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Review |
Address correspondence to Paolo Boscolo MD, Section of Occupational Medicine, Allergy, and Clinical Immunology; G. D’Annunzio University, Faculty of Medicine and Surgery, Via dei Vestini, 66013 Chieti Scalo, Italy; tel and fax 39 0871 355 6704; e-mail: boscolo{at}unich.it.
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Abstract |
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, TNF-
, and IL-5 release were the focus of this study. Spontaneous PBMC proliferation was inhibited by all 10–4 M salts (with the exception of PtCl2), while it was enhanced by 10–5 M PtCl2 as well as by 10–5 and 10–6 M (NH4)2[RhCl6] and RhCl3 (but not by 10–7 M salts). Pt, Pd, and Rh compounds showed similar effects on PHA-stimulated PBMC proliferation and cytokine release; however, the effects on IFN- release were stronger. Thus, 10–4 and 10–5 M (NH4)2[PtCl6] and 10–4 M (NH4)2[PtCl4] inhibited the PHA-stimulated immune activity;10–4 M PtCl2 did not exert activity, while10–6 M (NH4)2[PtCl6] and 10–5 and 10–6 M (NH4)2[PtCl4] and PtCl2 enhanced PBMC proliferation and/or cytokine release. (NH4)2[PdCl6] showed stronger dose-related inhibitory effects (present also at 10–7 M concentration) on PHA-stimulated proliferation and cytokine release than (NH4)2-[PdCl4], PdCl2, or Rh salts; the inhibitory activity of (NH4)2[RhCl6] was slightly higher than that of RhCl3. In conclusion, this study shows that: (a) the immune capacity of Pt, Pd, and Rh depends on speciation; (b) low concentrations of Pt salts stimulate spontaneous and PHA-stimulated immune responses; (c) the in vitro activity of Pd compounds (which are only inhibitory) is higher than that of Pt and Rh salts. These findings are consistent with the observations that sensitization and allergic contact dermatitis in response to Pd are increased in the general population, although the roles of cross-sensitization to Pd and Ni are difficult to determine. (received 19 April 2004; accepted 1 May 2004)
Keywords: platinum, palladium, rhodium, lymphocyte proliferation, cytokines, immunotoxicity, allergy
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Introduction |
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The available emission data suggest that Pt derived from catalytic converters is almost exclusively in metallic or oxidic forms, with very low sensitising potential [1,5]. However, halogenated Pt salts may possibly be emitted or secondarily formed from mono-crystalline Pt particles under environmental conditions in waters and sediments, and thereby may potentially enter the food chain [1,2].
Workers exposed to PGE include miners, employees in foundries, dental technicians, and workers in chemical industries that produce catalytic converters, ornaments, and electrical appliances [1,2, 3,6]. Halogenated and ammonium-halogenated PGE compounds are formed during refining and treatment of PGE in factories. PGE levels in the air of these plants are much higher than in environmental air. For example, Pt in air samples from catalyst production areas in Germany ranged from 12 to 64 ng/m3 [1], whereas Pt levels in environmental air were below 15 pg/m3 [7].
Epidemiological evidence suggests that the sensitizing potential of Pt compounds is restricted to halogenated and ammonium-chlorinated compounds formed during Pt treatment or refining, which have lower chemical stability and higher reactivity than the element alone [8–10].
Sources of Pt and Pd exposures for the population include dental alloys [11]; there is a difference between Pt and Pd in regard to sensitization capacity, since only metallic Pd, but not Pt in absence of chlorinated Pd salts, seems to induce allergic contact dermatitis (ACD) [1,12]. Patch tests of German children have shown numerous positive reactions to Pd, but not to Pt salts [13]; similar results were found in unselected Austrian and Italian patients with ACD [14,15]. On the other hand, workers in catalyst production who developed asthma, rhino-conjunctivitis, and contact urticaria showed a higher percentage of sensitization to Pt salts than to Pd salts [1,2,10–12]; cases of workers with respiratory allergy to Pt alone are frequent, while only a single case of asthma induced by sensitization to Pd alone has been reported [16].
There are no reports of occupational allergy induced by Rh alone in PGE-exposed workers. However, Rh can induce allergic reactions, including ACD, with concomitant sensitization to other PGE salts [17].
With the exception of CisPt [18], there are few experimental studies on the immune effects of environmental PGE compounds. Schuppe et al [19,20] investigated immunomodulatory effects of Pt compounds; Dearman et al [21] evaluated the type 2 immune response of mice to Pt salts; Pistoor et al [22] and Rustemeyer et al [23] demonstrated cross-reactivity of nickel (Ni) salts with Pd and copper (Cu) compounds.
