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. Author manuscript; available in PMC: 2021 Mar 18.
Published in final edited form as: Inhal Toxicol. 2020 Mar 18;32(2):68–78. doi: 10.1080/08958378.2020.1737762

Physiological Responses to Cisplatin Using a Mouse Hypersensitivity Model

DM Lehmann 1,#, WC Williams 1
PMCID: PMC7455881  NIHMSID: NIHMS1621638  PMID: 32188332

Abstract

The physiological mechanisms underlying the development of respiratory hypersensitivity to cisplatin (CDDP) are not well-understood. It has been suggested that these reactions are likely the result type I hypersensitivity, but other explanations are plausible and the potential for CDDP to induce type I hypersensitivity responses has not been directly evaluated in an animal model. To investigate CDDP hypersensitivity, mice were topically sensitized through application of CDDP before being challenged by oropharyngeal aspiration with CDDP. Before and immediately after oropharyngeal aspiration (OPA) challenge, pulmonary responses were assessed using whole body plethysmography (WBP). CDDP did not induce an immediate response or alter the respiratory rate in sensitized mice. Two days later, baseline Penh values were significantly elevated (p<0.05) in mice challenged with CDDP. When challenged with methacholine (Mch) aerosol, Penh values were significantly elevated (p<0.05) in sensitized mice and respiratory rate was reduced (p<0.05). Lymph node cell counts and IgE levels also indicated successful sensitization to CDDP. Irrespective of the sensitization state of the mice, the number of neutrophils increased significantly in bronchoalveolar lavage fluid (BALF) following CDDP challenge. BALF from sensitized mice also contained 2.46 (±0.8) × 104 eosinophils compared to less than 0.48 (±0.2) × 104 cells in non-sensitized mice (p<0.05). These data provide previously unknown insights into the mechanisms of CDDP hypersensitivity.

Keywords: Platinum, cisplatin, occupational asthma, respiratory hypersensitivity, whole-body plethysmography, pulmonary hyperresponsiveness

Introduction

Cisplatin (CDDP), a halogenated platinum compound, is an antineoplastic agent used successfully for treating a variety of malignancies. However, in some patients, therapeutic use of CDDP is limited by the development of hypersensitivity. Hypersensitivity to a chemotherapeutic agent is an unforeseen adverse reaction that cannot be explained by the known toxicity profile of the drug. Commonly, CDDP hypersensitivity reactions are associated with skin rash, flushing, abdominal cramping, itchy palms and back pain. More seriously, potentially fatal cardiovascular and respiratory complications can develop (Makrilia et al. 2010). Since hypersensitivity usually appears after multiple infusions, allergic reactions are believed to be one of the causes of these reactions.

Allergic hypersensitivity reactions occur when the immune system responds abnormally to an exogenous substance. As organized by Gell and Coombs, there are four classes of allergic hypersensitivity responses (Murphy et al. 2012). Types I, II and III are mediated by antibodies whereas type IV reactions are cell-mediated. Irrespective of the type, all hypersensitivity reactions develop in two stages (i.e., induction and elicitation). During the induction phase, the immune system is primed and sensitized such that during subsequent exposures the allergic response is triggered (Murphy et al. 2012).

Allergic hypersensitivity reactions to halogenated platinum compounds is a well-known phenomenon (reviewed in (WHO 2000; IPCS 2012)). In the workplace, chloroplatinates are considered potent sensitizers capable of causing occupational asthma in up to 50% of exposed workers (Roberts 1951; Hughes 1980). In the clinical setting, hypersensitivity responses to CDDP, for example, range from 5 – 20% with risk increasing concomitantly with the number of infusions (Makrilia et al. 2010). These adverse reactions occur within minutes of CDDP infusion, but typically don’t appear until between the 4th and 8th infusion cycle (Makovec 2019). In life-threatening cases, use of the platinum compound may have to be ceased entirely. In less severe circumstances, alternative platinum compounds may be substituted for the offending agent. While this approach has been successfully employed, cross-reactivity complicates this course of action (Makrilia et al. 2010). Cross-reactivity among platinum-containing anticancer drugs represents a serious concern for medical practitioners because patients are at risk of potential life-threating hypersensitivity reactions to related platinum agents (Hartmann and Lipp 2003). Furthermore, changing therapeutic agents can alter disease evolution since tumor response may not be the same with other drugs (Cetean et al. 2015). Clearly, there could be medically important implications for patients affected by this serious condition. For this reason, assessing the potential of platinum agents to cause allergic hypersensitivity reactions is paramount.

