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Managing Health Effects of Beryllium Exposure 4 Mechanisms, Genetic Factors, and Animal Models of Chronic Beryllium Disease This chapter provides an overview of the pathogenesis of chronic beryllium disease (CBD) and the mechanism of action of beryllium in causing it. It also provides a summary of studies to identify the genetic components involved in susceptibility to CBD and of attempts to develop animal models to study the disease. PATHOGENESIS AND MECHANISMS OF ACTION As early as 1951, Sterner and Eisenbud proposed that CBD was an immune-mediated hypersensitivity reaction directed against the inhaled beryllium antigen. Even the earliest accounts of the disease described it as hypersensitivity of delayed onset, which fits with the present understanding of the cellular immune mechanisms underlying CBD. Although alterations in humoral immune characteristics have been described in CBD patients (Resnick et al. 1970; Cianciara et al. 1980), by and large the immunopathology of the disease involves cellular immune mechanisms. Moreover, beryllium is not defined by the Occupational Safety and Health Administration as a chemical sensitizer. That is, repeated exposure to beryllium does not cause an immediate immunoglobulin E–mediated allergic reaction. Beryllium-induced disease is believed to be contingent on cell-mediated (delayed-type hypersensitivity) immunopathology. Therefore, the term beryllium sensitization (BeS) refers to the CD4+ T-cell immune response, which is measured with in vitro assays discussed elsewhere in this report. Understanding of the immunologic basis of CBD and the immunopathogenic mechanisms that contribute to it has advanced, but many questions about
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Managing Health Effects of Beryllium Exposure the details of interactions between exposure and host factors remain. The literature of CBD is extensive, and this section consists of a selective review of the primary pertinent literature that has shaped current understanding of the immune mechanisms involved and of genetic factors that might contribute to susceptibility to the disease. CBD is a systemic granulomatous disorder that affects the lungs predominantly. The mechanism underlying CBD pathogenesis involves an immune response to beryllium (Figure 4-1). CD4+ T lymphocytes recognize beryllium as an antigen that triggers cell proliferation and release of cytokines and inflammatory mediators. The release of inflammatory mediators results in an accumulation of mononuclear-cell infiltrates and fibrosis that lead to the lesion typical of the disease—a noncaseating granuloma. Critical Role of CD4+ T Cells Beryllium acts as a major histocompatability complex (MHC) class II restricted antigen that stimulates the proliferation and accumulation of beryllium-specific CD4+ T cells in the lungs (Saltini et al. 1989, 1990). Two observations illustrate the primary importance of CD4+ T cells in the pathogenesis of CBD: the development of granulomatous inflammation in the lungs is associated with the accumulation of CD4+ T cells in bronchoalveolar-lavage (BAL) fluid, and sensitization to beryllium is detected in the ability of CD4+ T cells to proliferate in response to beryllium salts in culture. FIGURE 4-1 Immune response to beryllium. Source: Fontenot and Maier 2005. Reprinted with permission; copyright 2005, Trends in Immunology.
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Managing Health Effects of Beryllium Exposure The immunobiology believed to be associated with CBD provides a diagnostic test for BeS. As noted earlier, the beryllium lymphocyte proliferation test (BeLPT) involves an in vitro challenge of either BAL-derived or peripheral-blood–derived mononuclear cells with beryllium salts. In beryllium-responsive people, the challenge induces an oligoclonal proliferation of sensitized lymphocytes that is measured in a standard assay in which tritiated-thymidine incorporation occurs in proportion to DNA synthesis and blastogenesis (Rossman et al. 1988; Kreiss et al. 1989). Because beryllium drives the proliferation and expansion of CD4+ T cells in an antigen-restricted manner, T-cell lines and clones have been derived from the BAL fluid and blood of CBD patients. There are important differences between the antigen-specific T-cell clones found in the lungs of CBD patients and those in the circulation of beryllium-sensitized people, and the differences may have implications for the progression from BeS to CBD. For example, the T-cell receptor (TCR) repertoire in beryllium-reactive peripheral blood cells appears to be more diverse than that in the lungs of CBD patients (Fontenot et al. 1999). That suggests that a subset of T-cell clones expressing homologous TCRs has pathogenic potential. In many people, particularly CBD patients in the ceramics industry exposed to beryllium oxides, the T cells found in the BAL fluid express TCRBV3 genes with identical or homologous complementary-determining region 3 sequences. As further evidence that these are oligoclonal expansions, the beryllium-responsive T cells coexpress only a few homologous TCRα genes (Fontenot et al. 1999). That means that there is selective expansion or accumulation of some CD4+ T-cell subsets in the lungs of CBD patients. The selectivity is probably related to the antigenicity of beryllium and probably provides clues to conventional antigen peptides that are modified by beryllium. Antigen Processing and Presentation of Beryllium As discussed above, sensitization to beryllium can be readily demonstrated in the ability of CD4+ T cells to proliferate in response to beryllium salts in culture. The proliferative response has characteristics of a response to antigen, but the nature of the antigen recognized by CD4+ T cells is not known. In studies of mouse lymphocytes, Newman and Campbell (1987) found that beryllium sulfate was mitogenic for B lymphocytes but not T lymphocytes. They did not address the potential for endotoxin contamination of the beryllium-salt preparation to drive the polyclonal B-cell response. On the basis of many human studies, it is reasonable to conclude that beryllium is not a mitogen for human lymphocytes. Proliferation of beryllium-specific CD4+ T cells requires the engagement of clonotypic TCRs with an unknown beryllium antigen bound by MHC class II molecules on the surface of antigen-presenting cells. The physicochemical properties of beryllium ions offer few clues to a better understanding of its immunogenicity. The immunogenicity of beryllium probably lies mainly in its ability to haptenate and thereby alter the structure of
