In response to infections and irritants, the respiratory epithelium releases the alarmin interleukin (IL)-33 to elicit a rapid immune response. However, little is known about the regulation of IL-33 following its release. Here we report that the biological activity of IL-33 at its receptor ST2 is rapidly terminated in the extracellular environment by the formation of two disulphide bridges, resulting in an extensive conformational change that disrupts the ST2 binding site. Both reduced (active) and disulphide bonded (inactive) forms of IL-33 can be detected in lung lavage samples from mice challenged with Alternaria extract and in sputum from patients with moderate–severe asthma. We propose that this mechanism for the rapid inactivation of secreted IL-33 constitutes a ‘molecular clock' that limits the range and duration of ST2-dependent immunological responses to airway stimuli. Other IL-1 family members are also susceptible to cysteine oxidation changes that could regulate their activity and systemic exposure through a similar mechanism.
Autoantibodies directed against citrullinated epitopes of proteins are highly diagnostic of rheumatoid arthritis (RA), and elevated levels of protein citrullination can be found in the joints of patients with RA. Calcium-dependent peptidyl-arginine deiminases (PAD) are the enzymes responsible for citrullination. PAD2 and PAD4 are enriched in neutrophils and likely drive citrullination under inflammatory conditions. PADs may be released during NETosis or cell death, but the mechanisms responsible for PAD activity under physiological conditions have not been fully elucidated. To understand how PADs citrullinate extracellular proteins, we investigated the cellular localization and activity of PAD2 and PAD4, and we report that viable neutrophils from healthy donors have active PAD4 exposed on their surface and spontaneously secrete PAD2. Neutrophil activation by some stimulatory agents increased the levels of immunoreactive PAD4 on the cell surface, and some stimuli reduced PAD2 secretion. Our data indicate that live neutrophils have the inherent capacity to express active extracellular PADs. These novel pathways are distinguished from intracellular PAD activation during NETosis and calcium influx-mediated hypercitrullination. Our study implies that extracellular PADs may have a physiological role under non-pathogenic conditions as well as a pathological role in RA.
Activation-induced deaminase (AID) catalyses deamination of deoxycytidine to deoxyuridine within immunoglobulin loci, triggering pathways of antibody diversification that are largely dependent on uracil-DNA glycosylase (uracil-N-glycolase [UNG]). Surprisingly efficient class switch recombination is restored to ung−/− B cells through retroviral delivery of active-site mutants of UNG, stimulating discussion about the need for UNG's uracil-excision activity. In this study, however, we find that even with the overexpression achieved through retroviral delivery, switching is only mediated by UNG mutants that retain detectable excision activity, with this switching being especially dependent on MSH2. In contrast to their potentiation of switching, low-activity UNGs are relatively ineffective in restoring transversion mutations at C:G pairs during hypermutation, or in restoring gene conversion in stably transfected DT40 cells. The results indicate that UNG does, indeed, act through uracil excision, but suggest that, in the presence of MSH2, efficient switch recombination requires base excision at only a small proportion of the AID-generated uracils in the S region. Interestingly, enforced expression of thymine-DNA glycosylase (which can excise U from U:G mispairs) does not (unlike enforced UNG or SMUG1 expression) potentiate efficient switching, which is consistent with a need either for specific recruitment of the uracil-excision enzyme or for it to be active on single-stranded DNA.
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