Although hypersensitivity to CDDP reactions have been mainly attributed to the development of type I hypersensitivity, the mechanisms underlying these reactions are not well-understood (Makrilia et al. 2010). Available evidence suggests that immunological and non-immunological mechanisms are at play. Although some idiosyncratic reactions have been reported (Santini et al. 2001), most often multiple exposures are required before symptoms occur, which are reversed by treatment with epinephrine (Shepherd 2003). This, and positive skin prick tests point towards type I hypersensitivity being the root cause, but no studies have identified platinum-specific IgE antibodies (Makrilia et al. 2010). Hemolysis, thrombocytopenia, chronic urticaria, joint pain and proteinuria suggest type II and III hypersensitivity (Cetean et al. 2015). The occurrence of inflammatory reactions presenting days after platinum agent infusion suggests type IV hypersensitivity or that the direct release of histamine may also be factors in these responses. After observing high levels of pro-inflammatory mediators associated with oxaliplatin infusion, Santini et al. (2001) speculated that oxaliplatin may act as a superantigen resulting in non-specific activation of T cells resulting in excessive cytokine release (Santini et al. 2001). The unpredictable presentation pattern of these reactions leaves many unanswered questions.

Very limited research using experimental animal models has been conducted to elucidate the mechanisms of CDDP hypersensitivity. Topical treatment of mice with CDDP results in immune responses consistent with sensitization (Dearman et al. 1998, 2013; Williams et al. 2014). For example, topical application of CDDP induced proliferation of lymph node cells in mice (Dearman et al. 2013; Williams et al. 2014). Most recently, the dermal sensitizing potency of CDDP was shown to be 0.19% w/v (Williams et al. 2014). Lymph node cell proliferation is common to the development of type I and type IV hypersensitivity. Consequently, since neither of these studies included an antigen challenge phase, it is not possible to differentiate between the two types of hypersensitivity in these studies. However, dermal exposure of mice to CDDP resulted in an increase in lymph node cell Th2 cytokines (e.g. IL4 and IL-10) along with an increase in the Th1 cytokine IFNγ (Dearman et al. 1998). However, Th2 cytokine profiles are not necessarily universally predictive of type I sensitizers (Traidl et al. 1999; Ulrich et al. 2001; Vanoirbeek et al. 2004; Vanoirbeek et al. 2006; Tarkowski et al. 2007; Ku et al. 2008). These reports illustrate the need to confirm allergen-induced cytokine secretion with an evaluation of changes in lung function as well as additional indicators of respiratory hypersensitivity (Murphy et al. 2012). The ability of CDDP to trigger type I respiratory responses has not been investigated in an animal model.

Recently, we developed the first mouse model of platinum respiratory hypersensitivity. Using this model, we showed that mice dermally sensitized with ammonium hexachloroplatinate (AHCP) or ammonium tetrachloroplatinate (ATCP) (1) exhibit an immediate pulmonary response (2) are responsive to methacholine, (3) experience inflammatory cell infiltration of the lung following respiratory tract challenge with platinum compound and (4) have significantly elevated total serum immunoglobulin E (IgE) (Williams et al. 2015; Lehmann and Williams 2018). This model was also used to perform proof-of-concept studies demonstrating cross-reactivity between AHCP and ATCP (Lehmann and Williams 2018). Here, we use this model to investigate the development and manifestation of hypersensitivity reactions to CDDP.

Materials and Methods

Animals

All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee of NHEERL, US EPA. Female BALB/c mice, 8–9 weeks of age at the time of study initiation, were used for these studies (Charles River Laboratories, Wilmington, MA). All mice were housed in an Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC)-approved facility (21.5 °C ± 1.5 °C, relative humidity of 55% ± 5%, and a 12h light/dark cycle). Mice were group-housed by treatment groups in polycarbonate cages with hardwood chip bedding (NEPCO, Warrensburg, NY) and were provided a balanced diet mouse chow (5POO Prolab RMH3000, PMI Nutrition International, Richmond, IN) and water ad libitum.

Chemicals

Test substances used for these studies, including ammonium hexachloroplatinate (AHCP; 99.995% purity), ammonium tetrachloroplatinate (ATCP; 99% purity)), cis-diamminedichloroplatinum (II) (CDDP; >99.9% purity) and acetyl-β-methacholine chloride (Mch), were purchased from Sigma Aldrich (St. Louis, MO). AHCP, ATCP and CDDP were prepared in dimethyl sulfoxide (DMSO; Wilmington, DE) for dermal dosing or pyrogen-free 0.9% sodium chloride, injection, USP (Hospira, Inc, Lake Forest, IL) for delivery to the airways.

