Prostaglandins regulate melanoma-induced cytokine production in macrophages

Tumor-secreted products can affect macrophage cytokine expression and in that way alter the immune response. Prostaglandins (PGs) are found in the tumor microenvironment and have been associated with local and regional immunosuppression. We investigated whether tumor-secreted factors could induce PG synthesis in macrophages and whether these PGs could alter macrophage production of immunoregulatory cytokines. In both murine and human models, melanoma conditioned medium (MCM) induced macrophage production of PGE(2), IL-6, and TNF-alpha. PGE(2) production increased over 24 h and was accompanied by an increase in cyclooxygenase-2 (COX-2) expression, while COX-1 expression remained unchanged. In the presence of 10 microM NS398, a selective COX-2 inhibitor, MCM-stimulated PGE(2) synthesis was almost completely suppressed, while production of IL-6 and TNF-alpha proteins and mRNA also was partially abrogated. In the murine model, 200 microM NS398 resulted in more significant inhibition of cytokine protein and mRNA production. Although MCM induced NFkappaB and NF-IL-6 activation, neither dose of NS398 altered this effect. We conclude that melanoma-secreted products stimulate COX-2 expression and PGE(2) synthesis in macrophages and that inhibition of COX-2-derived PG synthesis results in partial abrogation of macrophage cytokine production.     

R- and S-isomers of nonsteroidal anti-inflammatory drugs differentially regulate cytokine production

2-arylpropionic acids, a well known class of non-steroidal anti-inflammatory drugs (NSAIDs), exist as a racemic mixture of their enantiomeric forms, with S-isomers primarily responsible for inhibition of prostaglandin (PG) production and of inflammatory events. In this study we show that S-isomers are also responsible for the paradoxical up-regulation of tumor necrosis factor (TNF) induced by ketoprofen, flurbiprofen and ibuprofen in murine peritoneal macrophages stimulated by bacterial endotoxin (LPS). This effect is in close correlation with cyclooxygenase inhibitory capacity of S-isomers and, from Northern blot analysis, seems to be mediated by the up-regulation of TNF mRNA. In addition, up-regulation of TNF production by S-isomers is associated with inhibition of interleukin-10 (IL-10) production. Conversely, we have observed that S-enantiomers reduce IL-6 production at a concentration 100 times higher than that able to inhibit cyclooxygenase activity. The unwanted pro-inflammatory effects of S-isomers through TNF and IL-10 production could therefore hinder their analgesic effect, that is, at least in part, related to IL-6 inhibition. In addition, TNF amplification by S-isomers could be correlated to the clinical evidence of their gastric toxicity. On the other hand, R-isomers did not affect TNF and IL-10 production even at cyclooxygenase-blocking concentration, while they reduced IL-6 production to the same levels as S-isomers. It is concluded that the regulation of cytokine production by S-isomers of 2-arylpropionic acids could partially mask their therapeutic effects and could be correlated to the clinical evidence of their higher gastric toxicity. On the other hand, IL-6 inhibition without the unwanted effects on TNF and IL-10 production shown by R-isomers could be correlated to the analgesic effect reported for R-2-arylpropionic acids.     

Opioid and nociceptin receptors regulate cytokine and cytokine receptor expression

Opioids were originally discovered because of their ability to induce analgesia, but further investigation has shown that the opioids regulate the function of cells involved in the immune response. We suggest that the regulation of cytokine, chemokine, and cytokine receptor expression is a critical component of the immunomodulatory activity of the opioids. In this paper we review the literature dealing with the regulation of cytokine and cytokine receptor expression by agonists for the three major opioid receptor types (mu, kappa, and delta), and nociceptin, the natural agonist for the orphanin FQ/nociceptin receptor. Although the opioid receptors share a high degree of sequence homology, opposing roles between the kappa opioid receptor (KOR) and the mu opioid receptor (MOR) have become apparent. We suggest that activation of the KOR induces an anti-inflammatory response through the down-regulation of cytokine, chemokine and chemokine receptor expression, while activation of the MOR favors a pro-inflammatory response. Investigation into the opioid receptor-like (ORL1)/nociceptin system also suggests a role for this receptor as a down-regulator of immune function. These effects suggest a broad role for opioids in the modulation of the function of the immune system, and suggest possible targets for the development of new therapeutics for inflammatory and infectious diseases.     