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Managing Health Effects of Beryllium Exposure peptides that occupy the antigen-binding cleft of MHC class II molecules. Other metal ions—including nickel, cobalt, mercury, and gold—may elicit T-cell reactivity by similar mechanisms (Lawrence and McCabe 2002); however, the specific peptides and MHC molecules (pMHC) involved in all cases are different from those attributed to immune reactivity to beryllium. As with immune reactivities to other metal:pMHC, the response to beryllium:pMHC is exquisitely specific and lacks cross-reactivity with other metal:MHCs. Knowing that susceptibility to CBD was associated with particular alleles of the class II human leukocyte antigen-DP (HLA-DP) molecule, Fontenot et al. (2000) examined whether the CD4+ T-cell proliferation accompanying CBD involved the presentation of beryllium by HLA-DP. Beryllium-specific T-cell lines isolated from the lungs of CBD patients showed that the response to beryllium was almost completely and selectively blocked by monoclonal antibodies directed at HLA-DP. Additional studies with fibroblasts engineered to express only specific HLA-DP alleles demonstrated that the response to beryllium was restricted to haplotypes previously implicated in susceptibility to the disease. Hence, beryllium presentation by some HLA-DP alleles to CD4+ T cells is the underlying mechanistic basis of CBD. Analysis of the amino acid residues shared by HLA-DP alleles that present beryllium revealed that those with a negatively charged glutamic acid residue at the 69th position of the β chain (Glu69) were especially capable of inducing a T-cell response (Richeldi et al. 1993; Wang et al. 1999; Fontenot et al. 2000; Lombardi et al. 2001; Bill et al. 2005). Not all CBD patients have a Glu69 containing HLA-DP allele. Indeed, t early work by Fontenot et al. (2000) demonstrated that anti-HLA-DR reagents partially inhibited T-cell responsiveness to beryllium in some cases. Recent work by Bill et al. (2005) reported an increased frequency of HLA-DR13 in some CBD patients who lacked a Glu69 HLA-DP allele. These HLA-DR13 alleles have a glutamic acid at position 71 of the β chain (which corresponds to position 69 of HLA-DP). Beryllium presentation to CD4+ T cells might occur through an alternative HLA-DR Glu71 pathway that is capable of inducing beryllium-specific proliferation and interferon-gamma (IFN-γ) production by CD4+ T cells. Genetic susceptibility to CBD is discussed later in this chapter. Amicosante et al. (2001) conducted beryllium-binding assays with purified soluble HLA-DP molecules and beryllium sulfate and showed that the HLA-DPβGlu69 residue played a role in beryllium binding. Whether that involves a direct interaction between Glu69 and beryllium ions or beryllium modifies an unknown peptide that then preferentially interacts with the HLA-DPβGlu69 alleles is unknown (reviewed by Amicosante and Fontenot ). Homozygosity, as opposed to heterozygosity, in the expression of the HLA-DPβGlu69 supratypic variant allele did not impart increased responsiveness, so the cell-surface density of class II molecules charged with beryllium-modified antigenic peptides does not dictate the intensity of responsiveness (Amicosante et al. 2005). The nature of the beryllium antigen remains one of the key issues that require further study with respect to the immunopathogenesis of CBD. Amico-
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Managing Health Effects of Beryllium Exposure sante et al. (2001) demonstrated that beryllium binds to HLA-DPβGlu69 at a pH of 5.0 and at a pH of 7.5. pH 5.0 mimics the acidic microenvironment where peptides are loaded onto HLA class II molecules, whereas pH 7.5 represents the extracellular environment where beryllium might bind to HLA-DP molecules directly at the cell surface. That beryllium binds to HLA-DPβGlu69 at a pH of 7.5 suggests that it binds to HLA-DP in the absence of antigen processing. Furthermore, Fontenot et al. (2006a) demonstrated that paraformaldehyde-fixed beryllium-pulsed antigen-presenting cells stimulated the proliferation of CD4+ T-cell lines derived from the lungs of CBD patients. That suggests that the presentation of soluble beryllium does not require antigen processing. Although direct antigen presentation of beryllium from soluble beryllium salts may occur, Stefaniak et al. (2005) reported that dissolution of beryllium oxide particles in macrophage phagolysosomes may be an important source of dissolved beryllium for input into the cell-mediated immune reaction characteristic of beryllium disease. The physicochemical state of beryllium (single-constituent vs multiconstituent material) influences its bioavailability, which may be tied to the initiation or sustainment of immune reactivity. Stefaniak et al. (2006) found that the dissolution rate stimulated by phagolysosomal fluid was greater for beryllium-copper–alloy fume than for beryllium oxide; this suggests that the physicochemical form of beryllium encountered in the workplace may have a bearing on initiating the sensitization process. Beryllium complexed with ferritin may be an important source of beryllium taken up by macrophages (Sawyer et al. 2004a). The uptake of beryllium may lead to aberrant apoptotic processes and the release of beryllium ions, which will continue the stimulation of T-cell activation (Sawyer et al. 2000; Kittle et al. 2002; Sawyer et al. 2004a). Beryllium uptake may be accompanied by oxidative stress and generation of reactive oxygen species that lead to the apoptotic response (Sawyer et al. 2005). It has been hypothesized that the interaction between the innate and acquired immune systems leads to the cyclical rerelease of beryllium into the lungs, where it elicits proinflammatory cytokine production and T-cell proliferation (Sawyer et al. 2002). The beryllium-antigen–presenting cells themselves have not been well defined (L.A. Maier, National Jewish Medical and Research Center, personal communication, April 5, 2007). They may be macrophages, dendritic cells, or other professional antigen-presenting cells. Recently, self-presentation of beryllium by HLA-DP–expressing BAL CD4+ T cells has been reported (Fontenot et al. 2006b). Self-presentation by BAL T cells in the granuloma results in activation-induced cell death, which may lead to the oligoclonality of the T-cell populations characteristic of CBD. Th1 Cytokine Secretion by Beryllium-Specific T Cells The CD4+ T cells that accumulate in the lungs of CBD patients exhibit a Th1 phenotype and secrete such cytokines as interleukin-2 (IL-2), IFN-γ, and tumor-necrosis factor-alpha (TNF-α) (Tinkle and Newman 1997; Tinkle et al.