Sensitization and challenge protocols

Mice were sensitized according to the protocol originally reported by Dearman and colleagues (1998) and subsequently utilized by Williams et al. .2015 and Lehmann and Williams 2015. Briefly, mice were dosed topically with 1% CDDP (prepared in DMSO) on the shaved back (100 μL) on experimental days 0, 5 and 15 and on the ears (25 μL/ear) on experimental days 10, 11 and 12 (Figure 1B). Control mice received vehicle (i.e., DMSO) only.

Figure 1. Test compounds and exposure regimen.

Figure 1.

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(A) Halogenated platinum salts used in this study. (B) 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 oropharyngeal aspiration (OPA) with 100 μg CDDP on experimental day 24. Immediate responses (IR) were measured by whole-body plethysmography. On experimental day 26 pulmonary responses to aerosolized methacholine (Mch) were measured by WBP. Mice were euthanized 1-hour after Mch exposure.

Effects of CDDP sensitization on lung physiology were evaluated as described previously (Williams et al. 2015; Lehmann and Williams 2018). Briefly, on experimental day 24 mice were anesthetized (3.5% isoflurane in oxygen) before being suspended by their frontal incisors to allow dosing to the oropharynx with 100 μg CDDP prepared in saline (Figure 1B). After test material placement, the nose was briefly occluded to compel the mouse to inhale through its mouth (Williams et al. 2015; Lehmann and Williams 2018). Oropharyngeal aspiration (OPA; 50 μL inoculum) has been previously shown to reliably deliver material directly to the lungs (Foster et al. 2001). Directly after OPA delivery, immediate respiratory responses (IR) were measured in unrestrained, unanesthetized mice using whole-body plethysmography (WBP; DSI, St. Paul, MN). Enhanced pause (Penh), a derived parameter that yielded results consistent with direct/invasive measures of pulmonary hyperresponsiveness (Hamelmann et al. 1997), was calculated using Buxco BioSystem XA software (DSI). Respiratory responses to the non-specific bronchoconstrictor Mch were also measured using WBP on experimental day 26. For both pulmonary responses, average values were collected for 10 minutes prior to OPA delivery of CDDP. Immediately after OPA delivery, measurements were collected for 60 minutes and were used to determine Penh. Average Penh values were calculated for each animal over the entire measurement period (50 measurements for the 10-minute baseline period and 300 measurements for the 60-minute post-instillation period). Average values for each animal were used subsequently to calculate the group average Penh values (expressed +/− standard error of the mean). The same mice evaluated for assessing pulmonary responses were used for all other endpoint assessments.

Blood collection and euthanasia

One hour after Mch challenge and prior to blood collection on day 26, mice were administered Euthosol (Virbac AH, Inc) and then exsanguinated by cardiac puncture. Blood was collected in serum collection tubes (BD Falcon, San Jose, CA) and processed to serum according to the manufacturer’s instructions. Serum was stored at −80°C.

Lymph node cell collection

As previously described (Williams et al. 2015; Lehmann and Williams 2018), the auricular lymph nodes were collected using aseptic technique and placed in room temperature RPMI 1640 containing 25 mM HEPES, 2.05 mM L-glutamine (Invitrogen, Grand Island, NY), 10% fetal bovine serum (Hyclone, Logan, Utah), and 2% penicillin/streptomycin (Cellgro, Manassas, VA). Using a disposable plastic pestle, lymph nodes were mechanically disaggregated, passed through a 100 μm Celltrics filter (Partec, Munster, Germany) into a sterile 15 mL collection tube and pelleted by centrifugation (300 x g for 7 min. at room temperature). Lymph nodes were resuspended in 1 mL complete RPMI 1640 before being counted using a Coulter Counter (Beckman Coulter, Brea, CA), and viability was determined by trypan blue dye exclusion.

Bronchoalveolar lavage fluid (BALF) recovery and processing

BALF was collected after euthanasia as previously described (Williams et al. 2015; Lehmann and Williams 2018). Briefly, the trachea was exposed and cannulated, and the left lung was clamped off. The right lung was washed three times with 0.6 mL room temperature Ca++, Mg++ and phenol red-free HBSS (Life Technologies, Bethesda, MD). Approximately 85% of the instilled volume was recovered in all treatment groups. The second two washes were pooled. All washes were temporarily stored on ice. BALF cells pelleted by centrifugation (360 x g for 15 minutes at 4°C). The supernatant from the first wash was used for lactate dehydrogenase (LDH) and total protein measurements. The cell pellets from all three washes were pooled and resuspended in 1 mL Ca++, Mg++ and phenol red-free HBSS for cell count, viability and differential determinations.

