Nickel, palladium and rhodium induced IFN-gamma and IL-10 production as assessed by <it>in vitro </it>ELISpot-analysis in contact dermatitis patients

1471-2172-9-19 1471-2172 Research article Nickel, palladium and rhodium induced IFN-gamma and IL-10 production as assessed by <it>in vitro </it>ELISpot-analysis in contact dermatitis patients Bordignon Valentina bordignon@ifo.it Palamara Francesca cristaudo@ifo.it Cordiali-Fei Paola cordiali-fei@ifo.it Vento Antonella cordiali-fei@ifo.it Aiello Arianna cordiali-fei@ifo.it Picardo Mauro picardo@ifo.it Ensoli Fabrizio ensoli@ifo.it Cristaudo Antonio cristaudo@ifo.it

Laboratory of Clinical Pathology, Department of Allergology and Laboratory of Skin Physiopathology, Institute San Gallicano, IRCCS, IFO, Rome, Italy

BMC Immunology 1471-2172 2008 9 1 19 http://www.biomedcentral.com/1471-2172/9/19 18482439 10.1186/1471-2172-9-19 01 10 2007 15 5 2008 15 5 2008 2008 Bordignon et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

Recent attempts to diminish nickel use in most industrial products have led to an increasing utilization of alternative metal compounds for destinations such as the alloys used in orthopaedics, jewellery and dentistry. The present study was undertaken with the aim to evaluate the potential for an allergic response to nickel, palladium and rhodium on the basis of antigen-specific induction of inflammatory/regulatory cytokines, and to characterize, according to the cytokine profiles, the nature of simultaneous positive patch tests elicited in vivo.

Peripheral blood mononuclear cells (PBMC) from 40 patients with different patch test results were kept in short term cultures in the presence of optimized concentrations of NiSO₄× 6H₂O, PdCl₂and Rh(CH₃COO)₂. The production of IFN-γ and IL-10 elicited by metal compounds were analyzed by the ELISpot assay.

Results

We found a specific IFN-γ response by PBMC upon in vitro stimulation with nickel or palladium in well recognized allergic individuals. All controls with a negative patch test to a metal salt showed an in vitro IL-10 response and not IFN-γ production when challenged with the same compound. Interestingly, all subjects with positive patch test to both nickel and palladium (group 3) showed an in vitro response characterized by the release of IFN-γ after nickel stimulation and production of IL-10 in response to palladium.

Conclusion

These results strongly suggest that the different cytokine profiles elicited in vitro reflect different immune responses which may lead to the control of the allergic responses or to symptomatic allergic contact dermatitis. The development of sensitive and specific in vitro assays based on the determination of the cytokine profiles in response to contact allergens may have important diagnostic and prognostic implications and may prove extremely useful in complementing the diagnostic limits of traditional patch testing.

Background

Allergic contact dermatitis (ACD) affects a large fraction of the general population being most frequently elicited by nickel (Ni), which is responsible of a delayed-type hypersensitivity clinically revealed by the demonstration of eczematous reactions upon patch testing and by laboratory testing of antigen-specific T-cell proliferation 123. These alterations are the result of the interaction between the metal ions, which act as haptens, and the skin surface, thus favouring the contact and binding with cellular and extracellular matrix proteins 456, and the induction of a cellular immune response against the hapten-carrier protein complex 78. Recent attempts to diminish Ni use in most industrial products have led to an increase in the use of different metal compounds for specific destinations such as the alloys used in orthopaedics, jewellery and dentistry 9. For instance, the use of palladium (Pd) increased in the past 10 years 10 also due to the fact that dentistry alloys containing Pd appear more resistant to mechanical wearing effects 1112. Similarly, rhodium (Rh), which is primarily used as an alloying agent to harden platinum and Pd, it is now employed in electrical material because of its low electrical and contact resistance, in jewellery (platings of silver or white gold because of its silvery white colour), in optical instruments and dental prostheses.

Nevertheless, it has been recently recognized that also exposure to metal ions such as Pd, chromium, cobalt and gold, can induce a cellular immune response and clinical manifestations of ACD, even if less frequently than Ni 131415. In fact, positive patch testing to PdCl₂and clinical manifestation of ACD to Pd have been described with increasing frequency 1617. Despite its low sensitization potential, also Rh has been reported as sensitizer in the form of salt 18.

