Blockade of CCR7 leads to decreased dendritic cell migration to draining lymph nodes and promotes graft survival in low-risk corneal transplantation.

The chemokine receptor CCR7 is essential for migration of mature dendritic cells (DCs) to the regional lymph nodes, and it has been shown that blocking of CCR7 improves graft survival after high-risk corneal transplantation in vascularized recipient corneas. However, it is so far unknown whether blocking of CCR7 reduces migration of DCs from the avascular cornea to the draining lymph nodes and whether this leads to improved graft survival also in the low-risk setting of corneal transplantation, which accounts for the majority of perforating transplantations performed. Therefore, in this study, pellets containing Freund's adjuvant and bovine serum albumin (BSA) conjugated to Alexa488 fluorescent dye were implanted into the corneal stroma of BALB/c mice to analyze antigen uptake by corneal DCs and their migration to the regional lymph nodes. After pellet implantation, mice were either treated by local administration of a CCR7 blocking fusion protein that consisted of CCL19 fused to the Fc part of human IgG1 or a control-IgG. In vivo fluorescence microscopy showed uptake of Alexa488-conjugated BSA by corneal DCs within 8 h. Furthermore, analysis of single cell suspensions of draining lymph nodes prepared after 48 h revealed that 2.1 ± 0.3% of CD11c(+) cells were also Alexa488(+). Importantly, DC migration was significantly reduced after topical administration of CCL19-IgG (1.2 ± 0.2%; p < 0.05). To test the effect of CCR7 blockade on graft rejection after allogeneic low-risk keratoplasty, corneal transplantations were performed using C57BL/6-mice as donors and BALB/c-mice as recipients. Treatment mice received two intraperitoneal loading doses of CCL19-IgG prior to transplantation, followed by local treatment with CCL19-IgG containing eye drops for the first two weeks after transplantation. Control mice received same amounts of control-IgG. Kaplan-Meier survival analysis showed that in the CCL19-IgG treated group, 76% of the grafts survived through the end of the 8 week observation period, whereas 38% of the grafts survived in the control group (p < 0.05). Taken together, our study shows that blockade of CCR7 reduces the migration of mature corneal DCs to the draining lymph nodes and leads to improved graft survival in low-risk corneal transplantation.

Necroptosis in immunity and ischemia-reperfusion injury.

Transplantation is invariably associated with ischemia-reperfusion injury (IRI), inflammation and rejection. Resultant cell death has morphological features of necrosis but programmed cell death has been synonymous with apoptosis until pathways of regulated necrosis (RN) have been described. The best-studied RN pathway, necroptosis, is triggered by perturbation of caspase-8-mediated apoptosis and depends on receptor-interacting protein kinases 1 and 3 (RIPK1/RIPK3) as well as mixed linage kinase domain like to form the necroptosome. The release of cytosolic content and cell death-associated molecular patterns (CDAMPs) can trigger innate and promote adaptive immune responses. Thus, the form of cell death can substantially influence alloimmunity and graft survival. Necroptosis is a key element of IRI, and RIPK1 interference by RN-specific inhibitors such as necrostatin-1 protects from IRI in kidney, heart and brain. Necroptosis may be a general mechanism in response to other forms of inflammatory organ injury, and will likely emerge as a promising target in solid organ transplantation. As second-generation RIPK1 and RIPK3 inhibitors become available, clinical trials for the prevention of delayed graft function and attenuation of allograft rejection-mediated injury will emerge. These efforts will accelerate upon further identification of critical necroptosis-triggering receptor(s).

Ras-dependent and -independent pathways target the mitogen-activated protein kinase network in macrophages.