BALF cell counts and differentials

The total number of cells in BALF was determined using a Coulter Counter (Coulter, Hialeah, FL), and viability was assessed by trypan blue dye exclusion. Cells (200 μL of suspension) were cytocentrifuged (Shandon Southern Instruments, Sewickley, PA) onto duplicate slides for 10 minutes at 200 rpm. After drying, slides were stained with Wright Giemsa Stain Pack (Fisher Scientific, Suwanee, GA) using a Hema-tek 2000 (Miles Inc., Elkhart, IN). BALF differential cell counts and percentages were determined by counting 200 total cells per slide.

In Vitro EpiDerm™ Skin Irritation Test

The in vitro dermal irritation potential of CDDP was investigated using the EpiDerm Skin Irritation Test kit and protocol (Figure 1D; MatTek, Ashland, MA). On experimental day 0 (i.e., upon receipt), EpiDerm tissues were pre-incubated in culture medium overnight (37°C, 5% CO2, 95% RH) to release transport-stress related compounds and debris. After pre-incubation, the tissues (3/test substance) were topically exposed to the test substances (1% CDDP prepared in 12.5% DMSO) or manufacturer provided positive control (5% SDS) or known skin irritant (Williams et al. 2014) lactic acid (prepared in 12.5% DMSO) for 60 minutes at 37°C, 5% CO2, 95% RH. After the exposure period, the tissues were thoroughly rinsed, blotted to remove residual test substances, and transferred to fresh culture medium. After a 24-hour incubation period, the medium was replaced with fresh medium and the tissues were incubated for an additional 18 hours. Afterwards, the tissues were transferred to fresh plates containing MTT medium (1 mg/ml). After a 3-hour MTT incubation, the blue formazan salt was extracted with isopropanol (2.0 ml/tissue) and the optical density of the extracted formazan was determined using a spectrophotometer at 570 nm. The relative cell viability was calculated for each tissue as % of the mean of the negative control tissues. Skin irritation potential of the test material is predicted if the remaining relative cell viability is below 50% (https://www.jove.com/video/1366/an-vitro-skin-irritation-test-sit-using-epiderm-reconstructed-human).

Total serum IgE detection

Total serum IgE was determined as previously described (Williams et al. 2015; Lehmann and Williams 2018) using a commercial ELISA-based colormetric assay kit according to the manufacturer’s instructions (BD Pharmingen, San Diego, CA).

Total protein and LDH detection

Lactate dehydrogenase (LDH) activity and total protein content of BALF were assessed as previously described (Williams et al. 2015; Lehmann and Williams 2018) using commercially available kits (Thermo Fisher Diagnostics) adapted for use with the Konelab 30 clinical chemistry analyzer (Thermo Clinical Lab Systems, Espoo, Finland).

Statistical analysis

Statistical significance was defined as p < 0.05 as evaluated by one-way analysis of variance (ANOVA) and Tukey’s post hoc multiple comparisons test.

Results

Indicators of Dermal Sensitization to CDDP

CDDP has not been previously evaluated for effects on respiratory function in this mouse model of platinum hypersensitivity. To address this research gap, BALB/c mice dermally sensitized to CDDP were challenged by OPA instillation of CDDP (Figure 1B). Consistent with other published studies (Dearman et al. 2013; Williams et al. 2014), challenge with CDDP (100 μg) resulted in an increase in lymph node cells present in the ALN draining the site of topical exposure of sensitized mice (Figure 2A; p<0.05).

Figure 2. Indicators of skin sensitization.

Figure 2.

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(A) Cells harvested from the auricular lymph nodes (ALN) were counted using a Coulter counter. Data shown are +/− SEM (n = 6 mice/group. *p < 0.05 compared to non-sensitized (NS) mice challenged with saline (ANOVA#p < 0.05 compared to non-sensitized mice challenged with 100 μg CDDP (ANOVA). #p < 0.05 compared to sensitized mice challenged with saline (ANOVA). (B) The potential for CDDP to induce local skin irritation was assessed using the EpiDerm Skin Irritation Test (n = 3 tissues/ exposure group). (C) Total serum IgE levels were determined by ELISA. Data shown are +/− SEM (n = 6 mice/group. *p < 0.05 compared to non-sensitized (NS) mice challenged with saline (ANOVA). #p < 0.05 compared to non-sensitized mice challenged with 100 μg CDDP (ANOVA).

To demonstrate that the observed increase in lymph node cell proliferation was the consequence of sensitization and not excessive local inflammation, we topically exposed human reconstructed epidermis (RHE) tissues to 1% CDDP for 24 hours before evaluating tissue viability as an indicator of skin irritation. Responses to the negative control (TC-PBS), known skin irritant (25% lactic acid) and manufacturer recommended positive control (5% SDS) were as expected (Figure 2B). CDDP reduced the viability of the EpiDerm tissues compared to control by only 25% (skin irritants reduce relative viability by ≥ 50% (Figure 2B). Similarly, there were no indications of dermal irritation (i.e., redness/swelling) following administration of CDDP to the back and ears.

