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Abstract 1348 Poster Board I-371 Aberrant expression of CD40 ligand (CD40L) represents a strong endogenous danger signal associated with chronic inflammatory disease. However, CD40 activation alone is unable to induce interleukin (IL)-12p70 secretion, whereas IL-6, IL-12p40, TNF-alpha and IL-10 are produced. We demonstrate that cytokine-induced Janus kinase (JAK)-1 is unique in mediating a complementary pro-inflammatory signal for the production of IL-12p70 whilst at the same time inhibiting IL-10 secretion in human APCs. CD40- and JAK/STAT signals recruit complementary pathways: CD40 signals involve RelA and cRel for the induction of IL-12p70 and RelA for IL-10. In contrast, RelB inhibited IL-12p70 production. JAK1 complemented CD40-induced NF-kB signals by phosphorylating STAT-1 and inducing IRF-1 and IRF-8 expression resulting in IL-12p70 production. JAK-1-dependent inhibition of IL-10 transcription was mediated either by STAT-1 directly binding to the IL-10 promotor or by IRF-8 that in a complex with PU.1 (Spi-1) binds to inhibitory IFN-gamma-activated sequence (GAS)-sites of the IL-10 promotor. Our results demonstrate that specific inhibition of the “second signal” JAK1 will shift the balance of CD40-induced cytokine panels from IL-12p70 towards IL-10 in human APCs. Disclosures No relevant conflicts of interest to declare.

CD40Ligand (CD40L) represents a strong endogenous danger signal associated with chronic inflammatory disease. CD40L induces pro-inflammatory activation of CD40 expressing antigen-presenting cells (APC) such as DCs, monocytes, B-cells and endothelial cells; however, CD40 activation alone is insufficient to induce interleukin (IL)-12p70 secretion. Using quantitative Western blotting and siRNA inhibition of protein expression we demonstrate that cytokine-induced Janus kinase (JAK)1 and/or JAK2 activation mediates a complementary second signal for the production of IL- 12p70, whereas tyrosine kinase (Tyk)2 activation has inhibitory effects. Furthermore, JAK1 activation reciprocally reduced IL-10 production. The mechanism of JAK modulation of CD40 signals in dendritic cells (DCs) involved transcriptional regulation. The Th1 and Th2 cytokines interferon (IFN)-γ and IL-4 both enhance IL-12p70 via activation of the same JAKs. However due to a weaker JAK activity, IL-4 signalling has to persist much longer than IFN-γ signalling in order to induce similar amounts of IL-12p70. This different dependence on signalling persistence may give rise to the distinct biological characteristics of Th1/Th2 cytokines and may explain the pleiotropic in vivo and in vitro effects of IL-4. Complementary CD40 and JAK activity is essential for IL-12p70 secretion by all human APC studied. This strict requirement of two complementary signals opens a new way of interfering with CD40L induced APC activation indirectly by modulating the “second signal” JAK.

CD40L represents a strong endogenous danger signal that induces pro-inflammatory activation of CD40-expressing cells such as dendritic cells (DC), monocytes, and B cells. However, since CD40 activation alone is insufficient to induce pro-inflammatory cytokines such as IL-12p70, we studied whether CD40-mediated pro-inflammatory activity might be dependent on co-signalling pathways involving JAK/STAT. Using quantitative Western blotting, we demonstrate that JAK/STAT signalling is induced by cytokines such as IL-4, GM-CSF and IFNg, whereas CD40 activation mediates NFkB signalling. CD40L-induced IL-12p70 and IL-10 secretion in human DC, monocytes, B cells, and chronic lymphocytic leukemia (CLL) cells was measured upon complementary JAK/STAT activation by IL-4, GM-CSF and IFNg in the presence and absence of specific inhibitors of JAK2, JAK3, and pan-JAK. Whereas IL-12p70 could not be induced by CD40 ligation or by cytokines alone, IL-12p70 secretion and suppression of IL-10 was reproducibly observed after co-stimulation of CD40L with IL-4, GM-CSF, or IFNg. This effect could be completely reversed by pan-JAK inhibition. Persistence of IL-4/GM-CSF/IFNg-mediated JAK/STAT signalling as late as 12 hours following cellular activation via CD40 was required for IL-12p70 secretion as shown by the effects of delayed JAK inhibition. Similarly, persistence between 12 and 24 hours of IL-12p35 and p40 mRNA expression correlated best with the level of IL-12p70 secretion. Specific inhibition of JAK2 and JAK3 further revealed a context-dependent action of the distinct JAK family members: JAK2 showed a strong co-dominant effect in the setting of IL-4-induced JAK/STAT activity. Both, JAK2 and JAK3 were required for IL-12p70 secretion, whereas JAK2 alone was sufficient to modulate IL-10 secretion. However, in the context of IFNg-induced JAK/STAT signalling in DC, neither JAK2 nor JAK3 inhibition had effects on IL-12p70. Here, only inhibition by the pan-JAK inhibitor involving JAK1 abrogated IL-12p70 secretion, indicating that in IFNg-dependent signalling, JAK2 is apparently sub-dominant to JAK1 and had only a small enhancing effect on IL-10. This context dependence markedly differed in myeloid cells and B cells, as normal and malignant (CLL) B cells maintain a co-dominant JAK2 activity in the context of IFNg-induced JAK/STAT-signalling. In conclusion, complementary JAK/STAT signalling is required for the pro-inflammatory effects of CD40 ligation in humans, with different JAK subset predominance in myeloid and B cells. These results may open new ways of lineage-specific interfering with CD40 signals by modulating JAK/STAT activity using tyrosine kinase inhibitors.

SPARC-like protein 1 (SPARCL1), a member of the SPARC family, is downregulated in various tumors. In the present study, the expression and localization of SPARCL1 were analyzed in a wide range of nontumorous and neoplastic pancreatic tissues by quantitative reverse transcription-polymerase chain reaction, laser capture microdissection, microarray analysis, and immunohistochemistry. For functional analysis, proliferation and invasion assays were used in cultured pancreatic cancer cells. Pancreatic ductal adenocarcinoma (PDAC) and other pancreatic neoplasms exhibited increased SPARCL1 mRNA levels compared to those of the normal pancreas. SPARCL1 mRNA levels were low to absent in microdissected and cultured pancreatic cancer cells, and promoter demethylation increased SPARCL1 levels only slightly in three of eight cell lines. SPARCL1 was observed in small capillaries in areas of inflammation/tumor growth and in some islet cells. In PDAC, 15.4% of vessels were SPARCL1-positive. In contrast, the percentage of SPARCL1-positive vessels was higher in chronic pancreatitis and benign and borderline pancreatic tumors. Recombinant SPARCL1 inhibited pancreatic cancer cell invasion and exerted moderate growth-inhibitory effects. In conclusion, SPARCL1 expression in pancreatic tissues is highly correlated with level of vascularity. Its antiinvasive effects and reduced expression in metastasis indicate tumor-suppressor function.