Receptor activator of NF-kappa B ligand and osteoprotegerin regulate proinflammatory cytokine production in mice

Receptor activator of NF-kappaB ligand (RANKL) is a membrane-bound or soluble cytokine essential for osteoclast differentiation, whereas the decoy receptor osteoprotegerin (OPG) masks RANKL activity. In mouse serum, both soluble RANKL and OPG are detectable. We observed that mice injected with LPS showed significantly down-regulated serum RANKL levels, whereas serum OPG levels were up-regulated. However, the roles of RANKL and OPG in innate immunity remain obscure. We found that RANKL pretreatment suppressed production of proinflammatory cytokines in macrophages in response to stimulation by bacteria and their components. Furthermore, such RANKL-induced tolerance in macrophages was inhibited by GM-CSF treatment, which blocks RANKL signaling. RANKL-induced tolerance occurred in the absence of c-Fos, which is essential for osteoclast differentiation. In mice lacking OPG, LPS-induced production of proinflammatory cytokines was reduced, whereas in mice lacking RANKL, it was increased, and lethality following LPS injection was also elevated, suggesting that constitutive activities of RANKL suppress cytokine responsiveness to LPS in vivo. Strikingly, prior administration of RANKL protected mice from LPS-induced death. These data reveal prophylactic potential of RANKL in acute inflammatory diseases.     

Donor lung derived myeloid and plasmacytoid dendritic cells differentially regulate T cell proliferation and cytokine production

Background

Direct allorecognition, i.e., donor lung-derived dendritic cells (DCs) stimulating recipient-derived T lymphocytes, is believed to be the key mechanism of lung allograft rejection. Myeloid (cDCs) and plasmacytoid (pDCs) are believed to have differential effects on T cell activation. However, the roles of each DC type on T cell activation and rejection pathology post lung transplantation are unknown.

Methods

Using transgenic mice and antibody depletion techniques, either or both cell types were depleted in lungs of donor BALB/c mice (H-2^d) prior to transplanting into C57BL/6 mice (H-2^b), followed by an assessment of rejection pathology, and pDC or cDC-induced proliferation and cytokine production in C57BL/6-derived mediastinal lymph node T cells (CD3+).

Results

Depleting either DC type had modest effect on rejection pathology and T cell proliferation. In contrast, T cells from mice that received grafts depleted of both DCs did not proliferate and this was associated with significantly reduced acute rejection scores compared to all other groups. cDCs were potent inducers of IFNγ, whereas both cDCs and pDCs induced IL-10. Both cell types had variable effects on IL-17A production.

Conclusion

Collectively, the data show that direct allorecognition by donor lung pDCs and cDCs have differential effects on T cell proliferation and cytokine production. Depletion of both donor lung cDC and pDC could prevent the severity of acute rejection episodes.     

STAT4 isoforms differentially regulate Th1 cytokine production and the severity of inflammatory bowel disease

STAT4, a critical regulator of inflammation in vivo, can be expressed as two alternative splice forms, a full-length STAT4alpha, and a STAT4beta isoform lacking a C-terminal transactivation domain. Each isoform is sufficient to program Th1 development through both common and distinct subsets of target genes. However, the ability of these isoforms to mediate inflammation in vivo has not been examined. Using a model of colitis that develops following transfer of CD4(+) CD45RB(high) T cells expressing either the STAT4alpha or STAT4beta isoform into SCID mice, we determined that although both isoforms mediate inflammation and weight loss, STAT4beta promotes greater colonic inflammation and tissue destruction. This correlates with STAT4 isoform-dependent expression of TNF-alpha and GM-CSF in vitro and in vivo, but not Th1 expression of IFN-gamma or Th17 expression of IL-17, which were similar in STAT4alpha- and STAT4beta-expressing T cells. Thus, higher expression of a subset of inflammatory cytokines from STAT4beta-expressing T cells correlates with the ability of STAT4beta-expressing T cells to mediate more severe inflammatory disease.     

