HLA-DP-unrestricted TNF-α release in beryllium-stimulated peripheral blood mononuclear cells

Study population

Eleven individuals with berylliosis were enrolled in the study after giving informed consent: 10 males and one female, all Caucasians, mean age 45±7 yrs and average time of employment 15±6 yrs. The study protocol was approved by the Cleveland Clinic IRB (Cleveland, OH, USA). As a control, five healthy non-Be-exposed individuals were enrolled: four males and one female, all Caucasians and mean age 32±3 yrs.

Lymphocyte proliferation

Peripheral blood mononuclear cells (PBMCs) obtained from patients with berylliosis were isolated from heparinised whole blood by density centrifugation on a Ficoll Hypaque gradient. PBMCs (2×10⁵ cells·well⁻¹) were then cultured in 96-well flat-bottomed microtitre plates in Roswell Park Memorial Institute (RPMI) 1640 tissue culture medium supplemented with 2 mM L-glutamine, 10% foetal bovine serum, 100 U·mL⁻¹ penicillin, and 100 µg·mL⁻¹ streptomycin in the presence of beryllium sulfate (BeSO₄[4H₂O]) at 10, 50 and 100 µM (all reagents form Sigma Co., St. Louis, MO, USA). Phytohaemoagglutinin (PHA; 5 µg·mL⁻¹; Sigma) and Candida albicans (10 µg·mL⁻¹) were used as positive controls. T-lymphocyte proliferation was measured by thrytiated deossiribonucleotide thymidine ([³H]TdR) incorporation. After 5 days of culture, cells were pulsed with 1 µCi of [³H]TdR (Amersham International, Amersham, UK) and harvested onto glass-fibre filters 18 h later. Proliferation was measured as [³H]-TdR incorporation by liquid scintillation spectroscopy. The results are expressed as the mean of triplicate cultures. The stimulation index was calculated as the ratio of mean counts per minute (cpm), in antigen-stimulated wells, to the mean cpm in control wells.

In order to evaluate possible contamination with the bacterial endotoxin product of the BeSO₄ solution used as antigen, the BeSO₄ batch solution used throughout the study was tested with the Limulus Amebocyte Lysate test (CAPE CODE Inc., Woods Hole, MA, USA). The stock solution of BeSO₄ (0.2 M) showed undetectable levels of endotoxin contamination (<0.03 U·mL⁻¹ of Escherichia coli endotoxin, similar to endotoxin-free water). Similarly, undetectable endotoxin levels were revealed for BeSO₄ at the working concentration in RPMI complete medium.

Measurement of cytokines in supernatants

The levels of IFN-γ, TNF-α, RANTES (regulated on activation normal T-cell expressed and secreted), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-4, IL-6, IL-8, IL-10 and IL-12 release in the culture supernatants of Be-stimulated PBMCs were evaluated with commercially available solid-phase, two-site enzyme-linked immunosorbent assays (CytElisa; CytImmune Science, San Diego, CA, USA). Cell supernatants were collected after 3 days (RANTES, GM-CSF, IL-4 and IL-12) and 5 days (IFN-γ, TNF-α, IL-6, IL-8 and IL-10) of PBMC culture before [³H]TdR incorporation, and frozen at −80°C until use. Cytokine concentrations were evaluated in triplicate and the results expressed as the mean of triplicate cultures.

Monoclonal antibody inhibition of lymphocyte activation

Protein-A sepharose purified mAb directed against HLA-DR (L243) 10, HLA-DP (B7/21) 10, HLA-DQ (L2) 10, HLA-class I (W6/32) 10 and the 19 kDa Mycobacterium tuberculosis protein (HYT6) 15 were used at increasing concentrations (10, 20 and 50 µg·mL⁻¹) to inhibit antigen presentation, lymphocyte proliferation and cytokine production as previously described 5.

HLA typing

HLA-DP and TNFA1/A2 typing was carried out as previously described 13.

Statistical analysis

All the data were expressed as mean±sd. Comparisons between groups were made with t-tests. Comparisons between frequencies were made using the Chi-squared test with the Yates and Bonferroni corrections where necessary.

