INTRODUCTION

TOP ABSTRACT INTRODUCTION REFERENCES

Occupational asthma shares many characteristics with IgE-mediated asthma: in both, the responsible agent is known and affected patients' clinical presentation, response to inhalation challenge in the laboratory, and response to antiasthma drugs are similar. Although occupational asthma induced by high-molecular-weight compounds is often mediated by an immunoglobulin E (IgE) mechanism and is more frequent in atopic subjects, in most subjects with occupational asthma induced by low-molecular-weight compounds it is not possible to demonstrate an IgE-mediated mechanism (1). Nonatopic asthma has many features of chronic cell-mediated disease, which occurs independently of an IgE-mediated immunologic mechanism. Thus, while it has not been firmly proved that asthma is causally related to immunologic abnormalities, there is little doubt that "allergic" asthma is by far more frequent in atopic subjects (2), and particularly that atopic subjects are at higher risk of developing allergic asthma, and particularly asthma induced by high-molecular-weight occupational sensitizers (1). However, activated T lymphocytes, methacromatic cells and eosinophils are increased in the bronchial mucosa of allergic and nonallergic asthmatics and of subjects with occupational asthma induced by low-molecular-weight sensitizers (e.g., isocyanates), suggesting similar, probably immunologically mediated mechanisms for both nonoccupational and occupational asthma (5). Immunologic mechanisms of disease have been intensively explored in recent years, and excellent reviews are available (8).

After activation by antigen, T lymphocytes secrete a number of lymphokines that attract, activate, and promote the growth and the differentiation of other leukocytes. Based on murine studies, T helper (Th) clones may be subdivided on the basis of their cytokine profile (11). The use of antigen-specific T lymphocyte clones confirmed the results (12). Th1 clones produced predominantly interleukin (IL)-2 and interferon-gamma (IFN-), whereas Th2 clones produce mainly IL-4 and IL-5. Both Th1 and Th2 produce IL-3 and granulocyte/macrophage colony-stimulating factor (GMCSF). In vivo studies in humans confirm the existence of functionally distinct subsets of T lymphocytes. In addition to their role as helper cells for the production of humoral antibodies by B cells, activated CD4+ lymphocytes may be considered inflammatory cells. Activated T cells secrete IL-8, which is a chemotactant cytokine for polymorphonuclear leukocytes. They are also an important source of GMCSF and IL-5. The first is important in eosinophil development and activation, and it plays a significant role in the amplification of eosinophilic inflammation. Interleukin-5 appears to be specific in promoting the development, adhesion, and activation of eosinophils, and it is the predominant eosinophil-active cytokine present in bronchoalveolar lavage (BAL) fluid during allergen-induced late phase inflammation (13). Activated T cells may therefore initiate and propagate allergic inflammation in the airways and participate directly in the events responsible for asthma exacerbations (14). However, a number of other cells produce IL-3, IL-4, IL-5, GMCSF and other relevant cytokines (11, 12, 18- 20). There is now compelling evidence to confirm that T lymphocyte activation and local accumulation of activated eosinophils in the bronchial wall are present in asthma of diverse severity and etiology, i.e., IgE-mediated, intrinsic, or occupational (20). Regarding CD8+ T cells, different subsets participate directly in the B-cell suppressor function or act as cytotoxic cells for exogenous and endogenous antigens (25). It has been reported that soluble exogenous antigens or haptens are able to mount a major histocompatibility complex (MHC) class I restricted response (26, 27).

