Autocrine hemokinin-1 functions as an endogenous adjuvant for IgE-mediated mast cell inflammatory responses.

BACKGROUND: Efficient development of atopic diseases requires interactions between allergen and adjuvant to initiate and amplify the underlying inflammatory responses. Substance P (SP) and hemokinin-1 (HK-1) are neuropeptides that signal through the neurokinin-1 receptor (NK1R) to promote inflammation. Mast cells initiate the symptoms and tissue effects of atopic disorders, secreting TNF and IL-6 after FcεRI cross-linking by antigen-IgE complexes (FcεRI-activated mast cells [FcεRI-MCs]). Additionally, MCs express the NK1R, suggesting an adjuvant role for NK1R agonists in FcεRI-MC-mediated pathologies; however, in-depth research addressing this relevant aspect of MC biology is lacking. OBJECTIVE: We sought to investigate the effect of NK1R signaling and the individual roles of SP and HK-1 as potential adjuvants for FcεRI-MC-mediated allergic disorders. METHODS: Bone marrow-derived mast cells (BMMCs) from C57BL/6 wild-type (WT) or NK1R(-/-) mice were used to investigate the effects of NK1R signaling on FcεRI-MCs. BMMCs generated from Tac1(-/-) mice or after culture with Tac4 small interfering RNA were used to address the adjuvancy of SP and HK-1. WT, NK1R(-/-), and c-Kit(W-sh/W-sh) mice reconstituted with WT or NK1R(-/-) BMMCs were used to evaluate NK1R signaling on FcεRI-MC-mediated passive local and systemic anaphylaxis and on airway inflammation. RESULTS: FcεRI-activated MCs upregulated NK1R and HK-1 transcripts and protein synthesis, without modifying SP expression. In a positive signaling loop HK-1 promoted TNF and IL-6 secretion by MC degranulation and protein synthesis, the latter through the phosphoinositide 3-kinase/Akt/nuclear factor κB pathways. In vivo NK1R signaling was necessary for the development of passive local and systemic anaphylaxis and airway inflammation. CONCLUSIONS: FcεRI stimulation of MCs promotes autocrine secretion of HK-1, which signals through NK1R to provide adjuvancy for efficient development of FcεRI-MC-mediated disorders.

In situ generation of milk protein-derived peptides in drying-off cows.

Our previous studies demonstrated prompt elevation of proteinase activity in mammary secretion of drying-off cows and goats. The current study examined the progressive changes in composition of cow mammary secretion following drying-off and, in parallel, characterized the mode of peptide neogenesis using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and liquid chromatography-electrospray-ionization (LC-ESI) MS/MS. The results show that the percentage of casein of total milk protein at time of drying-off was 76%, which dropped to 41%, 24%, and 16%, respectively, 1, 2, and 3 weeks after drying-off. Levels of ß-lactoglobulin and α-lactoalbumin in mammary secretions of drying-off cows decreased prominently while levels of lactoferrin, BSA, and casein derived-proteins increased concomitantly compared with regular milk. A fractionation procedure was applied to remove molecules larger than 10 kDa before MALDI-TOF MS and LC-ESI MS/MS and the results show that the MALDI-TOF MS peptide profile of mammary secretion ranging from m/z 600 to 4000 was apparently modified after drying-off for 1 week, whereas species 1590 m/z and 2460 m/z were most obviously enriched compared with regular milk. LC-ESI MS/MS results were used to map peptide sequence with Mascot search server and under no post translational modification to reduce database size and 202 novel ß-casein-derived peptides were successfully identified in mammary secretion after drying-off for 1 week in contrast to regular milk. Accordingly at least 48 additional cleavage positions were assigned on ß-casein for mammary secretion. Among the 202 novel peptides, 5 are homologous with confirmed opioid agonists, angiotensin 1-converting enzyme inhibitors, or immuno-modulators. In conclusion, peptides are released in situ from milk proteins within short intervals following drying-off in cows. They might play roles in the transition of mammary glands from lactating to non-lactating. With specified post-translational modifications and focused functional screening, novel peptides are yet to be discovered in dry cow mammary secretion.