PECAM-1 Ligation Negatively Regulates TLR4 Signaling In Macrophages

As one of pathogen-associated molecular patterns (PAMPs), LPS (lipopolysaccharide) can be recognized by antigen-presenting cells (APC) including macrophages and then activate innate immune response. LPS-mediated activation of macrophages involves presentation of LPS by CD14 to members of the Toll-like receptor (TLR) family, particularly TLR4. Upon LPS recognition and activation of TLR4 siganling, macrophages produce a cascade of proinflammatory cytokines and chemokines including TNF-α, IL-6 , IFN-β and IL-1β through intracellular MyD88-dependent and MyD88-independent signaling cascades. These proinflammatory cytokines can mediate innate response and prime the adaptive immune response and also induce inflammation.It is well accepted that such response needs tight control because excessive or inappropriate inflammatory processes would be harmful or even fatal. Many negative regulators of LPS response have been reported. ST2, a Toll/IL-1 receptor family protein, was one of them. Macrophages from ST2-deficient mice produced more proinflammatory cytokines in response to LPS stimulation, suggesting that ST2 may be important in negative regulation of LPS response. Overexpression of ST2 inhibited TLR4-mediated NF-kB activation. The further study about ST2L co-precipitation assay showed thatST2 functions as an inducible transmembrane regulator through directly acting on MyD88-dependent pathway. SOCS1 (Suppressor of cytokine signaling 1) was also an inducible negative regulator of LPS response. Macrophages from SOCS1-deficient mice produce heightened levels of NO and pro-inflammatory cytokines in response to LPS stimulation.SOCS1-deficient mice were susceptible to endotoxin. But the inhibitory effect of SOCS1 on TLR4 signaling is indirect by inhibiting MyD88-independent pathway. Despite these inducible negative regulators, constitutively expressed negative regulators may inhibit the early phase of LPSresponse and more effective to reduce severity and pathology of inflammatory diseases.Platelet endothelial cell adhesion molecule-1 (PECAM-1), first identified as a adhesion molecule, is a 130-kDa member of the immunoglobulin (Ig) superfamily. However recent studies show that it can participate in signal transduction as a transmembrane protein. Furthermore, PECAM-1 contains intracytoplasmic immunoreceptor tyrosine inhibitory motifs (ITIMs), and more and more evidences prove it to be a member of the Ig-ITIM family, which can moderate or attenuate tyrosine kinase-mediated signaling pathways. More recently, PECAM-1-deficient mice were demonstrated to be more sensitive to LPS and their survival were reduced during endotoxic LPS-induced shock. As compared to wild-type controls, PECAM-1-deficient mice have elevated levels of serum TNF-cu IL-6 and IFN-P and reduced levels of phosphorylated STAT3. These findings suggest that PECAM-1 may play an important role in negative regulation of LPS response possibly via STAT3 signaling pathway.Here we observed the effect of LPS stimulation on PECAM-1 expression on APC including macrophages, demonstrated that PECAM-1 was a negative regulator of LPS-induced proinfiammatory cytokine production by macrophages. Finally, we found that inhibition of IkB(x and JNK pathways may be involved in the negative regulation of TLR4 response by PECAM-1.Part I. Effects of TLR agonists on the PECAM-1 expression on antigen-presenting cellsTo explore the role of PECAM-1 in macrophages response to LPS stimulation, we first examined whether PECAM-1 was expressed on antigen-presenting cells including macrophages. By using RT-PCR, we found that the mRNA expression of PECAM-1 isoforms in the murine macrophage-like cell line Raw264.7. The PECAM-1 isoforms included the full lengths A14exon and Al4,15exons of cytoplasmic domain of PECAM-1. Furthermore, PECAM-1 was also expressed on themurine macrophage-like cell line J774A. 1 and dendritic cells (DC). We also examined the PECAM-1 expression on RAW264.7 cells induced by LPS at various dosages. We found that 0.lug/ml LPS could induce significant increase in the PECAM-1 expression on RAW264.7 cells. With higher dose of LPS (0.5ug/ml or lug/ml) used, there was no difference on the maximal expression level of PECAM-1 as compared with that induced by 0. lug/ml LPS. While, the duration of high PECAM-1 expression was prolonged when higher dose of LPS used.In order to better understand the up-regulation of PECAM-1 expression