In a previous study by our group [24], spontaneous and phytohaemagglutinin (PHA)-stimulated proliferation of peripheral blood mononuclear cells (PBMC) and the in vitro release of IFN-, TNF-, and IL-5 were assessed in the presence of 10–4 M and 10–7 M Pt salts; the immune activity of Pt salts was in the following order: (NH4)2[PtCl6] > (NH4)2[PtCl4] > Na2PtI6 and CisPt > PtCl4 > PtCl2. Following this investigation, the aim of the present work was to compare the in vitro immune effects of Pt, Pd and Rh compounds with different chemical speciations and at graded concentrations on lymphocyte proliferation and the release of cytokines from human PBMC.
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Methods |
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Subjects. Healthy male volunteers (N = 9, mean age 34 yr, range 24–58 yr) were recruited for this study. They were not taking any drugs; their routine blood analyses, including white blood cell count (WBC), were within the normal ranges. Fasting samples of EDTA-treated whole blood were obtained from each subject at 8 am and PBMC were purified by Ficoll-Hypaque (BioSpa, Milan, Italy) density gradient centrifugation (20 min, 400 x g). After 3 washings with Hanks’ balanced salt solution (HBSS), PBMC were resuspended in RPMI 1640 medium that contained 10% fetal calf serum, 2 mM L-glutamine, 25 mM HEPES, 100 U/ml penicillin, and 100 µg/ ml streptomycin (Sigma, St Louis, MO). This is designated as complete medium.
PBMC proliferation. PBMC were suspended at 106 cells/ml in complete medium. Aliquots (100 µl) of cell suspension were placed in each well of a 96-well microtiter plate (Falcon, Oxnard, CA) under the following conditions: (a) no other reagents added (control sample); (b) with 20 µg/ml PHA (Sigma); (c) with 10–4, 10–5, 10–6, or 10–7 M (NH4)2[PtCl6], (NH4)2[PtCl4], PtCl2, (NH4)2[PdCl6], (NH4)2-[PdCl4], PdCl2, (NH4)2[RhCl6], or RhCl3, both in the presence and absence of PHA. The cells were incubated 72 hr at 37°C in a humidified atmosphere with 5% CO2.
Enzymatic immunoassay of PBMC proliferation. Proliferation was evaluated by a 5'-bromo-2'-deoxy-uridine (BrdU) cell proliferation assay (Oncogene Research Products, Darmstadt, Germany). BrdU was added to wells of microtiter plates during the final 24 hr of culture. Cells were fixed and permeabilized and their DNA was simultaneously denatured by treatment for 30 min at room temperature with a fixative/denaturing solution. Anti-BrdU monoclonal antibody was pipetted into the wells and incubated for 1 hr. Unbound antibody was washed away and horseradish peroxidase-conjugated goat anti-mouse antibody was added for 30 min at room temperature. Contents of wells were removed by inverting over a sink and tapping on paper towels. Chromogenic substrate solution, tetra-methylbenzidine (TMB), was added to each well and incubated in the dark at room temperature for 15 min. Stop solution was added to each well in the same order as the previously added substrate solution. All reagents were provided with the kit and were used according to the manufacturer’s protocol. Experiments were performed in triplicate. The absorbance of the contents of each well was measured using a spectrophotometric plate reader at dual wavelengths (450 and 540 nm). The color intensity was proportional to the cellular incorporation of BrdU and thus to the degree of cell proliferation.
Production and measurement of cytokines.
Cultures were set up in 1-ml wells of 24-well Costar plastic plates, using 0.8 ml of PBMC (containing 106 cells) in complete medium as follows: (a) no other reagent added (control sample); (b) with 10 µg/ml PHA (Sigma) only; and (c) with 10–4, 10–5, 10–6, or 10–7 M (NH4)2[PtCl6], (NH4)2[PtCl4], PtCl2, (NH4)2-[PdCl6], (NH4)2[PdCl4], PdCl2, (NH4)2[RhCl6], or RhCl3, in the presence of PHA. The cultures were incubated at 37°C in humidified atmosphere with 5% CO2 for 24 hr; afterwards, cells were checked for viability by trypan blue dye exclusion using an inverted Leica microscope. Aliquots of supernatants were collected and stored at –70°C until analysis. Interferon (IFN)-, IL-5, and tumor necrosis factor (TNF)- levels in culture supernatants were determined by Quantikine colorimetric ELISA kits (Benfer-Scheller, Keystone Labs) following the manufacturer’s instructions. All experiments were performed in triplicate.
