Beryllium Hypersensitivity Research Articles

MyD88 dependence of beryllium-induced dendritic cell trafficking and CD4⁺ T-cell priming.

Beryllium exposure results in beryllium hypersensitivity in a subset of exposed individuals, leading to granulomatous inflammation and fibrosis in the lung. In addition to its antigenic properties, beryllium has potent adjuvant activity that contributes to sensitization via unknown pathways. Here, we show that beryllium induces cellular death and release of IL-1α and DNA into the lung. Release of IL-1α was inflammasome-independent and required for beryllium-induced neutrophil recruitment into the lung. Beryllium enhanced classical dendritic cell (cDC) migration from the lung to draining LNs in an IL-1R-independent manner, and the accumulation of activated cDCs in the LN was associated with increased priming of CD4+ T cells. DC migration was reduced in TLR9KO mice; however, cDCs in the LNs of TLR9-deficient mice were highly activated, suggesting a role for more than one innate receptor in the effects on DCs. The adjuvant effects of beryllium on CD4+ T cell priming were similar in WT, IL-1R, caspase-1, TLR2, TLR4, TLR7, and TLR9 deficient mice. In contrast, DC migration, activation and the adjuvant effects of beryllium were significantly reduced in MyD88KO mice. Collectively, these data suggest that beryllium exposure results in the release of DAMPs that engage MyD88-dependent receptors to enhance pulmonary DC function.

Beryllium hypersensitivity and chronic beryllium lung disease

This review aims to present the clinician with a synthesis of recent studies that have enhanced our understanding of the epidemiology and pathogenesis of beryllium hypersensitivity (BeH) and chronic beryllium disease (CBD). Lower occupational limit levels to beryllium exposure and more stringent preventive measures can decrease the risk for development of BeH and CBD. Beryllium sensitization is determined by a positive beryllium lymphocyte proliferation test (BeLPT). Longitudinal data suggest that BeH progresses to CBD. Together with a comprehensive history the BeLPT may help identify berylliosis in patients erroneously diagnosed to have sarcoidosis. HLA-DPB1-Glu69 marker is associated with increased susceptibility to development of BeH and CBD but poor positive predictive value limits its use; other genetic markers are being investigated. Recent investigations augment our understanding on the role of T-lymphocytes and chemokines in the pathogenesis of beryllium-associated disease. However, the basis for treatment strategies remains scarce. Our enhanced understanding of beryllium-associated lung disease potentially provides a window to unraveling other granulomatous diseases. However, even more questions beg to be elucidated and additional efforts are needed to translate this body of knowledge into better prevention and treatment.

Diagnoses of chronic beryllium disease within cohorts of sarcoidosis patients

An increase in chronic beryllium disease (CBD) has been suggested due to higher industrial use of beryllium alloys. Since occupational CBD is a perfect phenocopy of sarcoidosis, it might be misdiagnosed as sarcoidosis. In the current it was hypothesised that CBD exists in cohorts of sarcoidosis patients. In a prospective case study, sarcoidosis patients were evaluated for potential beryllium exposure. In those patients in whom beryllium exposure was confirmed and beryllium hypersensitivity demonstrated, the diagnosis of sarcoidosis was rejected and corrected to CBD. In 84 patients seen for re-evaluation or making a diagnosis of sarcoidosis, beryllium exposure was recognised and a diagnosis of CBD was made in 34 out of 84 patients. The time lag between clinical diagnosis of sarcoidosis and the final diagnosis of CBD ranged 0-18 yrs (median 3 yrs) and the mean (range) age at time of diagnosis of CBD was 43.9(25-80) yrs. Beryllium-contaminated workplaces causing disease encompassed a wide spectrum of industries and technical trades in which beryllium-exposure is generally not perceived as a health hazard. In conclusion, chronic beryllium disease still belongs to the spectrum of differential diagnoses of granulomatous disorders.

