Human CD180 Transmits Signals via the PIM-1L Kinase

<div><p>Toll-like receptors (TLRs) are important sensors of the innate immune system that recognize conserved structural motifs and activate cells via a downstream signaling cascade. The CD180/MD1 molecular complex is an unusual member of the TLR family, since it lacks the components that are normally required for signal transduction by other TLRs. Therefore the CD180/MD 1 complex has been considered of being incapable of independently initiating cellular signals. Using chemogenetic approaches we identified specifically the membrane bound long form of PIM-1 kinase, PIM-1L as the mediator of CD180-dependent signaling. A dominant negative isoform of PIM-1L, but not of other PIM kinases, inhibited signaling elicited by cross-linking of CD180, and this effect was phenocopied by PIM inhibitors. PIM-1L was directed to the cell membrane by its N-terminal extension, where it colocalized and physically associated with CD180. Triggering CD180 also induced increased phosphorylation of the anti-apoptotic protein BAD in a PIM kinase-dependent fashion. Also in primary human B cells, which are the main cells expressing CD180 in man, cross-linking of CD180 by monoclonal antibodies stimulated cell survival and proliferation that was abrogated by specific inhibitors. By associating with PIM-1L, CD180 can thus obtain autonomous signaling capabilities, and this complex is then channeling inflammatory signals into B cell survival programs. Pharmacological inhibition of PIM-1 should therefore provide novel therapeutic options in diseases that respond to innate immune stimulation with subsequently increased B cell activity, such as lupus erythematosus or myasthenia gravis.</p></div

Cytokine release induced by TLR family members depends on intact PIM kinase activity.

<p>(a,b) THP-1 and U937 cells were treated with 10ug/ml LPS in the presence of logarithmic dilutions of Compound 5c. (c) JeKo-1 cells were treated with 1ug/ml anti-CD180 mAb G28 in the presence of logarithmic dilutions of Compound 5c. Representative results from more than five independent experiments covering different dose ranges are shown. (d) Various synthetic siRNA pairs were introduced into THP-1 cells, which were then left untreated (empty bars) or exposed to 10ug/ml LPS (filled bars). (e) Stable cell lines were generated by introducing plasmids encoding fully functional and kinase dead variants (K67M mutation for PIM-1, K61A mutation for PIM-2) into THP1 cells. Cells were then left either untreated (empty bars) or exposed to 10ug/ml LPS (filled bars). Representative results from at least three independent experiments are shown. In all assays overnight release of cytokines was measured by ELISA.</p

CD180 colocalizes and associates with Pim-1L.

<p>(a-c) HEK cells were grown on a coverslip, and were cotransfected with plasmids encoding fluorescently labelled CD180 (green, panel a) and PIM-1L (red, panel b). Confocal fluorescence microscopy indicated colocalization of CD180 and PIM-1L preferentially in the plasma membrane, and also in some subcellular compartments. (d) Coimmunoprecipitation of transfected cells. Various combinations of plasmids encoding APP-tagged PIM-1L and EGFP-tagged CD180 were introduced into HEK cells as indicated by the (+) marks. Equal amounts of cellular proteins were immunoprecipitated with the respective anti-tag antibodies as indicated. The total precipitate was then resolved on a denaturing gel, and blotted with an anti-PIM-1 antibody. Double and single arrowheads indicate the long and the short forms of the PIM-1 protein, respectively. Representative results from three independent experiments are shown.</p

CD180 stimulation induces PIM-dependent phosphorylation and proliferation.

<p>(a) Phosphorylation of BAD(Ser112) was determined from equal amounts of whole cell lysates using a MesoScale immunoassay. JeKo-1 cells were tested without (empty bars) or with stimulation by CD180-specific antibodies (filled bars); and in parallel, the same was done in the presence of 1uM Compound 5c (thin and thick stripes, respectively). K562 cells, either untreated (empty bars) or also treated with 5c (thin stripes) were used for comparison. Representative results from three independent experiments are shown. Significant differences between tested groups are indicated by brackets and p-values. (b) Primary human B cells purified by negative magnetic selection were stimulated by adding CD180-specific antibodies (striped bars = clone G28; filled bars = clone MHR23). A multimeric CD40L was included in increasing concentrations (0, 3ng/ml, 10ng/ml, and 30ng/ml), with or without the presence of Compound 5c (1uM). Proliferation was measured by 3H-thymidine incorporation after four days. Stars (***) indicated significant differences p<0.001 between pairs of treated vs. untreated samples. Results from one donor out of four tested with similar results shown.</p