Importantly, topical application of 1% CDDP induced significant increases in total serum IgE levels in CDDP-sensitized mice challenged with either saline or 100 μg CDDP on experimental day 24 (Figure 2C; p<0.05).

Pulmonary Responses in Mice Sensitized to CDDP

Immediately following OPA challenge with CDDP, respiratory responses were assessed by whole-body plethysmography. Irrespective of the sensitization state, baseline Penh values, were unaffected by sensitization and challenge with CDDP (Figure 3A). Average baseline maximum Penh values and IR maximum Penh values were also unaffected by treatment (Figure 3B). Similarly, sensitization and challenge with CDDP had no effect on baseline respiratory rate (Figure 3C) and tidal volume (data not shown).

Figure 3. Lung instillation of CDDP did not affect the immediate response (IR).

Figure 3.

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Pulmonary responses were measured using whole-body plethysmography. CDDP did not induce an IR (A), significantly alter maximum Penh (B) or alter percent baseline respiratory rate (RR) on experimental day 24 (C). CDDP altered responsiveness of sensitized mice challenged with 100 μg CDDP in the presence of 12.5 mg/mL methacholine (D). 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 sensitized mice challenged with saline (ANOVA).

Two days after assessing the antigen-specific IR (i.e. on experimental day 26), we measured respiratory responsiveness to non-specific stimuli (i.e., Mch) was evaluated in the same mice used for the IR assessment. Baseline Penh values were obtained prior Mch exposure. Baseline Penh values were significantly elevated for sensitized mice challenged with 100 μg CDDP averaging 3.3 (+/− 0.7) Penh (Figure 4C; p<0.05). Compared to non-sensitized and sensitized mice challenged with saline, baseline respiration rate was significantly reduced in non-sensitized and sensitized mice challenged with 100 μg CDDP (Figure 4D; p<0.05).

Figure 4. Lung instillation of CDDP altered responsiveness to methacholine (Mch).

Figure 4.

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Pulmonary responses were measured using whole-body plethysmography. (A) CDDP-sensitized mice exhibit a dose-dependent increase in Mch responsiveness on experimental day 26. (B) CDDP-sensitized mice exhibit a significant increase in maximum Penh, (C) average baseline Penh and (D) dose-dependent decrease in percent baseline respiratory rate (RR) 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 sensitized mice challenged with saline (ANOVA). S = sensitized; NS = non-sensitized.

After collecting baseline data, mice were exposed to increasing doses of Mch aerosol. Average Penh values dose dependently increased in sensitized mice challenged with CDDP (Figure 4A and Figure 3D). Average Penh peaked at 4.6 (+/−1.3) and 7.6 (+/−1.8) for sensitized mice challenged with CDDP when exposed to 12.5 mg/mL or 25 mg/mL Mch, respectively (Figure 4A; p<0.05). Similarly, statistically significant increases in maximum Penh were observed for the 12.5 and 25 mg/mL Mch groups when compared to mice challenged with saline (Figure 4B; p<0.05). Exposure to 12.5 mg/mL Mch produced dramatic penh values in all 6 of the sensitized mice challenged with CDDP and approached statistical significance (p = 0.06) whereas penh values for 3 of 6 mice in the non-sensitized group challenged with CDDP were comparable to control values (i.e., penh ~2). Tidal volume was unaffected by CDDP sensitization (data not shown).

Inflammatory cells infiltrate the airways of mice exposed to platinum compounds

Irrespective of the sensitization state, BALF total cell counts in mice challenged with CDDP were significantly increased compared to mice challenged with saline (Figure 5A; p<0.05). The number of neutrophils in the lung increased from 0.26 (±0.2) × 104 in non-sensitized mice challenged with saline to 22.3 (±9.4) × 104 in sensitized mice challenged with CDDP (Figure 5B; p<0.05). The number of eosinophils, a marker of allergic inflammation (Akuthota et al. 2011), found in BALF increased significantly in both non-sensitized and CDDP-sensitized mice challenged with 100 μg CDDP (Figure 5B; p<0.05). There were no statistically significant differences in the number of macrophages and lymphocytes in any experimental group.

Figure 5. Lung instillation of CDDP results in differential cellular infiltration into BALF.

Figure 5.

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(A) BALF was collected from non-sensitized and CDDP-sensitized mice on experimental day 26 and enumerated using a Coulter counter. (B). Neutrophils and eosinophils infiltrate the BALF of CDDP-sensitized mice. *p < 0.05 compared to non-sensitized mice challenged with saline (ANOVA). #p < 0.05 compared to non-sensitized mice challenged with 100 μg CDDP (ANOVA). ^p < 0.05 compared to sensitized mice challenged with saline (ANOVA). $p < 0.05 compared to sensitized mice challenged with 100 μg CDDP (ANOVA).

