Cytotoxic pulmonary injury
Multiple mechanisms may be responsible for cytotoxic pulmonary injury due to drugs, including reactive oxygen species (ROS) [
21,
23‐
25], reduction in deactivation of metabolites of the lung [
26‐
28], impairment of alveolar repair mechanisms [
29‐
31], and release of various cytokines [
23]. Many agents may be toxic to the lungs. These include cytotoxic drugs, such as bleomycin, MTX, and cyclophosphamide, and non-cytotoxic drugs, such as nitrofurantoin, sulfasalazine, and amiodarone [
32].
Chemotherapy lung is one representative example of cytotoxic lung injury. It is a severe type of pulmonary reaction that develops during or shortly after treatment with chemotherapeutic agents, such as antibiotics, alkylating agents, anti-metabolites, nitrosamines, rapamycin analogs, and podophyllotoxins [
4]. Histologically, chemotherapy lung corresponds to DAD [
1,
3]. Concurrent radiation or oxygen therapy increases the risk of developing chemotherapy lung. Moreover, chemotherapy lung can sometimes develop because of previously unresolved chemotherapy- or radiation-induced damage with additional chemotherapy [
3].
Mechanisms of cytotoxic pulmonary injuries
The pathogenesis of cytotoxic lung injury may include direct injury to pneumocytes or the alveolar capillary endothelium, with subsequent release of cytokines and recruitment of inflammatory cells. The systemic release of cytokines induced by chemotherapeutic agents (e.g., gemcitabine) may also result in capillary leakage and pulmonary edema. Early events in lung injury induced by tricyclic antidepressants may be related to endothelial damage [
21] because of impaired tight junctions mediated by amitriptyline-induced perturbations in intracellular calcium [
21].
MTX-induced pulmonary toxicity may induce the release of free oxygen radicals, such as nitric oxide, and various cytokines, such as IL-1β, TNF-α, and TGF-β. Kim et al. reported that the p38MAPK signaling pathway was associated with a pulmonary inflammatory response [
23].
By impairing alveolar repair mechanisms, gefitinib may potentiate the effects of lung injury [
29,
30]. Suzuki et al. have suggested that gefitinib therapy may augment any underlying pulmonary fibrosis through decrease in epidermal growth factor receptor phosphorylation with coincident regenerative epithelial proliferation [
31].
The toxic mechanism of amiodarone leads to the disruption of the lysosomal membranes of molecules through protein C activation and the subsequent release of toxic oxygen radicals, which may induce activation of caspase pathways and lead to apoptosis of lung epithelial cells [
24]. An additional mechanism reduces deactivation of toxic metabolites of the drug [
26,
27].
Pulmonary toxicity may also be caused by the generation of free oxygen radicals by mitomycin C, nitrofurantoin, and bleomycin. These drug-induced ROS generate substances such as H
2O
2, O
2-, and OH [
21]. In vivo and in vitro studies showed bleomycine, a cancer chemotherapeutic agent, to be the cause of pulmonary toxicity, which was mediated, at least partly, by a bleomycin-iron complex, generating toxic O
2-derived species within the lung [
25]. Particular susceptibility to bleomycin toxicity in the lung may depend on the fact that bleomycin is preferentially distributed in lung tissue and that the lung is relatively deficient in the hydrolase enzyme that detoxifies bleomycin [
28].
Nitrofurantoin and bleomycin share the ability to generate O
2 radicals and to cause lung damage. The reason that these drugs affect the lungs as their predominant site of toxicity remains unclear. One possibility is the rate of gas exchange and high oxygen load in the lungs, which enables damage due to these drugs [
21].
Diagnosis of cytotoxic pulmonary injury
Drug-induced pulmonary toxicity can be difficult to diagnose because cancer patients are usually administered multiple anti-neoplastic agents; thus, identifying the causative agent becomes difficult. Unfortunately, no single diagnostic test or tissue biopsy is currently available that can definitively confirm a diagnosis of chemotherapy-associated lung disease [
33].
Currently, in vitro drug challenge is not a readily available or clinically validated diagnostic assay for cytotoxic lung injury. Reactive drug metabolites are believed to play a role in many drug reactions. Differences in the capacity of cells to detoxify the reactive metabolites of drugs are important determinants in drug toxicity reactions, and these differences could be used as the basis of a diagnostic assay. Microsomes are a source of oxidative enzymes, primarily cytochrome P450 (CYP). Cell viability can be determined after incubating microsomes with PBMCs and the suspected drug in the presence or absence of a microsomal activating system. This assay has not been used for the diagnosis of cytotoxic lung injury. However, this approach may enhance our understanding of selected drugs that cause DILD, paving the way for development of clinically useful assays [
34,
35].
Mechanisms and diagnosis of immune-mediated DILD
Lungs provide a barrier to illness in which the immune system is chronically activated to provide optimal host defense. This constant, low-level activation provides a milieu that may facilitates pro-inflammatory signals and subsequent immune system responses.
Minocycline-induced pneumonia (MIP) is generally manifested as EP. A central role for T lymphocytes in the immunologic reaction to MIP was suggested by Gillon et al., who identified lymphocyte-mediated specific cytotoxicity against minocycline-bearing alveolar macrophages in vitro [
42]. Their findings, however, do not explain the presence of pulmonary eosinophilia, which is a characteristic feature of MIP. Thus, further investigations are needed to elucidate the pathophysiology of MIP. Three different mechanisms of amiodarone induced lung disease have been suggested: a direct toxic effect, an immune-mediated mechanism, the angiotensin enzyme system activation [
9]. From the immunological perspective, Kuruma et al. reported that the Th1/Th2 balance may influence amiodarone metabolism and may be a powerful indicator of amiodarone-induced subclinical lung toxicity [
43].