Comparative analysis between CFSE flow cytometric and tritiated thymidine incorporation tests for beryllium sensitivity

In this study, we evaluated alternative possibility for CFSE beryllium flow cytometric test against beryllium blood lymphocyte proliferation test (BeLPT) as a standard radioactive clinical screening method to identify sensitization to beryllium. Delta PD (the ratio of divided cell population to the total number of cells with subtracted counts of unstimulated cells) of specific beryllium-induced pathogenic CD3+ CD4+ T-lymphocytes and stimulation index (SI) in CFSE proliferation test was compared with delta counts per minute (mean test CPM minus mean control CPM) and SI in radioactive blood BeLPT. Comparison analysis of CFSE and BeLPT demonstrated excellent agreement between delta PD and delta CPM (kappa = 0.845, P << 0.0001). We determined 6.8% positive subjects in the beryllium-exposed, Be-LPT-negative group. The decreased mean difference of these indexes to percentage of average and the long tail in the plot reflects increased sensitivity. CFSE/CD4+ T-cell proliferation assay has 100% specificity, significantly higher sensitivity and efficiency than BeLPT. Both delta PD, measured by the precursor frequencies method in CFSE assay and delta CPM, defined by tritiated thymidine in BeLPT, can be used for the enumeration of beryllium specific CD4+ T-cell proliferation and may substantially improve the quality of the early diagnosis of beryllium hypersensitivity.

Diagnoses of chronic beryllium disease within cohorts of sarcoidosis patients

An increase in chronic beryllium disease (CBD) has been suggested due to higher industrial use of beryllium alloys. Since occupational CBD is a perfect phenocopy of sarcoidosis, it might be misdiagnosed as sarcoidosis. In the current it was hypothesised that CBD exists in cohorts of sarcoidosis patients. In a prospective case study, sarcoidosis patients were evaluated for potential beryllium exposure. In those patients in whom beryllium exposure was confirmed and beryllium hypersensitivity demonstrated, the diagnosis of sarcoidosis was rejected and corrected to CBD. In 84 patients seen for re-evaluation or making a diagnosis of sarcoidosis, beryllium exposure was recognised and a diagnosis of CBD was made in 34 out of 84 patients. The time lag between clinical diagnosis of sarcoidosis and the final diagnosis of CBD ranged 0-18 yrs (median 3 yrs) and the mean (range) age at time of diagnosis of CBD was 43.9(25-80) yrs. Beryllium-contaminated workplaces causing disease encompassed a wide spectrum of industries and technical trades in which beryllium-exposure is generally not perceived as a health hazard. In conclusion, chronic beryllium disease still belongs to the spectrum of differential diagnoses of granulomatous disorders.

Role of the HLA-DP Glu 69 and the TNF-alpha TNF-alpha 2 gene markers in susceptibility to beryllium hypersensitivity.

Berylliosis is an environmental chronic inflammatory disorder of the lung caused by inhalation of beryllium dusts, characterized by the accumulation of CD4+ T cells and macrophages in the lower respiratory tract. Beryllium presentation to CD4+ T cells from patients with berylliosis results in T cell activation and these Be-specific CD4+ T cells undergo clonal proliferation and Th1-type cytokine production such as interleukin-2, interferon-gamma and tumor necrosis factor-alpha. In exposed workers, genetic susceptibility to this granulomatous disorder is associated with major histocompatibility gene and the TNF-alpha gene. The HLA-DP glutamic 69 residue was shown to be the MHC genetic marker associated with disease susceptibility; furthermore the TNF-alpha TNFA-308*2 allele was found to be independently associated with HLA-DP Glu69 in the determination of berylliosis risk.

Beryllium Detection in Human Lung Tissue using Electron Probe X-Ray Microanalysis

Chronic berylliosis is an uncommon disease that is caused by the inhalation of beryllium particles, dust, or fumes. The distinction between chronic berylliosis and sarcoidosis can be difficult both clinically and histologically, as both entities can have similar presentations and exhibit nonnecrotizing granulomatous inflammation of the lungs. The diagnosis of chronic berylliosis relies on a history of exposure to beryllium, roentgenographic evidence of diffuse nodular disease, and demonstration of beryllium hypersensitivity by ancillary studies, such as lymphocyte proliferation testing. Additional support may be gained by the demonstration of beryllium in lung tissue. Unlike other exogenous particulates, such as asbestos, detection of beryllium in human lung tissue is problematic. The low atomic number of beryllium usually makes it unsuitable for conventional microprobe analysis. We describe a case of chronic berylliosis in which beryllium was detected in lung tissue using atmospheric thin-window energy-dispersive X-ray analysis (ATW EDXA). A woman with a history of occupational exposure to beryllium at a nuclear weapons testing facility presented with progressive cough and dyspnea and a nodular pattern on chest roentgenograph. Open lung biopsy showed nonnecrotizing granulomatous inflammation that was histologically indistinguishable from sarcoidosis. Scanning electron microscopy and ATW EDXA demonstrated particulates containing beryllium within the granulomas. This application of EDXA offers significant advantages over existing methods of beryllium detection in that it is nondestructive, more widely available, and can be performed using routine paraffin sections.