	ANTIBODY-MEDIATED HYPERSENSITIVITY

Some occupational agents, particularly high-molecular-weight sensitizers, act through an IgE-mediated mechanism (1). According to this mechanism, inhaled sensitizing agents bind to specific IgE on the surface of mast cells, basophils, and probably also macrophages, eosinophils, and platelets. High-molecular-weight sensitizing agents may act as complete antigens. By contrast, low-molecular-weight sensitizers probably need to react with autologous or heterologous proteins to produce a complete antigen. The reaction between antigen and IgE causes the cascade of events responsible for the activation of inflammatory cells and for the synthesis or release of a wide variety of preformed or newly formed inflammatory mediators that trigger the inflammatory reaction (10, 28). Other classes of antibodies may have a role in asthma (10, 28). The importance of the antibody-mediated immunity can be investigated in humans with in vivo and in vitro tests, most frequently with skin-prick tests and radioallergosorbent (RAST) or ELISA tests (1). Most studies in occupational asthma have concentrated on antibody-mediated immunity. Skin-prick tests and circulating specific antibodies against extracts or conjugates of occupational sensitizers have been researched. Positive skin-prick tests, specific IgE, and IgG have been shown to be present mainly in atopic subjects sensitized to high-molecular-weight sensitizing agents (29), but also in some subjects sensitized to low-molecular-weight agents (e.g., anhydrides, platinum salts, nickel, plicatic acid, and isocyanates) (30). In subjects with occupational asthma induced by both high- and low-molecular-weight compounds, specific immunoglobulins (IgE, IgG) may be found (30). Skin tests and specific antibodies may, therefore, be helpful in the diagnosis of occupational asthma due to high-molecular-weight compounds (35). However, positive skin tests and/or specific antibodies against occupational allergens are often present in subjects both with and without symptoms of asthma, suggesting that their presence may reflect exposure more than disease (1). These subjects require monitoring with the aim of confirming sensitization. Regarding occupational asthma due to low-molecular-weight compounds, it has been reported that in asthma induced by diisocyanates such as hexamethylene diisocyanate (HDI) and diphenylmethane diisocyanate (MDI) specific IgG antibodies are more important than IgE antibodies (32). These antibodies are also present in some subjects with no history of asthma and a negative inhalation challenge. As in isocyanate-induced asthma (32), specific antibodies against plicatic acid and against morphine have been found, respectively, in symptomatic and asymptomatic workers exposed to red cedar or to morphine (33, 36). Under certain circumstances (exposure to high-molecular-weight compounds and to certain low-molecular-weight compounds), the presence of specific IgE or IgG is useful in the diagnosis of occupational asthma, whereas in others, their presence is a biological marker of exposure and must be associated with pulmonary function tests to detect the clinical onset of the disease.

	CELL-MEDIATED HYPERSENSITIVITY

Until recently, T lymphocytes were considered to play a role in IgE-mediated immunity mainly through the induction and regulation of IgE by B lymphocytes (8). However, T lymphocytes may release potent cytokines that recruit and activate other inflammatory cells, and thus directly cause inflammation, suggesting that T lymphocytes may act as effector cells in atopic allergic inflammation and asthma through pathways clearly distinct from B-cell regulation and IgE production (25). T lymphocytes are capable of modulating eosinophil adherence, chemotaxis, and activation, and of stimulating these cells to cause tissue damage. Poorly studied in nonoccupational asthma, cell-mediated immunity and its role in airway inflammation has not been extensively examined in occupational asthma. However, as previously mentioned, similarly to patients with atopic asthma, an increased number of activated CD25+ T lymphocytes (i.e., lymphocytes expressing the IL-2R), of activated eosinophils, and of mast cells have been observed in patients with occupational asthma induced by toluene diisocyanate (TDI) and other small-molecular-weight sensitizers (5), suggesting that similar immunologic mechanisms may be involved in asthma of occupational as well as nonoccupational origin (25). The presence of activated lymphocytes and eosinophils in bronchial biopsies suggests that a T lymphocyte-eosinophil interaction may be important in asthma of different origins, a hypothesis further supported by the finding of cells expressing IL-5 messenger RNA in bronchial biopsies of atopic asthmatics (37). Interleukin-5 is in fact the most important eosinophil-regulating cytokine, and its concentration in the airway mucosa of asthmatics correlates with markers of T-lymphocyte and eosinophil activation (37). There have been a number of reports of alterations in T-cell subsets, either in blood or in BAL fluid, following specific bronchoprovocation, all supporting an immune-mediated mechanism for the development of airflow obstruction (38, 39). Circulating CD8+ cells and eosinophils increase significantly at 48-72 h after a specific inhalation challenge with TDI, at a time when airflow obstruction has resolved (38), suggesting that in sensitized subjects exposure to TDI not only causes airflow obstruction but also the recruitment and activation of lymphocytes and eosinophils, probably through an immunologic mechanism. Nickel-specific T-lymphocyte clones have been isolated in subjects with nickel-induced asthma (39), suggesting that T-cell-mediated immunity may be involved in this type of occupational asthma. Cobalt-sensitized lymphocytes proliferating after incubation in vitro with cobalt have been found in subjects with hard metal asthma (40).