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Managing Health Effects of Beryllium Exposure 1997; Fontenot et al. 2002). Bost et al. (1994) were the first to show that alveolar macrophages from CBD patients produced increased concentrations of mRNAs for TNF-α and IL-6 but not for IL-1β, and the increase in mRNA was accompanied by an increase in TNF-α in BAL fluid. Tinkle et al. (1996) extended those observations and showed that the cytokines were released in response to beryllium stimulation and contributed to the unchecked inflammatory responses of effector macrophages and lymphocytes that are characteristic of the disease. The frequency of beryllium-specific Th1-cytokine–secreting CD4+ T cells in the blood of beryllium-exposed people may prove to be a useful biomarker in discriminating between BeS and progression to CBD (Pott et al. 2005). The release of chemokines, including macrophage inflammatory protein-1 alpha and growth-related oncogene-1, may also lead to the migration of lymphocytes to the lung and the formation of the microenvironment that contributes to the development of CBD (Hong-Geller et al. 2006). The polarized Th1-like response to beryllium results in macrophage activation, accumulation, and aggregation and in the perpetuation of granulomatous inflammation seen in CBD. Immunopathogenic Hallmarks of Chronic Beryllium Disease The immunologic mechanisms underlying the progression of BeS to CBD are not well understood. Beryllium-sensitized people demonstrate a beryllium-specific immune response and show no evidence of lung disease. In contrast, CBD is characterized by granulomatous inflammation and the accumulation of beryllium-responsive CD4+ T cells in the lungs. As mentioned above, the development of granulomatous inflammation in the lungs is associated with the accumulation of CD4+ T cells in BAL fluid. Saltini et al. (1989, 1990) showed that increased frequency of mononuclear cells (macrophages and lymphocytes) in BAL fluid was a characteristic of CBD. Most of the BAL lymphocytes were CD4+ T cells, the majority of which express markers consistent with an effector-memory T-cell (TEM-cell) phenotype (such as CD45ROhi, CD62Llo, and CCR7lo). These TEM cells recognize the beryllium antigen in a CD28-costimulation–independent fashion, unlike beryllium-reactive cells in the periphery that require CD28 costimulation (Fontenot et al. 2003). A recent report by Palmer et al. (2007) extends that analysis of phenotypic characterization of CD4+ subsets implicated in CBD by showing that expression of the CD57 marker is associated with inflammation and functional competence of the T cells in the lungs. Progression from BeS to CBD is characterized by an increase in the frequency of beryllium-specific, Th1-cytokine–secreting CD4+ T cells in the lung and granulomatous tissue. There appear to be important differences between beryllium-reactive memory CD4+ T cells found in the lungs and in the peripheral blood of CBD patients (Fontenot et al. 2003). The differences include maturational differences in the memory T-cell compartment, as indicated by CD28-costimulation dependence of the CD4+ beryllium-specific T cells in the periph-
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Managing Health Effects of Beryllium Exposure ery, and dissociation between Th1-cytokine secretion and lymphoproliferation in the periphery. Fontenot et al. (2005) compared the memory-cell phenotype of beryllium-reactive cells from CBD and BeS subjects and found that progression from sensitization to disease was associated with a differentiation of memory cells to an effector-cell phenotype (TEM). Thus, an accounting of the frequency of TEM cells in the blood of sensitized people may provide a means of monitoring disease progression. In other words, the beryllium-reactive CD4+ T cells in the lungs of CBD patients are more differentiated than those in the blood of people who have BeS. Understanding the functional differences in CD4+ T cells between the two compartments may be the key to understanding the immunopathogenesis of CBD and conversion from BeS and may lead to the development of biomarkers to identify people at greatest risk. GENETIC SUSCEPTIBILITY As noted in Chapter 3, not all people exposed to beryllium become sensitized, and not all who do become sensitized progress to develop CBD. Development of CBD appears to depend not only on the history of exposure to beryllium but on the genotype and phenotype of the person exposed. Attempts to identify the genetic components involved in susceptibility have centered primarily on the definition of CBD as a cell-mediated MHC class-II-restricted inflammatory disease. Accordingly, most studies have focused on specific genetic polymorphisms in MHC class II and proinflammatory genes, and a few others have considered the role of TCR-expression repertoires and other potential modifier genes. Human Leukocyte Antigen Class II In humans, the most gene-dense and polymorphic region of the genome is the MHC, which resides on chromosome 6p21.31. At the centromeric end of the MHC, spanning about 800 kilobases of DNA, sits the class II region (Acton 2001). It codes for HLA-DP, HLA-DQ, and HLA-DR—three heterodimeric proteins with limited tissue distribution (for example, to macrophages, monocytes, dendritic cells, and B lymphocytes) that are involved in antigen presentation and processing. The notion of a role of these genes in CBD arose from experiments that used lymphocytes derived from blood and BAL fluid of patients with the disease. Several studies demonstrated that antibodies directed against class II molecules blocked proliferation of lymphocytes in response to beryllium stimulation. The studies led to the idea that some HLA class II molecules may bind to beryllium and present it to T cells. Each class II molecule consists of an α chain and a β chain, and the α1 and β1 domains of these chains form the peptide-binding domain of each molecule. Genes coding for those domains, which can be highly polymorphic, have been attractive candidates in genetic-association studies of CBD. Functional studies have also been used to study