In Ni sensitized subjects a simultaneous patch test reactivity to Pd has been frequently reported 1920 and a double sensitization can be hypothesised especially in individuals wearing dental prosthesis. Nevertheless, a cutaneous cross-reactivity cannot be excluded for the chemical and structural similarities of Ni and Pd 21. Metal ions are incomplete antigens which have to bind to endogenous peptides to be "visible" to the immune system 7 and Ni and Pd encountering the same cellular environment might form in vivo complexes with similar proteins thus eliciting cross-recognizing immune responses 17. Moreover, two independent in vitro studies demonstrated an immunologic cross reactivity between these metals at the T cell clonal level 2223.

Identifying both a double sensitization and a lymphocyte cross-reactivity to different metal ions in vivo is an important issue for clinicians in directing the choice of implant materials in order to avoid incompatible reactions. Consequently, the diagnosis of ACD to both Ni and Pd needs in vitro assays complementary to patch testing 2425. Indeed, the variability of the readouts obtained with patch testing have stimulated a number of studies aimed at developing assays characterizing the immune response to metal salts. Recently, Minang et al. 26 analyzed the different cytokine profiles induced by nickel in allergic or tolerant subjects by enzyme-linked immunospot (ELISpot) assay. In fact, the sensitivity and specificity of the ELISpot assay allow the measurement of very low frequencies of cytokine-producing cell, therefore offering a powerful tool for the detection and characterization of Ni sensitization with important implications for diagnostic strategies 2728.

The present study was undertaken with the aim to evaluate the potential of the ELISpot assay in determining sensitivities to Ni or Pd and to Ni and Pd on the basis of specific induction of inflammatory/regulatory cytokines. Since all subjects were skin challenged to Ni, Pd and Rh salts, and none of them had positive skin reactivity to Rh, this metal was employed to assess the cytokine profile in non allergic subjects and to explore the possibility to identify a cellular immunity against materials with traces of heavy metals.

Results

Patch tests

To analyse the cytokine response to metals, we considered four different groups of subjects according to their skin responses. As summarized in Table 1, group 1 included 10 patients with a strong positive reaction to Ni; group 2 included 10 patients with strong (n = 7) or moderate (n = 3) hypersensitivity to Pd; group 3 included 10 patients showing a strong reactivity to Ni and a simultaneous weak (n = 4) or moderate (n = 6) response to Pd; and, finally, group 4 included 10 patients with negative patch testing to all metals investigated. All patients enrolled in the study (n = 40) had a negative patch test response to Rh.

Table 1

Characteristics of patients and healthy controls.

Group Subject n.

Sex

Age range (median)

Patch test to Ni

Patch test to Pd

Patch test to Rh

1 n = 10

9 F

28 – 40 (30)

9 +++

9 -

1 M

1 +++

1 -

2 n = 10

9 F

28 – 38 (33)

9 -

7+++/2 ++

9 -

1 M

1 -

1 ++

1 -

3 n = 10

9 F

29 – 40 (32)

9 +++

6 ++/3 +

9 -

1 M

1+++

1 +

1 -

4 n = 10

9 F

28 – 38 (33)

9 -

1 M

1 -

As described in the methods, patch test reactivity to the different metal salts was defined as strong (+++: oedema, erythema, papules and vesicles), moderate (++: oedema, erythema and papules), weak (+: oedema and erythema) or no reaction (-).

IFN-γ and IL-10 production by PBMC in response to Ni, Pd and Rh

To determine the production of IFN-γ and IL-10 upon in vitro stimulation, PBMC from patients with different patch test results were kept in short term cultures in the presence of NiSO₄× 6H₂O, PdCl₂and Rh(CH₃COO)₂and analyzed by the ELISpot assay.

IFN-γ and IL-10 basal response or elicited by metal salts or PHA in PBMC from different groups of patients are shown in Figure 1. The primary readouts of spot forming cells for each experimental condition in individual patients are also shown in Table 2.