Mitogen-activated protein kinases (MAPKs) are activated upon a variety of extracellular stimuli in different cells. In macrophages, colony-stimulating factor 1 (CSF-1) stimulates proliferation, while bacterial lipopolysaccharide (LPS) inhibits cell growth and causes differentiation and activation. Both CSF-1 and LPS rapidly activate the MAPK network and induce the phosphorylation of two distinct ternary complex factors (TCFs), TCF/Elk and TCF/SAP. CSF-1, but not LPS, stimulated the formation of p21ras. GTP complexes. Expression of a dominant negative ras mutant reduced, but did not abolish, CSF-1-mediated stimulation of MEK and MAPK. In contrast, activation of the MEK kinase Raf-1 was Ras independent. Treatment with the phosphatidylcholine-specific phospholipase C inhibitor D609 suppressed LPS-mediated, but not CSF-1-mediated, activation of Raf-1, MEK, and MAPK. Similarly, down-regulation or inhibition of protein kinase C blocked MEK and MAPK induction by LPS but not that by CSF-1. Phorbol 12-myristate 13-acetate pretreatment led to the sustained activation of the Raf-1 kinase but not that of MEK and MAPK. Thus, activated Raf-1 alone does not support MEK/MAPK activation in macrophages. Phosphorylation of TCF/Elk but not that of TCF/SAP was blocked by all treatments that interfered with MAPK activation, implying that TCF/SAP was targeted by a MAPK-independent pathway. Therefore, CSF-1 and LPS target the MAPK network by two alternative pathways, both of which induce Raf-1 activation. The mitogenic pathway depends on Ras activity, while the differentiation signal relies on protein kinase C and phosphatidylcholine-specific phospholipase C activation.

Involvement of the protein tyrosine phosphatase SHP-1 in Ras-mediated activation of the mitogen-activated protein kinase pathway.

Ubiquitously expressed SH2-containing tyrosine phosphatases interact physically with tyrosine kinase receptors or their substrates and relay positive mitogenic signals via the activation of the Ras-mitogen-activated protein kinase (MAPK) pathway. Conversely, the structurally related phosphatase SHP-1 is predominantly expressed in hemopoietic cells and becomes tyrosine phosphorylated upon colony-stimulating factor 1 treatment of macrophages without associating with the colony-stimulating factor 1 receptor tyrosine kinase. Mice lacking functional SHP-1 (me/me and me(v)/me(v)) develop systemic autoimmune disease with accumulation of macrophages, suggesting that SHP-1 may be a negative regulator of hemopoietic cell growth. By using macrophages expressing dominant negative Ras and the me(v)/me(v) mouse mutant, we show that SHP-1 is activated in the course of mitogenic signal transduction in a Ras-dependent manner and that its activity is necessary for the Ras-dependent activation of the MAPK pathway but not of the Raf-1 kinase. Consistent with a role for SHP-1 as an intermediate between Ras and the MEK-MAPK pathway, Ras-independent activation of the latter kinases by bacterial lipopolysaccharide occurred normally in me(v)/me(v) cells. Our results sharply accentuate the diversity of signal transduction in mammalian cells, in which the same signaling intermediates can be rearranged to form different pathways.

Suppression of growth factor-mediated MAP kinase activation by v-raf in macrophages: a putative role for the MKP-1 phosphatase.

Many tyrosine kinase growth factor receptors activate the MAP Kinase (MAPK) pathway by stimulating the activity of the RAF kinase. In some, but not all cell types, the expression of activated RAF is sufficient to induce constitutive MAPK activation. In BAC-1.2F5 macrophages the expression of virally activated RAF does not correlate with constitutive MAPK activation; on the contrary, growth factor-mediated stimulation of MAPK activity is suppressed in these cells. Suppression correlates with v-RAF expression, as MAPK activation is normal in a revertant cell line that stopped expressing v-RAF. Inhibition of MAPK activation is associated with lack of ERK-2 tyrosine phosphorylation, and is not due to the suppression of CSF-1-mediated MEK activation. Pretreatment with vanadate restores growth factor-stimulated activation and tyrosine phosphorylation of MAPK in v-RAF-expressing macrophages, indicating the involvement of a tyrosine phosphatase. Interestingly, v-RAF-expressing macrophages contain low constitutive levels of MKP-1 mRNA, an immediate early gene that encodes a MAPK-specific phosphatase and is induced in the parental cell line by CSF-1 treatment. The restoration of MAPK activation by vanadate pretreatment and the presence of MKP-1 mRNA in v-RAF-expressing macrophages raise the intriguing possibility that in macrophages RAF may be feeding back on the MAPK pathway by participating in the control of MKP-1 expression.

A cellular screen identifies ponatinib and pazopanib as inhibitors of necroptosis.