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Managing Health Effects of Beryllium Exposure whether identified polymorphisms will result in differences in binding affinity and specificity for beryllium. HLA-DP In the HLA-DP heterodimer, the β chain displays far more polymorphism than the α chain. Some 23 alleles of HLA-DPα1 and 126 alleles of HLA-DPβ1 have been described as of April 2007 (EBI 2007). In a seminal study, Richeldi et al. (1993) first demonstrated the role of variants in the HLA-DPβ1 domain in CBD. That remains the best-studied and strongest genetic association in this disease. They identified 33 CBD patients defined by a history of occupational exposure, x-ray abnormalities, abnormal lung function, presence of granulomas, and a positive BeLPT result. The patients had a higher frequency of the HLA-DPB*0201 allele than 44 similarly exposed workers who had no manifestations of CBD (52% vs 18%) and a lower frequency of the DPB*0401 allele (27% vs 68%). The two alleles differ at position 69, where HLA-DPB*0201 has the amino acid glutamic acid instead of lysine. Further analysis showed that when all the alleles were considered, this Glu69 single-nucleotide polymorphism (GAG instead of AAG) was expressed in 97% of the CBD patients examined and 30% of the controls. HLA-DPB1 Glu69 appeared to be a definitive marker of susceptibility to beryllium disease. Later studies, many by the same group, have reaffirmed the predominant role of the Glu69 variant in CBD but have suggested that its frequency is lower than originally thought (see Table 4-1). For example, Saltini et al. (2001) found HLA-DPB1 Glu69 to be present in only 73% of 22 cases studied. Given the relatively small samples involved in the studies, such a discrepancy is to be expected. HLA-DP1 Glu69 and Sensitization The original Richeldi et al. (1993) study left open the question of whether HLA-DP1 Glu69 was a marker of an immune response to beryllium—specifically, recognition and presentation—or simply a marker of disease susceptibility. Several studies have now evaluated HLA-DP1 Glu69 in BeS rather than in CBD itself. Wang et al. (2001) found the Glu69 substitution in 22 (88%) of 25 BeS people but in only 61 (37%) of 163 nonsensitized people. One study reported a much lower frequency of Glu69 in BeS subjects than in CBD subjects (Saltini et al. 2001), but other, larger studies have confirmed the initial finding and have shown Glu69 frequency to be similar in people with BeS and those with CBD (Rossman et al. 2002; Maier et al. 2003b; McCanlies et al. 2004). HLA-DPB1 Glu69 is present in up to 48% of beryllium-exposed people who do not have CBD (McCanlies et al. 2003). Given the low frequency of the disease, that implies that most people with the Glu69 substitution do not develop
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Managing Health Effects of Beryllium Exposure TABLE 4-1 Summary of Association Studies on HLA-DPB1 Glu69 and TNF-α as Susceptibility Factors in Chronic Beryllium Disease and Beryllium Sensitization Reference Subjects Number Subjects Frequency Homozygosity Alleles HLA-DPB1 Glu69 Glu69 Richeldi et al. 1993 CBD 33 97% — 0201: 52% Controls 44 30% — 18% Richeldi et al. 1997 CBD 6 83% — — Controls 121 30% — — Wang et al. 1999 CBD 20 95% 30% 0201: 42% Non-0201: 80% Controls 34 45% 1.3% 68% Saltini et al. 2001 CBD 22 73% — 0201: 36% Non-0201: 41% BeS 23 39% — 22% 17% Controls 93 40% — 29% 11% Wang et al. 2001 BeS 25 88% 24% 0201: 44% Non-0201: 52% Controls 163 38% 3% 25% 13% Rossman et al. 2002 CBD 25 84% Not associated with CBD Non-0201: associated with CBD vs controls BeS 30 90% Controls 82 47% Data not shown — Maier et al. 2003b CBD 104 86% 26% 0201: 39% Non-0201: 63% BeS 50 85% 15% 40% 56% Controls 125 38% 1.7% 24% 14% McCanlies et al. 2004 CBD 90 82% 21% — BeS 64 68% 16% — Controls 727 33% 4% —
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Managing Health Effects of Beryllium Exposure Reference Subjects Number Subjects Frequency Homozygosity Alleles TNF-α-308 Other Polymorphisms Saltini et al. 2001 BeS, CBD 45 51% — — Controls 93 16% — — Dotti et al. 2004 BeS, CBD 73 27% — TNF-α-1031, -863, -238; all not associated vs controls; TNF-α-857T increased in CBD Controls 43 5.8% — Gaede et al. 2005 Europe, Israel CBD 13 15% 0% — Controls 216 34% 4.6% — United States CBD 39 44% 13% — Controls 67 16% 1.5% — McCanlies et al. 2007 CBD 91 29% 2.2% TNF-α-238: 8.9% BeS 63 38% 6.4% 13% Controls 722 28% 2.6% 12% Sato et al. 2007 CBD 147 30% 0% TNF-α-1031, -863, -857, -238; all not associated vs controls BeS 112 36% 2.5% Controls 323 30% 2.3% Abbreviations: BeS, beryllium sensitization; CBD, chronic beryllium disease.
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Managing Health Effects of Beryllium Exposure the disease. Their failure to get CBD may be due in part to undocumented differences in workplace exposure to beryllium, coexposure to other environmental factors, or an inability to identify people in the early stages of the disease. Alternatively, other genetic considerations may be important. Using allele-specific DNA sequencing, Wang et al. (1999) showed that the specific allele carrying the Glu69 might be important. The most common HLA-DPB1 Glu69 allele is *0201; however, in a comparison of 20 people who had CBD and 75 controls, the strongest association with CBD was found with the rarer non-*0201 Glu69 alleles. Furthermore, the specific alleles for the α chain (HLA-DPA1) in the HLA-DPB1 Glu69 carriers were associated with disease development. The disparity in the importance of HLA-DPB1*0201 between this study and that of Richeldi et al. (1993) was attributed to the small number of probes and the less sensitive technique (partial regional-group–specific hybridization) used in the earlier study. Wang et al. (2001) studied the role of the alleles in BeS in a followup study of the same 20 CBD patients, 25 patients with positive BeLPT results but without CBD, and 163 BeLPT-negative controls. The frequency of the rare non-*0201 Glu69 alleles was higher in BeS subjects (52%) than in controls (13%), and it appeared lower in BeS subjects than in CBD patients although this was not statistically significant. In particular, HLA-DPB1*1701 was overrepresented in CBD (30%) and BeS (16%) groups but rare in the controls (2%). Although those results are suggestive, there have been some concerns about misclassification of subjects. Studies by Rossman et al. (2002) and Maier et al. (2003b) have largely confirmed that the HLA-DPB1 non-*0201 Glu69 allele is more prevalent than HLA-DPB1*0201 in both CBD and BeS. One study found no differences between BeS subjects and controls (Saltini et al. 2001), but it had a smaller study population and this smaller group was included in other genetic studies. Using computational chemistry and molecular modeling, Weston et al. (2005) studied the HLA-DPB1 gene variants that were shown to code for Glu69. They assigned odds ratios for specific alleles on the basis of the studies cited above and found a strong correlation between the reported hierarchic order of risk of CBD and the predicted surface electrostatic potential and charge of the corresponding isotypes. They concluded that alleles associated with the most negatively charged proteins carry the greatest risk of BeS and CBD. Another unresolved issue is whether copy number affects sensitization and disease. In the studies by Wang et al. (1999, 2001), HLA-DPB1 Glu69 homozygotes were seen only at very low frequencies in the control groups (1.3-3%) but at 24% and 30% in the BeS and CBD groups, respectively. Maier et al. (2003b) showed a similar frequency (26%) in CBD patients and concluded that Glu69 homozygosity conferred the greatest risk for CBD; however, they did not find that it was a risk factor for BeS. That led to the conclusion that Glu69 homozygosity may be important in disease progression. McCanlies et al. (2004), in a study of 884 beryllium workers (including 90 with CBD and 64 with BeS), also found increased HLA-DPB1 Glu69 homozygosity in those with CBD (21%) or BeS (16%). However, they argued, on the grounds that the HLA-DPB1 Glu69