on macrophages by LPS, we used real-time quantitative PCR and Western Blot to detect the change of PECAM-1 expression on macrophages stimulated with 0.5ug/ml LPS for various time. Expression of PECAM-1 on RAW264.7 cells reached to approximately maximal level when stimulated with 0.5ng/ml LPS for 6h (by real-time quantitative PCR) or 9h (by Western Blot). Then the expression was decreased to the normal level. The similar results were obtained when the freshly isolated peritoneal macrophages used, however, the LPS-induced PECAM-1 expression on these cells was highest at 12h. The results demonstrate that LPS can induce PECAM-1 expression on macrophages, suggesting that PECAM-1 may be invovled in regulation of TLR4 signaling in macrophages.In addition to TLR4 agonist, we also observed the effects of other TLR agonists on the PECAM-1 expression on macrophages. By Western Blot, we demonstrated TLR9 ligand CpG ODNn PMA and heat shock promoted PECAM-1 expression on RAW264.7 cells. Interestingly, lOug/ml TLR3 agonist polyI:C was not able to induce PECAM-1 expression, because only one concentration of polylrC was used, and whether other concentrations of polyI:C can regulate PECAM-1 expression needfurther investigation.*Part II. Inhibitory effect of PECAM-1 on LPS-induced inflammatory cytokine production from macrophages and the involved signal pathwaysTo better understand the role of PECAM-1 in TLR4 signaling, we examined theproduction of LPS-induced inflammatory cytokine TNF-cu IL-6 and IFN-|3 in PECAM-1-overexpressing or silenced macrophages. CD38 is one ligand of PECAM-1, so, we expressed the fusion protein (mexCD38-Fc) of the extracellular domain of CD38 fused with IgG4 CH2 and CH3 fragments in COS-7 cells, then purified the fusion protein, and used it to bind PECAM-1 and activate PECAM-1 signaling, finally examined the role of PECAM-1 ligation in the LPS response in macrophages. The expression of mexCD38-Fc protein about 60-kDa was confirmedby Western Blot. Compared with human IgG protein pretreated control group, after pretreatment with mexCD38-Fc fusion protein (PECAM-1 ligation), RAW264.7 cells and thioglycollate-elicited peritoneal macrophages produced less TNF-cu IL-6 and IFN-P levels response to LPS stimulation.In order to identify the inhibitory effects of PECAM-1 ligation on macrophage production of proinflammatory cytokines, we designed and constructed an RNAi vector that specifically down-regulated PECAM-1 expression on Raw264.7 cells. The levels of PECAM-1 expression in PECAM-1 stably-silenced Raw264.7 cells were decreased about 70%, as compared control cells. As expected, the production of LPS-induced TNF-cu IL-6 and IFN-p from the PECAM-1 stably-silenced Raw264.7 cells was significantly increased, compared with that from the wild type cells or RNAi control cells. The similar results of the LPS-induced TNF-a production in PECAM-1 stably-silenced Raw264.7 cells were also obtained by ELISA. Furthermore, TLR4 expression remained unchanged in PECAM-1 silenced or overexpressing macrophages, indicating that the inhibitory effect of PECAM-1 ligation on TLR4 signaling was not due to the down-regulation of TLR4 expression on macrophages. Taken together, these results suggest that PECAM-1 is an indispensable negative regulator of LPS response in macrophages.To address the molecular mechanisms of the PECAM-1 ligation-mediated negative regulation of LPS-evoked responses in macrophages, we examined the phosphorylation state of related signaling molecules downstream of TLR4 pathway in Raw264.7 cells stimulated with LPS. It has been shown that LPS-induced cytokineproduction by macrophages is through MyD88 dependent and MyD88 independent pathways, hence we detected the phosphorylation levels of JNK> IkBcu ERK> P38 and IRF-3 in LPS-activated Raw264.7 cells when PECAM-1 was overexpressed or silenced. Our results suggested that PECAM-1 mediated negative regulation of LPS response probably through inhibiting activation of IkBcx and JNK pathways. Moreover, compared to control, the phosphorylation level of ERK was significantly increased when PECAM-1 was ligated. So, further experiments need to be performed to confirm the relationship between ERK activation and the negative regulation of TLR4 signaling by PECAM-1.Furthermore, the signal pathways we analyzed above are involved in many biological processes, so, the findings that PECAM-1 can inhibit or activate the signal pathway indicate that PECAM-1 may affect other functions of macrophages.