Necroptosis is a form of regulated necrotic cell death mediated by receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and RIPK3. Necroptotic cell death contributes to the pathophysiology of several disorders involving tissue damage, including myocardial infarction, stroke and ischemia-reperfusion injury. However, no inhibitors of necroptosis are currently in clinical use. Here we performed a phenotypic screen for small-molecule inhibitors of tumor necrosis factor-alpha (TNF-α)-induced necroptosis in Fas-associated protein with death domain (FADD)-deficient Jurkat cells using a representative panel of Food and Drug Administration (FDA)-approved drugs. We identified two anti-cancer agents, ponatinib and pazopanib, as submicromolar inhibitors of necroptosis. Both compounds inhibited necroptotic cell death induced by various cell death receptor ligands in human cells, while not protecting from apoptosis. Ponatinib and pazopanib abrogated phosphorylation of mixed lineage kinase domain-like protein (MLKL) upon TNF-α-induced necroptosis, indicating that both agents target a component upstream of MLKL. An unbiased chemical proteomic approach determined the cellular target spectrum of ponatinib, revealing key members of the necroptosis signaling pathway. We validated RIPK1, RIPK3 and transforming growth factor-ß-activated kinase 1 (TAK1) as novel, direct targets of ponatinib by using competitive binding, cellular thermal shift and recombinant kinase assays. Ponatinib inhibited both RIPK1 and RIPK3, while pazopanib preferentially targeted RIPK1. The identification of the FDA-approved drugs ponatinib and pazopanib as cellular inhibitors of necroptosis highlights them as potentially interesting for the treatment of pathologies caused or aggravated by necroptotic cell death.

Transforming growth factor-beta1 induces activation of Ras, Raf-1, MEK and MAPK in rat hepatic stellate cells.

The transdifferentiation of hepatic stellate cells into myofibroblast-like cells and the proliferation of the transdifferentiated cells are controlled by TGF-beta1. Little is known about the intracellular signal transducers of TGF-beta1. In this paper we show that in cultured hepatic stellate cells TGF-beta1 induces activation of Ras, Raf-1, MEK and MAPK p42 and p44. The activation of MAPK depends on the activation of MEK. Our data exclude that the observed effects are mediated by a bFGF or PDGF autocrine loop.

IL-16 activates the SAPK signaling pathway in CD4+ macrophages.

IL-16 has been reported as a modulator of T cell activation and was shown to function as chemoattractant factor. The chemotactic activity of IL-16 depends on the expression of CD4 on the surface of target cells, but the intracellular signaling pathways are only now being deciphered. This report describes IL-16 as an additional activator of the stress-activated protein kinase (SAPK) pathway in CD4+ macrophages. Treatment of these cells with recombinant expressed IL-16 leads to the phosphorylation of SEK-1, resulting in activation of the SAPKs p46 and p54. IL-16 stimulation also leads to the phosphorylation of c-Jun and p38 MAPK (mitogen-activated protein kinase), without inducing MAPK-family members ERK-1 and ERK-2. Interestingly, the IL-16-mediated activation of SAPKs and p38 MAPK in macrophages alone induces no detectable apoptotic cell death. These observations suggest specific regulatory functions of IL-16 distinct from the proinflammatory cytokines TNF-alpha and IL-1beta.

Bacterially expressed murine CSF-1 possesses agonistic activity in its monomeric form.

CSF-1 is a dimeric peptide growth factor, stabilized by disulfide bonds. We expressed mouse CSF-1 in bacteria as a fusion protein either with glutathione S-transferase (GST) or with a six histidine tag (His-tag). Large amounts of recombinant material were obtained and purified by a single affinity chromatography step. Purified CSF-1-His-tag monomers efficiently dimerized in vitro, but the presence of variable amounts of GST-moiety in CSF-1 preparations obtained by thrombin cleavage of GST-fusion proteins (thrombin-released CSF-1) interfered with dimerization. However, the thrombin-released CSF-1 monomers possessed agonistic activity, being capable of stimulating tyrosine phosphorylation of the CSF-1 receptor and of an array of cellular proteins in living macrophages and of supporting their growth. These results show that CSF-1 dimerization is not essential for receptor activation in vivo.

Ecto-alkaline phosphatase activity identified at physiological pH range on intact P19 and HL-60 cells is induced by retinoic acid.

