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In vitro effects of nickel-sulphate on immune functions of normal and nickel-allergic subjects: a regulatory role for zinc

Posted on: Tuesday, 6 January 2004, 06:00 CST

Abstract

Nickel hypersensitivity represents a very common human disease state, mainly occurring in females, defined as allergic contact dermatitis. Ni is a transition metal whose activity may be modulated by congeners. Zinc, an essential component for living organisms, has been shown to counteract Ni effects in patients with Ni hypersensitivity. We analysed immune responses to both Ni and Zn in healthy subjects and patients with allergic contact dermatitis to Ni.

Our in vitro results show that Ni modulates surface receptors expression, reduces phytohemagglutinin (PHA)-driven lymphoproliferation, and upregulates some proinflammatory cytokines production, including Interferon (IFN)-gamma. Zn also induced CD4+ lymphocyte proliferation, but it abolished or reduced most Ni- mediated effects. Our data are consistent with the hypothesis that Zn and Ni, as part of the heavy transition metals, may exchange roles in immune-mediated phenomena leading to expression of allergic contact dermatitis.

Key words: nickel, allergic contact dermatitis, cytokines, T lymphocytes

Introduction

Heavy metals are widely used in industrial procedures as well as in everyday consumers goods and this contributes to an increase of harmful effects to human subjects (1, 2). Direct contact and/or ingestion of transition elements such as nickel is difficult to avoid (1, 3) and lifelong exposure carries increased risk of sensitization. Ni represents the most pervasive and common sensitizing agent (2) resulting in allergic contact dermatitis (ACD), a condition characterized by erythematous macular eruptions, followed by squamous lesions and accompanied by intense itching, sometimes evolving to vesicles or papulae with eczematous aspects. Diagnosis is easily performed by cutaneous patch tests with Ni- sulphate and reading at 48-72 hours, which in case of positivity reproduce the features of ACD. Since about 10% of adult females are thought to suffer from ACD, and this frequent disorder has both professional and environmental causes, it deserves special attention with respect to social and healthcare costs, research of pathogenesis and development of remedies (1-3). Until recently, the immunopathogenesis of ACD was believed to be a prototype of type IV reactions (delayed-type) according to Gell and Coombs' classification (4). There are at least two main differences between classical type IV (tuberculin-type) reactions and ACD: first, the effector lymphocyte type, which is CD4+ in classical type IV reactions, but CDS+ in ACD; second, the role of accessory (nonimmune) cells in the sensitization phase, namely the role of keratynocytes and skin Langerhans cells in ACD (4, 5). In support of the first distinction is the fact that delayed-type reactions are often abrogated in AIDS patients with CD4 depletion, but ACD is frequently observed in the same patients (4). The other variables involved in pathogenesis (i.e. genetic predisposition, chemical configuration of Ni salts, complexing with self proteins to acquire the status of sensitizer and metal-metal interactions) are still object of study and some reviews have been published (1, 5, 6). The relevance of nickel chemistry has been investigated by in vivo and in vitro studies. Nickel, being a transition metal and possessing empty orbitals available to host electrons, strongly interacts with nucleophilic molecules like proteins and nucleic acids. In particular, the formation of labile complexes with proteins may promote their modification. Furthermore, nickel is able to oxidatively modify unsaturated lipids, especially in cell membranes, generating cytotoxic derivatives like hydroperoxydes and aldehydes (1). Nickel adverse effects are prevented or diminished by diverse divalent ions. In particular, zinc exhibits the strongest effect in inhibiting biological modifications attributable to nickel (7).

Zinc is another transition metal, which is essential for normal functioning of several biological systems, both enzymatic and not, including the immune system. Zinc is readily absorbed via the oral route, it does not cause sensitization, and it cures the zinc- deficient immune defect of enteropathic acrodermatitis. Its in vivo administration displays anti-inflammatory properties and in situ application in a Ni-sulphate patch test has been shown to abrogate positive responses (1, 7).