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Managing Health Effects of Beryllium Exposure genotypic distribution did not conform to Hardy-Weinberg population laws in CBD cases but did in BeS cases and controls, that it is the presence of those alleles rather than homozygosity itself that confers risk. The mechanism by which homozygosity would enhance an immune response is unclear. The issue is complicated by the finding that expression of HLA-DP Glu69 in the BeLPT determines higher T-cell proliferation rates but that homozygotes do not show greater proliferation than heterozygotes (Amicosante et al. 2005). Gene-Environment Interaction In a cross-sectional study of 127 workers, Richeldi et al. (1997) found that CBD was 8 times more likely in machinists (who have the greater exposure to beryllium) with HLA-DPB1 Glu69 than in those without this variant and was 7 times more prevalent than in nonmachinists with HLA-DPB1 Glu69. Those results suggest a potent additive gene-environment interaction, but the number of cases was very small (six), and this issue has yet to be addressed adequately in a larger setting. HLA-DQ and HLA-DR The original report that identified the importance of HLA-DPB1 Glu69 in CBD found no relationship between CBD and HLA-DR or HLA-DQ (Richeldi et al. 1993). However, because many CBD patients (3-27%) do not have Glu69, other MHC class II molecules have been investigated. The huge number of alleles involved, the small populations studied, and the relative lack of appropriate tools have limited the studies, and their results have been equivocal. The most consistent finding has been an increased frequency of HLA-DR13 alleles in those lacking HLA-DPB1 Glu69 (Rossman et al. 2002; Maier et al. 2003b; Amicosante et al. 2005). Support for this association comes from the finding that those with the alleles have a glutamic acid at position 71 of the β chain, which corresponds to Glu69 of HLA-DP. Functional experiments show that this Glu71 is essential for beryllium presentation by HLA-DR to CD4+ T cells (Bill et al. 2005). Associations between HLA-DQ markers and BeS or CBD in people lacking HLA-DPB1 Glu69 have been reported, but they have been attributed primarily to linkage disequilibrium with HLA-DR (Amicosante et al. 2005; Maier et al. 2003b). Tumor-Necrosis Factor-α The gene for TNF-α is telomeric to the class II loci. This proinflammatory cytokine is thought to play a key role in CBD. High TNF-α concentrations have been associated with more severe pulmonary disease in CBD. In addition, beryllium stimulation of CD4+ T cells from the BAL fluid of CBD patients, but not
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Managing Health Effects of Beryllium Exposure BeS or sarcoidosis patients, will potentially induce TNF-α production (Sawyer et al. 2004b). The process appears to be transcription-dependent in that beryllium exposure specifically upregulates the AP-1 and NF-κB transcription factors (Sawyer et al. 2007). Accordingly, several studies have evaluated functional polymorphisms in the promoter of the TNF-α gene and their role in BeS and CBD. The most commonly studied is the polymorphism with a G to A transition at the 308 position, which has been shown by many to be associated with increased TNF-α production and disease severity in a variety of conditions. In a small study, Maier et al. (2001) confirmed that this polymorphism was also associated with increased beryllium-stimulated BAL-cell TNF-α production by studying CBD patients who had been classified as high (20 cases) or low (10 cases) TNF-α producers. Saltini et al. (2001) saw associations between the TNF-308A polymorphism and both BeS and CBD in a population of 639 workers. In a followup study of the same cohort, Dotti et al. (2004) extended the results and reported that TNF-308A alleles were more prominent in the 73 subjects with either BeS or CBD (26.7%) than in the 43 controls (5.8%). Moreover, a similar association was observed for another polymorphism, TNF-857T. Jonth et al. (2007) studied polymorphisms in the transforming growth factor β1 (TGF-β1) gene and found no significant differences in TGF-β1 variants or haplotypes between CBD patients and controls. Within the CBD group, however, the TGF-β1 variants were found to be associated with a more pronounced decline in lung-function and gas-exchange measures. TGF-β polymorphisms have been associated with lower TGF-β production in other models, so those variants may be mechanistically linked to the immune dysregulation underlying CBD. Gaede et al. (2005) suggested that genetic background might also play a role in the importance of the TNF-308 allele. They reported that the high TNF-α–producing variant was present at increased frequency in CBD patients in the United States but not in those in Europe and Israel, but it is likely that the two groups had different beryllium exposure and disease severity. Recent large-scale studies have cast doubt on earlier findings of the importance of polymorphisms in CBD. McCanlies et al. (2007) found no relationship between CBD and either TNF-308 or TNF-238 in a large population-based study (886 beryllium workers, including 92 with CBD and 64 with BeS). Furthermore, contrary to previous reports by one group (Saltini et al. 2001; Amicosante et al. 2001; Rogliani et al. 2004), no interaction between HLA-DP1 Glu69 and either allele could be seen. Similarly, in probably the most thorough examination of the question to date, Sato et al. (2007) compared CBD patients (147), BeS subjects (112), and healthy beryllium-exposed controls (323) and studied five TNF-promoter single-nucleotide polymorphisms (including all those studied previously) and six relevant haplotypes. They reported that although some alleles and haplotypes might be associated with constitutive and beryllium-stimulated BAL-cell production, they were not risk factors for either CBD or BeS. The discrepancies between past studies showing associations and the more recent studies may be due to misclassification, exposure dif-
