Elsevier

Toxicology

Volume 165, Issue 1, 13 August 2001, Pages 27-38
Toxicology

Beryllium sensitivity is linked to HLA-DP genotype

https://doi.org/10.1016/S0300-483X(01)00410-3Get rights and content

Abstract

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.

Introduction

Beryllium, due to its unique chemical and physical properties, is being used increasingly in almost every modern industry. However, inhalation of Be-containing vapor mist or dust can cause two types of toxicity; an acute inflammation response and a chronic immune response (Sterner and Eisenbud, 1951). The latter is the basis for chronic beryllium disease (CBD), which was first described in 1946 (Hardy and Tabershaw, 1946). CBD is a lung granulomatous disease characterized by a Type IV, delayed hypersensitivity, cell-mediated immunity (Newman, 1995). Some occupations that have higher risk for greater exposure to beryllium also have a higher incidence of CBD, providing evidence that the amount of beryllium exposure is related to the development of disease (Kreiss et al., 1993). However, not all beryllium workers exposed to higher doses of beryllium develop CBD; and workers with apparently minimal beryllium exposure history also can develop CBD (Eisenbud, 1998). Overall, CBD affects 1–5% of all beryllium workers (Kreiss et al., 1994).

The mechanism by which CBD develops is currently thought to be that beryllium mediates the binding of an antigenic peptide (endogenous or exogenous) to the human leukocyte antigen (HLA)-DP heterodimers formed on the cell surface of antigen-presenting cells (macrophages, B lymphocytes, and other cells) by the products of the HLA-DPA1 and HLA-DPB1 genes, HLA-DP α chain and HLA-DP β chain. It is then believed that this complex of HLA-DP dimers and antigenic peptide is recognized by the T Cell Receptors (TCR) on the cell surface of specific T cell clones. This antigen recognition process activates these specific T cells, which in turn results in an autoimmune response, and finally causes CBD. Recent evidence lends strong support to the hypothesis that a genetic risk factor influences the individual response to beryllium exposure. The 69th amino acid (Glu) in the mature β chain of HLA-DP gene, a member of the major histocompatibility complex (MHC) Class II gene family for immune responses in humans, has been reported to be highly associated with CBD (Richeldi et al., 1993, Wang et al., 1999). Moreover, it was found that certain rare Glu69 HLA-DPB1 alleles such as *1701, *0901, 1001, and rare HLA-DPA1 *02 alleles containing the Glu69 substitution, as well as Glu69 homozygotes, occurred with higher frequency in CBD patients compared to controls representing a normal population (Wang et al., 1999).

The lymphocytes isolated from a lung lavage or peripheral blood of CBD patients can proliferate in vitro in response to beryllium stimulation, a response that has been used to distinguish CBD from other lung granulomatous diseases such as sarcoidosis (Newman, 1995). The Beryllium (Be)-Lymphocyte Proliferation Test has been widely used in the detection of CBD (Rossman et al., 1988, Newman et al., 1989, Mroz et al., 1991); but also for the early identification of subclinical beryllium-induced disease and beryllium sensitivity in the absence of demonstrable disease (Kreiss et al., 1989, Newman et al., 1989). The beryllium sensitivity response involves the proliferation of the CD4+ T cells (Saltini et al., 1989, Farris et al., 2000), which are known to interact with HLA class II molecules (Saltini et al., 1989). Therefore, we hypothesized that the same HLA-DP variations that have been found with higher frequency in CBD patients should also be found in individuals with beryllium sensitivity, but without CBD. In this study, we used a lymphocyte proliferation test combined with immunophenotyping by flow cytometry to define a population of beryllium sensitive, but non-CBD, individuals; and examined the association between lymphocyte proliferation response to beryllium and HLA-DP genotype. If a positive response on the blood LPT is predictive of CBD, then the individuals who have a positive blood LPT will likely have HLA genotypes similar to those observed in CBD.

Section snippets

Sample collection

Blood samples from 20 CBD patients, 25 individuals characterized as beryllium-sensitive (blood LPT positive but not CBD), and 163 beryllium-exposed controls were all obtained with their informed consents. The 20 CBD samples were the same used previously (Wang et al., 1999). CBD diagnosis was based on (1) history of beryllium exposure, (2) non-caseating granulomas on lung biopsy, and (3) abnormal bronchoalveolar lavage beryllium lymphocyte proliferation test. The 25 blood LPT-positive, non-CBD

Association between Glu69 and positive blood LPT

The 69th amino acid (Glu) in the mature β chain of HLA-DP heterodimer has been reported to be highly associated with CBD (Newman, 1993, Richeldi et al., 1993, Wang et al., 1999). We compared the Glu69 frequencies in the beryllium-exposed, blood LPT negative, non-CBD individuals and the blood LPT positive, non-CBD individuals (hereafter called the ‘Be-sensitive’ group). As predicted, the Glu69 frequency in the Be-sensitive group was significantly higher than that in the LPT negative group (88%,

Discussion

The development of beryllium sensitivity is the hallmark of CBD, and the blood lymphocyte proliferation test is used to screen beryllium workers for beryllium sensitivity, which is considered an early sign of the disease (Kreiss et al., 1989, Newman et al., 1989). This is the first report to relate a specific outcome on the LPT (i.e. a CD4+ response as determined by the Immuno-LPT) with HLA-DP loci demonstrated to have a strong association with CBD. The present results obtained in a group of

Acknowledgements

This work was supported by the US Department of Energy, and the National Flow Cytometry Resource (NIH grant No.P41-RR013150), the National Institutes of Health [Grant ES-06538 (LSN), GCRC Grant M01 RR 00051 (LSN), and Grant K08 HL-03887 (LAM)], and the National Institute for Occupational Safety and Health Centers for Disease Control and Prevention, US Public Health Service [Cooperative Agreement U60 CCU 812221 (LSN)].

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