Critical paracrine interactions between TNF-alpha and IL-10 regulate lipopolysaccharide-stimulated human choriodecidual cytokine and prostaglandin E2 production

Increased production of PGs by gestational membranes is believed to be a principal initiator of term and preterm labor. Intrauterine infection is associated with an inflammatory response in the choriodecidua characterized by elevated production of cytokines and PGs. The precise physiological significance of enhanced choriodecidual cytokine production in the mechanism of preterm labor remains uncertain. These studies were undertaken to dissect the roles and regulation of endogenous cytokines in regulating PG production by human choriodecidua. We used LPS treatment of human choriodecidual explants as our model system. In choriodecidual explant cultures, LPS (5 microg/ml) induced a rapid increase in TNF-alpha production, peaking at 4 h. In contrast, IL-10, IL-1beta, and PGE2 production rates peaked 8, 12, and 24 h, respectively, after LPS stimulation. Immunoneutralization studies indicated that TNF-alpha was a primary regulator of IL-1beta, IL-10, and PGE2 production, while IL-1beta stimulated only PGE2 production. Neutralization of endogenous IL-10 resulted in increased TNF-alpha and PGE2 production. IL-10 treatment markedly decreased TNF-alpha and IL-1beta production, but had no effect on PGE2 production. Taken together, these results demonstrate that the effects of LPS on choriodecidual cytokine and PG production are modulated by both positive and negative feedback loops. In the setting of an infection of the intrauterine, TNF-alpha may be a potential target for treatment intervention; IL-10 could be one such therapeutic.     

Cytolinker cross-talk: periplakin N-terminus interacts with plectin to regulate keratin organisation and epithelial migration

Periplakin is a cytoskeletal linker protein that participates in the assembly of epidermal cell cornified envelope and regulates keratin organisation in simple epithelial cells. We have generated a stably transfected MCF-7 subclone expressing HA-tagged periplakin N-terminus to identify molecular interactions of periplakin. Co-immunoprecipitation with anti-HA antibodies and mass spectrometry identified a 500-kDa periplakin-interacting protein as plectin, another plakin family member. Plectin-periplakin interaction was confirmed by immunoblotting of complexes immunoprecipitated by either anti-HA or anti-plectin antibodies. Transient transfections of periplakin deletion constructs indicated that first 133 amino acid residues of the N-terminus are sufficient for co-localisation with plectin at MCF-7 cell borders. Immunofluorescence analysis demonstrated that periplakin and plectin isoforms 1, 1f and 1k co-localise at cell borders of MCF-7 epithelia and that plectin-1f and 1k co-localise with periplakin in suprabasal epidermis. Ablation of plectin by siRNA in HaCaT keratinocytes resulted in aggregation of periplakin to small clusters. Scratch-wounded MCF-7 epithelia expressing periplakin N-terminus showed accelerated keratin re-organisation that was inhibited by siRNA knock-down of plectin. Finally, ablation of either periplakin or plectin, or both proteins simultaneously, impaired migration of MCF-7 epithelial sheets. Thus, we have identified a novel functional co-localisation between two plakin cytolinker proteins.     

Adenosine receptors regulate exosome production

Exosomes, small-sized extracellular vesicles, carry components of the purinergic pathway. The production by cells of exosomes carrying this pathway remains poorly understood. Here, we asked whether type 1, 2A, or 2B adenosine receptors (A₁Rs, A_2ARs, and A_2BRs, respectively) expressed by producer cells are involved in regulating exosome production. Preglomerular vascular smooth muscle cells (PGVSMCs) were isolated from wildtype, A₁R^?/?, A_2AR^?/?, and A_2BR^?/? rats, and exosome production was quantified under normal or metabolic stress conditions. Exosome production was also measured in various cancer cells treated with pharmacologic agonists/antagonists of A₁Rs, A_2ARs, and A_2BRs in the presence or absence of metabolic stress or cisplatin. Functional activity of exosomes was determined in Jurkat cell apoptosis assays. In PGVSMCs, A₁R and A_2AR constrained exosome production under normal conditions (p?=?0.0297; p?=?0.0409, respectively), and A₁R, A_2AR, and A_2BR constrained exosome production under metabolic stress conditions. Exosome production from cancer cells was reduced (p?=?0.0028) by the selective A_2AR agonist CGS 21680. These exosomes induced higher levels of Jurkat apoptosis than exosomes from untreated cells or cells treated with A₁R and A_2BR agonists (p?=?0.0474). The selective A_2AR antagonist SCH 442416 stimulated exosome production under metabolic stress or cisplatin treatment, whereas the selective A_2BR antagonist MRS 1754 reduced exosome production. Our findings indicate that A_2ARs suppress exosome release in all cell types examined, whereas effects of A₁Rs and A_2BRs are dependent on cell type and conditions. Pharmacologic targeting of cancer with A_2AR antagonists may inadvertently increase exosome production from tumor cells.