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Managing Health Effects of Beryllium Exposure ferences, linkage disequilibrium between HLA-DRB1 and genes, or statistical power. Other Modifier Genes Despite the assumption that CBD is a multigenetic disease, few genes outside the MHC loci have been carefully studied. Maier et al. (1999) studied polymorphisms in the gene for angiotensin-1–converting enzyme (ACE), a vasodilatory proinflammatory peptide, because of the observation that serum ACE activity is associated with CBD severity (Newman et al. 1992). They did not find any differences in ACE genotype between CBD patients and controls, nor did they find any statistically significant associations between ACE genotype and markers of disease severity or BeLPT results. Gaede et al. (2005) did find an association between polymorphisms in the transforming growth factor β1 (TGF-β1) gene and CBD. However, TGF-β has not been measured in serum or BAL fluid of CBD patients, so the functional relevance of the association is unknown. Bekris et al. (2006) compared 29 healthy beryllium-exposed people, 27 BeS subjects, and 30 CBD subjects and observed associations between functional polymorphisms in the gene for glutamate cysteine ligase (GCLC TNR 7/7 and GCLM-588 C/C), an enzyme involved in glutathione synthesis, and CBD but not BeS. Because CBD is characterized by a Th1 cytokine response in the lungs and increased glutathione is thought to favor a Th1 response and is observed in the lungs of CBD patients, the results are functionally plausible; but they need to be confirmed in larger studies. Recent gene-expression studies of beryllium-naive peripheral-blood mononuclear cells stimulated with beryllium have shown upregulated expression in many inflammation-related genes (Hong-Geller et al. 2006). Similar studies of CBD lung tissues will provide likely candidates. ANIMAL MODELS OF PULMONARY IMMUNOTOXICITY AND SENSITIZATION Beginning in the early 1950s, studies were conducted with beryllium to determine its chemical toxicity (see Table 4-2). Generally, the studies evaluated effects in several species given beryllium in various doses and chemical forms, via different exposure routes, and over different periods. Most studies provided evidence of beryllium-induced chemical toxicity in the lungs, such as lipid and enzyme changes indicative of lung damage and nonspecific inflammation (e.g., Hart et al. 1984; Sendelbach and Witschi 1987; Sendelbach et al. 1989; Finch et al. 1994). Foreign body granulomas have also been reported in some species, such as the rat (Robinson et al. 1968; Haley 1991). The studies have also generally shown that more soluble forms of beryllium are more toxic than insoluble forms (Hall et al. 1950; Schepers 1964; ATSDR 2002). Below we review the
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Managing Health Effects of Beryllium Exposure TABLE 4-2 Selected Pulmonary and Immunologic Toxicity Studies in Animals Reference Species Route Dose Findings Single Exposure Robinson et al. 1968 Dog Inhalation Be at 115 mg/m3 from BeO, BeF2, BeCl2 (single); particle size 30% > 5µm; detection limit, 1 µm Ultrastructure changes in lungs indicative of foreign-body reaction Kang et al. 1977 Rabbit Intradermal 10 mg BeSO4 (single) Sensitization, skin granulomas Barna et al. 1981 Guinea pig Endotracheal 10 mg BeO (single); mean particle size, 5 µm Granulomas, interstitial infiltrate with fibrosis with thickening of alveolar septae Barna et al. 1984 Guinea pig Endotracheal 5 mg BeO (single) Granulomatous lesions in strain 2 but not strain 13 guinea pigs Hart et al. 1984 Rat Inhalation Be at 500 ± 4.1 ng from BeO (single, lung burden; particle size, 90% with mean diameter ≤ 1 µm Lipids and enzymes increased in BAL fluid Sendelbach and Witschi 1987 Rat Inhalation Be at 3.3 or 7.0 µg/L from BeSO4 (single); particle size, 1.95 µm, GSD = 1.38, at 3.3 µg/L; 2.0 µm, GSD = 1.85, at 7.0 µg/L Enzyme changes in BAL indicative of lung damage Mouse Inhalation Be at 7.2 µg/L from BeSO4 (single); particle size, 1.85 µm, GSD =1.59 Votto et al. 1987 Rat Intratracheal 2.4 mg BeSO4 (after subcutaneous immunization with 8 mg/mL BeSO4 at 2-wk intervals) Granulomas, T-cell subsets in BAL and lung tissue not correlated Haley et al. 1989 Dog Inhalation 17 and 50 µg/kg BeO (single, lung burden) Granulomas, evidence of immune response, low-fired beryllium more toxic Sendelbach et al. 1989 Rat Inhalation Be at 4.05 µg/L from BeSO4 (single); particle size, 1.9 µm, GSD = 1.89 Focal interstitial pneumonitis Haley et al. 1990 Rat Inhalation 800 µg/m3 (single; initial lung burden, 625 µg) Acute pneumonitis Haley et al. 1992 Dog Inhalation 17 and 50 µg/kg BeO (two doses, lung burden) Granulomatous pneumonia with focal septal fibrosis
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Managing Health Effects of Beryllium Exposure Reference Species Route Dose Findings Huang et al. 1992 Mouse Intratracheal BeSO4: immunization with 5 µg Be (3 doses), challenge with 1-5 µg Be BeSO4: immunization with 5-50 µg Be (3 times biweekly), challenge with 1-5 µg Be BeO: 4, 20, and 100 µg Granulomas produced in A/J strain but not BALB/c or C57BL/6 Finch et al. 1994 Rat Inhalation 0.32, 1.8, 10, and 100 µg Be (single, lung burdens) Acute pulmonary toxicity, progression of acute to chronic toxic responses Haley et al. 1994 Monkey Intrabroncheal 1, 50, and 150 µg Be 2.5, 12.5, and 37.5 µg BeO; particle size, for BeO, 1.6 µm; GSD = 1.9; for Be, 1.4 µm; GSD = 1.4 Pulmonary toxicity differed between chemical form of beryllium (oxide less toxic than metal) Nikula et al. 1997 Mouse Inhalation 62 ± 18 µg Be (lung burden) for C3H/HeJ strain (normalization for body weight, 3.6 ± 0.5 µg/g Be); 49 ± 16 µg Be (lung burden) for A/J strain (normalization for body weight, 3.2 ± 1.3 µg/g; particle size, 1.4 µm, GSD = 1.9 Pneumonitis Benson et al. 2000 Mouse Intratracheal 12.5, 25,and 100 µg BeCu; 2 and 8 µg Be (single) Acute lung toxicity associated with alloy exposure by not metal exposure Tinkle et al. 2003 Mouse Dermal 25 µL of 0.5 M BeSO4 70 µg BeO in petrolatum Lung microgranulomas; some resolved Repeated Exposure Hall et al. 1950 Cat, dog, guinea pig, rabbit, rat, monkey Inhalation 10-88 mg/m3 BeO powders (6 h/d, 5 d/wk, for 56-360 h total) BeSO4•4H2O fired at 1,350°C (median particle size, 5 µm) BeSO4•4H2O fired at 1,150°C (median particle size, 12.7 µm) BeO•2H2O fired at 1,150°C (median particle size, 0.45 µm) Hematologic and pulmonary toxicity observed in all species. Rats had highest acute toxicity. Be4O(C2H3O2)6 at 400°C was the most toxic, authors suggest likely due to small particle size and more extensive distribution.