Figure 1

Cytokine response elicited by metals or PHA by ELISpot analysis

Cytokine response elicited by metals or PHA by ELISpot analysis. Elispot results of detection of IFN-γ or IL-10 producing PBMC upon stimulation with Ni (20 μg/ml), Pd (2.5 μg/ml), Rh (5 μg/ml) or PHA (1 μg/ml) were expressed as Index values. Index is expressed by the ratio between the number of spot forming cells (IFN-γ and IL-10 producing PBMC) upon stimulation with metal salts and those present in the absence of stimuli (spontaneous cytokine production). The mean values of Index values ± SD were obtained for the 4 different groups of 10 subjects according to their in vivo reactivity to metal salts. Cytokine producing cells in control triplicate wells with medium alone was the baseline value (Index = 1). Panel A: patch test positive to Ni (group 1); B: patch test positive to Pd (group 2); C: patch test positive to Ni and Pd (group 3); D: negative patch test (group 4). The statistical analysis between IFN-g and IL-10 producing cells upon stimulation with Ni or Pd between patients belonging to group C and A or B was performed using the Mann-Whitney test. The highly significant differences (P < 0.0001), found between group C and B, are indicated by symbols (*, **, °, °°).

Table 2

Individual values of spot forming cells for each experimental condition expressed as mean values and Standard Errors of triplicate wells.

IFN-γ

IL-10

Subject n.

Mean

Std. Error

W/O

PHA

W/O

PHA

Patch test positive to Ni

Mean

18,67

151

116

14,33

5,667

132

79,67

(Group 1)

Std. Error

2,404

4,359

3,055

1,202

0,8819

0,5774

3,606

2,082

3,93

3,786

Mean

25,17

164,3

150

29,33

17,67

157,3

36,5

86,33

Std. Error

1,74

6,766

3,215

2,333

1,453

2,082

6,36

0,2887

2,309

2,333

Mean

27,67

203

136,5

19,33

16,67

11,67

22,5

Std. Error

2,333

3,606

0,866

0,8819

1,453

0,8819

3,606

1,443

3,606

2,887

Mean

26,67

203

168,3

18,67

5,333

33,33

270

62,67

51,33

132,7

Std. Error

2,404

9,074

3,48

1,202

6,351

6,333

1,667

5,207

Mean

30,67

246,7

168,3

27,33

289

65,33

143,3

147,7

Std. Error

0,8819

1,856

2,603

0,8819

1,764

2,517

6,083

2,603

2,963

7,311

Mean

13,33

100,3

6,667

177

32,67

79,33

93,67

Std. Error

2,186

3,18

3,055

1,732

1,202

5,568

6,658

2,906

0,8819

4,667

Mean

21,33

171,7

119,3

10,83

10,5

21,67

147,3

35,67

85,33

Std. Error

0,3333

3,667

4,667

0,6009

0,2887

2,848

5,925

3,283

2,603

2,082

Mean

158

129,3

10,33

15,5

265,7

46,5

136

132,7

Std. Error

1,155

3,464

3,18

1,764

1,443

2,309

11,98

0,866

6,245

5,487

Mean

20,67

160,7

118,3

4,333

8,333

33,5

233

129,3

130

Std. Error

1,764

5,239

4,41

1,453

0,866

7,506

2,887

5,783

3,215

Mean

13,33

105,3

14,67

2,667

15,5

107,7

25,33

Std. Error

2,404

5,696

2,082

1,453

1,202

1,607

3,844

2,082

1,732

Patch test positive to Pd

Mean

69,33

45,33

2,333

197,3

84,33

129,3

(Group 2)

