Cross-reactivity between halogenated platinum salts in an immediate-type respiratory hypersensitivity model

Abstract

Halogenated platinum salts can trigger the development of occupational asthma. Until recently, laboratory research into the development and manifestation of platinum hypersensitivity responses were hindered by the lack of an animal model suitable for assessing the functional consequences of allergic sensitization. We employed a newly developed mouse model to assess the potential allergenicity of ammonium tetrachloroplatinate (ATCP), compare the relative potency of ATCP and another platinum salt, ammonium hexachloroplatinate (AHCP) and assess potential cross-reactivity. Mice were topically sensitized with ATCP before being challenged by intratracheal aspiration (IA) with ATCP. Ventilatory responses were assessed using whole-body plethysmography (WBP). An immediate response (IR) was observed in ATCP-sensitized and challenged mice. Two days later, responsiveness to the nonspecific stimuli methacholine (Mch) was detected in ATCP-sensitized mice using WBP. Bronchoalveolar lavage fluid collected from sensitized mice contained an average of 3.3% eosinophils compared to less than 0.5% in non-sensitized mice (p<.05). Serum harvested from sensitized mice also contained increased total serum immunoglobulin E (p<.05). These data are the first to demonstrate that topical exposure to ATCP is sufficient to develop immediate type hypersensitivity and that a single intra-airway challenge is capable of triggering pulmonary responses. To investigate potential cross-reactivity, mice were sensitized to AHCP and, challenged by a single IA with a second platinum compound, ATCP. Compared to non-sensitized mice challenged with ATCP, these mice exhibited an IR, responsiveness to Mch, and eosinophilic infiltration in the lungs similar to that achieved with AHCP challenge, thus demonstrating cross-reactivity.

Keywords: Platinum; ammonium hexachloroplatinate; ammonium tetrachloroplatinate; cross-reactivity; occupational asthma; pulmonary hyperresponsiveness; respiratory hypersensitivity; whole-body plethysmography.

Figure 1.. Exposure regimen.

BALB/c mice were dosed with test article/vehicle topically on the shaved back on experimental days 0, 5 and 15 (*). Test article/vehicle was applied to the dorsum of both ears on experimental days 10, 11 and 12 (#). Mice were challenged by intratracheal aspiration (IA) with AHCP on experimental days 24. Immediate responses (IR) were measured by whole-body plethysmography. On experimental days 26, pulmonary responses to aerosolized methacholine (Mch) were measured by WBP.

Figure 2.. Assessment of ATCP treatment on lymph node cell proliferation by direct cell counts.

Cells harvested from intrapulmonary/tracheobronchial (mediastinal) lymph nodes (LLN) (A) and auricular lymph nodes (ALN) (B) were counted using a Coulter counter. Data shown are +/− SD (n = 6). *p < 0.05 compared to non-sensitized (Non-sens.) mice challenged with saline (ANOVA). ^#p < 0.05 compared to non-sensitized mice challenged with 100 μg ATCP (ANOVA).

Figure 3.. Pulmonary physiology.

Pulmonary responses were measured using whole-body plethysmography. (A) ATCP-sensitized mice exhibit an immediate response (IR) following ATCP challenge on experimental day 24. (B) ATCP-sensitized mice challenged with ATCP exhibit a dose-dependent increase in methacholine (Mch) responsiveness on experimental day 26. (C) AHCP-sensitized mice challenged with ATCP exhibit an immediate response (IR) on experimental day 24. (D) AHCP-sensitized mice challenged with ATCP exhibit a dose-dependent increase in methacholine (Mch) responsiveness on experimental day 26. For all panels, data shown are +/− SEM (n = 6). *p < 0.05 compared to non-sensitized mice challenged with saline (ANOVA). ^#p < 0.05 compared to non-sensitized mice challenged with 100 μg AHCP (ANOVA). ^{^}p < 0.05 compared to non-sensitized mice challenged with 100 μg ATCP (ANOVA). Non-sens. = non-sensitized.

Figure 4.. Lung instillation of AHCP results in cellular infiltration into BALF.

(A) BALF was collected from non-sensitized and ATCP-sensitized mice on experimental day 26. Collected cells were enumerated using a Coulter counter. (B) Eosinophils selectively infiltrate the BALF of ATCP-sensitized mice following IA challenge. (C) Neutrophils infiltrated the BALF of non-sensitized and ATCP-sensitized mice challenged with AHCP. (D) BALF was collected from non-sensitized and AHCP-sensitized mice on experimental day 26. Collected cells were enumerated using a Coulter counter. (E) Neutrophils infiltrated the BALF of non-sensitized and AHCP-sensitized mice challenged with ATCP. (F) Eosinophils selectively infiltrate the BALF of AHCP-sensitized mice following IA challenge. For all panels, data shown are +/− SEM (n = 6). *p < 0.05 compared to non-sensitized (Non-sens.) mice challenged with saline (ANOVA). ^#p < 0.05 compared to sensitized mice challenged with corresponding platinum compound (ANOVA). Non-sens. = non-sensitized.

Figure 5.. Protein levels are elevated in BALF collected from AHCP-challenged mice.

Protein levels in BALF collected from ATCP-sensitized (A) and AHCP-sensitized (C) mice were determined by Coomassie blue. Lactate dehydrogenase levels in BALF collected from ATCP-sensitized (B) and AHCP-sensitized (D) mice were determined using Thermo LD-L Kit reagent and Thermo Trace Data-Trol controls. For all panels, data shown are +/− SEM (n = 6). *p < 0.05 compared to non-sensitized (Non-sens.) challenged with saline (ANOVA). ^#p < 0.05 compared to non-sensitized mice challenged with 100 μg AHCP (ANOVA). Non-sens. = non-sensitized.

Figure 6.. Total serum IgE levels were significantly elevated in mice sensitized to AHCP.

Total serum IgE levels from ATCP-sensitized (A) and AHCP-sensitized (B) mice were determined by ELISA on experimental day 26. For all panels, data shown are +/− SEM (n = 6). *p < 0.05 compared to non-sensitized (Non-sens.) mice challenged with saline (ANOVA). ^#p < 0.05 compared to non-sensitized mice challenged with 100 μg ATCP (ANOVA). ^$p < 0.05 compared to non-sensitized mice challenged with 100 μg AHCP (ANOVA). ^p < 0.05 compared to non-sensitized mice challenged with 100 μg AHCP (ANOVA). Non-sens. = non-sensitized.