Statistical analyses were performed with Statistica software (release 4.5). The Kolmogorov-Smirnov test was used to assess the data distributions.
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Results |
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, TNF-, and IL-5 released from PBMC of control cultures averaged 1165 ± 215, 1634 ± 331, and 96 ± 18 pg/ ml, respectively.
The following observations were made: 10–4 (NH4)2[PtCl6] and 10–4 M (NH4)2[PtCl4] (ammonium hexachloro-Pt(IV) and tetrachloro-Pt(II) salts, respectively) significantly inhibited spontaneous PBMC proliferation, while 10–4 M PtCl2 did not exert immune effect and 10–5 M PtCl2 enhanced spontaneous PBMC proliferation (Table 1
).
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); 10–5 M (NH)2[PtCl6] also exerted an inhibitory action (p <0.05) at 10–5 M concentration, while PtCl2 showed significant stimulatory action (p<0.01) at 10–6 M concentration. At 10–7 M, Pt salts did not affect the PHA-stimulated PBMC proliferation.
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). The inhibitory effect of (NH4)2[PdCl6] was higher than (NH4)2[PdCl4] and PdCl2; however, at 10–5, 10–6, and 10–7 M, Pd salts did not affect spontaneous PBMC proliferation.
At 10–4 M, (NH4)2[PdCl6] showed higher capacity (p <0.001) for inhibiting PHA-stimulated PBMC proliferation than the other 10–4 M Pd salts (p <0.01) (Fig. 2). PHA-stimulated PBMC proliferation was significantly inhibited by 10–5 and 10–6 M (NH4)2[PdCl6] (p <0.001) and by 10–5 M (NH4)2[PdCl4] and PdCl2 (p <0.05), with dose-response effects, while lower concentrations of Pd salts did not exert significant effects.
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). On the contrary, 10–5 and 10–6 M RhCl3 as well as 10–5 M (NH4)2[RhCl6], enhanced spontaneous PBMC proliferation (Table 1).
PHA-stimulated PBMC proliferation was significantly inhibited by 10–4 M (NH4)2[RhCl6] and RhCl3 (p <0.01 and p <0.05, respectively) (Fig. 3) and by 10–5 M Rh salts (p <0.05), while 10–6 and 10–7 M Rh salts did not exert effects.
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, TNF-, and IL-5 release from PBMC was significantly inhibited by 10–4 M and 10–5 M (NH4)2[PtCl6], whereas 10–4 M (NH4)2[PtCl4] significantly reduced IFN- and IL-5 release, but not TNF- release (Table 2); finally, 10–4 M PtCl2 did not modify PHA-stimulated cytokine release. Lower concentrations of Pt compounds (10–6 M (NH4)2[PtCl6], 10–5 and 10–6 M (NH4)2-[PtCl4] and PtCl2 significantly enhanced IFN-, TNF-, and IL-5 release from PBMC; no significant effects were exerted by 10–7 M Pt salts.
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release from PBMC with a dose-response effect at concentrations ranging from 10–4 to 10–7 M (Table 2). A similar lower inhibitory effect (with a dose-response effect from 10–4 to 10–6 M) on IFN- release was exerted by (NH4)2[PdCl4] and PdCl2. (NH4)2[PdCl6] showed greater inhibitory effect than (NH4)2[PdCl4] and PdCl2 in reducing PHA-stimulated TNF- release, with a dose-response effect from 10–4 to 10–6 M (Table 2).
(NH4)2[PdCl6] significantly inhibited IL-5 release from PBMC with a dose-response effect at concentrations from 10–4 to 10–6 M, while 10–4 and 10–5 M were the only active concentrations of (NH4)2[PdCl4], and PdCl2 (Table 2)
Among Rh salts, (NH4)2[RhCl6] significantly inhibited the PHA-stimulated IFN- release from PBMC with a dose-response effect at concentrations from 10–4 to 10–6 M; the inhibitory effects of this salt on TNF- and IL-5 release were significant only at 10–4 to 10–5 M. On the other hand, RhCl3 significantly inhibited IFN- release at 10–4 and 10–5 M concentrations and TNF- and IL-5 at 10–4 M.