This may represent a novel and useful approach to treatment, since Ni avoidance either by contact and/or by ingestion represents a very difficult task, given its ubiquitous presence or environmental contamination (1, 2).

We addressed the issue of immunological responses to Ni-sulphate in ACD patients and normal healthy subjects, and the in vitro effects of Zn salts on lymphomononuclar cells from the same individuals.

Materials and methods

Subjects

In two different locations, but according to the same protocol, healthy subjects were selected as non-atopic adults with no history of ACD and no drug interferences. These were both females (n = 20) and males (n = 10) aged from 22 to 52 years. Patients with confirmed Ni hypersensitivity were selected after patch testing with Ni- sulphate and positive readings at 48 hours. These were recruited only in Rome, with 8 females and 2 males enrolled, with the same age range as the controls. All had recent episodes of ACD and were not receiving any drug. After informed consent 15 mL of venous blood was obtained from all volunteers and patients in EDTA containing vials. This study was approved by the ethical committees of both clinical institutions involved (Rome and Chieti).

Lymphomononudear cells isolation

Density gradient centrifugation using Ficoll solution (BioSpA, Milan, Italy) at 400 g was used to isolate lymphomononuclear cells (PBMC). After extensive washing in phosphate Dulbecco's saline, cells were resuspended in RPMI-1640 medium enriched of L-glutamine and supplemented with pen-strep antibiotics and 10% v/v of fetal bovine serum (all products purchased from Sigma Chem. Co., St.Louis, USA).

Culture conditions and proliferation and cytokine assays 10^sup 6^ lymphomononuclear cells (PBMC: 85% lymphocytes, 15% monocytes by light microscopy evaluation) in 1 mL of complete culture medium were cultured in vitro for 48-72 hours with/without phytohaemoagglutinin (PHA, Sigma) stimulation at 10 g/mL, with/without addition of different doses of Ni-sulphate and/or Zn-sulphate (reagent grade, *6 H2O, provided from ECVAM and IRCCS S. Gallicano internal chemistry reagent departments). Cells were plated in 24-well Costar plates and incubated at 37 C at 5% CO2/95% air in an insulated humidified incubator for 48-72 hours. Cells and supernatants were collected at the end of culture, and processed. Cells were stained with monoclonal antibodies (mAbs) to CD4, CDS, CD56 and CD3 surface antigens in double fluorescence analysis (4 parameters including forward and side scatters for lymphocytes gating) and analysed by cytofluorimetry using a Facscan (Beckton-Dickinson Immunocytometry Systems, San Jose, USA). Cell proliferation was assessed by MTB reduction assay (Oncogene Research Products, Darmstadt, Germany) of BrdU-treated cultures, and spectrofluorometry detection or (in Rome) by tritiated methyl-thymidine (Amersham, Islington, UK) incorporation in the final 18 hours of culture, determided by liquid scintillation (Packard Inst., USA). Stimulation indexes were calculated by dividing OD readings or cpm of experimental cultures by those obtained in control (unstimulated) cultures. Identification of proliferating cells was carried out by triple labelling at the end of culture with mAbs to CD3, CD4, CD8 and Ki-67, a marker for cell proliferation (all mAbs from Beckton Dickinson and BD Bioscences Pharmingen, San Diego, USA, differently labelled with fluorescent tags). Mean fluorimetric detection of surface antigens expression was obtained by cytofluorimetric linear analysis of fluorescence using the Cell Quest software and MFI (mean fluorescence index). Intracellular cytokine content was assessed by permeabilization of cultured cells according to previously published methods (8) and cytofluorimetric analysis using mAbs to cytokines IL- 4 and IFN-gamma obtained from Becton-Dickinson Bioscience. Culture supernatants, deprived of cellular components by centrifugation at 800 g, were tested for cytokines content by enzymelinked immunosorbent assay (ELISA) purchased from R&D Systems, Minneapolis, USA. Procedures were carried out according to the manufacturer's instructions and results reported as interpolated from kit included standard references. Data analysis was performed using SSPS standard statistical software.