The activity of membrane-bound alkaline phosphatase (ALP) expressed on the external surface of cultured murine P19 teratocarcinoma and human HL-60 myeloblastic leukemia cells was studied at physiological pH using p-nitrophenylphosphate (pNPP) as substrate. The rate of substrate hydrolysis catalyzed by intact viable cells remained constant for eight successive incubations of 30 min and was optimal at micromolar substrate concentrations over the pH range 7.4-8.5. The value of apparent K(m) for pNPP in P19 and HL-60 cells was 120 microM. Hydrolytic activity of the ecto-enzyme at physiological pH decreased by the addition of levamisole, a specific and noncompetitive inhibitor of ALP (K(i) P19 = 57 microM; K(i) HL-60 = 50 microM). Inhibition of hydrolysis was reversed by removal of levamisole within 30 min. Retinoic acid (RA), which promotes the differentiation of P19 and HL-60 cells, induced levamisole-sensitive ecto-phosphohydrolase activity at pH 7.4. After its autophosphorylation by ecto-kinase activity, a 98-kDa membrane protein in P19 cells was found to be sensitive to ecto-ALP, and protein dephosphorylation increased after incubation of cells with RA for 24 h and 48 h. Orthovanadate, an inhibitor of all phosphatase activities, blocked the levamisole-sensitive dephosphorylation of the membrane phosphoproteins, while (R)-(-)-epinephrine reversed the effect by complexation of the inhibitor. The results demonstrate that the levamisole-sensitive phosphohydrolase activity on the cell surface is consistent with ecto-ALP activity degrading both physiological concentrations of exogenously added substrate and endogenous surface phosphoproteins under physiological pH conditions. The dephosphorylating properties of ecto-ALP are induced by RA, suggesting a specific function in differentiating P19 teratocarcinoma and HL-60 myeloblastic leukemia cells.

Lipopolysaccharide induces activation of the Raf-1/MAP kinase pathway. A putative role for Raf-1 in the induction of the IL-1 beta and the TNF-alpha genes.

Bacterial LPS is a potent macrophage activator. The early steps in LPS signal transduction involve the tyrosine phosphorylation and activation of a number of kinases of the src family, and inhibition of this pathway causes a severe impairment in the production of the cytokines TNF-alpha and IL-1 beta. We find that LPS-induced macrophages activation also involves the Raf-1 kinase, a key component in mitogenic signal transduction. Treatment of BAC-1.2F5 macrophages with LPS causes phosphorylation and activation of Raf-1. This is paralleled by the stimulation of MEK-1 and MAP-kinase activity and by the phosphorylation of the transcription factor Elk-1, a nuclear target of MAP-kinase. Activation of the Raf/MAP-kinase pathway was inhibited upon pretreatment of the cells with genistein, a tyrosine kinase inhibitor. Raf-1 must thus lie downstream of tyrosine kinase in LPS signal transduction. However, Raf-1 is not a direct substrate of a LPS-induced tyrosine kinase, because Raf-1 immunoisolated from LPS-induced cells contains only phosphoserine. This resembles the situation after CSF-1-stimulation of macrophages, in which Raf-1 clearly transduces a signal generated by the CSF-1 receptor kinase, but is phosphorylated exclusively in serine. Phosphopeptide maps of Raf-1 immunoprecipitated from LPS- or CSF-1-treated cells are indistinguishable, suggesting that these agents activate Raf-1 by similar mechanisms. Finally, v-raf-infected BAC-1.2F5 macrophages were found to constitutively express low levels of IL-1 beta and TNF-alpha. These data argue that Raf-1 functions downstream of tyrosine kinases in LPS-mediated macrophage activation and cytokine production.

Ectopic expression of a chimeric colony-stimulating factor-1/TrkB-receptor promotes CSF-1-dependent survival of cultured sympathetic neurons.

The regulation of the density of innervation and the promotion of survival of neurons are the original effects depending on neurotrophins. Here we analyse such effects evoked by trkB tyrosine kinase in transfected PC12 cells and transfected sympathetic neurons. In order to exclude the previously described modulation of trk kinase activity by the extracellular activation of the low-affinity p75 neurotrophin receptor, we applied a chimeric receptor approach: The extracellular domain of colony-stimulating factor-1 (CSF-1) receptor was fused to the transmembrane and cytoplasmic domain of the trkB tyrosine kinase receptor, allowing its selective activation by the heterologous ligand. Protein expression and CSF-1-induced tyrosine phosphorylation of the chimeric receptor protein was demonstrated in transfected COS cells. After stable transfection into nerve growth factor (NGF)-responsive PC12 cells, CSF-1 mediated the K252a-sensitive induction of fiber outgrowth. Furthermore, we were able to show by heterologous expression of the chimeric receptor, that activation of trkB tyrosine kinase activity is sufficient to promote survival of neurotrophin deprived sympathetic neurons.