Human leukocyte antigen Class II amino acid epitopes: susceptibility and progression markers for beryllium hypersensitivity.

Chronic beryllium disease (CBD) is a hypersensitivity granulomatosis characterized by beryllium hypersensitivity (BH) and mediated by CD4+ T cells. However, all individuals with BH may not develop CBD. To examine the role of the three different human leukocyte antigen (HLA) Class II isotypes in BH with (CBD) and without clinical disease (BHWCD), we performed DNA-based typing of HLA-DPB1, HLA-DQB1, and HLA-DRB1 loci on 55 subjects with BH (25 with established CBD and 30 with BHWCD), and compared this with the results for 82 beryllium-exposed workers with no evidence of BH. The allele distribution was utilized to identify candidate amino acid epitopes that differed between the study groups. HLA-DPB1-E69 was the most important marker for BH, and did not differentiate BHWCD from CBD. A significant association with CBD was observed with HLA-DQB1-G86 (p(corr) < 0.04), and HLA-DRB1-S11 was significantly increased in CBD as compared with BHWCD (p < 0.03). These observations suggest that HLA-DPB1-E69 is a marker for susceptibility to BH and not just a progression marker for CBD. In addition, HLA amino acid epitopes on HLA-DRB1 and -DQB1, in concert with or independently of HLA-DPB1-E69, may be associated with progression to CBD.

Beryllium sensitivity is linked to HLA-DP genotype

Chronic beryllium disease (CBD) appears to arise from a combination of both exposure and genetic risk factors. A distinguishing feature of CBD is beryllium hypersensitivity, which can be measured in vitro by a lymphocyte proliferation test. The objective of this study was to determine whether certain allelic variations of the HLA-DPB1 gene, which had been observed previously in CBD, could be found in a group of individuals having beryllium hypersensitivity, but no symptoms of CBD. A flow cytometry-based Lymphocyte Proliferation Test combined with immunophenotyping (Immuno-LPT) was used to detect CD4+ and CD8+ T cell proliferation in response to in vitro stimulation with beryllium. The HLA-DPB1 haplotypes of the same individuals were determined by automated DNA sequencing. Twenty-two out of 25 beryllium-sensitive, non-CBD individuals were found to be carriers of the HLA-DPB1 gene having a substitution of a glutamic acid at position 69 in Exon 2 (Glu69), and a significantly high percentage (24%) were Glu69 homozygotes. Most of the CD4+ responders on the Immuno-LPT (10/14) carried rare, non-*0201 Glu69 DPB1 alleles; while most of the non-CD4+ responders (9/11) were common Glu69 carriers (*0201 or *0202) or non-Glu69 individuals (non-Glu69/non-Glu69). This is the first direct evidence that HLA-DP genotype is linked to a phenotypic response that occurs in beryllium sensitization in the absence of clinical CBD.

Chronic beryllium disease: uncommon disease, less common diagnosis.

Chronic beryllium disease (CBD) is typically considered only when occupational exposure to beryllium is a certainty; however, CBD has also occurred in occupational and environmental settings where exposure was unexpected. When the etiology of a case of granulomatous pulmonary disease is not determined, sarcoidosis is the "diagnosis of exclusion." This diagnosis does not communicate much information about the patient's prognosis, the disease's etiology, or even what disease etiologies were specifically excluded. Some cases of CBD have been called sarcoidosis, allowing exposure to continue for the patient and (at times) other individuals. The granulomatous changes of sarcoidosis are thought to result from an abnormal immune response. While the etiologic agents that can initiate this response are largely unknown, the immunopathogenesis of CBD has been well described, and laboratory methods are available in a few centers that can (if used) identify beryllium hypersensitivity. The potential for exposure and disease to be widely separated in time and location makes it important for health-care and environmental health professionals to be aware of these new diagnostic methods.