Although an immunologic mechanism has never been firmly established, occupational asthma induced by TDI shares several features with allergic asthma. For example, as previously mentioned, circulating CD8+ lymphocytes and eosinophils increase significantly after exposure to TDI and remain increased even when FEV₁ returns to baseline. This observation suggests that the events triggered by exposure to TDI in sensitized subjects include changes in lung function and systemic effects that last longer than bronchoconstriction and involve suppressor/cytotoxic lymphocytes and eosinophils. These findings, taken together, suggest that TDI-induced late asthmatic reactions may be associated with an immunologic response to TDI or to its products (38). In addition, isocyanate-induced asthma and atopic (extrinsic) asthma have a similar pattern of inflammatory cell infiltrate (5, 24), including mast cells, eosinophils, and mononuclear cells. Mononuclear cells are probably activated lymphocytes, as shown by the expression of IL-2 receptor in the cell surface. Interestingly, in collaborative studies (41, 42) we observed thatat variance with atopic asthma, in which the majority of T-cell clones derived from the atopic patients are CD4+ with a Th2 pattern of cytokine productionthe majority of T-cell clones derived from patients with TDI-induced asthma are CD8+ and capable of producing IL-5, a cytokine that attracts, activates, and increases survival of eosinophils. Thus, while CD4+ Th2-like T cells, because of their pattern of cytokine production, may play a central role in determining the nature of inflammatory response seen in bronchial mucosa of asthmatic atopic patients through the induction of both allergen-specific IgE (via IL-4) and eosinophilia (via IL-5), CD8+ T cells may play a similar role in nonatopic subjects with TDI asthma, not through the induction of both TDI-specific IgE and eosinophilia, but by directly causing bronchial eosinophilia via local production of IL-5. Interestingly, an increased percentage of CD8+ T cells have been recently reported in BAL of intrinsic nonatopic asthmatic subjects (43). These few studies, although preliminary, and studies conducted in other occupational lung diseases such as hypersensitivity pneumonitis (44) and berylliosis (45) reveal potential information that could be obtained from cell immunology studies in occupational asthma.

	GENETIC MECHANISMS IN OCCUPATIONAL ASTHMA

In trying to resolve the genetic basis of various diseases, including asthma, two basic approaches have been used: "forward" and "reverse" genetic mapping. The former focuses on prospective candidate genes. By using the second approach, termed "positional cloning," researchers aim to establish a linkage between the asthma phenotype and a series of highly polymorphic genetic markers. Specific immune responsiveness to an antigen is affected by HLA class II molecules encoded by well-defined MHC genes on human chromosome 6p, which are required for the presentation of an antigen to a T-cell receptor (TcR) in order to initiate the chain of events that leads to an antibody response, including IgE. Individual differences in HLA class II molecules may alter the ability of the molecules to bind peptides and thereby change the nature of T-cell recognition. There is now compelling evidence to suggest that antigens derived from extracellular sources are recognized in the context of HLA class II molecules by CD4+ T cells. It is likely that the expression of genetic factors, regulating both overall and specific responsiveness toward specific allergenic epitopes, is affected by the intensity and frequency of allergen exposure, as well as by the ability of the allergen to enter the lung (46).

Recently, it has been proposed that specific HLA class II alleles could be involved in susceptibility or resistance to isocyanate-induced asthma (47), suggesting that the basic molecular and cellular mechanisms of occupational asthma induced by low-molecular-weight compounds are immunologic, as in IgE-mediated asthma, in which the association with HLA class II genes is well documented (48). Occupational asthma induced by isocyanates and IgE-mediated asthma differ in terms of their association with particular HLA class II alleles or haplotypes. Although in IgE-mediated asthma a strong positive association with haplotype DR4 is found, in isocyanate-induced asthma the frequency of haplotype DR4 is low and does not differ from control subjects. Recent studies reported other HLA associations in occupational asthma, and particularly a significantly increased frequency of DQB1*302 in subjects with western red cedar asthma (50) and an excess of HLA-DR3 with specific IgE to acid anhydrides in exposed workers (51), suggesting that MHC II proteins may be an important determinant of the specificity of the IgE response to an inhaled low-molecular-weight sensitizer. The exact nature of the antigen related to sensitization to isocyanates is unknown. Isocyanates themselves may be involved in antibody binding or in the induction of structural changes in an unidentified protein. It has been proposed that the mechanism of low responsiveness to foreign antigens is linked to DQ alleles, whereas DR haplotypes are implicated in the upregulation of the immune response (48, 49). Low responsiveness could be mediated by specific CD4+ T cells, which in turn might activate CD8+ suppressor cells. Recently in a collaborative study, Bignon and colleagues carried out HLA typing in isocyanate- induced asthma (47). They found that the allelic combination DQB1*0503 was associated with susceptibility to the disease and that the combination DQB1*0501 was associated with resistance. More recently we confirmed these results and extended those observations (52). In fact, we found the presence, at the 57 position, of aspartic acid in DQB1*0503 allele and valine in DQB1*0501 allele, and we suggested that the residue 57 of HLA-DQB1 could be used in risk assessment of isocyanate-induced asthma.