Biochemical markers of inflammation in response to AHCP and CDDP

Changes in BALF LDH levels are often used as an indicator of non-specific cellular damage. BALF LDH levels were significantly increased from 38.3 (+/− 5.9) U/I to 109.2 (+/−12.4) U/I and 35.5 (+/− 4.3) U/I to 83.3 (+/− 22.4) U/I in non-sensitized and sensitized mice challenged with 100 μg CDDP, respectively. However, the effect was only significant in non-sensitized mice (Figure 6A & B; p<0.05). Elevated levels of protein in BALF indicate increased permeability of the lung epithelium, which is a measure of pulmonary edema. BALF protein levels were only significantly elevated in non-sensitized mice challenge with 100 μg CDDP (Figure 4B; p<0.05).

Figure 6. LDH and protein levels are elevated in BALF collected from CDDP-challenged mice.

Figure 6.

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(A) Protein levels in BALF were determined by Coomassie blue. (B) Lactate dehydrogenase levels were determined using Thermo LD-L Kit reagent and Thermo Trace Data-Trol controls. *p < 0.05 compared to non-sensitized mice challenged with saline (ANOVA). ^p < 0.05 compared to sensitized mice challenged with saline (ANOVA).

Discussion

The physiological mechanisms underlying the development of hypersensitivity to medically-useful platinum agents are not well-understood. It has been suggested that these reactions are likely the result of the development of type I hypersensitivity (Makrilia et al. 2010), but other explanations are plausible and the potential for CDDP to induce type I hypersensitivity responses has not been directly evaluated in an animal model. Consequently, investigators must apply a weight of the evidence approach, assessing a variety of complementary endpoints along the path from exposure to development of sensitization and ultimately manifestation of symptoms following re-exposure (i.e., challenge).

In this exploratory study, mice were topically sensitized and later subjected to intra-airway challenge with CDDP. Using this established model, we demonstrated that mice topically exposed to CDDP 1) exhibit increased lymph node cell proliferation, a characteristic consistent with allergic sensitization, (2) did not experience an IR following targeted delivery of CDDP to the lung, 3) are responsive to non-specific stimuli (i.e. Mch), 4) experience neutrophilic and eosinophilic infiltration of the lung and, 5) have significantly elevated levels of total serum IgE that persisted in CDDP-sensitized mice for 11 days after the final topical exposure. To our knowledge, this is the first laboratory investigation to report changes in serum IgE induced by CDDP sensitization. While that is the case, it must be recognized that we quantified total serum IgE, not antigen-specific IgE. Taken together, these data indicate that dermal exposure to CDDP induces immunological changes consistent with the development of type I hypersensitivity and that a single respiratory challenge is enough to trigger pulmonary responses in dermally sensitized mice.

Compared to other halogenated platinum salts tested in this model (i.e., AHCP and ATCP), CDDP behaved somewhat uniquely (Table 1). Targeted lung delivery of CDDP did not produce an IR in sensitized mice or alter maximum or baseline Penh values. Respiratory rate was also unaffected. The presence of an IR is not a universal finding in studies of this type. In some instances, it may be necessary to prime the lungs of mice previously sensitized to an allergen by the dermal route through targeted airway exposure (Scheerens et al. 1999; Matheson et al. 2001; Matheson et al. 2005; Selgrade et al. 2006). In the clinical setting, time delay between therapeutic platinum agent exposures is a risk factor for the development of platinum hypersensitivity (Makrilia et al. 2010). However, as indicated by responsiveness to Mch, eosinophil number, and IgE levels, priming the lung was not essential to the development and manifestation of responses consistent with hypersensitivity in our model. Still, modifying study design parameters to distribute the dermal sensitization doses over a longer time frame or to incorporate additional IR challenges may be necessary for the development of an IR. The likelihood of detecting an IR may also be increased by extending the post-OPA observation period was beyond 1 hour to 3 or 4 hours, as was the case in a report published Anderson and colleagues (Anderson et al. 2013).

Table 1.

Summary data and comparisons to previously published studies.

