INTRODUCTION

TOP ABSTRACT INTRODUCTION CONCLUSION REFERENCES

Asthma is presently considered a chronic inflammatory disease (1, 2). It involves complex interactions between exogenous factors (primary and secondary exposures to allergens, intercurrent infections, noxious environmental stimuli) and genetically determined endogenous factors.

The purpose of this short review is to discuss the nature and the role of the interactions between lymphocytes and eosinophils on one side, and between macrophages and neutrophils on the other side, in inducing bronchial hyperresponsiveness (BHR) in guinea pigs and mice upon provocation with allergen or bacterial endotoxin (lipopolysaccharide [LPS]), respectively. In both instances, induction and perpetuation of BHR involve cell-to-cell interactions. The central role of lymphocytes in immunological reactions is universally recognized, and the participation of eosinophils in the allergic reaction (via receptor-mediated adhesion, chemotaxis, and synthesis/ release of mediators, particularly of cationic proteins, PAF and eicosanoids, oxygen metabolites, and cytokines) is generally accepted. Lipopolysaccharide is a primary activator of monocytes/macrophages that may operate via cytokines, chemokines, and neutrophils. Interactions between environmental LPS and allergy is a subject of increasing awareness.

	BRONCHOPULMONARY ALLERGY

Lymphocytes and Eosinophils

Bronchial hyperresponsiveness, an increased sensitivity of the bronchial smooth muscle to nonspecific stimuli, and airway infiltration by eosinophils and T lymphocytes are hallmarks of asthma (1, 2). The severity of asthma correlates with the presence of activated T lymphocytes and eosinophils in the bronchoalveolar lavage fluid (BALF) (3), with cytokines generated in the lungs being essential for pathogenesis (4, 5). The accumulation and activation of inflammatory cells at sites of antigenic deposition requires the mobilization, adherence, transendothelial passage, activation, and proliferation of T lymphocytes. CD4+ T lymphocytes are presently classified in at least three subsets: Th1 cells, which produce interleukin (IL)-2 and interferon (IFN)-; Th2 cells, which produce IL-4, IL-5, and IL-10; and Th0 cells, which are less selective in producing cytokines (6). More recently, Tc1 and Tc2 subsets have been distinguished among CD8+ cytotoxic lymphocytes. The Th2 product IL-4 promotes IgE synthesis by B lymphocytes (7) and plays an essential role in inducing lymphocyte differentiation to the Th2 subtype in vivo (8, 9). The BALF of asthmatic subjects is enriched with Th2 lymphocytes, which also produce IL-5 (10). Interleukin-5 initiates and perpetuates eosinophilia (11) and the selective proliferation and differentiation of eosinophils, at distance in the bone marrow or in situ in the lungs (12).

Early Work with the Guinea Pig Model

Guinea pigs have been particularly useful for studying allergic pathophysiology because of the marked reactivity of their bronchial smooth muscle to allergen and to histamine and lipid mediators (15), which for many years were considered major mediators of allergic diseases, particularly for immediate asthmatic response. In fact, the potential role of histamine, leukotrienes, and platelet-activating factor (PAF) in the pathogenesis of asthma has been overestimated (16). We demonstrated that injection of PAF into the rat pleural cavity is followed by the in situ formation of a transferable activity, capable of recruiting eosinophils to the pleural cavity of recipient rats made refractory to the carry-over effects of PAF itself (17). This hypothesized secondary mediator was initially named eosinophilotactin, and later identified as IL-5 (18). Retrospectively, it is likely to be a mixture, including chemokines such as eotaxin or RANTES. Even though the chemokines are likely to be involved with eosinophilia and/or BHR, more evidence has accumulated implicating IL-5.

