Covalent Binding, Protein leads to Increased proinflammatory mediators

Multiple cell types in the lung and skin express the necessary pattern recognition receptors for this KER, including epithelial cells, endothelial cells, macrophages, fibroblasts, and dendritic cells. It is likely that different cell types are involved in the process. 

It has been demonstrated in lung cell lines, 3D human airway epithelial cell cultures, and human lung slices that exposure to haptens induces proinflammatory cytokine and chemokine (e.g. IL-1α, TNF-α, IL-6, IL-8, CCL2, CXCL1, CCL5, etc.) release. (Huang et al., 2013, Lauenstein et al., 2014, Verstraelen et al., 2009)

Exposure of reconstituted three-dimensional (3D) human airway epithelia (MulcilAir™) to respiratory sensitizers trimellitic anhydride (TMA) and methylene diphenyl diisocyanate (MDI) elevated the levels of proinflammatory cytokines and chemokines interleukin (IL)-6, IL-8, monocyte chemoattractant protein-1 (MCP-1)/chemokine ligand (CCL)2, growth regulated oncogene-a (GRO-a)/C-X-C motif (CX)CL1, and regulated on activation, normal T cell expressed and secreted (RANTES)/CCL5. (Huang et al., 2013) Similarly, typical respiratory sensitizers caused an elevation of proinflammatory cytokines IL-1a (TMA) and tumor necrosis factor (TNF)-a (glutaraldehyde) in precision-cut lung slices. (Lauenstein et al., 2014)

(Hur et al., 2009) found that toluene diisocyanate (TDI)-human serum albumin (HSA) conjugates increased reactive oxygen species (ROS) production in A549 cells. The significance of the Nrf2-Keap1 pathway in respiratory sensitization is not as extensively studied compared to skin sensitization, but in vitro data for a limited number of respiratory sensitizers showed that these are able to activate Nrf2-dependent genes both in airway and skin epithelium. (Emter et al., 2010, Natsch et al., 2013, Remy et al., 2014) Activation of Nrf2-Keap1 by skin sensitizers has been explained by covalent interaction of cysteine residues on Keap1 with cysteine-reactive chemicals, leading to Nrf2 association and transcriptional activation of genes. It is not fully understood how respiratory sensitizers activate this pathway. Although respiratory sensitizers are more likely to bind to hard nucleophiles such as lysine, (Enoch et al., 2010) in chemico studies show that cysteine binding occurs as well. (Lalko et al., 2011, 2013) Hence, Nrf2 activation may be a direct result of covalent interaction with cysteine residues or an indirect result of GSH depletion and an altered redox balance. The indirect activation of Nrf2-dependent genes was shown in THP-1 cells exposed to acid anhydrides, which had a preference to lysine in the direct peptide reactivity assay (DPRA) (Migdal et al., 2013); however, actual Nrf2 and heme oxygenase-1 proteins accumulated only minimally in the cells.

Both qualitative and quantitative characterization of inflammatory pathway activation in response to haptenation remains an area of investigation for sensitization.

Respiratory Sensitizers To elucidate which pathways respiratory sensitizers regulate, in vitro DNA microarray studies were performed in different human lung cell lines exposed to a limited set of respiratory sensitizers. These studies were not able to identify specific molecular pathways that were regulated by respiratory sensitizers. They could identify activation of genes, related to innate immune response. In human alveolar epithelial cells (A549 cell line), for example, genes encoding for TLR2, TNF-a, IL-1 receptor, and cytokine signaling pathways were upregulated by hexamethylene diisocyanate (HDI) and TMA. (Verstraelen et al., 2009) NLRP3 has been demonstrated to be important in respiratory sensitization by proteins, (Besnard et al., 2012) but the involvement in the induction of respiratory sensitization by low-molecular-weight chemicals is unknown. In human keratinocytes, the respiratory sensitizers MDI and TMA failed to elevate intracellular proinflammatory IL-18 levels. (Corsini et al., 2009) Conflicting reports as to whether IL-18 is associated with a Th1 or Th2 immune response hamper interpretation of this result.

Additionally, the canonical phosphatase and tensin homolog (PTEN)-signaling pathway might be relevant for respiratory sensitization. (Verstraelen et al., 2009) This pathway regulates cell survival signaling pathways and plays a protective role in the pathogenesis of asthma. (Kwak et al., 2003) In a mouse model of TDI-induced asthma, the PTEN pathway was shown to play a protective role in asthma pathogenesis, because it was involved in the regulation of IL-17 induction and NF-kB activation. (Kim et al., 2007) A more recent in vitro study showed that the PTEN pathway was not consistently induced by all respiratory sensitizers, since maleic anhydride and 7-aminocephalosporanic acid failed to induce this pathway but another diisocyanate, HDI, did. (Remy et al., 2014)

There is some evidence to support the hypothesis that the binding behavior of respiratory sensitizers is related to the eventual Th2-skewed immune response, with binding to lysine on serum albumin in particular, as well as secretion of type 2 cytokines, being associated with known respiratory sensitizers. (Hopkins et al., 2005) The biological hypothesis that lysine is the primary nucleophile responsible for respiratory sensitization is supported by the preference for harder electrophiles compared with those that cause skin sensitization (lysine is a harder nucleophile than cysteine). (Enoch et al., 2010) This is evidenced by the difference in the coverage of the various mechanistic domains that show typical respiratory sensitizers to be chemicals acting via the acylation and Schiff base mechanisms rather than Michael addition (a significantly important mechanism for skin sensitization). Structure/activity analysis has shown the importance of electrophilicity and protein crosslinking for respiratory sensitization for low-molecular-weight organic chemicals. (Hopkins et al., 2005, Agius et al., 1991, 1994, Seed and Agius, 2010, 2017)

One proposed explanation (Kimber et al., 2018) for the association between peptide selectivity and deviation between respiratory and dermal sensitization is based on the observation that respiratory sensitizers, in a co-culture including both U937 cells and serum, preferentially react with serum proteins such as albumin, which has a high number of lysine residues. This behavior was observed for TMA, fluorescein isothiocyanate (FITC), and dinitrobenzenesulfonyl chloride (DNBSCl). Concordantly, skin sensitizers dinitrochlorobenzene (DNCB), dinitrofluorobenzene (DNFB) preferentially bound to cellular proteins in the same co-culture. (Hopkins et al., 2005) This is corroborated by the observation that serum albumin is a major target protein of the respiratory sensitizer hexahydropthalic anhydride in humans. (Johannesson et al., 2001) Further, this is a reasonable hypothesis for the biological mechanism of deviation between skin and respiratory sensitizers, particularly in the case of dermal exposure, as the distribution of antigen formation of chemical allergens in the in vitro model system segregates with the type (Th1- or Th2-activating) of cytokines secreted from activated lymph node cells in an in vivo mouse model.

Haptenation is essentially instantaneous, and in order to protect the in vivo organism from further damage, local cellular responses to induce inflammation to haptens are rapid. As a result, relevant in vitro readouts to detect proinflammatory mediators (e.g. cytokine/chemokine secretion, redox responses) may be quantifiable within minutes to a few hours, but sensitivity and precision vary based on the assay detection method. Haptenated peptides generated in vitro can be quantified after 15 minutes. (Hettick, et al., 2009) Cellular assay protocols commonly quantify inflammatory secretion 24 – 48 hours after exposure.