Abstract 1200: CD40 Ligand Induces Pentraxin 3 (PTX3) Expression In Human Macrophages
Background CD40 ligand (CD40L) and its receptor CD40 promote cell activation and mediate inflammatory responses. Recent clinical evidence links PTX3 with vascular inflammation, suggesting its role as a new biomarker for cardiovascular risk. Various cell types produce the long pentraxin PTX3, unlike the short pentraxin CRP, a liver product.
Methods and Results We tested the hypothesis that CD40L/CD40 signaling induces PTX3 expression in primary macrophages. CD40L (0.3–3.0μg/ml) consistently increased PTX3 mRNA expression by human peripheral blood-derived macrophages in vitro in a concentration-dependent manner, although the magnitude of maximum induction varied among 15 donors (4.7–55.7 fold, real-time RT-PCR). PTX3 induction peaked 2–4h after stimulation and returned to the basal level at 8h. CD40L also induced PTX3 protein secretion by human macrophages, assessed by Western blot analysis and ELISA. To examine whether CD40 mediates this response, we used small interfering RNA (siRNA) oligonucleotides targeting CD40. siRNA silenced CD40 mRNA expression by >95% and abrogated CD40L-induced PTX3 mRNA expression by 73.2±4.4% (vs. control siRNA, n=3), while this treatment did not affect IL-1β-induced PTX3 expression. Members of the TNF receptor associated factor (TRAF) family mediate CD40 signaling positively or negatively. TRAF2, 3, 5, or 6 siRNA treatment suppressed CD40L-induced PTX3 expression substantially, whereas TRAF1 siRNA enhanced PTX3 induction. CD40L induced phosphorylation of MAP kinases including ERK, JNK, and p38, and nuclear translocation of the NF-κB p50 subunit, indicating activation of signaling mechanisms downstream of TRAFs. ERK or JNK inhibition, but not p38 inhibition or NF-κB inhibitor peptide SN50, reduced CD40L-induced PTX3. These data indicate that the TRAF-ERK/JNK pathways mediate CD40L-induced PTX3 expression in human macrophages. Immunohistochemistry further colocalized PTX3, CD40L, and CD40 in macrophages (CD68-positive cells) of human carotid plaques. Quantitative analysis correlated immunoreactive PTX3 with lesional macrophages closely (31 areas in 7 cases, r=0.88).
Conclusions PTX3 may play a role in CD40L/CD40-mediated inflammatory diseases including atherosclerosis.