Novel function of DUSP14/MKP6 (dual specific phosphatase 14) as a nonspecific regulatory molecule for delayed‐type hypersensitivity

Summary Background  Nonspecific unresponsive states of delayed‐type hypersensitivity (DTH) to unrelated antigens are induced in mice by a single administration of hapten. In these studies, we found a unique regulatory mechanism of contact hypersensitivity (CHS) mediated by nonspecific suppressor factor (NSF) induced by the intravenous injection of hapten‐conjugated syngeneic spleen cells. NSF is a ≈ 45‐kDa protein released from the macrophage‐like suppressor cells and binds selectively to dendritic cells (DCs). Moreover, NSF‐treated DCs release a second ≈ 20‐kDa NSF (NSF int ). Objectives  To try and identify NSF and characterize its function. Methods  The suppressor activity was evaluated by inhibition of the passive transfer of CHS by the effector cells sensitized with hapten and the antigen‐presenting cell (APC) activity of hapten‐primed draining lymph node cells (DLNCs) to induce CHS. NSF‐containing supernatants obtained from the culture of spleen cells from mice that had been injected intravenously with oxazolone‐conjugated syngeneic spleen cells 7 days before were prepared and purified with a Green A dye‐affinity column, DEAE column and Sephacryl S‐200 column. Then, samples of molecular mass of ≈ 45 kDa were separated by native‐PAGE (polyacrylamide gel electrophoresis) and nonreducing sodium dodecyl sulphate (SDS)‐PAGE. After confirming the suppressor activity of proteins of ≈ 45 kDa separated by native‐PAGE, samples were separated by nonreducing SDS‐PAGE, transferred onto polyvinylidene difluoride membranes and analysed using matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectrometry. Results  Proteins of ≈ 45 kDa eluted from a Sephacryl S‐200 column and the slice of native‐PAGE gel exhibited the strong suppressor activity. Analyses using MALDI‐TOF mass spectrometry and MASCOT algorithm of the protein bands around 45 kDa separated by nonreducing SDS‐PAGE identified NSF as a 22·5‐kDa protein, dual specific phosphatase 14/MAP‐kinase phophatase‐6 (DUSP14/MKP6), which functions as a negative regulator of the MAP‐kinase signalling. Western blot analyses revealed that recombinant DUSP14 (rDUSP14) exists as the mixture of 22·5‐kDa monomer and 45‐kDa dimer under nonreducing conditions, and monomers under reducing conditions. Treatment with rDUSP14 at 4 °C for 2 h suppressed the ability of effector cells to transfer CHS dose dependently and the APC function of DLNCs to induce CHS. Epicutaneous application of rDUSP14 immediately after challenge inhibited the subsequent CHS expression. rDUSP14 was bound specifically by major histocompatibility complex class II (Ia)‐positive spleen cells (presumably DCs). The suppressor activity of NSF was neutralized by anti‐DUSP14 monoclonal antibody. Expression of DUSP14 mRNA in the spleen was upregulated parallel to the unresponsive state induced by hapten‐conjugated cells. NSF, NSF int and rDUSP14 exhibited the phosphatase activity towards p ‐nitrophenyl phosphate in vitro as alkaline phosphatase. Conclusions  These studies indicate for the first time that NSF is a dimer of DUSP14 secreted by macrophage‐like suppressor cells by stimulation with hapten‐conjugated cells and exerts a regulatory function on CHS through DCs as a secreted phosphatase.