The rising interest in the role of IL-5 in allergic disease led us to demonstrate that human recombinant IL-5 (hrIL-5) moderately activates guinea-pig eosinophils and substantially primes their chemotactic responsiveness to PAF and leukotriene B4 (LTB4) (19). Priming was observed ex vivo as well, since isolated lungs from immunized and antigen-challenged guinea pigs, whose BALF contained increased numbers of eosinophils, responded more intensively to the bronchoconstrictor and secretagogue effects of PAF and LTB4 after the intratracheal administration of hrIL-5 (20). This finding suggests that IL-5 released into the pulmonary microenvironment amplifies the effects of inflammatory mediators and accounts for the intense infiltration of eosinophils in the lungs and airways of allergen-stimulated guinea pigs (21). In keeping with this suggestion, allergic airway eosinophilia and BHR in guinea pigs are suppressed by an anti-IL-5 antibody (22), even though the doses of the antibody required to suppress eosinophilia are much lower than those needed to inhibit the development of BHR. This raises the intriguing possibility that the protective effects of the anti-IL-5 antibody against BHR and eosinophilia in the guinea pig are not directly related. The low doses that were effective against antigen-induced eosinophilia in the lungs have been shown to suppress eosinophilia in the blood, peritoneal fluid, BALF, and bone marrow of Toxocara canis- infected guinea pigs (25).

A relevant question concerns the mechanisms of the recruitment of lymphocytes and eosinophils to the guinea pig airways. Eosinophils and T lymphocytes infiltrate the bronchi of asthmatics and allergen-challenged animals, sharing pathways of adherence to endothelial cells. Indeed, the adhesion of eosinophils and lymphocytes, but not of neutrophils, involves 4 integrins, particularly very late activation antigen-4 (VLA-4), which interacts with vascular cell adhesion molecule-1 (VCAM-1) expressed on the activated endothelial cells (26, 27). Different reports have stressed the importance of 4 integrins for leukocyte recruitment to allergic lungs (28). We showed that an anti-4 integrin monoclonal antibody administered to sensitized guinea pigs on the day of antigen challenge suppresses BHR, as well as BALF and bronchial tissue eosinophilia and infiltration by CD4+ T lymphocytes (32). Because cationic protein generation in the BALF was also inhibited, it is likely that the neutralization of 4 integrin inhibits eosinophil and T lymphocyte infiltration and, as a consequence, their activation within the airways.

That eosinophil infiltration alone does not account for BHR was clearly shown when the bronchial responses to methacholine and the cell distribution and concentration of eosinophil peroxidase (EPO), a secretion marker for eosinophils, in BALF were compared in guinea pigs sensitized to ovalbumin and exposed to single or repeated challenges (33). Both groups displayed increased eosinophilia in BALF and bronchial tissue and increased CD4+ T lymphocyte numbers in the lamina propria. The association of an increase in bronchial reactivity with a rise in the amounts of EPO in the BALF supernatants suggested that in situ eosinophil activation went hand-to-hand with BHR. If exposed to a single antigen inhalation, guinea pigs exhibited eosinophil infiltration in the BALF and bronchial tissues but no augmentation of the EPO content of the BALF nor, most importantly, enhanced reactivity to methacholine. Eosinophil infiltration into the airways by itself is thus not enough to trigger BHR.

Eosinophil degranulation is detected by the augmented levels of eosinophil-derived proteins, such as EPO, as mentioned, and major basic protein (MBP) in serum or in the BALF (34, 35). For years, a role for MBP has been advocated (36). Because antibodies against human MBP do not cross- react with guinea pig MBP, it took time before investigations were performed in guinea pigs. To do so, we raised a polyclonal rabbit antibody directed against guinea-pig MBP. Administration of this antibody into the airways of immunized guinea pigs suppressed BHR (37), strongly suggesting that MBP released in the airways by activated eosinophils induces BHR. Neutralization of endogenous MBP may therefore represent a therapeutic approach to suppressing allergen-induced functional changes in the airways.

The Murine Model

Because of the lack of genetically established strains and specific immunological reagents, the guinea-pig model is inadequate for studying the complexities of the mechanisms leading to BHR. Well-controlled murine strains are available; therefore, we (38) and others (39) developed models of allergic airway eosinophilia (43) and BHR in mice. As a rule, BHR was found to be associated with airway inflammation, but in at least one case (42) BHR was noted in female BALB/c mice without inflammation.