From Williams et al. 2015 From Lehmann and Williams 2018 Current study
AHCP-sensitized ATCP-sensitized CDDP-sensitized
100 μg AHCP challenge 100 μg ATCP challenge 100 μg CDDP challenge
IR
(Penh; +/−SEM)		 9.2 (0.6) 2.3 (0.6) NE
Response to 25 mg/mL Mch (Penh; +/−SEM) 5.3 (0.6) 5.5 (1.2) 7.6 (1.8)
Response to 25 mg/mL Mch (Maximum Penh; +/−SEM) ND ND 22.1 (4.4)
Total BALF cell count (x1O4, +/−SEM) 16.8 (2.2) 11.6 (2.1) 37.7 (9.4)
% Eosinophils
(+/−SEM)		 5.1% (1.4) 3.3 (1.0) 6.9 (2.7)
% Neutrophils
(+/−SEM)		 27.2 (6.3) 27.2 (6.4) 70.0 (8.7)
BAL protein
(+/−SEM)		 474.2 (87.0) 181.6 (31.9) 341.8 (102.6)
BAL LDH
(+/−SEM)		 75.8 (9.9) 53.8 (3.6) 83.3 (22.4)
Total serum IgE
(+/−SEM)		 19,204.5 (2376.8) 9,018.7 (927.0) 11,333.7 (1,400)
ALN counts (x 106; +/−SD) 4.1 (0.1) 5.5 (0.5) 11.4 (1.7)
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AHCP = ammonium hexachloroplatinate; ATCP = ammonium tetrachloroplatinate; CDDP = cis-diamminedichloroplatinum (II); IR = antigen-specific immediate response; Penh = enhanced pause; SEM = standard error of the mean; Mch = methacholine; BALF = bronchoalveolar lavage fluid; LDH = lactate dehydrogenase; IgE = immunoglobulin E; ALN = auricular lymph nodes; ND = not determined; NE = no effect.

CDDP-sensitized mice were, however, responsive to the non-specific stimulus Mch. Compared to non-sensitized mice, dramatically elevated maximum Penh values were observed in CDDP-sensitized mice challenged with 100 μg CDDP at the 12.5 and 25 mg/mL Mch levels. Interestingly, baseline Penh values were also significantly elevated in these mice two days after targeted lung delivery of CDDP. While we are not certain of the cause of this result, the high baseline Penh values observed could be the result of allergic mechanisms to CDDP or direct lung damage caused by CDDP. CDDP is known to be cytotoxic (Kosmidis et al. 2019) and it is plausible that the compound damaged the lung, an explanation supported by increased BALF LDH activity and total protein levels we observed. Lung irritation-may have also contributed to the high Penh values observed in non-sensitized mice challenged with CDDP. However, unlike with sensitized mice challenged with CDDP, responses to Mch were not significantly different from non-sensitized and sensitized mice challenged with saline. Airway hyperresponsiveness data collected during this exploratory study should be expanded on to include lower, less irritating CDDP challenge doses (e.g., 25 or 50 μg) and confirmed with specific measures of airway resistance and compliance using a larger sample size to enhance statistical power.

Using the same mice evaluated for pulmonary responses, we evaluated changes in BALF composition. Introduction of CDDP into the lungs of mice produced a dramatic influx of neutrophils into BALF. AHCP and ATCP increased the proportion of neutrophils in BALF from <5% in mice challenged with either platinum compound to approximately 25% (Williams et al. 2015; Lehmann and Williams 2018), whereas CDDP challenge increased the number of neutrophils in BALF in both non-sensitized and sensitized mice . Neutrophils are the most abundant leucocyte in blood and, by acting as first responders, are key mediators of acute inflammation (Rosales 2018). Accumulation of neutrophils in the airways has been reported in mouse models of allergic airway diseases, and neutrophil numbers have also been shown to correlate with the severity of airway obstruction in patients (Park et al. 2006). The observed influx of neutrophils into the lungs of both non-sensitized and sensitized mice suggests a non-specific inflammatory response. This view is supported by elevated protein levels and LDH activity in BALF and elevated Penh values in non-sensitized mice following a single challenge with CDDP.

Other insights into platinum hypersensitivity can be gleaned from this study. Of particular interest, early reports of platinum hypersensitivity suggested that platinum compounds containing more chlorines are more potent sensitizers (Cleare et al. 1976; Murdoch and Pepys 1984a, 1984b, 1985, 1986; Schuppe et al. 1992; Schuppe et al. 1997; Linnett and Hughes 1999). In contrast, we showed that the dermal sensitizing potency of halogenated platinum salts was not influenced by the number of chlorines the platinum compound contained when evaluated in the LLNA (Williams et al. 2014). The utility of LLNA potency values for respiratory sensitizers has been questioned (Basketter and Kimber 2011; Williams and Lehmann). Using our mouse model with the capacity to evaluate changes in lung function, we showed that the magnitude of the IR response to AHCP was greater than the response to ATCP (Lehmann and Williams 2018), an outcome consistent with the aforementioned chlorination hypothesis. In the current study, CDDP, which only contains two chlorines, did not induce an IR providing further evidence that the number of chlorines coordinated with platinum may influence the manifestation of type I hypersensitivity symptoms. In addition, these findings provide more evidence that LLNA-derived potency values may be of limited utility for respiratory sensitizers. Follow-up studies are necessary to better understand the role chlorination plays in the manifestation of allergic effects in dermally sensitized mice.