The results of these two studies (47, 52) are consistent with the hypothesis that an immunologic mechanism is involved in TDI-induced asthma and that specific genetic factors play a role in conferring susceptibility to or protection against this disease. An immunologic mechanism in TDI-induced asthma is likely to occur because the inflammatory response of the airways in TDI-induced asthma is characterized not only by increased numbers of mucosal eosinophils and mast cells, but also by persistent activation of lymphocytes and chronic expression of proinflammatory cytokines (5, 24, 53). When associations between HLA class II genes and occupational asthma are investigated, the problems encountered in the field of atopic diseases are less important (54). In fact, the phenotype of occupational asthma due to TDI exposure may be defined with accuracy (55). In this study we found a positive and a negative association with two HLA class II markers. When we analyzed the differences between hypervariable amino acid residues in the DQB1 alleles, we found that residue 57 of HLA-DQB1 has a role in conferring susceptibility to the disease. It is well established that HLA class II molecules are involved in antigen presentation and that a correct binding between these glycoproteins and the antigen is necessary for functional antigen recognition by T lymphocytes. We do not know what the TDI antigen looks like. It seems unlikely that TDI itself is the antigen because of its low molecular weight (MW = 174.2). In animal studies, it has been shown that inhaled radiolabeled TDI rapidly accumulates in the lung and becomes potentially accessible to immunocompetent cells (56). We assume that TDI binds to endogenous or exogenous peptides that are recognized by the T lymphocytes as a foreign antigen. More than one mechanism may be put forward for TDI. It could behave as a hapten and alter the structure or the specificity of the TcR directly or by acting at the gene level, or it could react with membrane proteins such as adhesion molecules, amplifying T-cell-B-cell interactions. Residue 57 is a negatively charged residue that may directly interact with TDI itself. Finally, it could modify the structure of MHC class II molecules or self peptides at the surface of the antigen-presenting cells (APC) in such a way that the modified epitopes are recognized as foreign by the T cells.

Other possibilities include the hypothesis that, because DQ molecules act as dominant suppressor genes (Is) involved in active suppression against specific antigens (57), TDI could cause a dysfunction in the suppressor-inducer network. This behavior is supported by the finding of an increase in circulating CD8+ lymphocytes in sensitized subjects exposed to TDI in the laboratory (38) and by the finding that T-cell clones obtained from endobronchial biopsies of subjects with TDI- induced asthma exhibit the CD8 phenotype and produce IL-5 and interferon-gamma (41). Whatever the exact mechanism of the association DQ-TDI-induced asthma may be, the data are consistent with the hypothesis that at least two risk factors exist for TDI-induced asthma: exposure to the chemical and inheritance of the genetic marker. We do not know how the interaction works and what proportion of subjects exposed to TDI with the marker develop the disease. What is important is that most asthmatic subjects have the marker in both haplotypes. Because asthma is likely to be a multifactorial and polygenic disease, other genes or environmental factors may be necessary for expression of TDI-induced asthma. A multifactorial inheritance may explain the low incidence in the exposed subjects, the presence of healthy subjects carrying the predisposing marker, and asthmatics lacking the same marker. Therefore, it could be that the identification of a variability at the 57 position in the DQB1 chain is not enough to cause the expression of the disease, and other conditions, particularly environmental conditions, may be necessary besides HLA gene products to determine sensitization to a compound present in the workplace and cause asthma.

Taken together, these results suggest a role for HLA class II genes in conferring susceptibility or protection against the disease. However, on the basis of the previous considerations, at the present moment we do not know whether the recognition of the amino acid residue variation in the "beta chain" structure is a useful tool for secondary prevention of TDI- induced asthma.