Inbred strains of mice display different susceptibilities to cutaneous anaphylaxis (44). Thus, genetic factors determine differences in the intensity of the anaphylactic response. Levitt and Mitzner (45) demonstrated that the airway response to acetylcholine is at least sixfold larger in A/J than C3H/HeJ mice. Genetic factors also determine the intensity of the response of the respiratory smooth muscles to direct agonists in a nonmanipulated environment, i.e., in absence of sensitization to foreign proteins, which is required for the expression of BHR. Clearly, this strain difference is independent from acquired immunity, and Gavett and Wills-Karp (46) suggested that it results from a more efficient coupling of the muscarinic receptors in A/J lungs to G proteins, resulting in better signal transduction. The variations in intrinsic airway responsiveness should be kept in mind in studies of BHR; indeed, use of the term BHR should be restricted to acquired hyperreactivity within a given strain.

Variations in the intensity of the inflammatory response according to the immune status of an animal (within the strain) and to the strain itself has also been shown. Thus, more leukotriene C4 is released from isolated antigen-challenged lungs of Swiss mice than of BALB/c mice (47). Given similar primary immunization, antigen-boosted mice respond differently to the lethal and cell-recruiting effects of PAF than do unboosted mice (48).

Using CBA, Swiss, and IL-5 transgenic mice (38), and later BALB/c and the Biozzi selection named BP2 (Bons Producteurs 2), we demonstrated that, as in guinea pigs, eosinophil recruitment to the airways is not sufficient to induce BHR (49). Indeed, airway eosinophilia is readily induced in immunized animals after a single antigenic provocation, whereas BHR requires repeated provocations for BALB/c (50, 51), C57Bl/6 (52), or BP2 mice (49).

Factors other than eosinophilia per se are thus required for the expression of BHR. These factors can be visualized using the BP2 selection, which like other Biozzi mice have been selectively bred for modified antibody production (53, 54). The BP2 mice, which carry the H-2^q haplotype, were selected for high antibody responsiveness to sheep erythrocytes. They became routinely available thanks to Dr. D. Mouton (INSERM U255, Institut Curie, Paris, France; provider: Elevages Janvier, Le Genest Saint Isle, France). A single intranasal challenge with ovalbumin of immunized BP2 mice was followed by intense BALF and airway infiltration by eosinophils and CD4+ and CD8+ T lymphocytes. As in case of BALB/c mice, eosinophilia was accompanied by rises in IL-5 titers in serum and BALF, without increases in bronchial reactivity, further demonstrating that BHR is not due to IL-5 per se. By contrast, when the mice were challenged with ovalbumin twice a day for 2 d or once a day for 10 d, BHR was consistently induced in response to the intravenous injection of serotonin or to aerosolized methacholine. Bronchial hyperresponsiveness persisted for at least 3 wk, outlasting lung and BALF eosinophilia. It was suppressed by the anti-IL-5 antibody TRFK-5 (49), by dexamethasone (55), by the immunosuppressor agent FK506 (56), and by nonanaphylactogenic anti-IgE antibody 1-5 (57). These results suppport a role for T lymphocytes and IgE in allergen-induced airway inflammation and BHR. Dexamethasone and FK506 probably interfere with allergic airway inflammation by different mechanisms, because the latter did not reduce the number of infiltrating T cells but inhibited their activation (56), particularly the antigen-induced production of IL-5, whereas the former also reduced the elevated lymphocyte numbers in BALF following allergen provocation (55). Infiltration of CD4+, but not of CD8+, T lymphocytes was observed in murine bronchial tissue (44) and BAL (58) of immunized and antigen-challenged BALB/c mice.

Three important differences detected between BALB/c and BP2 mice may be relevant to the interactions between eosinophils and BHR. First, in BP2 mice made hyperreactive, recruited eosinophils were found in the bronchial submucosa as well as in the respiratory epithelium, but they were restricted to the submucosa in BALB/c or in nonhyperresponsive (single-challenged, for instance) BP2 mice, as if their passage into the bronchial lumen (sampled by bronchial lavage) was speedier in the absence of some chemoattractant activity of the allergic epithelium. This hypothetical factor has not yet been identified.