Results

Data reported in Fig. 1 show that Ni-sulphate at 10-30 g/mL induced a proliferative response only in patients affected by ACD. However Zn-sulphate at the same concentrations was able to elicit a response also in healthy non allergic controls. This resulted in enhanced lymphoproliferation detected by 3H-thymidine incorporation in both groups when the two salts were simultaneously present in the cultures, with an additive effect. The stimulation index was higher in ACD patients at all concentrations tested, for both metals.

In triple-labelling experiments with mAb to Ki-67, we found that CD4+ cells were mainly prol\iferating both in normal and ACD patients after Ni or Zn stimulation, but a few CD8+ cells were Ki- 67 positive in Zn-treated cultures of ACD cases (Fig. 2). Our analysis demonstrates that CD4-CD8- (double negative) T cells represent most of the proliferating cells. Also in this experiment lymphomononuclear cells from ACD patients were more active than those from healthy donors, and Zn induced a strong proliferative response in both groups.

Fig. 1. Proliferation of PBMC after exposure to Ni and/or Zn salts for 72 hours and revealed by 3H-methyl thymidine incorporation assay, expressed as stimulation index (cpm stimulated/cpm unstimulated cultures). Data report the means of 20 subjects, and show that Ni and Zn induce responses in ACD cases, whereas a weaker response to Zn only is observed also in healthy subjects. An additive effect in Zn+Ni stimulated cultures on PBMC proliferation is observed. PHA is shown as positive control.

Table 1. Changes of PHA-driven lymphoproliferation induced by NiSO^sub 4^

Fig. 2. Proliferative responses of gated CD3+ lymphocytes to various doses of metals and negative and positive (PHA) controls, assessed by triple fluorencence analysis for Ki-67 (a marker of proliferation) and CD4 or CD8 expression. In panel A are reported data of controls, in panel B of subjects with ACD. CD4+ cells are the main proliferating cells in response to either stimuli and more so in ACD patients, where a small fraction of CDS+ T lymphocytes also respond. The majority of Ki-67+ proliferating cells are therefore double negative for both CD4 and CD8 markers.

Table 2. Mean fluorescence intensity (MFI) of lymphocytes after exposure to PHA and NiSO^sub 4^

In MTB assays conducted in control healthy subjects selected in Chieti, 10^sup -4^ mol/L Ni-sulphate significantly decreased PHA- stimulated lymphoproliferative responses as shown in Table 1. A suboptimal dose of 10^sup -7^ mol/L did not affect this parameter, and even increased the index. No significant changes were observed in non-stimulated cultures (Table 1).

Intracellular cytokine staining for IFN-gamma showed a strong induction when cells from both normal and ACD subjects were cultured with Ni, but not with Zn (Fig. 3). We did not observe induction of IL-4 both in ACD and normal subjects with either metal. This type-1 inflammatory cytokine response is thought to play a major role in ACD pathogenesis, along with other cytokines whose production has been evaluated by ELISA in culture supernatants (i.e. TNF-alpha, data not shown).

NiSO^sub 4^ alone was able to induce significant levels of IFN- gamma and IL-1beta production but in ACD patients mainly (Fig. 4 and Fig. 5). Zn stimulation induced only IL-1beta, but at lower levels and in both groups studied. Simultaneous administration of both metals to lymphomononudear cell cultures resulted in a significant decrease of both proinflammatory cytokines secretion induced by NiSO^sub 4^ alone.

Fig. 3. Intracellular staining for IL-4 and IFN-gamma in gated lymphoid cells after culture without (upper panel) and with NiSO^sub 4^ (bottom left) or ZnSO^sub 4^ (bottom right). The percentage of positive cells is indicated. No IL-4+ lymphocytes were detected in any condition.

Fig. 4. Production in vitro of IFN-gamma in the experimental conditions is indicated. Results are expressed as % change of ACD cultures stimulated with metals from unstimulated cultures tested in parallel. Data represent means standard deviations of 12 subjects. *: p < 0.05.