Using a weight of the evidence approach, we arrived at the conclusion that CDDP exposure likely triggered a type I hypersensitivity reaction. However, the possibility remains that the effects we observed relate to other mechanisms. For example, the distinction between the different hypersensitivity classes is not always clear cut. Importantly, type IV reactions can occur in the lung triggering induction of airway hyperreactivity (Garssen et al. 1991). Although type IV reactions were traditionally aligned with Th1 cells, there are type IV hypersensitivity subtypes mediated by CD8+ and Th2 T-cells. When mediated by Th2 cells, a chronic asthma condition associated with eosinophil recruitment to the lung can develop (Czarnobilska et al. 2007). Given these points, additional investigations using this model and other approaches (e.g., T cell subset determinations) are necessary to better understand the contributions of different hypersensitivity types to CDDP hypersensitivity.

In addition to being useful for elucidating the mechanisms underlying CDDP hypersensitivity responses, this model will also be useful for investigating the contribution of different exposure routes to the development and manifestation of type I hypersensitivity and for evaluating cross-reactivity between platinum compounds. Knowing that topical exposure to CDDP induced sensitization and that re-exposure triggered reactions consistent with type I hypersensitivity, minimizing topical exposure during the manufacturing and processing of CDDP is necessary. Otherwise, exposed individuals may be at risk of developing systemic anaphylaxis.

Although we gained insights into platinum hypersensitivity using this model, our study design has some limitations that should be discussed. As evidenced by large numbers of neutrophils and elevated LDH levels in non-sensitized mice and the known cytotoxic properties of CDDP, the CDDP challenge dose used in this study (i.e., 100 μg) was likely too high thereby complicating our efforts to tease out subtle differences between non-sensitized and sensitized mice. Including histology in our study design would have enabled us to garner information about changes in airway morphology underlying the functional changes we observed. This type of evaluation would also provide an opportunity to corroborate observed changes in the cellular composition of BALF collected from non-sensitized and sensitized mice. The strength of our conclusions would have also been enhanced by a design that included more animals per exposure group.

Recently, we demonstrated cross-reactivity between two industry relevant halogenated platinum salts (Lehmann and Williams 2018). Cross-reactivity between therapeutic platinum agents is a well-known phenomenon and can alter the course of treatment. While switching to a different platinum compound is an option, the possibility of potentially life-threating hypersensitivity response remains (Makrilia et al. 2010). Use of this model presents the opportunity to evaluate the potential for cross-reactivity experimentally thereby promoting patient safety.

Conclusions

Herein, we applied a mouse model to investigate CDDP hypersensitivity responses. Using this model, we demonstrate in topically sensitized mice, a single airway challenge with CDDP is capable of inducing immunological changes consistent with type I hypersensitivity. We also identified potentially important points of mechanistic divergence between CDDP hypersensitivity and hypersensitivity to chloroplatinates. This model enables future investigations of the 1) variable allergenic potential of different therapeutic platinum compounds, 2) investigations of potential new delivery routes and delivery vehicles, and 3) cross-reactivity between therapeutic platinum compounds. Findings from future studies using this model may have important implications for human health risk assessment.

Acknowledgements:

The authors thank C. Copeland, L. Copeland, D. Andrews and J. Richards for their expert technical assistance. We also thank Drs. A. Farraj, R. Koethe and M.J. Selgrade for their thoughtful and critical review of this work.

Abbreviations:

AHCP

ammonium hexachloroplatinate

ATCP

ammonium tetrachloroplatinate

CDDP

cis-diamminedichloroplatinum (II)

LA

lactic acid

SDS

sodium dodecyl sulphate

IgE

immunoglobulin E

LLNA

local lymph node assay

WBP

whole-body plethysmography

Mch

methacholine

IR

immediate response

DMSO

dimethyl sulfoxide

OPA

oropharyngeal aspiration

TMB

tetramethylbenzidine

Penh

enhanced pause

BALF

bronchoalveolar lavage fluid

RR

respiratory rate

LDH

lactate dehydrogenase

NS

non-sensitized

S

sensitized

ANOVA

analysis of variance

Footnotes

Declaration of interest

This article has been reviewed by the U.S. Environmental Protection Agency and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency or of the US Federal Government, nor does the mention of trade names or commercial products constitute endorsement or recommendations for use of those products. The authors report no financial or other conflicts of interest. The authors alone are responsible for the content and writing of this article.

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