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Discussion |
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(NH4)2[PtCl6] and (NH4)2[PdCl6] (ammonium hexachloro-Pt(IV) and -Pd(IV), respectively) show more immune activity than (NH4)2[PtCl4] and (NH4)2[PdCl4] (ammonium tetrachloro-Pt(II) and -Pd(II), respectively). Ammonium hexachloro-Pt(IV) and -Pd(IV) are also more active on PBMC proliferation and cytokine release than (NH4)2-[RhCl6] (ammonium hexachloro-Rh(IV)). In this regard, the immune effects of this salt are only slightly more significant than those of RhCl3.
Ammonium tetrachloro-Pd(II) and -Pt(II), as well as PdCl2, show similar inhibitory immune capacity, while PtCl2 exert only a stimulatory immune effect at low concentration. The absence of inhibitory effects of PtCl2 may reflect either the low solubility of this salt (as reported in Material and Methods) or its low cellular uptake: a previous study of our group showed that the Pt content of BALB/3T3 cells was 2.1 fg/cell following exposure to (NH4)2[PtCl6] for 72 hr and only 0.07 fg/cell after similar exposure to PtCl2.
In the mouse popliteal lymph-node assay, Na2PtCl6 and Na2PtCl4 were active in modulating receptor-mediated endocytosis on Langherans cells [19,20,26]. Both the sensitizing capacity of PGE salts and their effects on PBMC may thus depend on mechanisms of intracellular uptake, including endocytosis.
It is interesting to note that PtCl2 and Rh salts enhance spontaneous and/or PHA-stimulated PBMC proliferation and that all the Pt salts enhance PHA-stimulated cytokine release at 10–5 or 10–6 M. A stimulatory effect of PtCl4 on IL-5 release from PBMC was also observed in a previous study of our group [24]; it is known that IL-5 stimulates the Th2-type immune response [27,28].
TNF- release was significantly reduced by all the 10–4 M chlorinated Pd and Rh compounds and (NH4)2[PtCl6], but not by other Pt salts. TNF- is produced by a variety of cell types including T and B lymphocytes, NK cells, astrocytes, and dendritic cells [28]; it represents a cytotoxic factor for many tumor cells also by inducing apoptosis [29].
The effects of PGE salts on PHA-stimulation of IFN- release were similar to those on TNF-and IL-5 release, but less pronounced. Moreover, PGE salts were more active on PHA-stimulated INF- release from PBMC than on PBMC proliferation. INF-, produced mainly by activated T lymphocytes, is a marker of Th1-cell mediated immune response [27,28], while PBMC proliferation needs the concomitant activation of several metabolic mechanisms, including those involving IL-2 production [30].
The present findings that ammonium hexachloro-Pt(IV) and -Pd(IV) salts exert marked immune effects are in agreement with those of an investigation on workers in a plant producing auto-catalysts: workers exposed to tetravalent Pt compounds showed a cumulative chance of 51% of being sensitized after several years of exposure, while those exposed to tetra-amine Pt dichloride alone were not sensitized [8]. However, it is unlikely that the general population, exposed to ambient concentrations of soluble Pt, may be sensitized and develop allergic diseases [1].
There is concern that environmental exposure to Pd may induce ACD. Double sensitization to cobalt salts or potassium dichromate and Pd salts was found in German children [13]. About 5% of positive patch test to Pd were recently found in Austrian patients (76% female), including children, adults, and the elderly [14]. A similar percentage of patch-test positive to Pd (6.7% in females and 2.3% in males) was observed in Trieste (Italy) [15].
Pd sensitization may occur either because of exposure to Pd or cross-reactions with other elements [13–15,30]. Pistor et al [22] reported that Ni-reactive T lymphocyte clones from donors reacted with Pd and Cu salts. They suggested that cross-reactivity could be favoured by the propinquity of Ni, Pd, and Cu in the periodic table and by their propensity to bind to histidine residues of surface peptides of major histocompatibility complex class II molecules. Rustemeyer et al [23] reported that Ni-primed T cells by haptenated autologous dendritic cells showed frequent cross-reactivity with Pd and Cu. Cross-reactivity of Pd salts with Ni salts was also found using patch test techniques in guinea pigs [31]
Studies on patients sensitized to Pd [13–15] suggest that sensitization to Pd is increasing in the general population. The present study, demonstrating that Pd salts are more active than Pt and Rh salts on human PBMC, suggests that environmental Pd exposure should be considered as a possible source of sensitization, although it is difficult to determine the role of cross-sensitization to Pd and Ni. Environmental Pd levels and the trends of Pd sensitization in exposed populations should be monitored in the years ahead. Further research on Pd cellular binding and metabolism is also required.
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Acknowledgements |
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in human cancer development, prevention and palliative care. Mech Ageing Dev 2002;123:1655–1663.[Medline]
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