HLA-DP restriction of beryllium-stimulated lymphocyte proliferation

All PBMCs obtained from berylliosis patients (n=11), carrying glutamate-69-positive HLA-DP alleles, showed positive responses to PHA and the recall antigen C. albicans (table 1⇓). Of these, nine had a proliferative response to BeSO₄ that was >3 times the mean SI of unstimulated cells (table 1⇓). In all cells the anti-HLA-DP specific mAb induced a marked inhibition of Be-stimulated proliferation (88±16%) that was significantly greater than that obtained with mAbs against HLA-DR (29±38%), HLA-DQ (2±2%), class-I major histocompatability complex (MHC; 1±2%), and the M. tuberculosis 19 kDa-specific protein (0±1%; p<0.05 for all comparisons with anti-HLA-DP; fig. 1⇓).

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Fig. 1.—

Inhibition of beryllium-induced a) proliferation, b) interferon-γ and c) tumour necrosis factor-α release by monoclonal antibodies directed against human leucocyte antigen (HLA)-DR, HLA-DP, HLA-DQ, HLA-class I and the 19 kDa Mycobacterium tuberculosis protein in peripheral blood mononuclear cells from berylliosis patients. DR: anti-HLA-DR; DP: anti-HLA-DP; DQ: anti-HLA-DQ; CI: anti-HLA-class I; Mtb: anti-19 kDa M. tuberculosis.

Finally, PBMCs from Be-non-exposed healthy subjects did not proliferate in response to BeSO₄ (data not shown).

HLA-DP restriction of interferon-γ beryllium-stimulated release by mononuclear cells

PBMCs from all 10 patients tested released IFN-γ in response to PHA and C. albicans (table 1⇑). Be-stimulated IFN-γ release was significantly higher than control in nine of 10 patients (table 1⇑). The release of this cytokine was strongly blocked by the anti-HLA-DP mAb (77±16%) but not by the other mAbs used (anti-HLA-DR, 14±10%; anti-HLA-DQ, 1±2%; anti-class-I, 1±1%; anti-M. tuberculosis 19 kDa-specific protein, 0±1%; p<0.05 for all comparisons with anti-HLA-DP; fig. 1⇑).

Finally, none of the five healthy non-Be-exposed controls released IFN-γ in response to BeSO₄ (data not shown).

Lack of HLA-DP restriction of tumour necrosis factor-α beryllium-stimulated release by mononuclear cells

PBMCs from all of the nine berylliosis patients tested released TNF-α in response to PHA and C. albicans (750.1±426.6). PBMCs from seven of the subjects evaluated also released TNF-α in response to Be stimulation at levels that were significantly higher than controls (table 1⇑). However, in contrast to IFN-γ, the release of TNF-α was not blocked by the anti-HLA-DP specific mAb (fig. 1⇑). Similarly to berylliosis patients, PBMCs obtained from four of five non-Be-exposed healthy subjects released TNF-α in response to BeSO₄ (195.3±76.2 pg·mL⁻¹, p=0.02 with respect to Be-unstimulated PBMCs (91.9±15.8); p=0.09 with respect to PBMCs from berylliosis patients stimulated with BeSO₄), while anti-HLA class II monoclonal antibodies had no effect on the cytokine release (data not shown).

No significant levels of the cytokines IL-4, IL-6, IL-8, IL-10, IL-12, GM-CSF and RANTES were found in Be-stimulated culture supernatants of PBMCs from any of the patients tested, although all cytokines were released in response to PHA stimulation (table 1⇑). Finally, when TNF-α release levels were analysed in relation to the carriage of the TNFA2 allele (TNFA2/TNFA1 or TNFA2/TNFA2) and those who carried only the TNFA1 allele, no differences were found either in the response to Be (TNFA2-negative, n=5, 281±90 pg·mL⁻¹; TNFA2-positive, n=4, 312±108 pg·mL⁻¹; p>0.05) or to PHA (TNFA2-negative, 1,232±778 pg·mL⁻¹; TNFA2-positive, 1,385±761 pg·mL⁻¹; p>0.05) and C. albicans (TNFA2-negative, 741±469 pg·mL⁻¹; TNFA2-positive, 633±546 pg·mL⁻¹; p>0.05).