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Managing Health Effects of Beryllium Exposure Be4O(C2H3O2)6 at 400°C (median particle size, 0.38 µm) Scheppers 1964 Monkey Inhalation 27µg/ft3 BeF2 (5.2 µg/ft3 Be) 66 µg/ft3 BeSO4 (5.6 µg/ft3 Be) 66 µg/ft3 BeHPO4 (5.6 µg/ft3 Be) 66, 373 µg/ft3 and 2.75 mg/ft3 BeHPO4 BeF2 more toxic than BeSO4 and BeHPO4. Marx and Burrell 1973 Guinea pig Intraperitoneal, intradermal 2.6 mg BeSO4 (1 injection/wk for 2 wk); 10 µg BeSO4 (12 biweekly injections) Sensitization Kang et al. 1977 Rabbit Intradermal 1.4 mg BeSO4 (cumulative dose, administered 2 time/wk for 6 wk) Sensitization Eskenasy 1979 Rabbit Intramuscular 10 mg/mL BeSO4 (5 injections at 7-d intervals) Sensitization, berylliosis Goel et al. 1980 Rat Oral 20 mg Be(N03)2 (40 doses over 2.5 mos) Pulmonary toxicity (histopathology and enzymology of the lungs) Freundt and Ibrahim 1990 Rat Oral 100 ppm BeSO4 for 91 d (drinking water) Increased body weight gain
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Managing Health Effects of Beryllium Exposure animal studies that have attempted to replicate the immunologic and pathologic features of human CBD. As it became apparent that the immune system plays an important role in CBD, numerous attempts were made to develop animal models representative of human CBD (e.g., see review by Finch et al. 1996; Mroz et al. 2001). The studies were conducted with well-characterized laboratory-produced aerosols so that the importance of the chemical form and particle size of the beryllium aerosols could be evaluated. Haley et al. (1989) evaluated the effects of inhaled beryllium oxide calcined at 500°C or 1,000°C in beagles. The beryllium was inhaled only once, but there was prolonged retention in the lungs because of the relative insolubility of beryllium oxide. Peribronchiolar and perivascular changes in the lungs that progressed to granulomatous pneumonia were observed. The changes were more severe in dogs exposed to the 500°C beryllium oxide than in dogs exposed to the 1,000°C beryllium oxide. Higher calcination temperatures generally decrease the solubility and toxicity of beryllium and probably contributed to the difference in response. Peripheral blood lymphocytes responded to beryllium challenge in vitro, but positive proliferation results for lung lymphocytes were observed only in samples taken from dogs with high lung burdens of 500°C beryllium oxide. The granulomatous lung response was thought to be similar to that observed in humans with CBD, but the granulomas appeared to resolve within a year after the single treatment. In a followup to that study, Haley et al. (1992) exposed the same dogs to the same forms of beryllium oxide (single inhalation exposure) 2.5 years after the first exposure and achieved lung burdens of 17 and 50 µg/kg. Lung histology showed perivascular and interstitial lymphocytic infiltrates with progression to patchy granulomatous changes and focal septal fibrosis. Beryllium-induced proliferation of blood and lung lymphocytes was also demonstrated. Beryllium cleared from the dogs according to a two- component negative exponential function. The first component had a half-life of 44 days, and the second a half-life greater than 1,000 days. The skeleton and the lungs each contained about half the beryllium in the body at the time of sacrifice (210 days after exposure). The authors concluded that “[beryllium]-induced granulomatous and fibrotic lung lesions are accompanied by [beryllium]-specific immune responses within the lung” (p. 400). However, there was no correlation between the presence or severity of lesions and previous exposure or pulmonary immune response to beryllium oxide. Also, the lesions were not cumulative with sufficient latency time between the exposures. Haley et al. (1994) conducted studies in cynomolgus monkeys given beryllium oxide (calcined at 500°C) or beryllium metal by bronchoscopic instillation. Lymphocytes were increased in the BAL fluid after 14, 30, or 90 days in monkeys treated with beryllium metal and after 60 days in monkeys treated with beryllium oxide. BAL lymphocytes from monkeys exposed to beryllium metal, but not monkeys exposed to beryllium oxide, responded positively in the BeLPT. The lungs of monkeys treated with beryllium metal showed inflammation, interstitial fibrosis, and type II cell hyperplasia; some also had discrete
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Managing Health Effects of Beryllium Exposure granulomas. Smaller and fewer lesions were found in monkeys treated with beryllium oxide. Whether the lesions resolved over time as in other animal models was not reported. In a review of the work described above, Finch et al. (1996) concluded that although dogs and monkeys respond to beryllium by developing granulomatous lung lesions with a substantial lymphocytic component and their lymphocytes exhibited a beryllium-specific proliferative response in vitro, the lesions failed to progress. Thus, neither species was a good model of the progressive CBD seen in people. Studies in guinea pigs exposed to beryllium oxide have also produced granulomatous lung disease and evidence of immune sensitization to beryllium. Barna et al. (1981, 1984) administered intratracheal injections of 5-10 mg of beryllium oxide to outbred Hartley guinea pigs and two inbred strains of guinea pigs (strains 2 and 13). Six weeks after exposure, the Hartley and strain 2 guinea pigs, but not strain 13, developed granulomatous lung changes typical of human CBD, with numerous granulomas, mononuclear interstitial infiltrates, and fibrosis. The Hartley and strain 2 guinea pigs also showed evidence of beryllium sensitization, with positive delayed-type hypersensitivity skin tests and in vitro proliferation of lymphocytes in response to beryllium sulfate. Intravenous or oral exposure to beryllium sulfate induced tolerance to the intratracheally administered beryllium oxide. The lung granulomas and beryllium sensitization were also mitigated by treatment with prednisone, L-asparaginase, or cytoxan. The authors concluded that the inbred strain differences in susceptibility to beryllium implicated genetically determined immune mechanisms similar to human CBD. The lung granulomas in strain 2 guinea pigs appeared to be resolving 1.5 years after exposure even though lung tissues still contained beryllium (Barna et al. 1984). Different mice strains have also been used in attempts to develop an animal model that mimics human CBD. For example, Nikula et al. (1997) demonstrated chronic granulomatous pneumonia and lymphocytic responses induced in A/J and C3H/HeJ mice by a single inhalation of beryllium metal at 1,030 mg/m3 for 90 min. Granulomas with increased numbers of CD4+ cells, epithelial hyperplasia, and inflammatory cells were detected in the lungs of both strains of mice at 28 weeks, but beryllium-specific lymphocytic proliferation could not be demonstrated. The authors suggested that this response in mice was associated with T-cell delayed hypersensitivity and not a foreign body reaction as observed in rats. Finch et al. (1998a) exposed C3H/HeJ mice to beryllium-metal aerosols in a single exposure to achieve lung burdens of 1.7-34 µg. Particle clearance was impaired at lung burdens of 12 and 34 µg through day 196. Increased numbers of inflammatory cells were detected in the BAL fluid of mice with the two highest lung burdens. Granulomatous changes were seen histologically beginning on day 8 in mice with lung burdens of 12 and 34 µg and on day 15 in mice with 2.6 µg. Beryllium sensitization and persistence of the granulomas were not observed.