(a)-(i) in vitro tests for DILD
DLST and LMT are bioassays that help to confirm the presence of drug-sensitized lymphocytes. The DLST verifies the growth of sensitized lymphocytes after a drug is used for antigen stimulation, while the LMT identifies the cytokines or chemokines produced by sensitized lymphocytes after a drug is used for antigen stimulation [
45].
The DLST is the most commonly used in vitro test for detecting the causative drug in cases of drug allergy. The causes of drug allergies determined using the DLST have been extensively reported in cases of drug eruption [
44,
46,
47]. Laboratory-based in vitro methods, such as the DLST, have numerous advantages, including absolute safety, ability to assess T cell responses to multiple drugs simultaneously, and absence of risk of developing additional drug allergies [
44]. This technique measures the uptake of a DNA precursor (tritiated
3 H] thymidine) after lymphocytes have been exposed to an antigen in vitro. This test is associated with blast formation by lymphocytes [
44].
The LMT demonstrates the presence of sensitized lymphocytes when granulocytes, either alone or mixed with normal lymphocytes, exhibit migration inhibition when cultured at optimal drug concentrations. Saito et al. reported that the LMT had a higher positive response rate than the DLST for several hypersensitivity symptoms, such as skin eruptions and hepatic injury [
45].
While DLST has been widely used in the diagnosis of DILD in Japan, this is not the case in other countries. Compelling data as to the sensitivity and specificity of the DLST for DILD is currently lacking. A cell-mediated hypersensitivity reaction on DLST is the basis for diagnosis of gold-induced pneumonia (GIP). Tomioka and King included CD8+ lymphocytic alveolitis and a positive DLST in their diagnostic scheme for GIP [
39]. However, GIP is rarely a clinical problem at the present time. Studies on the use of DLST in DIP are far from well controlled. Based on data that compared the results of the DLST and provocation tests for patients with DILD, DLST contributed little in detecting the causative agents in these patients [
48,
49].
DLST is believed to be insensitive to MIP. Toyoshima et al. reported the results of DLST for minocycline in six patients; in all cases, the results were negative [
50]. In addition, suppressive effects of minocycline on T cell proliferation have been described [
51,
52].
For MTX and Kampo (Japanese herbal) drugs, the results of DLST tend to be overestimated. Many reports have provided evidence that the uptake of
3 H thymidine into lymphocytes in the presence of MTX may be explained by the upregulated incorporation of thymidine from the extracellular space following the depletion of the intracellular thymidine pool caused by MTX. In one study, MTX created an early block in the cell cycle without reducing the cellular uptake of
3 H-thymidine [
53]. Hoffman also found a discrepancy between the formation of blast-like cells and high thymidine uptake in MTX-treated, mitogen-stimulated lymphocytes [
54]. Hirata et al. showed that DLST using MTX was inadequate in confirming MTX-induced DILD [
55].
Kampo drugs are generally contaminated with non-specific mitogens from plants. Mantani et al. reported positive DLST results for Kampo drugs in 85.7% of enrolled patients not taking any Kampo medicines [
56]. In addition, several studies reported discrepancies between DLST findings and results of provocation drug tests in Kampo drugs [
7,
18,
48,
49].
Leukocyte migration inhibition factor production has also been observed in well-established cases of hypersensitivity pneumonitis, such as that due to beryllium [
42,
57]. Various reports have drawn attention to the positive results of this test in cases of DILD due to different drugs, such as minocycline, amiodarone, propranolol, nitrofurantoin, gold salts, MTX, and paclitaxel [
41,
57‐
61]. However, the number of samples used in these reports were small.
CD69 upregulation on T cells has been reported as an in vitro marker for delayed-type drug hypersensitivity reactions [
62]. Compared with other early T cell activation markers, such as CD71 or CD25, CD69 appeared to be most suitable, as it was rapidly upregulated and showed the greatest difference from baseline values [
63].
CD69 upregulation was shown to be drug-specific and not an inherent property of cells derived from patients. It did not occur in unstimulated cultures or in drug-stimulated PBMCs from non-allergic donors. Cells from drug-allergic subjects reacted only to the causative drug, but not to any other tested drugs [
63]. This assay has not been used for the diagnosis of DILD. However, this approach may enhance our understanding of selected drugs that cause DILD.
(a)-(ii) drug provocation tests (DPT)
Rechallenge of patients with DILD is generally considered unethical as the pulmonary damage caused by DILD is largely irreversible, and re-challenge increases the risk to the patient. Re-test of drugs has been safely performed in some patients, although a fatality was reported following a re-test with MTX in one case [
64]. Several investigators have conducted re-tests with minocycline to establish an accurate diagnosis [
65] with no reported morbidity. Similar findings have been reported for EP induced by other drugs, such as sulfalazine [
3], amoxicillin [
7], and nimesulide [
66].
Recently, Yasui et al [
48]. proposed a DPT method for mild DILD. DPT was performed in 58cases, 41 of which showed positive results. This test was initiated by administering the lowest dosage of the suspected causal drug that could achieve a response, and then gradually increasing the dose at daily intervals until a normal daily dose was reached or symptoms occurred. This DPT protocol was deemed useful according to the criteria provided by Yasui et al [
48,
49]. Their diagnostic criteria for DIP included a 1°C increase in body temperature and one or more of the following an increase in the alveolar-arterial difference in oxygen tension (A-aDO2) of >10 mmHg; an increase >20% in white blood cell count; and positive conversion of C-reactive protein [
48,
49].