Fig. 5. Production of IL-1beta (pg/mL) in 72 hours cultures after exposure to metals at the concentrations shown. Controls and ACD cases responded to both Zn and Ni, but simultaneous exposure decreased Ni response in both groups. Data from 12 subjects expressed as means standard deviations. * and $: p < 0.05.

Modulation of surface receptor molecules was also analysed by tests for MFI after cytofluorimetric investigation using a panel of mAbs. A major down-modulation was observed for CD3 and CD56+ cells, either negative or coexpressing CD8 (Table 2, data on subsets not shown). Only minor variations were detected for CD4+ or CD8+ cell surface markers.

Discussion

Our studies have highlighted several previously undisclosed features of the immune response to Ni-sulphate and its modulation by Zn. First, lymphoproliferative response to Ni is exerted by Ni- allergic patients and it is confined to CD4+ cells, though CDS+ ones are thought to be the effectors in the pathogenesis of ACD (4, 9). Second, Mi-stimulated cells produce IFN-gamma, as confirmed by our study, and this is down-regulated by Zn, as well as IL-1beta secretion in vitro. Third, the CD3 antigen of T cells and NK-cell associated receptor CD56 are down regulated at the cell surface after exposure to high dose of NiSO^sub 4^. This finding indicates that high dose NiSO^sub 4^ might fundamentally alter the normal lymphocyte response to proliferative stimuli delivered through the T- cell receptor associated molecule CD3, which is normally down- regulated after cell activation (8). Since Ni negatively affects PHA- induced lymphoproliferation (Table 1), we conclude that over- stimulation is not the cause for excess reduction of the CD3 molecule, but this reduced expression might constitute a factor contributing to inhibition of PBMC proliferative responses. It is less clear why the NK adhesion molecule CD56 is also less expressed due to Ni, but chemical reasons are probably implicated, since CD56 is more labily anchored to the cell membrane via phosphatidyl residues.

One possible consequence of all these changes in immune cells membrane structure and functions is the susceptibility to viral and fungal infections, autoimmune diseases and cancer. Epidemiological data, although Ni hypersensitivity affects about 10% of the general population, are inconclusive (1, 2, 4, 6).

Zn elicits potent lymphoproliferation, despite being an essential non-toxic metal which has well-known immunopotentiating ability. The response is not accompanied by proinflammatory cytokine secretion, with the exceptions of TNF-alpha (not shown) and a smaller amount of IL-1beta, but its addition to cell cultures together with Ni reduces most of the proinflammatory effects of the latter, i.e. strong IFN- gamma and IL-1beta secretion. This is in agreement with clinical data showing that Zn oral supplementation may reduce ACD symptoms induced by Ni (1, 7).

The central role of IFN-gamma and other proinflammatory cytokines in the induction of symptoms of ACD has been clearly established (4, 9). Many proinflammatory cytokines such as IL-1beta and TNF-alpha are also locally secreted by keratynocytes and macrophages, but IFN- gamma is produced mainly by T lymphocytes, and mostly by CDS+ T cells which are thought to play a key role in the skin damage of ACD (4). Therefore our findings of Niinduced IFN-gamma intracellular accumulation and secretion, and its modulation by Zn, represent novel and fertile ground for further investigation of therapeutic options.

Other studies from our groups have indicated several other effects of Ni either by ingestion on intestinal epithelial cells, showing influx of CD4+ lymphocytes (10), or by cultures with keratynocytes, showing induction of keratynocyte growth factor receptor (11). All these effects of Ni should be studied in dose- response designed trials, and also with respect to the anti- inflammatory actions of Zn. One possible explanation of such effects might be the selective or preferential induction of anti- inflammatory cytokines, such as IL-10 or TGF-beta. Clinical and in vitro studies are currently under way.