     

Presynaptic N-type calcium channels regulate synaptic growth

Voltage-gated calcium channels couple changes in membrane potential to neuronal functions regulated by calcium, including neurotransmitter release. Here we report that presynaptic N-type calcium channels not only control neurotransmitter release but also regulate synaptic growth at Drosophila neuromuscular junctions. In a screen for behavioral mutants that disrupt synaptic transmission, an allele of the N-type calcium channel locus (Dmca1A) was identified that caused synaptic undergrowth. The underlying molecular defect was identified as a neutralization of a charged residue in the third S4 voltage sensor. RNA interference reduction of N-type calcium channel expression also reduced synaptic growth. Hypomorphic mutations in syntaxin-1A or n-synaptobrevin, which also disrupt neurotransmitter release, did not affect synapse proliferation at the neuromuscular junction, suggesting calcium entry through presynaptic N-type calcium channels, not neurotransmitter release per se, is important for synaptic growth. The reduced synapse proliferation in Dmca1A mutants is not due to increased synapse retraction but instead reflects a role for calcium influx in synaptic growth mechanisms. These results suggest N-type channels participate in synaptic growth through signaling pathways that are distinct from those that mediate neurotransmitter release. Linking presynaptic voltage-gated calcium entry to downstream calcium-sensitive synaptic growth regulators provides an efficient activity-dependent mechanism for modifying synaptic strength.     

Apoptotic cells and innate immune stimuli combine to regulate macrophage cytokine secretion

Macrophage interactions with apoptotic cells can suppress inflammatory responses, but cell death by apoptosis may also trigger inflammation. We now report that murine macrophages exposed to the combination of apoptotic cells and archetypal ligands for Toll-like receptors (TLRs) 2, 4, and 9 mount cytokine responses that differ importantly from those elicited by either class of stimulus alone. TLR ligands induced early and sustained secretion of TNF-alpha, macrophage-inflammatory protein (MIP) 1alpha and MIP-2 with later secretion of IL-10, IL-12, and TGF-beta1; apoptotic cells alone stimulated late TGF-beta1 secretion only. The combination of apoptotic cells and TLR ligands enhanced early secretion of TNF-alpha, MIP-1alpha, and MIP-2 and increased late TGF-beta1 secretion, while suppressing late TNF-alpha, IL-10, and Il-12 by mechanisms which could nevertheless be overridden by IFN-gamma. We propose that this combinatorial macrophage cytokine response to apoptotic cells and TLR ligands may contribute to recruitment and activation of innate immune defense when cell death occurs at infected inflamed sites while promoting later resolution with diminished engagement of adaptive immunity.     

Cramoll 1,4 lectin increases ROS production, calcium levels, and cytokine expression in treated spleen cells of rats

This study reports the in vivo stimulatory effects of Cramoll 1,4 on rat spleen lymphocytes as evidenced by an increase in intracellular reactive oxygen species (ROS) production, Ca²⁺ levels, and interleukin (IL)-1β expression. Cramoll 1,4 extracted from seeds of the Leguminosae Cratylia mollis Mart., is a lectin with antitumor and lymphocyte mitogenic activities. Animals (Nine-week-old male albino Wistar rats, Rattus norvegicus) were treated with intraperitoneal injection of Cramoll 1,4 (235 μg ml^?1 single dose) and, 7 days later, spleen lymphocytes were isolated and analyzed for intracellular ROS, cytosolic Ca²⁺, and IL-6, IL-10, and IL-1 mRNAs. Cell viability was investigated by annexin V-FITC and 7-amino-actinomycin D staining. The data showed that in lymphocytes activated by Cramoll 1,4 the increase in cytosolic and mitochondrial ROS was related to higher cytosolic Ca²⁺ levels. Apoptosis and necrosis were not detected in statistically significant values and thus the lectin effector activities did not induce lymphocyte death. In vivo Cramoll 1,4 treatment led to a significant increase in IL-1β but IL-6 and -10 levels did not change. Cramoll 1,4 had modulator activities on spleen lymphocytes and stimulated the Th2 response.     