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Managing Health Effects of Beryllium Exposure Huang et al. (1992) investigated beryllium-induced pulmonary granuloma with different mice strains and different immunization and challenge protocols. Granulomas and beryllium sensitization were found only in A/J mice treated with beryllium sulfate, and the granulomas regressed within 20 weeks. Similar experiments with beryllium sulfate in BALB/c and C57BL/6 mice did not produce any lung granulomas, nor were granulomas induced in mice treated with a single intratracheal instillation of beryllium oxide. In general, A/J mice appear more prone to develop granulomas in response to beryllium than BALB/c, C57BL/6, or other mice strains. Mice have also been sensitized to beryllium by intradermal and dermal exposures (Huang et al. 1992; Tinkle et al. 2003). Tinkle et al. (2003) sensitized C57L/6 mice to beryllium sulfate and beryllium oxide by using skin exposure followed by a single intratracheal instillation or nose-only inhalation of beryllium. Ear swelling was used as an indicator of a delayed-type hypersensitivity reaction. Some mice developed lung microgranulomas, but they resolved spontaneously. Unlike dogs, mice, and nonhuman primares, rats similarly exposed to beryllium do not appear to develop an immune granulomatous response. Haley et al. (1990) studied the toxicity of beryllium metal after a single nose-only exposure of F344 rats at 800 µg/m3 for 50 min. Rats developed acute, necrotizing, hemorrhagic, exudative pneumonitis and intra-alveolar fibrosis that peaked on day 14. The authors concluded that human CBD is “an immunologically mediated granulomatous lung disease, whereas beryllium-induced lung lesions in rats appear to be due to direct chemical toxicity and foreign-body-type reactions” (p. 767). Finch et al. (1994) studied the acute and chronic effects of beryllium metal administered nose-only to F344 rats to achieve lung burdens of 0.32-100 µg. Sacrifices were performed periodically for up to a year after exposure. The lowest lung burden of beryllium that induced pulmonary toxicity was 1.8 µg. At burdens of 10 and 100 µg, particle clearance from the lung was reduced, and pulmonary inflammation was observed, but typical immune-mediated lung granulomas were not observed. Lung lesions (fibrosis, chronic inflammation, and epithelial hyperplasia) were evident in the 100-µg group after 8 days of exposure. BAL fluid from rats with histologic alterations had nonspecific increases in neutrophils and proteins. Rats were affected at lung burdens below doses that affected mice. Few studies have investigated beryllium-metal alloys. Benson et al. (2000) performed biodistribution studies in C3H/HeJ mice given particles of beryllium-metal or beryllium-copper alloy (2% beryllium and 98% copper) intratracheally. The alloy was given at 12.5, 25, or 100 µg, and beryllium metal was given at 2 or 8 µg. That acute lung toxicity and death were associated with the alloy but not with the metallic beryllium powder suggested copper toxicity. Pulmonary clearance of beryllium was found to be much slower than clearance of copper. Zissu et al. (1996) found that beryllium-copper alloys were more potent sensitizers of outbred guinea pigs than were soluble beryllium salts after intradermal injection.
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Managing Health Effects of Beryllium Exposure CONCLUSIONS Although animal studies in different species have demonstrated immunologic responses or pathologic changes similar to human CBD, animal models developed to date have varied between species, and none has adequately replicated the key features of human CBD, especially the persistence of granulomatous changes in the lung. The reason for the differences remains unclear, but the specific genes responsible for CBD in humans might not be present in animals. Thus, animal studies cannot reliably predict exposure-response relationships, immunogenicity of different forms of beryllium, or mechanisms relevant to human CBD. Generally, more soluble forms of beryllium, such as beryllium salts as opposed to beryllium metal, have shorter half-lives in the lung and a greater potential for systemic absorption and sensitization; relatively insoluble forms of inhaled beryllium (beryllium metal and beryllium oxide) deposited in the lungs will be retained with much longer half-lives and may lead to sensitivity reactions or toxicity even after a single exposure. No animal model has characteristics of human CBD that are sufficient for it to be used to establish the relative importance of the various chemical forms (including alloys) or physical characteristics of inhaled beryllium encountered in the workplace. The mechanism underlying CBD pathogenesis involves an antigenic immune response to beryllium associated with beryllium-specific CD4+ T cells. Progress has made in characterizing the immune response to beryllium and host risk factors. More research is needed on the nature of the beryllium antigen recognized by the CD4+ T cells. Research has also indicated that an allele of HLA-DPβGlu69 is the most important marker of susceptibility to CBD. However, the presence of that marker alone does not necessarily confer susceptibility, nor is its absence a guarantee of nonsusceptibility. T-cell-receptor expression, inflammation-related genes, and other potential modifier genes also appear to play roles in disease progression and warrant further investigation. Efforts are under way to create humanized-mouse models in which human alleles are associated with a range of BeS and CBD risk that might be useful in experimental study of beryllium dose-response relationships, of beryllium types and characteristics that confer risk, and of therapeutic approaches to beryllium diseases. It is unclear whether expression of single target genes associated with sensitization will recapitulate the signs and symptoms of human CBD. There is no doubt that an animal model of immune-mediated CBD as it occurs in humans would aid substantially in developing an understanding of the mechanisms of CBD and the relative importance of the various forms of beryllium encountered in the workplace in the causation of CBD.