Second, BP2 mice had higher elevations in total serum IgE. Treatment with an anti-IgE antibody suppressed BHR and the accompanying eosinophilia (57). We have hypothesized that an enhanced availability of IgE in the microenvironment where the recruited eosinophils are located enhances their activitya form of priming. Against this hypothesis is the observation that the epithelial eosinophils, which might be exerting their deleterious secretion-dependent effects, are not degranulated when observed under electron microscopy. One explanation presently under investigation is that eosinophil priming by IL-5 should lead to an enhanced release of granule-independent mediators, such as leukotrienes. Alternatively, a role for the chemokine eotaxine has been advocated (59). Such a scheme would accommodate at the same time the evidence for cytokine participation (chronic disease) and for leukotriene participation (acute manifestations of the actual disease, whenever triggering factors are evoked).

The third important difference between BP2 and BALB/c mice is that the former respond with serotonin-dependent bronchoconstriction to the intravenous administration of antigen, whereas the latter respond only with an increase in vascular permeability (56). Anaphylactic bronchoconstriction in BP2 mice is not suppressed by dexamethasone FK-506 (55, 56) under conditions that suppress antigen-induced BHR, confirming that the mechanisms accounting for BHR and for anaphylaxis differ. By contrast, the anti-IgE antibody was efficient against both (57).

Endogenous mechanisms modulate airway inflammation and possibly BHR. Thus, the co-instillation of IL-10, a Th2 product, by the intranasal route significantly inhibits the peritoneal and bronchopulmonary eosinophilia induced by ovalbumin in immunized mice (60, 61). Interferon- also inhibits allergic eosinophilia (62) and BHR (63), probably by inducing formation of IL-10.

Bone Marrow as Source of Allergy-modified Eosinophils

The airway invasion by eosinophils that follows intranasal allergenic provocation in mice is preceded by an enhanced early distal eosinopoiesis. Indeed, by 2 h after provocation, the total number of EPO+ cells in the bone marrow is significantly increased (38). Eosinophils found in the airways after provocation may thus originate in the bone marrow, but in situ differentiation of precursor cells, recruited into the airways from circulating blood by the action of Th2 cytokines, may also occur. Whatever the source of the eosinophils, the mechanisms that lead the precursors or mature eosinophils to the airways are unclear. The factors responsible for their recruitment can probably be found in the circulation (64, 65). Alternatively, the number of circulating eosinophil progenitors available for recruitment to the tissues may be augmented in asthmatic subjects. The presence of these circulating progenitors may correlate with development of BHR (66). Because the modification in the number of eosinophilic progenitors in asthmatic subjects has been attributed to variations in the rate of their release from the bone marrow (66), it is clear that the marrow can respond to allergenic challenges either by producing more mature eosinophils or by releasing more progenitors into the circulation. It has been demonstrated that antigen inhalation in the dog is accompanied by an augmentation in the number of circulating progenitors capable of responding to Stem Cell Factor and to granulocyte/macrophage colony-stimulating factor (67). We recently demonstrated an exquisite sensitivity of eosinophilic precursors to IL-5 when collected from the bone marrow 24 h (but not 2 h) after allergenic provocation (65). Possibly important related observations are that lung tissue or bone marrow transplanted from asthmatic patients can transfer the disease to the recipients (68, 69).

Because of the intimate connections between BHR and pulmonary eosinophilia, it is likely that cytokines produced by the T lymphocytes of immunized animals augment the rate or intensity of bone marrow eosinopoiesis in strains that produce enough IgE. It is also possible that there is an as-yet undescribed connection between the capacity to produce IgE and the capacity to attract eosinophils after allergen challenge, a possibility consistent with the control of IgE synthesis by IL-4 and eosinophil attraction by IL-5, both Th2-borne cytokines.