The cGMP/protein kinase G pathway contributes to dihydropyridine-sensitive calcium response and cytokine production in TH2 lymphocytes

Th2 lymphocytes differ from other CD4+ T lymphocytes not only by their effector tasks but also by their T cell receptor (TCR)-dependent signaling pathways. We previously showed that dihydropyridine receptors (DHPR) involved in TCR-induced calcium inflow were selectively expressed in Th2 cells. In this report, we studied whether cGMP-dependent protein kinase G (PKG) activation was implicated in the regulation of DHPR-dependent calcium response and cytokine production in Th2 lymphocytes. The contribution of cGMP in Th2 signaling was supported by the following results: 1) TCR activation elicited cGMP production, which triggered calcium increase responsible for nuclear factor of activated T cell translocation and Il4 gene expression; 2) guanylate cyclase activation by nitric oxide donors increased intracellular cGMP concentration and induced calcium inflow and IL-4 production; 3) reciprocally, guanylate cyclase inhibition reduced calcium response and Th2 cytokine production associated with TCR activation. In addition, DHPR blockade abolished cGMP-induced [Ca2+]i increase, indicating that TCR-induced DHP-sensitive calcium inflow is dependent on cGMP in Th2 cells. Th2 lymphocytes from PKG1-deficient mice displayed impaired calcium signaling and IL-4 production, as did wild-type Th2 cells treated with PKG inhibitors. Altogether, our data indicate that, in Th2 cells, cGMP is produced upon TCR engagement and activates PKG, which controls DHP-sensitive calcium inflow and Th2 cytokine production.     

Modulation of cytokine production by carnitine

The ability of carnitine congeners to modulate cytokine production by human peripheral blood mononuclear cells (PBMC) was investigated. Modulation of cytokine production by PBMC of young (30 years of age or younger) and old (70 years of age or older) normal donors was first compared. The PBMC were collected over Ficoll-Hypaque and incubated in the presence of various concentrations of acetyl L-carnitine for 24 h. Subsequently the supernatants were collected and examined for cytokine production. The presence of cytokines in tissue culture supernatants was examined by ELISA. The cytokines measured included IL-1alpha, IL-1beta, IL-2, IL-4, IL-6, TNFalpha, GM-CSF, and IFNgamma. The results showed that at 50 mug/ml of acetyl L-carnitine the most significant response was obtained for TNFalpha. In this regard four of five young donors responded, but only one of five old donors responded. More recently these studies were expanded to examine the ability of L-carnitine to modulate cytokine production at higher doses, 200 and 400 mug/ml, in young donors. The results of these studies showed that in addition to TNFalpha, significant production of IL-1beta and IL-6 was observed. These preliminary studies provide evidence that carnitine may modulate immune functions through the production of selected cytokines.     

Critical sulfhydryls regulate calcium release from sarcoplasmic reticulum

Rapid Ca²⁺ release from the sarcoplasmic reticulum (SR) can be triggered by either binding of heavy metals to a sulfhydryl (SH) group or by catalyzing the oxidation of endogenous groups to a disulfide. Ca²⁺ release has been monitored directly using isolated vesicle preparations or indirectly by monitoring phasic contractions in a skinned fiber preparation. SH oxidation triggered by addition of Cu²⁺ /mercaptans, phthalocyanine dyes, reactive disulfides, and various anthraquinones appears to involve a direct interaction with the Ca²⁺ release protein from the SR. A model is presented in which reversible oxidation and reduction of endogenous SH groups results in the opening and closing of the Ca²⁺ release channel from the SR.Abbreviations SR sarcoplasmic reticulum - SH sulfhydryl - T-tubule transverse tubule - 2,2-DTDP 2,2-dithiodipyridine - 4,4-DTDP 4,4-dithiodipyridine - DTT dithiothreitol