Std. Error

1,155

3,48

0,5774

2,603

1,333

2,887

7,219

2,404

3,606

3,18

Mean

14,33

100,3

20,67

83,67

15,67

16,5

124,7

58,67

10,33

68,33

Std. Error

3,844

1,453

2,333

4,631

1,202

1,443

3,283

1,202

2,603

2,906

Mean

19,33

62,33

37,67

285

143

186

Std. Error

3,215

5,686

2,028

3,844

1,528

1,453

5,508

2,309

1,732

3,464

Mean

13,5

101

71,67

10,67

35,5

285

127,7

151

Std. Error

0,866

7,937

3,215

2,333

0,8819

0,2887

5,508

4,41

1,155

1,528

Mean

360,3

229,7

36,33

9,333

73,67

37,67

10,67

41,33

Std. Error

1,732

9,351

1,732

2,906

3,48

1,764

2,728

1,202

1,764

1,856

Mean

6,5

5,667

47,33

13,67

45,33

Std. Error

1,443

2,082

1,202

1,155

1,528

1,856

1,764

Mean

15,67

117,3

9,667

72,67

6,333

23,33

190

Std. Error

3,48

7,688

0,6667

1,453

0,8819

2,848

2,082

2,309

1,155

Mean

6,667

5,333

28,67

198,3

110,3

112

Std. Error

1,764

3,512

0,8819

2,082

1,856

10,4

9,171

1,155

7,371

Mean

32,67

273

170,3

13,33

10,33

70,33

34,33

9,667

Std. Error

1,202

3,606

2,646

5,548

1,202

0,8819

2,728

3,283

1,202

2,646

Mean

30,67

244

24,33

153,3

28,67

197,3

120,7

31,67

116

Std. Error

1,202

4,509

2,028

2,848

1,528

2,404

2,333

8,95

1,764

4,041

Patch test positive to Ni and Pd

Mean

105,7

89,67

17,67

10,67

20,33

153,7

32,67

72,33

(Group 3)

Std. Error

2,646

8,293

6,692

2,404

1,764

1,453

5,783

3,528

2,333

3,786

Mean

24,83

172

150

176

26,67

79,67

93,67

Std. Error

2,048

3,786

3,464

2,082

2,887

5,568

6,658

3,283

2,333

2,603

Mean

26,67

203,7

157

37,33

14,67

38,67

292,3

66,67

148,7

152

Std. Error

1,453

8,686

4,619

2,333

1,764

2,186

5,783

1,856

4,055

3,606

Mean

247

186

30,67

18,33

23,67

186,3

21,33

94,67

119,3

Std. Error

1,155

2,309

3,464

1,453

2,028

4,702

7,623

3,528

4,41

1,856

Mean

25,67

197

157,7

22,33

37,33

285

145,7

143

Std. Error

1,764

4,041

1,856

2,028

2,309

1,453

5,508

3,215

3,383

3,215

Mean

157,7

117

15,5

125,3

29,67

61,67

Std. Error

1,155

3,18

2,082

1,155

2,517

0,2887

4,667

1,667

2,887

1,764

Mean

12,33

95,67

70,33

9,667

30,33

210,7

39,67

114,7

118,3

Std. Error

1,453

10,37

3,383

1,528

2,028

3,18

6,984

4,978

3,48

3,756

Mean

361

244,3

49,67

52,67

31,67

215

124,7

133

Std. Error

1,732

9,292

4,096

3,283

2,333

2,963

5,132

2,646

3,48

2,309

Mean

45,5

373,3

285

38,33

264,3

72,67

161,7

153,3

Std. Error

2,021

2,963

6,429

1,732

3,512

4,055

12,25

2,028

4,055

3,48

Mean

15,67

118

6,333

16,33

33,67

254,3

132

131,3

Std. Error

2,333

3,215

6,928

2,028

2,728

2,963

8,413

2,309

5,292

2,963

Patch test negative

Mean

3,667

4,667

32,33

243

146,7

116,7

134,3

(Group 4)

Std. Error

0,8819

1,528

1,155

0,8819

1,202

2,028

4,041

4,631

6,173

5,608

Mean

17,33

130

27,67

21,33

47,33

377

237,3

181

201,3

Std. Error

2,333

2,887

1,764

3,283

1,732

2,728

2,646

2,603

3,215

6,064

Mean

6,667

53,33

10,33

4,333

5,667

18,33

140,3

69,67

66,33

90,67

Std. Error

1,202

2,728

0,8819

1,453

0,6667

1,453

2,603

2,404

4,842

4,631

Mean

11,33

82,67

24,33

193,3

108

Std. Error

0,8819

2,333

2,082

1,732

1,528

1,202

3,844

1,732

4,619

5,196

Mean

14,33

112,7

17,33

18,67

7,333

34,33

260,3

134

111,7

132

Std. Error

1,202

5,044

1,453

2,028

0,8819

2,404

3,18

1,528

4,41

4,359

Mean

3,333

0,6667

2,333

138

78,33

67,67

Std. Error

0,5774

1,528

1,453

0,6667

0,3333

2,646

4,163

1,453

3,055

2,404

Mean

141

23,33

7,333

4,333

109,7

57,33

58,67

Std. Error

1,732

5,033

1,453

1,202

0,8819

1,732

2,906

2,404

3,18

4,256

Mean

22,67

180,7

27,33

18,33

11,67

196,7

154,3

114,3

122,7

Std. Error

1,202

2,404

1,453

1,202

1,732

1,453

2,404

6,009

4,41

Mean

6,667

6,333