	HYPERREACTIVITY INDUCED BY BACTERIAL ENDOTOXIN

Awareness of the complex interactions between man-modified environment and individual factors (including, but not restricted to, genetic constitution) is rapidly increasing. The interactions of microorganisms and allergens are of particular interest. Bacterial endotoxin (LPS) is present in house dust (70) and may modify the expression of respiratory allergy. Schwartz and colleagues (71) demonstrated synergism between LPS and corn dust extracts, suggesting that responsiveness to LPS is critical to the development of grain dust-induced inflammation of the airways. In this example, desensitization to LPS by repeated exposures reduced the inflammation induced by corn dust extracts. These findings are consistent with our own study showing that LPS, administered systemically 24 h before ovalbumin reduces antigen-induced bronchoconstriction and the release of mediators from isolated guinea pig lungs (72). This peculiar inhibition was not related to the intense lung invasion by neutrophils that follows LPS injections to guinea pigs; it might instead have involved the endogenous formation of anti-inflammatory cytokines such as IL-10 (59, 60). We know now that LPS induces the release of secreted 14 kD phospholipase A2 into the circulation (73) and the BALF (74) of LPS-treated guinea pigs. The BALF activity originates from alveolar and interstitial macrophages; its relevance to inflammation is under scrutiny.

In other circumstances, LPS enhances chemoattractant-induced eosinophil recruitment to skin sites (75); no effects on allergen-induced recruitment were described. Most interestingly, Dubin and colleagues (76) demonstrated that extravasation of LPS-binding protein and soluble CD14 into the bronchoalveolar compartment after antigen inhalation may enhance the capacity of inhaled or aspirated LPS to activate an inflammatory cascade that may amplify the inflammatory response to inhaled antigen in some asthmatics. This may be related to a large number of potential targets of LPS; it is a very important finding. This finding may be related to the marked hyperreactivity to the bronchoconstrictor effects of serotonin induced by LPS in the guinea pig which we described, and which is neutrophil-independent and suppressed in platelet-depleted animals or after the administration of the platelet-protective agent prostacyclin (77). The presently recognized importance of chemokines, such as eotaxin and RANTES (the latter being produced by platelets), may provide the link between these different effects of LPS and allergen. In this connection, we demonstrated recently that serum from naive or immunized LPS-treated guinea pigs contains an activity that augments the production of eosinophils in the bone marrow (78).

The intranasal instillation or aerosolization of LPS to mice is followed by the production of tumor necrosis factor- (TNF-) and by an intense recruitment of neutrophils into the BALF (79); both are enhanced by pretreatment with nonsteroidal anti-inflammatory drugs (NSAIDs) and reduced by substances that increase the macrophagic content in cyclic adenosine monophosphate (cAMP). This suggests that a downregulating feedback by the stimulated macrophages, accounted for by the production of prostaglandins, probably prostaglandin E2, is suppressed by the NSAID (79). In addition, the intranasal administration of LPS to mice is followed by other relevant effects, including an augmentation of basal airway resistance, probably due to airway engorgement; augmented protein content of the BALF, indicating increased vascular permeability; and BHR to inhaled methacholine. Bronchial hyperresponsiveness is also observed after the systemic administration of LPS, even though no neutrophil recruitment to the airways is observed. As we have stated before (77), systemic LPS also induces BHR to serotonin in guinea pigs. The direct effects of both LPS and BHR in mice are reduced by dexamethasone, but it is unclear at this stage whether the suppressive effect results from similar or different targets and mechanisms.

	CONCLUSIONS

TOP ABSTRACT INTRODUCTION CONCLUSION REFERENCES

The targets for therapeutic intervention in asthma, specified according to the present concepts of its physiopathology, can be listed as follows:

1. Modulation of mast cell function
2. Inhibition or antagonism of lipid mediators, leukotrienes, and PAF
3. Interactions between antigen-presenting cells and T lymphocytes
4. T lymphocyte recruitment and homing in the airways
5. Activation of T lymphocytes and production of interleukins
6. Induction of IgE production
7. Interference with eosinophil differentiation in the bone marrow and/or precursors in lymphoid tissue or airways
8. Recruitment and homing of eosinophils in the airways
9. Eosinophil activation (production of lipid mediators and release of preformed mediators, including major basic protein)
10. Epithelial modifications (lesions) with exposure of subepithelial structures
11. Interactions with the cholinergic or nonadrenergic noncholinergic system

Nonallergenic causes of BHR, such as those involving LPS, involve other targets, particularly monocytes/macrophages and their respective cytokines. Bronchial hyperresponsiveness is thus at the center of a complex network of cells and cytokines. Unraveling the interactions involved will undoubtedly bring new therapeutic targets for lung and airway diseases.