Ni and Zn are just two players in the field of immunotoxicology, where chemical status, binding to proteins and, most of all, doses, represent key features. Moreover, individual characteristics (e.g. female gender) (2, 5) and environmental exposure (by contact, food or else) (2, 10) should be also more deeply investigated.

Acknowledgements

The work described in this paper was funded by a grant from the Italian Ministry of Health to R.P. and M.P. (2000-2002) and partly by EU contract no. 14660-1998-12 FIED ISP-IT.

References

1. Santucci B, Camera E, and Picardo M (2002) Biochemical aspects of nickel hypersensitivity: factors determining allergenic action. In: Hostynek JJ, Mailbach HI (Eds) Nickel and the skin. CRC Press, Boca Raton, pp. 201-217

2. Emmett EA, Risby TH, Jiang L, Ng SK, and Feinman S (1998) Allergic contact dermatitis to nickel: bioavailability from consumer products and provocation threshold. J Amer Acad Dermatology 19: 314- 322

3. Manaham SE (1994) Fundamentals of chemistry. In: Environmental Chemistry. 6th edition, CRC Press, Boca Raton, p. 741

4. Grabbe S, and Schwarz T (1998) Immunoregulatory mechanisms involved in elicitation of allergic contact hypersensitivity. Immunology Today 19: 37-44

5. Robinson MK (1999) Population differences in skin structure and physiology and the susceptibility to irritant and allergic contact dermatitis: implications for skin safety testing and risk assessment. Contact Dermatitis 41: 65-79

6. Mc Fadden JP, and Basketter DA (2000) Contact allergy, irritancy and danger. Contact Dermatitis 42: 123-127

7. Santucci B, Cristaudo A, Mehraban M, Valenzano C, Camera E, and Picardo M (1999) ZnS04 treatment of NiS04-positive patients. Contact Dermatitis 40: 281-282

8. Carbonari M, Tedesco T, Del Porto P, Paganelli R, and Fiorilli M (2000) Human T cells with a type-2 cytokine profile are resistant to apoptosis induced by primary activation: conseguences for immunopathogenesis. Clin Exp Immunol 120: 454-462

9. Budinger L, and Hertl M (2000) Immunologic mechanisms in hypersensitivity reactions to metal ions: an overview. Allergy 35: 108-115

10. Di Gioacchino M, Boscolo P, Cavallucci E, Verna N, Di Stefano F, Di Sciascio M, Masci S, Andreassi M, Sabbioni E, Angelucci D, and Conti P (2000) Lymphocyte subset changes in blood and gastrointestinal mucosa after or\al nickel challenge in nickel- sensitized women. Contact Dermatitis 43: 206-211

11. Marchese C, Visco V, Almati L, Cardinali G, Buttari B, Bellocci M, Torrisi MR, and Picardo M (2003) Nickel-induced keratynocyte proliferation and up-mudulation of the keratynocyte growth factor expression. Exp Dermatol 11, 12: 497-505

Roberto Paganelli1,*, Brigitta Buttari4, Emanuela Camera4, Maria Lucia Dell'Anna4, Arianna Mastrofrancesco4, Luca Di Giampaolo1, Marcella Reale2, Cosima Schiavone1, Nicola Verna1, Mario Di Gioacchino1, Enrico Sabbioni3, Paolo Boscolo1 and Mauro Picardo4

1 Departments of Medicine and Sciences of Aging, University,,G. d'Annunzio", Chieti, Italy

2 Oncology and Neurosciences, University,,G. d'Annunzio", Chieti, Italy

3 ECVAM, JRC, Ispra, Italy

4 S. Gallicano Dermatological Institute - IRCCS, Rome, Italy

*Correspondence to: Roberto Paganelli, Dept. of Medicine and Sciences of Aging, University,,G. d'Annunzio", via dei Vestini, 66013 Chieti scalo, Italy,

Phone: +39-0871-3554144 or 347-1656298 (cell),

Fax: +39-0871-3554145, E-mail: rpaganel@unich.it

Copyright Urban & Fischer Verlag 2003

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