SHP-1 differentially regulates LPS-mediated TNF-alpha and IL-10 production in murine splenic macrophages

Abstract

Host-Pathogen interactions are characterized by inflammatory responses, aimed at eliminating invading pathogens. During inflammatory responses, immune cells secrete cytokines and chemokines, with a broad range of immuno-regulatory roles. The role of the signaling protein, SHP-1 in regulating signaling pathways activated by cytokine, antigen and growth factor receptors has been studied for some time. However, the involvement of SHP-1 in the regulation of Lipopolysaccharide (LPS)-activated signal transduction pathways leading to cytokine production is less well understood. Loss of SHP-1 leads to development of complex inflammatory disorder(s). To understand the role of SHP-1 during the inflammation process, the SHP-1-deficient moth-eaten (me/me) mouse model was used to investigate LPS-induced TNF-alpha and IL-10 production, representing pro and anti-inflammatory cytokines, respectively. Results show that me/me splenic macrophages, when challenged with LPS in-vitro , secreted lower levels of IL-10 and concomitantly elevated TNF-alpha levels, compared to normal healthy littermate controls. Deregulated LPS-induced TNF-alpha and IL-10 production in SHP-1 deficient splenic macrophages were observed irrespective of LPS dose and time of stimulation as demonstrated by ELISA. These findings were further confirmed by RT-PCR, SHP-1 antisense experiments in normal splenic macrophages and adenoviral reconstitution of SHP-1 expression in me/me macrophages, suggesting a dual role of SHP-1 as a negative regulator of LPS induced TNF-alpha and a positive regulator of IL-10. To delineate the precise role of SHP-1 in regulating LPS-mediated IL-10 production, LPS-activated signaling proteins, which represent SHP-1 targets were examined. Results obtained established a role of p38 MAPK in LPS-mediated IL--10 secretion, but also demonstrate that P38 involvement is independent of SHP-1. Results of this study further reveals a role for tyrosine kinase Src and an adhesion molecule Pyk2 in SHP-1 dependent LPS-mediated IL-10 production and infer that optimal production of LPS-induced IL-10 requires an assembly of a signalosome, consisting of Src-Pyk2-SHP-1 proteins. In conclusion, we show for the first time that: SHP-1 is a positive regulator of LPS-induced IL-10 production in splenic macrophages and, LPS-mediated IL-10 production is governed by two distinct signaling pathways, only one of which is SHP-1 dependent.