensitized tissues for 60 min prior to allergen provocation and during the administration of allergen produced no significant attenuation of the allergen-induced smooth muscle contraction. Secondly, although 17-9 abolished specific allergen responses conferred by IgE-sensitization of airways, it had no effect on the increase in nonspecific responsiveness induced by atopic serum, as illustrated by the persistence of the enhanced response to histamine. These potential restrictions may, however, not be relevant in vivo when non-anaphylactogenic anti-IgE antibodies can act for prolonged periods of time. It has been shown that treatment of humans with a humanized non-anaphylactogenic anti-IgE antibody results in an effective dose-dependent reduction of serum IgE levels (58). The IgE level, in turn, seems to regulate the degree of Fc RI receptor expression, as concluded from a study in which patients treated with humanized non-anaphylactogenic anti-IgE antibody for several weeks showed a 96% reduction in basophil Fc RI expression, associated with a markedly decreased response of these cells to IgE-mediated triggering ex vivo (59). Thus, as has been shown by studies in animals (60), it is likely that such anti-IgE antibodies may prevent allergic reactions in sensitized individuals. It has been demonstrated that a corresponding non-anaphylactogenic antibody to murine IgE was able to inhibit antigen-induced skin reactions and bronchoconstriction in sensitized mice (61). Moreover, anti-IgE also caused a marked inhibition of pulmonary eosinophil infiltration induced by allergen inhalation in these animals (62). This latter effect of anti-IgE has been shown to be mediated by blockade of IgE interaction with CD23, the low affinity receptor for IgE (Fc RII). Thus, it is likely that non-anaphylactogenic anti-IgE antibodies could also be effective in allergic asthma. On the other hand, it is not expected that these antibodies would reverse the nonspecific serum factor-mediated airway hyperresponsiveness to histamine. In this context it is noteworthy that two recently published studies using the anti-IgE antibody E25 demonstrated marked effects on allergen responses but variable effects against nonspecific hyperresponsiveness (63, 64).

In summary, it was found that human airways passively sensitized with human sera with elevated concentrations of IgE have increased shortening velocity and increased shortening capacity. These data suggest that passive immune sensitization of human tissue confers an inherent change in the contractility of airway smooth muscle that may contribute to hyperresponsiveness.

Passive sensitization of human airways also induces and augments myogenic contractile activity of smooth muscle from seventh generation airways. We also suggest that serum alone does not affect the myogenic contractile response but that the presence of high concentrations of IgE is necessary for this response and that allergic bronchospasm in vivo may be manifested, at least in part, to the augmentation of myogenic responses of the smooth muscle from intrapulmonary conducting airways.

Furthermore, our data suggest that while the specific allergen responsiveness is entirely IgE-dependent, the nonspecific histamine hyperresponsiveness is either independent of IgE or requires IgE in the presence of some other factor(s) present in the sensitizing serum. Further studies are required to determine the nature of the serum factor(s) and whether histamine hyperresponsiveness is conferred by the factor(s) alone or in combination with IgE.

Finally, novel monoclonal anti-human-IgE antibodies, such as 17-9, block passive IgE sensitization of human airways by either atopic serum or by a chimeric anti-NIP IgE, without affecting the responses of the tissues to histamine. This confirms that the specific responses of passively sensitized human airways to allergens is dependent on IgE, while increased responsiveness to histamine is mediated by a component of atopic serum other than IgE. This may limit the clinical efficacy of this class of drugs.