Statistical methods for the blood beryllium lymphocyte proliferation test.

The blood beryllium lymphocyte proliferation test (BeLPT) is a modification of the standard lymphocyte proliferation test that is used to identify persons who may have chronic beryllium disease. A major problem in the interpretation of BeLPT test results is outlying data values among the replicate well counts (approximately 7%). A long-linear regression model is used to describe the expected well counts for each set of Be exposure conditions, and the variance of the well counts is proportional to the square of the expected count. Two outlier-resistant regression methods are used to estimate stimulation indices (SIs) and the coefficient of variation. The first approach uses least absolute values (LAV) on the log of the well counts as a method for estimation; the second approach uses a resistant regression version of maximum quasi-likelihood estimation. A major advantage of these resistant methods is that they make it unnecessary to identify and delete outliers. These two new methods for the statistical analysis of the BeLPT data and the current outlier rejection method are applied to 173 BeLPT assays. We strongly recommend the LAV method for routine analysis of the BeLPT. Outliers are important when trying to identify individuals with beryllium hypersensitivity, since these individuals typically have large positive SI values. A new method for identifying large Sls using combined data from the nonexposed group and the beryllium workers is proposed. The log(SI)s are described with a Gaussian distribution with location and scale parameters estimated using resistant methods. This approach is applied to the test data and results are compared with those obtained from the current method.

Genetic Markers in Beryllium Hypersensitivity

Chronic beryllium disease (CBD) is a pulmonary granulomatous hypersensitivity that is mediated by the proliferation liferation of CD4+ T cells. In vitro assays have shown that CD4+ T cells respond to beryllium salts in the context of class II molecules. An association between a specific motif on human leukocyte antigen (HLA)-DP (Glu-69) and beryllium hypersensitivity (BH) has been observed. Sequence specific oligonucleotide probes (SSOPs) were used to identify HLA-DRB1, -DRB3, -DRB4, and -DRB5 and HLA-DPB1 alleles in 29 persons with demonstrated BH and in individuals with beryllium exposure (BE) but no hypersensitivity.

Novel in vitro method for identification of individuals at risk for beryllium hypersensitivity.

Beryllium-specific lymphocytes were generated by in vitro immunization of peripheral blood mononuclear cells (PBMC) from healthy unexposed individuals. Measurement of blastogenic responses of PBMC by [3H]thymidine uptake demonstrated that sensitization of PBMC with beryllium salts followed by stimulation with unrelated salts resulted in a negative response, whereas sensitization and restimulation of PBMC with beryllium salts produced a positive response. Flow cytometric and cell depletion analyses showed that all of the responding cells were CD4+ T cells. The in vitro immunization system was used to screen 52 human subjects for susceptibility to beryllium sensitization in vitro. The results show that of the 52 healthy unexposed subjects tested, only 1 (2%) was highly responsive, 4 subjects (8%) were moderately responsive, 20 subjects (39%) were low-level responders, and 27 subjects (52%) were nonresponders. The results showing 2% high-level responsiveness to beryllium sensitization in vitro correlate with the 1 to 5% prevalence of chronic beryllium disease in individuals sensitized to beryllium dust in vivo and thus support the thesis that the in vitro immunization system may permit the identification of individuals at risk for beryllium hypersensitivity.

Lymphocyte beryllium binding: relationship to development of delayed beryllium hypersensitivity.

Beryllium binding to human and both inbred and outbred guinea pig lymphocytes has been characterized by Scatchard binding analysis. In isotonic media, only one class of binding sites, presumed to be located on the outer cell surface, was identified with a relatively low affinity (KA3-4 X 10(5) M-1). In hypotonic media in which the cells rupture, an additional class of binding sites, probably in the cell nucleus, is revealed with a higher affinity (KA1-2 X 10(6) M-1). Preliminary investigation of the high affinity binding in strain XIII guinea pig peripheral blood lymphocytes provides evidence that a greater level of beryllium binding may be predictive of the potential to express delayed beryllium hypersensitivity.